In an article entitled "Surgical Glove Perforation" Br. J. Surg. 1988, Vol 75, October, 966-968, it was stated "the surgical glove was introduced in 1889 by Halsted to protect his nurse assistant, Caroline Hampton, from dermatitis caused by mercuric chloride used for Listerian antisepsis. It was soon realized that sterile gloves provided a barrier between the surgeons, hands and the patient and could thus reduce iatrogenic infection. Lord Moynahan pioneered their use in the UK and the wearing of gloves during surgery has long been standard practice.
Modern surgical scrub technique dramatically reduces the bacterial counts on surgeons remain. It has often been stated that when glove perforation occurs, bacteria can escape and contaminate the wound, thus leading to an increased risk of wound sepsis. For this reason surgeons change their gloves during an operation when perforation has occurred although supporting bacteriological evidence is "scant". While the technology of surgical gloves has made tremendous advances in terms of quality of the touch retained by the user as well as a high residual manipulative capability of the hand and fingers including wearer comfort, the current American (voluntary) Standard ASTM D3577-78 permits new gloves to have 1/2% holes. Even more hazardous, at the end of operations 10-50% of surgeon's gloves are reported to have holes (Church, J., Sanderson, P.: Surgical Glove Punctures. Journ. Hosp. Infect. Control 1980, 1:84).
In the Br. J. Surg. publication identified hereinabove, the authors also reported on a study to investigate the incidence and significance of surgical glove perforation, bacterial contamination of surgeons' hands and gloves before and after operation was measured and the gloves tested for damage. Perforations were found in 74 of 582 gloves (12.7 per cent) and occurred in 34.5 per cent of operations. Glove perforation did not influence bacterial counts on the surgeons' hands or on the outside of their gloves. A separate clinical study of 100 adult hernia repairs gave no evidence that perforation increased wound sepsis. After standard clinical significance to the patient, but their high incidence should alert surgeons to the need for protection against pathogens transmissible during surgery, such as hepatitis B and the human immunodeficiency virus. Protection of the surgeon is an indication for preoperative change of damaged gloves throughout the course of the operation or surgical procedure. Increased incidence of risk for the persons performing activities with respect to persons having dreaded communicable diseases which are transmitted via the bodily fluids, make protection of the user of sharp instruments such as surgical knives and/or needles a major concern and has emphasized the need for the proper use of surgical gloves. Proper use is also particularly important for the person using the surgical instrument or conducting any activity inside the body of the infected or potentially infected patient and particularly when the surgical or protective gloves are penetrated and at the same time the user of the gloves is provided with little protection from such sharp objects as a needle, knife or scissor's edge, etc. While no evidence has yet established that a surgeon can contract the AIDS virus by penetrating his surgical glove and his own skin with a needle or similarly penetrating instrument, there is an overwhelming concern about the possibility which is to significant to ignore. The teachings of the present invention respond to this concern.
The problem with holes in surgical gloves has been studied by others in addition to the report sited hereinabove, one such report is by H. Matta, et al, entitled "Does wearing two pairs of gloves protect operating theatre staff from skin contamination?", pages 597-598--BMJ volume 290--Sept. 3, 1988. Therein, ten surgeons and nine scrub nurses in a surgical unit wore two pairs of gloves during general surgical operations on 144 consecutive patients. The gloves were tested at the end of the operation by a recognized method detecting perforation. The following table was reported: Numbers of punctures detected at different sites in 728 outer and inner gloves
______________________________________ Left hand Right hand Outer glove Inner glove Outer glove Inner glove ______________________________________ Thumb 6 1 Index 33 5 finger Third 6 1 4 1 finger Fourth 3 2 1 finger Fifth 1 finger Palm 16 6 5 1 ______________________________________
It is notable that three quarters of the perforations have occurred on the index finger of the left hand. It may therefore be presumed, particularly if both gloves frequently contained holes, that these were punctured holes produced by sharp instruments rather than tears or other violence; or, part of the permitted quality level original holes in the glove.
While this reported study confirms that wearing two pairs of surgical gloves confers some protection against contamination of the surgeons or nurses from the patients tissue and fluids, a very significant aspect of the results is that it identifies to a substantial degree where the glove punctures are located. For example, the non-dominant hand for the user of surgical instruments would be the left hand and therein is were the largest number of punctures are identified. That fact and the location of the punctures in the categories as set forth in the table leads to the conclusion that while all of the punctures or holes in the gloves are not the result of self inflicted punctures by the user through the use of the surgical instrument, many are. The locations of those punctures are identified and give the user an idea of where they may be causing punctures of the surgical gloves as well as the invasion of their own skin. The information described above is confirmed by an article entitled "Risk to Surgeons: A Survey of Accidental Injuries During Operations", Br. Journal of Surg. April 1988, pp:314-315.


VALLETTA (Reuters) - A million-euro ($1.2 million) reward for information on who ordered the murder of Malta’s most prominent journalist is still available, a year after her death.

Slideshow ( 9 images )

Daphne Caruana Galizia, 53, who wrote an anti-corruption blog, was killed on Oct. 16, 2017 by a car bomb. Three people arrested in December were charged with the killing, which they have denied.

Police believe an as-yet-unidentified person ordered the attack that Justice Minister Owen Bonnici called “the biggest crime that has ever happened in Malta”.

Prime Minister Joseph Muscat, a frequent target of Caruana Galizia’s writings, offered the reward last year.

The money would be paid for anything “which can unlock the facts behind the murder. So I reiterate the offer, it is still there,” Bonnici told Reuters in an interview.

The minister defended the daily cleanup of a national memorial opposite Valletta’s law courts that has become a shrine to the journalist’s memory and a center of protest against alleged corruption.

“We have the rule of law in Malta. We have procedures. You cannot simply wake up in the morning and appropriate yourself of a national monument,” he said, adding that he had asked the activists to apply for a permanent memorial to be set up.

Bonnici said the government had made strides to improve the freedom of journalists since the murder, including abolishing criminal libel.

But he declined to criticize the fact that Caruana Galizia’s family continued to face civil defamation lawsuits for what the journalist wrote, saying those who had been defamed felt they had the right to seek redress.

Local and international media groups, including Reuters, began following up stories covered by Caruana Galizia in the wake of her death, in an initiative called the Daphne Project.


---
title: JWT组件
meta:
  - name: description
    content: 基于EasySwoole组件实现的json web token
  - name: keywords
    content: swoole|swoole 拓展|swoole 框架|easyswoole|JWT组件
---

## 编码相关方法

设置加密方式，默认HMACSHA256
```php
    function algMethod(string $method):Jwt
```

设置秘钥，默认Easyswoole
```php
    function setSecretKey(string $key):Jwt
```

初始化一个没有附带信息的token的JwtObject
```php
    public function publish():JwtObject
```

设置加密方式, 默认HMACSHA256
```php
    public function setAlg($alg): self
```

设置用户
```php
    public function setAud($aud): self
```

设置过期时间
```php
    public function setExp($exp): self
```

设置发布时间
```php
    public function setIat($iat): self
```

设置发行人
```php
    public function setIss(string $iss): self
```

设置jwt-id，用于标识该jwt
```php
    public function setJti($jti): self
```

在此之前不可用
```php
    public function setNbf($nbf): self
```

设置主题
```php
    public function setSub($sub): self
```

设置其他数据
```php
    public function setData($data): self
```

获取token
```php
    function __toString()
```


## requires flask
## install by:
## pip install flask

from flask import Flask
from flask import request

app = Flask(__name__)

@app.route("/")
def get_agent():
    agent = request.user_agent.platform
    return f'hello {agent} user, ' \
           f'... a Flask WebServer, ' \
           f'made for Hacktoberfest'

if __name__ == "__main__":
    app.run(host='0.0.0.0', port='8080')


Irina Fedorovna Popova

Irina Fedorovna Popova (; born September 28, 1961) is a Russian sinologist and historian. Since April 2003 she has been the Director of the Institute of Oriental Manuscripts of the Russian Academy of Sciences (IOM RAS) at Saint Petersburg, Russia. She is also Head of the Department of Manuscripts and Documents of the IOM RAS, and Full Professor at St Petersburg State University.

Popova has been a visiting scholar with the Center for Chinese Studies in Taipei (Taiwan), University of Pennsylvania (USA), Nordic Institute of Asian Studies (Copenhagen, Denmark), and Peking University (China).

Education
Popova graduated in 1983 from the St Petersburg State University (Faculty of Asian and African Studies, Department of the History of Far East), and completed her Ph.D. in 1989 at the Russian Academy of Sciences. She holds a Doctor of Sciences degree in history (2000) from the Russian Academy of Sciences. The subject of her Ph.D. thesis (in Russian) is "Rules for Emperors" ("Ti-fan") by T'ang T'ai-tsung as a source on the Chinese Political Thought of the 7th century, and the subject of her Doctoral thesis (in Russian) is The Theory of the State Rulership in the Early T'ang China.

Research
Basing primarily on the documents of the Tang dynasty, Popova has contributed to the study of imperial rulership in medieval China and the study of the influence of the political practice and imperial ideology in pre-modern China. She has introduced into the academic use the original documents from Dunhuang and Turfan, Chinese epigraphic texts, visual sources on the history of Qing dynasty, archival material on the history of Russian expeditions in Central Asia and history of the Oriental studies.

Popova is a member of a number of editorial boards, including the Head of the Editorial Board of the periodicals Pis'mennye pamiatniki Vostoka, Written Monuments of the Orient, Strany i narody Vostoka (Countries and Peoples of Orient), Tulufan janjiu, Minzu guji yanjiu, Zhongguo lishi gudai wenxian (China), Studia Orientalia Slovaca (University of Bratislava, Slovakia), Shygys-Orient (Kazakhstan); and Deputy Head of the Editorial Board of the academic series Pamiatniki pismennosti Vostoka (Written Monuments in the Oriental Scripts).

Publications
She has written over 200 articles, 5 monographs (3 in cooperation with other researchers) and edited 10 collections of articles.

Awards
 2013 – St Petersburg City Government Prize in science and engineering (Prize in philology named after S. F. Oldenburg)
 2016 – Honorary rank "Yangzi Scholar" 长江学者 by Ministry of Education of China

Popova has been made an Honorary Professor of Lanzhou University (China), Ningxia University (China), Shandong University (China), Minzu University of China (Beijing, China), Shaanxi Normal University (Xi'an, China). She has also been awarded an honorary doctororate by the Institute of History of the Chinese Academy of Social Sciences.

References

External links
 Irina Popova
 Irina Popova. Searches and findings in Central Asia. Lecture 1, ACADEMIA

Category:1961 births
Category:Living people
Category:Russian historians
Category:Russian orientalists
Category:Russian sinologists


Gallien Krueger Fusion 550 Bass Head and RBX 810 cabinet. This stack is in absolute mint condition only about 6 months old, has only been played a handful of times, and has been in the same spot in my studio since it was purchased. The price is $1900.00. This is a savings of $400.00 over what you would pay for it in the store for a stack that is virtually never been used. Please contact me at 613-799-4762 or rob@uboat.ca

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The State of Bitcoin in Kenya

Following bitcoin’s birth in 2009 as a decentralized peer-to-peer payment network with fast transaction speeds and low transaction fees, one of its most predicted future application uses was for international remittance purposes. Many experts envisioned that bitcoin would be a driving force in reducing the costs of international money transfers to developing regions such as Sub-Saharan Africa and, thereby, create more financial inclusion for the unbanked.

While anticipated bitcoin remittances into Africa have still not taken off at a large scale, Africa has birthed several local Bitcoin economies spread across the continent.

In this article, you will be introduced to the state of bitcoin in Kenya to give you an insight into East Africa’s leading bitcoin hub.

Kenya’s Bitcoin Ecosystem

Kenya is among the four main Bitcoin economies in Africa next to South Africa, Nigeria, and Ghana. While bitcoin awareness and adoption are growing in many parts of Africa, only the countries mentioned above offer Bitcoin ecosystems that include startups, meetups, a local Bitcoin community, and bitcoin liquidity.

Kenya is the home to several bitcoin startups, regular bitcoin meetups, a blockchain-focused coworking space and accelerator; a population used to making mobile payments, and an increasing demand for bitcoin according to LocalBitcoins trading volumes.

BitPesa

You cannot talk about Bitcoin in Kenya without mentioning Kenya’s bitcoin startup poster child BitPesa. BitPesa was launched in 2013 with the aim to provide individuals and business with the opportunity to make low-cost international money transfers to and from Sub-Saharan Africa by leveraging the cryptocurrency bitcoin.

BitPesa has so far managed to raise over $6 million from leading blockchain-focused venture capital firms, including Blockchain Capital, Draper Associates, Digital Currency Group, and BitFury Capital, to grow its business and expands into new markets. BitPesa currently operates in Kenya, Uganda, Tanzania, Nigeria, Democratic Republic of Congo, Senegal as well as the UK and can process cross-border payments in over 30 currencies.

When BitPesa first started out, it focused on remittances for private individuals but has since changed its business model to focus primarily on B2B cross-border transactions. This decision was partly a result of BitPesa’s quarrel with Kenya’s leading telecoms company Safaricom, which switched off BitPesa’s access to its mobile money platform MPesa that it was using to turn bitcoin remittances into fiat currency for everyday users. BitPesa went to court against Safaricom to regain access to its mobile money platform but lost the case.

Nonetheless, through refocusing its business on B2B cross-border payments and by expanding into other regions in Africa, the BitPesa success story continues, and its recent $2.5 million funding round from January 2017 is a testament to that.

The Central Bank’s View on Bitcoin

Following the BitPesa vs. Safaricom court case in December 2015, the Central Bank of Kenya (CBK) issued a public warning against buying bitcoin by taking out newspaper ads to inform the public. The notice stated:

“virtual currencies such as bitcoin are not legal tender in Kenya and therefore no legal protection exists in the event that the platform that exchanges or hold the virtual currency fails or goes out of business…”

The CBK also suggests that the anonymous aspect of bitcoin transactions make them, “…susceptible to abuse by criminals in money laundering and terrorism financing.”

Since then, the Kenyan central bank has issued no official guidelines or public clarification on the use of bitcoin and other digital currencies.

At a meetup in Nairobi in summer of 2016, a leading member of Kenya’s Bitcoin community stated that the Kenyan Central Bank had informed local banks not to conduct business with bitcoin startups. Existing bitcoin startups have had their bank accounts shut down, making it very challenging for new bitcoin startups to open company bank accounts in Kenya.

Kenya’s Blockchain-focused Coworking Space and Incubator

In 2016, BitHub.Africa launched East Africa’s first bitcoin and blockchain-focused coworking space and startup incubator in Kenya’s capital, Nairobi.

Former BitPesa employee John Karanja founded BitHub.Africa in 2015 with the aim to become a center of excellence for blockchain technologies in Africa. BitHub.Africa offers two coworking spaces, an incubator for startups that are looking into developing blockchain-based solutions, and consultancy services for companies interested in learning more about blockchain technology.

Silicon Savannah Creates Promising Breeding Ground for Bitcoin, but Progress is Slow

The biggest hindrance for bitcoin adoption in Africa is the low level of internet penetration. Currently, internet penetration in Africa stands at 26.3 percent, but internet connectivity is mainly found in the major cities and is clustered across a few of Africa’s larger economies.

Kenya has the highest internet penetration, 66 percent, in Africa and is also one of the two leading Sub-Saharan countries in internet and telecommunication development next to South Africa. Consequently, the capital Nairobi has turned into a hub for tech startups, dubbed as Silicon Savannah. Furthermore, Kenya is leading Africa in mobile payment adoption with 86 percent of households having mobile money accounts according to McKinsey.

While these factors bode for a good breeding ground for bitcoin startups, only a few entrepreneurs are currently leveraging blockchain technology to develop solutions in Kenya. Aside from BitPesa and Bithub.Africa, other Kenyan blockchain startups include Remitano and Umati Blockchain.

Regarding merchant adoption, there is little to none in Kenya. According to CoinMap, a platform that collates and lists merchants that accept bitcoin, there are currently only seven vendors accepting bitcoin payments. Two travel agents, two tech companies, and three e-commerce stores.

Talking to several members of Nairobi’s Bitcoin community, they say that the main people using bitcoin are the young tech-savvy generation that is either receiving bitcoin as payments for freelance work or are buying bitcoin in an attempt to make an investment profit. Private peer-to-peer remittances using the cryptocurrency have not become a common occurrence in Nairobi.

With an unclear regulatory stance on bitcoin and the low level of bitcoin awareness and adoption, foreign blockchain startups are unlikely to enter the Kenyan market anytime soon. Local interest in the cryptocurrency is definitely on the rise, but like everything in this part of the world, it will take a little while longer for bitcoin to become a “thing” in Kenya.


[Pharmacological effects of Daphne genkwa and Chinese medical prescription, "Jyu-So-To"].
Daphne genkwa (Thymelaeaceae) has been used as a folk medicine in China. We investigated the effects of D. genkwa and Jyu-So-To on various pharmacologic models in mice including the azoxymethane (AOM)-induced colonic aberrant crypt focus formation assay, ornithine decarboxylase (ODC) activity assay, and two types of mouse ear swelling model. Administration of 236.3 ppm of Jyu-So-To in drinking water significantly suppressed AOM-induced colonic aberrant crypt focus formation (p < 0.05), with an inhibitory ratio of 46.7%. The effects of several extracts with organic solvents of D. genkwa on murine epidermal ODC activity were examined. In particular, the inhibitory ratio of the n-hexane extract was 30.8%. In the 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced ear edema model in mice, the methanol extract resulted in 56.3% inhibition compared with the control. On the other hand, there are two peaks of responses at 1 h (immediate-phase reaction; IPR) and 24 h (late-phase reaction) in biphasic cutaneous reactions, which are enhanced in the dinitrofluorobenzene model (DNFB). The water extract of D. genkwa clearly inhibited the IPR ear swelling. These results suggest that D. genkwa and Jyu-So-To should be a promising source of antitumor, antiinflammatory, and antiallergy agents.


General Programming In Julia Language From An Advanced Standpoint¶

Julia is a multi-paradigm language which is purely dynamic but supports optional typing.

As the introduction of some basic constructs like For-Loop , While-Loop and If-Else and Function akin to those of Python, R or MATLAB, Julia becomes simple and concise, and allows you to work fluently with a fraction of its language features.

Although People seldom focus on Julia’s language features other than its high performance(until April 2019), I’d show my point of view of the general programming in Julia.

First-Class¶ Almost all language constructs in Julia are first-class, upholding more flexible and advanced program composition. There’re quite a lot of people that has a prejudice against Python for the absense of multi-line lambdas. Although it’s pretty trivial and straightforward to compile multi-line lambdas into normal Python code objects, the frontend, more concretely the mandatory indentation, comfines Python to the expressive power as a result of that statements are rigidly separate from expressions(statements cannot occur in expressions), and then first-class gets missed. There’re other manners to achieve indentation without lossing expression-first like that of ML family and Haskell, but not adopted by Python community yet. Julia seems to be over-first-class, for all constructs there are expressions and, syntactically there’s no restriction to compose arbitrary constructs. Definitions and computations are distinct from each other in major functional languages, but each of both consists of the other. [ let y = f ( x ); ( x , y ) end for x in points ] map ( function map_fn ( x ) # do stuffs end , points ) let bind_maybe ( a , f ) -> a == nothing ? f ( a ) : nothing , ( |> ) = bind_maybe init |> a -> do1_stuffs ( a ) |> a -> ... |> a -> ... |> a -> ... end The reason why composability is crucial has been discussed often years on, so I won’t make an another about this topic. For Julia, first-class also helps when it comes to processing programs as data, which will be unfolded latter in this article.

Polymorphisms of Multiple Dispatch¶ Julia supports polymorphisms to achieve necessary abstractions and reduce self-repeating, though it does stand apart from those pooular industrial languages. Multiple dispatch looks like parametric polymorphism, but actually it’s more than the latter. function usum ( x :: Vector { T }) where T s = zero ( T ) for each in x s += each end s end Overloading is supported as well. uzero ( _ :: Type { Int }) = 0 uzero ( :: Type { Float64 }) = 0.0 However, multiple dispatch is more than what I listed above. In fact, type is exclusively a specialized instance of immutable data that could be taken advantage of dispatching, while you can make dispatches via immutable data no matter whether it is a type or others. struct Const { T } end flip ( :: Type { Const { : even }}) = Const { : odd } flip ( :: Type { Const { : odd }}) = Const { : even } f ( :: Type { Const { 0 }}) = Const { : even } f ( :: Type { Const { 1 }}) = Const { : odd } f ( :: Type { Const { N }}) where N = f ( Const { N - 1 }) |> flip With above codes, we can statically compute parity of numbers, just as expected. julia > @code_warntype f ( Const { 2 }) Body :: Type { Const { : even }} 1 ─ return Const { : even } Note that when multiple dispatch fails at static inferences, it’ll behave as dynamic dispatch like Python’s.

Full-Featured Macros¶ Macro is one of the quite few manners to achieve code reuse, also the reason of why some programmers can be thousands of times more efficient than others. julia > macro gen_var ( n :: Int , f ) defs = [ Expr ( : ( = ), Symbol ( "var" , i ), : ( $ f ( $ i ))) for i in 1 : n ] esc ( Expr ( : block , defs ... , nothing )) end @gen_var ( macro with 1 method ) julia > f ( x ) = x * 10 - 2 f ( generic function with 1 method ) julia > @gen_var f ( x * 10 - 2 ) julia > var1 8 julia > var2 18 julia > var3 28 Macro, the Function from AST to AST¶ Once you know macros are functions from ASTs to ASTs, there’s no mystery in Julia macros. macro f ( x ) println ( x ) : ( $ x + 1 ) end @assert ( @f 1 ) == 2 Above snippet shows a vivid example of Julia macros. Firstly macro keyword leads a definition of macro transformation rule, and @f marks a callsite of corresonding macro. You might ask why (@f 1) == 2 , for the return of macro @f is supposed to be an AST, it seems a bit magic that it equals to an integer 2 . Pay attention to the expression @assert (@f 1) == 2 . As the macro invocations are processed recursively from the inside out, we should firstly process @f 1 . ( function f ( x ) println ( x ) : ( $ x + 1 ) => : ( 1 + 1 ) end )( 1 ) Above step also writes stdio, when executing the AST to AST function f , a.k.a macro @f . Next, as we has already got the output, an AST :(1 + 1) , imagine that we displace @f 1 by it in the preceding codes, which produces @assert $(:(1 + 1)) == 2 , simplify it, we’ll get @assert (1 + 1) == 2 . You might ask why not @assert :(1 + 1) == 2 , good question, let’s dig into it. Think that if what you return from a macro invocation is always a runtime AST, it will not be transformed into codes to compile, so that such a macro becomes useless at all. However, if we “unquote” the macro return Unquote Rule ¶ Quoted Unquoted :(:(1 + 1)) :(1 + 1) :(1 + 1) 1 + 1 quote 1 + 1 end 1 + 1 quote $x + x end <x> + x , where <x> stands for some computated expression. 1 1 [1, 2, 3] [1, 2, 3] Above table unveils the rules of AST interpolations, and obviously there’s a law that if we say an expression is quoted N times, it’ll be interpolated as an expression quoted max(0, N - 1) times. Scope and Hygiene¶ The scoping rules of macros are simple enough when you are under the point of view that macros are functions from ASTs to ASTs. julia > module A var = 0 macro ma () quote var end end end julia > var = 5555 julia > A . @ma 0 julia > using . A : @ma julia > @ma 0 The first I’d present here is, the expression a macro return is evaluated by the module where the macro’s defined. When a macro is expanding inside the local scope of a function, a concept called hygiene comes up naturally. macro assign_y ( x ) : ( y = $ x ) end function f ( x ) @assign_y x y end f ( 1 ) You might expect it works, but unfortunately it won’t, and solely feed you with ERROR : UndefVarError : x not defined Stacktrace : [ 1 ] f ( :: Int64 ) at ./ REPL [ 6 ] : 2 [ 2 ] top - level scope at none : 0 The reason why for this is, AST interpolations will be always preprocessed to make sure all bare symbols(not boxed in QuoteNode or deeper quotation) are transformed into mangled names(a.k.a, gensym) that looks a bit weird like ##a#168 . Also, the reason why Julia does this is, to by default avoid generate new symbols visible in current local context. Just think about you want a macro to log the value just calculated: macro with_logging ( expr ) quote a = $ expr @info : logging a a end end We didn’t transform the symbol a into something like ##a#167 , what if you had already define a in your codes? function x5 ( a ) # take it as multiply 5 x2 = @with_logging 2 a x3 = @with_logging 3 a x2 + x3 end my_func ( 1 ) You can see that if macro with_logging didn’t transform a written in macro body, you’ll get x5(1) == 8 instead of x5(1) == 5 . That’s it, and we call this sort of macro the Hygienic Macros. But there does have some context-sensitive cases for code generation, that you want to share the context of multiple generator functions. A impressive example is my MLStyle, which I’m extremely proud of for it has reached a high performant pattern matching compilation with the extensibility(customizable) I’ve dreamed about since I started programming. In this package, when compiling patterns, I’d use manual gensym instead of depend on hygienic macros, for I might access specific symbols from separate functions that’ll generate some codes. In this case that people want to generate symbols that will contaminate scopes, Julia provides an escape mechanism to avoid gensym. macro assign_y ( x ) esc ( : ( y = $ x )) end function f ( x ) @assign_y x y end f ( 1 ) # 1 The previous code finally works after supplementing a esc invocation on returned AST. Other Useful Knowledge for Julia Macros¶ @__MODULE__ gets you current module. When you want to control which module to evaluate a given AST, you can use moduleX.eval(expr) or @eval moduleX expr . Although we already know macros are functions, something need to be stressed is, there’re 2 implicit arguments of a macro: __module__ and __source__ . __module is the module you invoke the macro in, __source__ is the line number node that denotes the number of the line you invoke the macro.

A Big Step Forward in AST Manipulations¶ Julia does a lot on ASTs, e.g., analysis, substitution, rewriting, and so on. As we’ve introduced the laws of AST interpolations, you might know that we can generate ASTs like following codes instead of in purely constructive manner. ex0 = [ 1 , 2 , 3 ] ex1 = : [ 1 , 2 , 3 ] ex2 = : ( $ ex0 + 1 ) # :([1, 2, 3] + 1), [1, 2, 3] here is already evaluated before interpolation. ex3 = : ( $ ex1 + 1 ) # :([1, 2, 3] + 1) That’s not that cool for there’re also other approximate supports in other languages in current stage. The advance is at the deconstructing of ASTs, which extremely impacts the way we could analyse ASTs. Think about a case that you’d like to collect positional arguments and keyword arguments from some function callsites. get_arg_info ( : ( f ( a , b , c = 1 ; b = 2 ))) # => ([:a, :b, :(c = 1)], [:(b = 2)]) get_arg_info ( : ( f ( args ... ; kwargs ... ))) # => ([:(args...)], [:(kwargs...)]) get_arg_info ( : ( f ( a , b , c ))) # => ([:a, :b, :c], []) How will you achieve this task? Attention! No matter how you’ll deal with it, think about whether you need to get a prerequisite about Julia AST structures? Say, you have to know Expr (a.k.a one of the most important Julia AST types) has 2 fields, head and args , or you have to understand the structure of a.b is Expr head : Symbol . args : Array { Any }(( 2 ,)) 1 : Symbol a 2 : QuoteNode value : Symbol b instead of Expr head : Symbol . args : Array { Any }(( 2 ,)) 1 : Symbol a 2 : Symbol b , or you have to make it clear that in vector literals, there’re Vector/Matrix Literals ¶ Julia code AST structure [1 2 3] Expr head : Symbol hcat args : Array { Any }(( 3 ,)) 1 : Int64 1 2 : Int64 2 3 : Int64 3 [1, 2, 3] Expr head : Symbol vect args : Array { Any }(( 3 ,)) 1 : Int64 1 2 : Int64 2 3 : Int64 3 [1; 2; 3] Expr head : Symbol vcat args : Array { Any }(( 3 ,)) 1 : Int64 1 2 : Int64 2 3 : Int64 3 [ 1 2 ; 3 4 ] or [ 1 2 3 4 ] Expr head : Symbol vcat args : Array { Any }(( 2 ,)) 1 : Expr head : Symbol row args : Array { Any }(( 2 ,)) 1 : Int64 1 2 : Int64 2 2 : Expr head : Symbol row args : Array { Any }(( 2 ,)) 1 : Int64 3 2 : Int64 4 To be honest, there’re so many detailed rules about the strcutrue, but is it really necessary to know them all if you’re planning to work with Julia ASTs(including analysis about their strcutrues)? No! Absolutely no! Although I know many of you older Julia guys are always checking and decomposing ASTs with your vast knowledge about Julia internals , I’d suggest you sincerely to start using MLStyle.jl, for the sake of your efficiency. At here, I’d introduce MLStyle’s AST Manipulations to you via giving some impressive examples. For sure this package will be displaced by some better alternative one day, but the underlying methodology wouldn’t change at all. Don’t open the link immediately now, just follow my introduction now, you don’t need to care much about a specific package or framework, but its core idea. A tremendous inspiration occurred to me on one day in the last year(2018) that, what if we can deconstruct ASTs just as how they’re constructed. In fact, You don’t have to know accurately about all AST structures when you start using corresonding syntaxes, like you just write a = [ 1 , 2 , 3 ] b = [ 1 2 3 ] c = [ 1 ; 2 ; 3 ] d = [ 1 2 ; 3 4 ] Does you have to know what will the their AST look like? No. A classmate of mine who knows only mathematics and has never got an experience in programming before can still write such codes fluently to complish his linear algebra homeworks, but he does feel annoyed when I try to explain the concepts of ASTs and how the ASTs he just written would look like. You might have notice the importance of using syntactic components, yes, it’ll simply makes progress in the history we manipulate programs as data. Pattern matching is an essential infrastructure in modern functional languages, which reduces the complexity of almost all logics via deconstructing data as how data is constructed. Okay, this sentence occurred twice now: deconstructing data as how data is constructed. Remember it, and it’s our principle in this section. Let’s think about how ASTs are constructed? Firstly, we can write raw ASTs, write them literally. ex = : ( a + 1 ) ex = : [ 1 2 3 ] Next, there are syntactic AST interpolations. ex = : [ 1 , 2 , 3 ] ex = : ( $ ex + 1 ) # :([1, 2, 3] + 1) That’s enough. Now, let’s introduce a @match . This syntax may be deprecated in the better alternative in your time, but you must be able to simply make an equivalence or superior via the better one with the new start-of-the-art pattern matching package at the time you’re reading this article. @match value begin pattern1 => value1 pattern2 => value2 end To support match literal ASTs, we must get a true with following codes, # rmlines is a function that removes all line number nodes in an AST. # you can fetch it here : https://github.com/thautwarm/MLStyle.jl#preview @match rmlines ( : ( let x = 1 ; x end )) begin : ( let x = 1 ; x end ) => true _ => false end Think about the principle we’ve figured out, okay, I’d stress it again here as I’m a shabby repeater: deconstructing data as how data is constructed. Then v = : [ 1 , 2 , 3 ] ex = : ( $ v + 1 ) @match ex begin : ( $ v + 1 ) => v == : [ 1 , 2 , 3 ] _ => false end Oooh! Do you understand it? Does it make sense in your opinion? AST interpolation corresponds to constructing, while AST interpolations occur in pattern it’s regarded as deconstructing. We can call it “AST extractions”. Now, let’s turn back to the original question, to implement get_arg_info mentioned previously. We should at first introduce some examples about constructing in the case of get_arg_info . If we want to pass arguments to f(a, b; c, d) , we can use f ( a , b ; c , d ) = a + b + c + d args = [ 1 , 2 ] kwargs = Dict ( : c => 1 , : d => 2 ) f ( args ... ; kwargs ... ) , which produces a result 6 . Notice about the form f(args...; kwargs...) , it might indicates that in AST level, positional arguments and keyword arguments are stored in arrays, respectively. Let’s have a try: args = [ : a , : b ] : ( f ( $ ( args ... ))) And you’ll get an output exactly as :( f ( a , b )) Good job, now we use MLStyle’s @match , following the rule deconstructing data as how data is constructed as well. args = [ : a , : b ] @match : ( f ( $ ( args ... ))) begin : ( f ( $ ( args ... ))) => args == [ : a , : b ] _ => false end Then you get a true as output. Think a while, and check the final implementation of get_arg_info : get_arg_info ( ex ) = @match ex begin : ( $ name ( $ ( args ... ); $ ( kwargs ... ))) || : ( $ name ( $ ( args ... ))) && Do ( kwargs = []) => ( args , kwargs ) _ => throw ( "invalid input" ) end || denotes the so-called Or-Pattern.


Quantitative proteomics in Friedreich's ataxia B-lymphocytes: A valuable approach to decipher the biochemical events responsible for pathogenesis.
Friedreich's ataxia (FRDA) represents the most frequent type of autosomal-recessively inherited ataxia and is caused by the deficiency of frataxin, a mitochondrial protein. It is known that frataxin-deficiency leads to alterations in cellular and mitochondrial iron metabolism and impacts in the cell physiology at several levels. Frataxin is thought to play a role in iron-sulfur cluster biogenesis and heme synthesis. Currently, cellular antioxidant defense is dysregulated when frataxin is deficient, which exacerbates oxidative damage in FRDA. Moreover, alterations in lipid metabolism have been observed in several models of the disease. To better understand the biochemical sequelae of frataxin reduction, global protein expression analysis was performed using quantitative proteomic experiments in Friedreich's ataxia patient-derived B-lymphocytes as compared to controls. We were able to confirm a subset of changes in these cells and importantly, we observed previously unreported signatures of protein expression. Among the novel protein signatures that we have identified, the decrease in CHCHD4 might partly explain some aspects of the molecular pathogenesis of FRDA. The identification of a core set of proteins changing in the FRDA pathogenesis is a useful tool in trying to decipher the function(s) of frataxin in order to clarify the mitochondrial metabolic disease process.


Smoking: It'll wreck your face.

Recently, researchers at Case Western University's Departments of Plastic Surgery and Otolaryngology set out to identify the effects of cigarette smoking on facial aging. To do so, they compared standardized photos of identical twins with different smoking histories. Their results reveal subtle but noticeable differences in the faces of smoking and nonsmoking twins.

From 2007 to 2010, researchers led by plastic surgeon Bahman Guyuron recruited a total of 79 pairs of twins, all of them attendees of the annual Twins Days Festival in Twinsburg, Ohio. Importantly, Guyuron and his team only recruited pairs in which one twin either smoked, or had smoked a minimum of five years longer than his or her sibling. Twins were photographed by professional photographers, and their photos were later analyzed by a panel of blinded judges, who graded wrinkles and ranked age-related facial features according to standardized scales.

Their results, which appear in the latest issue of Plastic and Reconstructive Surgery, were as follows:

Smoking twins compared with their nonsmoking counterparts had worse scores for upper eyelid skin redundancy [i.e. lax eyelid tissue, the result of gravity, loss of tissue elasticity, and weakening of the connective], lower lid bags, malar bags [aka "cheek bags"], nasolabial folds [the "smile-lines" that run from your nose to your mouth], upper lip wrinkles, lower lip vermillion wrinkles, and jowls. Lower lid hyperpigmentation [thought to contribute to dark under-eye circles] in the smoking group fell just short of statistical significance. Transverse forehead wrinkles, glabellar wrinkles [the vertical lines that form between the eyebrows, where the nose meets the forehead], crow's feet, and lower lip lines accentuated by puckering did not have a statistically significant differences in scores. Among twins with greater than 5 years' difference in smoking duration, twins who had smoked longer had worse scores for lower lid bags, malar bags, and lower lip vermillion wrinkles.

In other words: Smokers, unsurprisingly, tend to look worse for wear than their non-smoking counterparts. What makes the study interesting, of course, is that it provides the closest thing to a control group that researchers can get when conducting an investigation of this nature, allowing them to get a rough gauge for how much worse for wear the smokers appear. "This study details the specifics of facial aging brought on by smoking, which primarily affects the middle and lower thirds of the face," conclude the researchers. "It also demonstrates that a 5-year difference in smoking history can cause noticeable differences in facial aging in twins."

Which brings us to the photographs. Below are photos of four representative twin pairs who participated in the study. Can you guess which of these twins is the smoker, or has the longer smoking history?

All captions via Guyuron et al.

The twin on the left has smoked 17 years longer than the twin on the right. Note the differences in lower lid bags and upper and lower lip wrinkles.

Both twins are smokers. The twin on the right smoked 14 years longer than his brother.

The twin on the left is a nonsmoker and the twin on the right smoked for 29 years. Note the differences in periorbital [i.e. the eyelid or skin around the eye] aging.

The twin on the right is a smoker; the twin on the left is a nonsmoker. Notice differences in nasolabial creases [i.e. the lines running from the sides of the nose to the sides of the lips].

How'd you fare? I was four for four, but I'll be the first to admit that the differences were not as striking as I thought they'd be. It occurs to me that Guyuron and his colleagues would do well to continue the study, for completeness' sake; imagine, for example, a similar photo series comparing the twins' hearts and lungs, postmortem.

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Introduction {#s1}
============

One of the major clinical challenges in most patients with familial adenomatous polyposis (FAP) is the development of thousands of colorectal adenomas which can progress to carcinoma without proctocolectomy. Current medical management to prevent colon cancer is not sufficiently efficacious to eliminate the need for prophylactic surgery in FAP patients. Germline mutation of one allele of the *adenomatous polyposis coli* (*APC*) gene underlies the vast majority of FAP cases, with adenomas arising in large part from the somatic inactivation of the remaining wild type copy of the *APC* gene. *APC* inactivation is also seen in 80% of sporadic colorectal adenomas and colorectal cancers (CRCs). The primary means by which *APC* inactivation contributes to colon adenoma and CRC development is believed to be the resultant dysregulation in the levels and localization of the β-catenin protein, particularly the role of nuclear β-catenin as a binding partner for the T cell factor (TCF) family of transcription factors and the function of β-catenin/TCF complexes as key regulators of the expression of certain genes [@pone.0096023-SaitoDiaz1].

Rapamycin (RAP) is an inhibitor of the mammalian target of rapamycin protein (mTOR) which is strongly expressed in mouse small intestine polyps with *Apc* defects [@pone.0096023-Fujishita1] and in human colorectal adenomas and CRCs [@pone.0096023-Tuech1]. Additionally, a downstream phosphorylation target and likely physiological effector of mTOR signaling, the S6 ribosomal protein, shows increased phosphorylation (phospho-S6) in roughly 40 percent of colorectal carcinomas (CRCs) and adenomas [@pone.0096023-Nozawa1]. mTOR regulates cell growth, proliferation, and motility and inhibition inhibits cancer development or growth in various models [@pone.0096023-Easton1], [@pone.0096023-Alayev1].

The purpose of this study was to assess the ability of RAP to inhibit colon polyps in an *Apc* mutation-dependent colon polyposis model where both copies of the *Apc* gene were somatically inactivated in mouse colon epithelium. We found RAP treatment inhibited colon polyp formation, increased time to progression to dysplasia, and also increased survival. The tissue effects of RAP on *Apc* mutation-induced colon polyps included decreased proliferation and increased differentiation. Future studies may further bolster the case for use of mTOR inhibitors for polyposis inhibition in FAP patients.

Materials and Methods {#s2}
=====================

Antibodies and Reagents {#s2a}
-----------------------

Primary antibodies used in this study included: mouse anti-BrdU and mouse anti-β-catenin, BD Biosciences (San Jose, CA); rabbit anti-phospho-S6 ribosomal protein (Ser240/244) XP mAb, rabbit anti-phospho-mTOR (Ser2448) mAb, Cell Signaling (Danvers, MA); mouse anti- β-actin, Sigma-Aldrich (St. Louis, MO); rabbit anti-Sox9, Millipore (Bilerica, MA) and mouse anti-active β-catenin Milipore (Bilerica, MA). The secondary antibodies used were HRP-conjugated anti-rabbit IgG, Cell Signaling and HRP-conjugated anti-mouse IgG, Thermo Scientific (Cincinnati, OH). Dulbecco\'s Modifid Eagle Medium (DMEM), M199 and cosmic calf serum were from HyClone (Logan, UT). Tamoxifen, Alcian blue, human apo-transferin, hydrocortisone and sodium selenite were obtained from Sigma-Aldrich. Human epithelial growth factor was from Peprotech (Rocky Hill, NJ). Human insulin and 0.05% Trypsin-EDTA were from Invitrogen (Carlsbad, CA). Rapamycin (RAP) was purchased from LC Laboratories (Woburn, MA).

Cell Line and Culture {#s2b}
---------------------

The human colonic epithelial cell line (HCEC) was a kind gift from Dr. Jerry W. Shay [@pone.0096023-Roig1]. HCEC cell lines stably transformed with shAPC (HCEC/ptripzshAPC) or shScramble (HCEC/ptripzshSCR) were established. HCEC cells were seeded in DMEM + Medium 199 (4∶1) supplemented with 2% cosmic calf serum, 20 ng/ml human EGF, 10 ug/ml human recombinant insulin, 2 µg/ml human apo-transferrin, 1 µg/ml hydrocortisone and 5 nM sodium selenite. The cells were treated with Doxycycline (1 µg/ml) or vehicle for 48 hr, then, the cells were treated with RAP (0.001, 0.01, 0.1 and 1 µM) or dimethylsulfoxide vehicle for 24 hours in DMEM + Medium 199 (4∶1) with 0.2% cosmic calf serum, with or without 1 µg/ml of Doxycycline.

Mice {#s2c}
----

### Ethics {#s2c1}

We cared for mice and performed experimental procedures in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the University Committee on Use and Care of Animals at the University of Michigan and according to Michigan state and US federal regulations. All procedures were performed using isoflurane for anesthesia and all efforts were made to minimize suffering.

### Model {#s2c2}

*CDX2P-CreER^T2^* transgenic mice, expressing a tamoxifen-regulated Cre transgene under control of a roughly 9.5 kb human *CDX2* upstream regulatory element were used. The generation of the *CDX2P-CreER^T2^* transgenic mouse line \#741 used here has been described previously [@pone.0096023-Piso1], though as compared to the *CDX2P-CreER^T2^* transgenic line \#752 that was the focus of the paper of Feng and colleagues (9), transgenic line \#741 shows increased tamoxifen-regulated Cre function in distal colon epithelium. The *CDX2P-CreER^T2^* mice were intercrossed with mice homozygous for an *Apc* allele containing loxP sites flanking Apc exon 14 (*Apc^fl/fl^*, 580S) [@pone.0096023-Buunen1]. All the mice were housed in specific-pathogen free conditions. After weaning, rodent 5001 chow and automatically supplied water were provided *ad libitum* to mice. At 8--10 weeks of age, *CDX2P-CreER^T2^ Apc^fl/fl^* mice were injected with single dose of tamoxifen (TAM) \[0.07 g/kg, intraperitoneal (i.p.)\] to activate Cre recombinase. Following Cre-mediated targeting of the *loxP*-containing *Apc* gene, the targeted allele cannot express a functional Apc protein. Injection with RAP (3 mg/kg/day, i.p.) or vehicle was then started 2 days later. Administration of RAP or vehicle was continued daily for two weeks and then stopped for two weeks, and then cycled on and off in two-week intervals. Mice were weighed twice weekly. Mice were euthanized when near moribund or at various time points for tissue studies. In a second group of mice, dense polyps were induced (determined by endoscopy) and the mice were given RAP for 0, 1, 3, or 5 days and euthanized.

Endoscopy and assessment of polyps {#s2d}
----------------------------------

The colon was prepped by water enema introduced trans-anally via transfer pipette. The mice were anesthetized with 1.5% isoflurane. A small animal endoscope (Karl Storz Veterinary Endoscopy, Goleta, CA) was inserted trans-anally and advanced to the bend just proximal to the cecum and videos were recorded. Videos were later viewed and scored in a blinded fashion. Severity of the polyposis was scored according to the following polyp scoring system: 0, no lesion; 1, one small polyp; 2, few small distal polyps; 3, many small distal polyps; 4, many large distal polyps; 5, dense distal polyps. In mice with dense polyposis, the length of the dense area of polyps was measured in centimeters in the explanted colon after euthanasia and fixation of the colon under a dissecting microscope and compared to the total length of the colon excluding the cecum. Hematoxylin and eosin stained sections of the distal colon were assessed by a gastrointestinal pathologist (Joel Greenson, MD) to look for dysplasia.

Immunohistochemistry {#s2e}
--------------------

The mice were anesthetized with isoflurane and euthanized at defined time points or when moribund. Colon tissues were then removed and fixed in 10% Formalin overnight, prior to embedding in paraffin. One hr prior to euthanasia, BrdU (0.04 g/kg) was injected i.p. into the mice. Sections of formalin-fixed, paraffin-embedded mouse colon tissues were utilized for immunohistochemical analysis using the following primary antibodies: mouse anti-BrdU (1∶300), mouse anti-β-catenin (1∶800), rabbit anti-phospho-S6 (1∶100), rabbit anti-phospho-mTOR (1∶100) and rabbit anti-Sox9 (1∶1000). Antigen retrieval was performed by heating the sections in microwave in Antigen Retrieval Citra Solution (Biogenex, Fremont, CA) for 15 minutes prior to application of the primary antibodies. Appropriate secondary antibodies were used and the reaction was visualized using diaminobenzidine tetrahydrochloride (DAB) (Vector Laboratories), and cell nuclei were visualized by counterstaining with hematoxylin. BrdU staining was quantified by counting the number of positive and negative cells in at least 20 crypts and the percentage of positive cells per crypt was then calculated. The crypts were then divided into 3 equal parts (top, middle, and bottom) and the quantification was performed again.

Alcian blue staining {#s2f}
--------------------

Alcian blue was dissolved in 3% acetic acid to make 1% of Alcian blue solution. Sections were stained in Alcian blue solution for 20 minutes, and counterstained with hematoxylin. Alcian blue staining was quantified by counting the number of positive and negative cells in at least 20 crypts and the percentage of positive cells per crypt was then calculated.

Western Blot {#s2g}
------------

After treatment with RAP, cells were washed with cold PBS and lysed in RIPA buffer supplemented with complete Mini Protease Inhibitor Cocktail and phosphatase inhibitor cocktail, Roche Applied Science (Indianapolis, IN). Western blotting was performed as previously described [@pone.0096023-Knaus1]. The primary antibodies against active β-catenin [@pone.0096023-Staal1], phospho-S6, phospho-mTOR and β-actin were used at 1∶1,000 dilutions. Phospho-S6 or β-actin protein expression bands were detected with SuperSignal, West Pico chemiluminescent substrate (Thermo Scientific, Cincinnati, OH) and phospho-mTOR protein expression bands were detected with SuperSignal, West Femto Maximum sensitivity substrate (Thermo Scientific, Cincinnati, OH).

Quantitative Reverse-Transcription Polymerase Chain Reaction {#s2h}
------------------------------------------------------------

Total RNA from RAP treated HCEC\'s was extracted using the RNeasy Mini Kit (Qiagen). The following PCR primers were used: AXIN2, forward, 5′-TTCCCGAGAACCCACCGCCT-3′, reverse, 5′-GCCCCCTCCCGCGAATTGAG-3′; NKD1, forward 5′- CATGCACGACATGAAAATCC-3′, reverse 5′-TTGCCTGGATCTTGGAAAAC-3′; IRS1, forward 5′-CAAGACCATCAGCTTCGTGA-3′, reverse, 5′-AGAGTCATCCACCTGCATCC-3′. Real-time PCR was performed using the QuantiTect SYBR Green RT-PCR kit (Qiagen).

Reverse transcription of mRNA and real-time PCR were performed using the one step QuantiTect SYBR Green RT-PCR kit (Qiagen) according to the manufacturer\'s protocol. Relative expression of *AXIN2*, *NKD1* and *IRS1* were assessed and normalized with housekeeping gene U6.

Statistical Analysis {#s2i}
--------------------

Kaplan-Meier survival curves were generated and analyzed with log rank tests. For the cell culture experiment, two-way ANOVA was used. All other comparisons were performed using t-test or chi-square including 2 sample Z test where appropriate. P values of \<0.05 were considered significant. Statistical tests were performed using GraphPad Prism6 software (La Jolla, CA).

Results {#s3}
=======

Rapamycin Improves Survival in an *Apc* Mutation-dependent Mouse Colon Polyposis Model {#s3a}
--------------------------------------------------------------------------------------

We induced colon polyp formation in *CDX2P-CreER^T2^ Apc^fl/fl^* mice by administration of TAM to activate the function of the CreER^T2^ protein, so that the *Apc* alleles with loxP sites could be somatically mutated by Cre-mediated excision of *Apc* exon 14 in colon epithelial cells. Mice receiving TAM began forming macroscopic polyps within two-three weeks. Polyps progressed rapidly, with regions of dense polyposis apparent in the colon by three-four weeks. Mice were euthanized when near moribund or at defined time points for tissue studies. Mice were treated with 3 mg/kg RAP, which was given for 2 weeks and then stopped for 2 weeks and cycled on and off. The RAP dose and schedule used here was chosen to minimize the adverse effects of RAP. These effects included weight loss and diarrhea in preliminary studies. Treatment with RAP of the mice that had TAM-induced somatic *Apc* inactivation in colon epithelium significantly enhanced survival when compared to treatment with vehicle, p = 0.03, N = 6 mice per group ([Figure 1](#pone-0096023-g001){ref-type="fig"}). The median survival of mice receiving vehicle alone was 6.5 weeks versus 21.5 weeks for mice receiving RAP. We measured the body weight in the mice twice per week. We compared mice that were euthanized due to illness (excluding from the analysis those mice euthanized at defined time points), and we did not find significant differences in weight loss between the *CDX2P-CreER^T2^ Apc^fl/fl^* mice receiving vehicle or RAP (18 percent vs 25 percent weight loss) at the time of death. All of the mice that experienced significant weight loss had polyposis. In contrast, the mice in the RAP treatment group that were euthanized after 20 weeks at the completion of the study, rather than due to illness, did not experience weight loss and did not have polyposis. All of the mice that experienced weight loss had polyposis. However, the mice in the RAP group that were euthanized after 20 weeks due to completion of the study, rather than due to illness, did not experience weight loss and did not have many polyps, suggesting that the RAP treatment decreased the weight loss by reducing the polyp formation. We did not witness rectal bleeding in the mice but did note loose stools in both groups. The control mice that did not have Cre recombinase did not experience weight loss with or without RAP.

![Rapamycin (RAP) improves survival in a mouse model of polyposis.\
Kaplan-Meier survival curves where TAM administration was used to induce *Apc* mutation-dependent colon polyposis in *CDX2P-CreER^T2^ Apc^fl/fl^* mice and then mouse cohorts were treated with vehicle (dotted) or RAP (solid) and monitored for survival. Log-rank test comparing the groups treated with RAP or vehicle revealed a significant difference in survival (*p* = 0.03, N = 6 mice per group).](pone.0096023.g001){#pone-0096023-g001}

RAP Treatment Inhibits Polyp Formation in the Mouse Polyposis Model {#s3b}
-------------------------------------------------------------------

We performed endoscopies on each mouse every 1--2 weeks and recorded videos of the endoscopies which were later used to score the colon polyps in a blinded fashion. The cohort of mice that developed polyps following TAM administration and that were treated with RAP had lower polyp scores than the vehicle-treated polyposis cohort at every time point ([Figure 2A--C](#pone-0096023-g002){ref-type="fig"}). The polyp scores were statistically lower by 12 weeks of RAP treatment using 2-sample Z-tests of the proportions of mice with score 4 or higher (N = 6 per group; at 2 weeks, p = 0.12; at 6 weeks, p = 0.08; at 12 weeks, p = 0.01).

![RAP inhibits polyp formation and progression in a mouse model of colon polyposis.\
(A--C) *CDX2P-CreER^T2^ Apc^fl/fl^* mice were administered TAM to induce *Apc* mutation-dependent polyposis and were then monitored by video-endoscopy for polyp formation after treatment with vehicle (circle) or RAP (triangle). Scores of polyp severity in RAP- and vehicle-treated mice groups were determined at 2, 6, and 12 weeks for all mice that were alive. Each circle or triangle represents an individual mouse (N = 6 per group). The numbers of mice requiring euthanasia due to illness are represented at the top of each graph. The polyp scores were statistically lower by 12 weeks of treatment using 2-sample Z-tests of the proportions of mice with score 4 or higher (N = 6 per group, at 2 weeks, p = 0.12; at 6 weeks, p = 0.08; at 12 weeks, p = 0.01, \* signifies p = 0.01). (D) Representative endoscopic pictures of the mouse colons from a control wild-type mouse and TAM-treated and TAM- plus RAP-treated *CDX2P-CreER^T2^ Apc^fl/fl^* mice, with inhibition of polyposis by RAP treatment in the model. Polyps are indicated by black arrows.](pone.0096023.g002){#pone-0096023-g002}

[Figure 2D](#pone-0096023-g002){ref-type="fig"} shows representative endoscopic images from a control wild-type mouse and the *CDX2P-CreER^T2^ Apc^fl/fl^* mice, including the absence of colon polyps in a *CDX2P-CreER^T2^ Apc^fl/fl^* mouse with TAM-induced polyposis receiving RAP, as well as the presence of multiple polyps in a mouse in *CDX2P-CreER^T2^ Apc^fl/fl^* mice received only vehicle treatment. Selected RAP and vehicle-treated mice were euthanized at 7 weeks, and photomicrographs of the gross pathology of the large bowel were obtained. Inactivation of *Apc* alleles in colon and rectal epithelium via TAM administration to *CDX2P-CreER^T2^ Apc^fl/fl^* mice led to polyposis in the distal colon, and RAP treatment potently inhibited and delayed polyposis ([Figure 3A](#pone-0096023-g003){ref-type="fig"}). A group of mice was monitored and given TAM plus vehicle or RAP and euthanized when ill. The percentage of the colon covered with dense polyps at the time of death was found to be significantly lower in the mice receiving RAP ([Figure 3B](#pone-0096023-g003){ref-type="fig"}, [4](#pone-0096023-g004){ref-type="fig"}.3 +/− 2 vs 56.5 +/− 10.8 percent, p = 0.001, N = 6).

![Percentage of the colon with dense polyps is decreased in RAP-treated mice.\
A.) Gross appearance of colon tissue in *CDX2P-CreER^T2^ Apc^fl/fl^* mice with TAM-induced *Apc* mutation-dependent polyposis plus vehicle treatment for 7 weeks or RAP treatment for 7 and 20 weeks. Note distal colorectal mucosal thickening and visible polyps in tissue from mouse with TAM-induced polyposis treated only with vehicle. Individual polyps are indicated by black arrows. The areas of most dense polyps are indicated by brackets. B.) Mice were monitored and euthanized when ill. The percentage of the distal colon covered with dense polyps at the time of death was found to be significantly lower in the mice receiving RAP (4.3 +/− 2 vs 56.5 +/− 10.8 percent, \*p = 0.001, N = 6).](pone.0096023.g003){#pone-0096023-g003}

![RAP administration delays development of dysplasia in *CDX2P-CreER^T2^ Apc^fl/fl^* mice.\
*CDX2P-CreER^T2^ Apc^fl/fl^* mice were treated with TAM to induce polyposis and then treated with either vehicle or RAP. A.) Histology of vehicle and RAP treated mice. A group of mice were euthanized at 7, 16 and 20 weeks to compare histology. Arrows indicate areas of low and high grade dysplasia (LGD and HGD, respectively). B.) The second group of mice were allowed to continue until they became ill or beyond 20 weeks of treatment and then were euthanized. Mean number of weeks to visible high grade dysplasia in colonic polyps after TAM induction were recorded for individual mice treated with vehicle (circle) or RAP (square). We found that HGD was seen significantly earlier in the vehicle treated mice vs the RAP treated mice (5.1 vs 15.8 weeks, p = 0.003, N = 7 and 4 in vehicle and RAP group respectively).](pone.0096023.g004){#pone-0096023-g004}

High Grade Dysplasia Identified Later in RAP Treated Mice {#s3c}
---------------------------------------------------------

A gastrointestinal (GI) pathologist examined representative sections from 8 mice receiving vehicle and 7 mice receiving RAP treatments. Of the mice in the vehicle group, one was euthanized for a 7 week time point and the rest were euthanized because they were moribund with weight loss. Of the mice in the RAP group, 3 mice were euthanized because they were moribund with weight loss, one was a 7 week time point, and 3 were not ill but had lived beyond 20 weeks of treatment.

When the polyps at the time of death were assessed by a GI pathologist, 7 of the 8 vehicle treated colons were found to contain multiple regions of high grade dysplasia (HGD), and 4 of 7 mice receiving RAP had HGD (p = 0.16). Representative images are in [Figure 4A](#pone-0096023-g004){ref-type="fig"}. All mice that were euthanized due to weight loss and illness had HGD at the time of death. We then analyzed just the mice where HGD was found and compared the weeks of treatment between the vehicle and RAP treated mice. We found that HGD was seen significantly earlier in the vehicle treated mice vs the RAP treated mice (5.1 vs 15.8 weeks, p = 0.003, N = 7 and 4 in vehicle and RAP group respectively) ([Figure 4B](#pone-0096023-g004){ref-type="fig"}).

RAP Treatment Alters mTOR Signaling, Proliferation and Differentiation Markers, and Expression of β-catenin and Sox9 {#s3d}
--------------------------------------------------------------------------------------------------------------------

RAP has been shown to inhibit mTOR function, and the ribosomal S6 protein is among the proteins known to be phosphorylated by mTOR. When compared at 7 weeks after *Apc* inactivation (N = 5 mice per group), our immunohistochemistry studies revealed increased phospho-mTOR and phospho-S6 staining throughout the *Apc*-mutant adenomatous crypts in TAM-administered *CDX2P-CreER^T2^ Apc^fl/fl^* mice that were treated with vehicle. In comparison, we observed overall modest levels and more focal staining for phospho-S6 and phospho-mTOR in colon tissues of control mice or in colon tissues of *CDX2P-CreER^T2^ Apc^fl/fl^* mice with TAM-induced polyposis that had been treated with RAP ([Figure 5A-B](#pone-0096023-g005){ref-type="fig"}). Significantly increased bromo-deoxyuridine (BrdU) incorporation, reflecting increased cell proliferation, was noted throughout the TAM-induced, *Apc*-mutant adenomatous crypts of *CDX2P-CreER^T2^ Apc^fl/fl^* mice that were vehicle treated as compared to RAP treated mice(70.6+/− 1.5 vs 34.2+/−3.4 percent BRDU positive cells; p\<0.0001). In addition RAP treatment significantly limited cell proliferation to the lower half of the crypt, similar to the proliferation patterns seen in control colon crypts ([Figure 4C](#pone-0096023-g004){ref-type="fig"}). We divided each crypt into 3 equal parts: top, middle, and bottom, and counted BrdU-positive cells and calculated the percent of positive cells in each area in vehicle- and RAP-treated mice at 7 weeks after *Apc* inactivation. We found that the top and middle regions had significantly more positive cells for vehicle-treated vs RAP-treated mice but the bottom of the crypt was not significantly different between the groups (top: vehicle: 74.7+/−2.1 vs RAP: 6.8 +/− 3.1 percent with p\<0.0001; middle: vehicle: 70.0 +/−2.5 vs RAP 21.0 +/−5.3 percent with p\< 0.0001; bottom: vehicle: 63.4 +/− 2.2 vs RAP 57.8 +/− 3.5 percent with p = 0.1689).

![Long-term intermittent dosing with RAP suppresses markers of proliferation and increases differentiation after 7 weeks of treatment (N = 5).\
(A) and (B) Immunostaining of mouse distal colon shows increased phospho-mTOR (p-mTOR) and phospho-S6 (p-S6) expression in mouse polyps from *CDX2P-CreER^T2^ Apc^fl/fl^* mice injected with TAM. Both increases are suppressed by RAP treatment. (C) BrdU immunostaining showed increased incorporation of BrdU in mouse polyps and RAP partially reversed the effect. (D) Alcian blue staining shows loss of most of the differentiated goblet cells in mouse polyps and RAP restores the goblet cell differentiation. (E) Immunostaining shows increased expression of Sox9 (a β-catenin-regulated cell fate marker) expression in polyps and RAP decreased the expression. (F) Immunostaining shows increased nuclear and cytoplastic β-catenin staining in polyps and RAP treatment restores the membrane staining in most of the cells except a few strongly staining crypts. Mice assessed in the RAP group when moribund had rare areas of polyps with HGD that generally stained like mice in the vehicle group for all markers assessed (column 4, A--F). Scale bars  =  100 µm. The inserts are at two times higher magnification than the original.](pone.0096023.g005){#pone-0096023-g005}

Intestinal mucin, present in differentiated goblet cells, is detected by Alcian blue staining. In TAM-induced, *Apc*-mutant adenomatous crypts of *CDX2P-CreER^T2^ Apc^fl/fl^* mice, we found that Alcian blue staining was largely absent, and RAP treatment restored some of the differentiated goblet cells in *Apc*-mutant adenomatous crypts, albeit not entirely to the numbers seen in control colon epithelium (N = 5) ([Figure 5D](#pone-0096023-g005){ref-type="fig"}). The percentage of Alcian blue-positive cells was significantly higher in the RAP-treated group at 7 weeks after TAM injection (vehicle: 1.6 +/−0.3 vs RAP: 86.6 +/−1.2 percent, P\<0.0001).

Increased cytoplasmic and nuclear staining for β-catenin and increased nuclear staining for the β-catenin-regulated Sox9 transcription factor were seen throughout the TAM-induced, *Apc*-mutant adenomatous crypts of vehicle-treated *CDX2P-CreER^T2^ Apc^fl/fl^* mice (N = 5) ([Figure 5E and 5F](#pone-0096023-g005){ref-type="fig"}). In contrast, in control colon crypts, β-catenin staining was largely confined to the membrane and Sox9 staining was only seen in cells at the crypt base ([Figure 5E, 5F](#pone-0096023-g005){ref-type="fig"}). RAP treatment of *CDX2P-CreER^T2^ Apc^fl/fl^* mice largely restored β-catenin and Sox9 staining to the patterns seen in control mice, with the exception of a few strongly staining crypts (N = 5) ([Figure 5E, 5F](#pone-0096023-g005){ref-type="fig"}). Neither lysozyme staining for Paneth cell differentiation nor TUNEL staining for apoptotic cells were significantly different when vehicle- and RAP-treated groups of TAM-administered *CDX2P-CreER^T2^ Apc^fl/fl^* mice were compared (data not shown). The polyps with dysplasia at the time of death in the mice receiving RAP who became moribund were assessed histologically and by immunohistochemistry and were found to be similar to those in the mice receiving vehicle in staining patterns (fourth column, [Figure 5](#pone-0096023-g005){ref-type="fig"}). The staining in these polyps was not quantified as these areas of dysplasia were rare (n = 2--3). Additionally, as seen in [Figure 2A--C](#pone-0096023-g002){ref-type="fig"}, the polyps took longer to be visible on endoscopy.

To address the potential means by which RAP might be acting to inhibit polyposis, in a second group of mice (N = 5 per time point), we inactivated *Apc* in the colon epithelium of *CDX2P-CreER^T2^ Apc^fl/fl^* mice by TAM administration and followed the mice endoscopically until they had dense large polyps. The colon epithelium of the mice was then examined via immunohistochemical approaches after polyposis was induced (no RAP treatment) and after the mice had been treated with RAP for periods of one, three, or five days. The mice receiving RAP were found to have decreased staining for phospho-mTOR and phospho-S6 in colon crypts, along with decreased incorporation of BrdU and increased Alcian blue staining, particularly so by day 5 of RAP treatment ([Figure 6A--D](#pone-0096023-g006){ref-type="fig"}). Staining for days 1 and 3 were intermediate. We quantified the percent BrdU-positive cells in the mice that had developed polyposis and that had been treated with vehicle or RAP for 5 days, and we found that the colon epithelium of the vehicle-treated mice had significantly higher percent BrdU-positive cells as compared to the RAP-treated group (86.1 +/−1.2 vs 38.3 +/−2.0, p\<0.0001) (number of mice  = 5). We divided each crypt into top, middle and bottom compartments and we found significantly greater mean percent BrdU-positive cells in each compartment (top: 76.2 +/− 2.9 vs 5.1 +/− 1.5 percent for vehicle vs. RAP, p\<0.0001; middle: 84.8 +/− 2.4 vs 13.8 +/−3.2 percent for vehicle vs. RAP, p\< 0.0001; bottom: 85.5 +/− 1.8 vs 54.8 +/− 2.5 percent for vehicle vs. RAP, p = 0.0001). In addition, we quantified the percent of alcian blue-positive cells per crypt and found that after 5 days of RAP, there were significantly more alcian blue-positive cells in RAP-treated mice (vehicle: 1.9 +/−0.6 vs RAP: 86.8 +/−3.0 percent, P\<0.0001). Lysozyme and TUNEL staining showed no differences between vehicle- and RAP-treated groups (data did not shown).

![A brief course of RAP treatment decreases markers of proliferation and increases markers of differentiation.\
*CDX2P-CreER^T2^ Apc^fl/fl^* mice were injected with TAM and monitored until polyposis was seen endoscopically, then the mice were treated with vehicle or RAP at 3 mg/kg concentration for periods of one, three, or five days (N = 5 per timepoint). (A) and (B) Immunostaining of distal colons from these mice or Cre negative *Apc^fl/fl^* mice for phospho-mTOR and phospho-S6. Both phospho-mTOR and phospho-S6 show increased expression in mouse polyps due to *Apc* inactivation, which is suppressed by 5 days of RAP treatment. (C) Immunostaining of BrdU showed increased incorporation of BrdU in mouse polyps and RAP partially reversed the effect. (D) Alcian blue staining shows loss of most of the differentiated goblet cells in mouse polyps and RAP restore the goblet cell differentiation. (E) Immunostaining shows increased Sox9 (a cell fate marker) expression in polyps and RAP decreased the number of cells expressing Sox9. (F) Immunostaining shows increased nuclear and cytoplastic expression of β-catenin in polyps and RAP treatment for 5 days decreased the expression. Scale bars = 100 µm. The inserts are at two times higher magnification than the original.](pone.0096023.g006){#pone-0096023-g006}

RAP Does Not Antagonize the β-catenin-dependent Wnt Signaling Pathway in Intestinal Epithelial Cells {#s3e}
----------------------------------------------------------------------------------------------------

To further address the possible means by which RAP acts to inhibit Apc mutation-dependent colon polyp formation and progression, we studied immortalized human colon epithelial cells (HCECs) where APC expression was markedly inhibited via shRNA-mediated approaches. HCECs stably expressing a shRNA targeting APC or a control scrambled shRNA were treated with vehicle or various concentrations of RAP. Western blot showed that phospho-mTOR and phospho-S6 were significantly decreased by RAP in a dose-dependent manner but that active β-catenin [@pone.0096023-Staal1], which is presumably responsible for Wnt signaling, is not decreased ([Figure 7A](#pone-0096023-g007){ref-type="fig"}). Using real-time PCR, we found that the expression of downstream transcriptional targets of the β-catenin/TCF signaling pathway that are known to be elevated in colorectal adenoma and carcinomas, such as *AXIN2*, *NKD1*, and *IRS1*, were not decreased by RAP treatment in the HCECs ([Figure 7B--7D](#pone-0096023-g007){ref-type="fig"}). These findings suggest that the inhibitory effects of RAP on polyposis occur via mechanisms independent of direct effects on the β-catenin/TCF-dependent signaling pathway.

![RAP inhibits mTOR signaling in HCECs in a β-catenin/TCF transcription-independent manner in vitro.\
HCEC lines stably transformed with shAPC (HCEC/ptripzshAPC) or shScramble (HCEC/ptripzshSCR) were treated with Doxycycline (DXC) at 1 µg/ml to induce shRNA expression for 48 hr or untreated (-). The cells were then treated with RAP (0.001--1 µM) or vehicle (−) for 24 hr in the presence of Doxycycline under serum starving condition (0.2% serum). Total cell lysates were analyzed by Western blotting for phospho-S6 and phospho-mTOR. β-actin protein levels served as loading control (A). Significant decrease in phospho-S6 and phospho-mTOR levels was observed in cells treated with RAP. Expression of selected β-catenin/TCF-regulated target genes *AXIN2*, *NKD1* and *IRS1* were analyzed in the same cells by real time PCR (B--D). The results represent the mean ± SEM; n = 5 for each treatment group. There were no statistical differences between groups.](pone.0096023.g007){#pone-0096023-g007}

Discussion {#s4}
==========

The studies and data here demonstrate the utility of RAP in polyp prevention in a mouse colonic model of FAP. In this model, mice are given an inducing agent and both copies of *Apc* are inactivated in many colon crypts, leading to severe polyposis. We found significantly improved survival, decreased polyp formation, and prolonged time to development of dysplasia with RAP treatment. We found changes linked to increased proliferative features, such as increased β-catenin and Sox9 expression and increased BrdU labeling in polyps, and these changes were countered by short or long term dosing with RAP. We also saw decreased Alcian blue staining, indicating a loss of differentiated goblet cells, and the decrease in goblet cells was markedly reversed by as little as 5 days of RAP treatment. To further investigate the relationship between the mTOR pathway and the Wnt signaling pathway, we pursued focused studies of the effects of RAP on β-catenin/TCF-regulated target genes in cultured, immortalized HCECs where endogenous *APC* expression was dramatically inhibited by shRNA-based approaches. We did not find evidence that RAP directly affected the expression of β-catenin/TCF-regulated genes in the cultured HCECs, and the means by which RAP dramatically inhibits *Apc*-mutation dependent polyposis in mouse colon epithelium *in vivo* is likely independent of direct effects on the β-catenin-dependent Wnt signaling pathway.

Prior cell culture-based studies of RAP in colon cancer-derived cell lines have offered mixed results. Nozawa and co-workers treated eight different colon cancer cell lines with various concentrations of RAP and did not find any significant change in proliferation [@pone.0096023-Nozawa1]. However, Shao and colleagues did find RAP inhibited proliferation in an intestinal epithelial cell line with an inducible expression construct expressing a K-Ras^Val12^ mutant protein [@pone.0096023-Hildebrandt1]. Yet another study from Ponnurangam and colleagues reported that Tandutinib, which inhibits mTOR signalling, decreased proliferation in SW480 cells and in HCT116 xenografted tumors growing in immunocompromised mice, but the effect of Tandutinib was not purely through the Akt/mTOR pathway [@pone.0096023-Ponnurangam1].

Prior studies in Apc^min/+^ mice, where polyp formation is predominantly in the small intestine and animals die of anemia and weight loss, showed that adding RAP to their food decreased polyp formation, improved anemia, decreased weight loss, and improved the defects in colon electrolyte transport [@pone.0096023-Koehl1]. In this study by Koehl, the authors acknowledged that they did not see an effect on colon polyps, which are rare in Apc^min/+^ mice. Our mouse model produces extensive polyposis in the distal colon and our findings demonstrate substantive effects of RAP in inhibiting colon polyposis and the development of high grade dysplasia in colon epithelium. As such, our findings indicate that quite dramatic inhibitory effects of RAP on Apc mutation-dependent tumorigenesis are also clearly evident in colon epithelium.

Prior animal studies have found that Everolimus, another mTOR inhibitor, decreased polyp formation in the small intestine and reduced mortality in APC^Δ716^ mice [@pone.0096023-Fujishita1]. The mechanism for the in vivo effects of Everolimus on intestinal polyp formation in the APC^Δ716^ mice was not elucidated. The authors of that work did in fact provide data indicating that siRNA mediated inhibition of β-catenin expression in the SW480 and DLD-1 colorectal cancer cell lines decreased both total and phosphorylated mTOR levels. Studies and data on the possible effects of mTOR inhibition on downstream β-catenin/TCF-regulated transcriptional targets were not addressed. While endoscopic evaluation of the regression of individual *Apc*-mutant mouse colon polyps following RAP dosing has been described [@pone.0096023-Miller1], [@pone.0096023-Hinoi1], the studies in this paper are to the best of our knowledge the first to show dramatic prevention of polyposis formation and/or progression in an *Apc* mutation-dependent colonic polyposis model. The advantages of the model of colon polyposis used in the studies here relative to previously described mouse models include the following: I) dramatic colon polyposis is induced in adult mice at a defined time point by TAM administration; II) a relatively predictable pattern and number of colon polyps arise within a three-four week period; and III) individual mice can be serially studied by endoscopy to assess changes in polyposis over time, including the effects of potential inhibitors of polyposis development and/or progression.

There are currently no definitive medical management alternatives to proctocolectomy for the long-term clinical management of FAP patients. Because proctocolectomy can have potentially significant complications and life-altering effects, improved medical management strategies to forestall surgery would be highly desirable. RAP is unlikely to be a strong candidate for in-depth clinical studies in FAP patients, due to its side effects such as immune suppression and inhibition of healing [@pone.0096023-Rodel1], [@pone.0096023-Kenniston1]. Other clinically useful inhibitors of mTOR, with less significant side effects than RAP, may have greater potential promise. We used RAP for the studies here as a proof-of-principal reagent for mouse colon polyposis inhibition due in large part to cost consideration issues. Another potential use for drugs inhibiting mTOR in FAP patients might be to prevent upper gastrointestinal tract polyps, especially as pancreaticoduodenectomy, which is necessary when upper tract polyposis becomes too extensive to manage endoscopically, is associated with considerable morbidity.

Further studies that better discern the mechanism of inhibition of colon polyposis by mTOR inhibitors in mouse models are expected to be of considerable benefit in developing new approaches and agents for the inhibition of polyposis in FAP patients and perhaps in other patient populations with APC-mutant colorectal tumors.

We thank Thomas Wang, MD, PhD for generously allowing us to use his mouse endoscopy equipment for the studies presented here and Rork Kuick, PhD for his assistance with the statistical analysis. This was presented at the American Society of Colon and Rectal Surgery Meeting as a Podium presentation in Phoenix, Arizona, April 27 -- May 1, 2013

[^1]: **Competing Interests:**The authors have declared that no competing interests exist.

[^2]: Conceived and designed the experiments: KMH JL YF ERF JKG. Performed the experiments: KMH JL JKG. Analyzed the data: KMH JL YF ERF JKG. Contributed reagents/materials/analysis tools: YF ERF. Wrote the paper: KMH JL YF ERF JKG.


acidity

Marisa McClellan, of Food In Jars, talks about sugar’s role in canning, the purpose of acid, when a boiling water bath works and when it doesn’t and why, the advantages of canning in small batches, and more.

The USDA recommends always using bottled lemon juice in preserving recipes. The acidity is consistent, but it's also full of sulfites. So is the juice of fresh lemons acidic enough to use? One blogger decided to get some answers.


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Welcome to our country house bed & breakfast. Hempstead Hall is a 19th Century farm house steeped in history with records dating the farm back to the 13th century. Enjoy a relaxed stay with the Mack family on our working arable farm that stretches to 300 acres of tranquil North Norfolk countryside.

Arriving down our private 1/2 mile long drive you will notice the hall emerging from the end of our front meadows where cattle, horses and donkeys can often be seen grazing, or the resident barn owl hunting . Arriving in the farm yard you will know you are in for a relaxing time, with no sound of traffic, just the chatter of the farmyard ducks. Home to the Mack family since the 1930's Hempstead Hall is a fine example of 19th century architecture. The interior is decked with period furniture to maintain that historic feel and guests dine in the large dining room around a grand dining table when tucking into one of Lynda's famous traditional farmhouse breakfasts.

Take walks to see the local wildlife, cycle to the coast for delicious dining or shop till you drop in the Georgian town of Holt. Quite simply you cannot beat Hempstead Hall for location. Just 2 miles from the delightful town of Holt and 5 miles from the coast, the possibilities are endless. You can relax in the kitchen garden amongst the well maintained flower and vegetable patches watching ducks and chickens strutting their stuff. Once you have enjoyed a breath of fresh air why not plan that evening out at one of the many top class pubs and restaurants that North Norfolk has to offer.

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The Farm and its History

Hempstead Hall Farm is run as a family partnership and currently headed by William Mack. The farm is steeped in history with many historical finds mapped on the farm, impressive farm buildings dating from the 1700’s and a crater caused by a German bomb being dropped during WWII. The Mack family have farmed at Hempstead Hall since William’s Grandfather and Father purchased the farm from the Gurney family in the 1940’s. When the farm was purchased it was in a very poor state (as was UK agriculture). Henry Mack (William’s late Father) took it upon himself to tidy up the farm and built a business based on a mixed system of arable crops combined with beef & dairy herds.The herds gave way in the mid 1960’s and the business utilised the light North-Norfolk soils to concentrate on growing malting barley. The Mack family take conservation and the environment very seriously and this is notable by the many species of bird that can be found on the farm. William was presented with a wildlife conservation award by John MacGregor (agriculture minister) in 1987. This tradition of farmland conservation has continued and the farm is currently part of the Higher Level Stewardship scheme with grasslands, wild bird seed mixes, pollen and nectar mixes and many bird and bat boxes all contributing to flourishing wildlife numbers on the farm.

Guests are welcome to walk around the farm and try to spot the resident deer population, buzzards and red kites riding the thermals, numerous brown hares playing in the fields plus many many more.

Tariff and Booking Info

Large Double/ Twin room with en-suite bathroom £42 per person per night

Double Room with private bathroom £39 per person per night

Freeview flatscreen Television, hairdryer,alarm clock and tea/coffee facilities are provided in the rooms. Our bathrooms have a bath and a hand held shower. Rates are inclusive of a traditional farmhouse breakfast utilising free range eggs from the farmyard hens and locally sourced bacon & sausages. We use home grown tomatoes from the greenhouse when in season. Sorry adults only and no pets. Minimum stay 2 nights. Access statement available on request. Wifi is available.

If you have any questions please do not hesitate to telephone Lynda Mack on 01263 712224 or click here to email. Please note bookings can only be made by telephone.

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Gallery of Hempstead Hall and Local Area

IMG_5255.JPG

Dining Room.JPG

Staircase.JPG

DoubleTwin.JPG

3donkeys.JPG

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Kitchen Garden.JPG

Bluebells.JPG

Relax.JPG

farmyard.JPG

Chicken.JPG

Barn Owl.JPG

Donkeys.JPG

Blakeney Harbour.JPG

beans seal trips.jpg

Blickling Hall.jpg

Cley Beach.JPG

Cley Marshes.JPG

Cley Windmill.JPG

Cromer Lifeboat Station.jpg

Felbrigg Hall.jpg

Pensthorpe Nature Reserve.jpg

The Poppy Line.jpg

MAP & LOCATION

Hempstead Hall is quietly set half a mile down a private well made drive.

When arriving at Holt roundabout take the A148 towards Cromer, in about 1/4 mile take the first right onto the Hempstead Road also signposted to Baconsthorpe.

After 1.5 miles at the green with the Hempstead village sign fork right at the crossroads towards the village.

After approx 400 yards take the first right into Hempstead Hall's private drive. Post code for SATNAV users: NR25 6TN


Can We Help You?

More about 392 Harrison Lane, Henderson, NC 27537Lot joins Kerr Lake Property with access to water, has slip in Community Dock at no additional cost. gated community, paved roads, storage area for boats, street lights,great lot for walk out basement, close to I-85 and Hwy.1. Lot already cleared for House.Also includes Lot 30A.

Location for 392 Harrison Lane

Drive direction to 392 Harrison Lane, Henderson, NC 27537

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