Monday, October 29, 2018

sustainable piece does not come about by chance

United Nations peacekeeping is a concrete example of the multilateral at work. you demonstrate how the Global community can address some of today's most complex and dangerous issues with a mixture of creativity and Magnetism.

since the first blue helmet well deployed in 1948 peacekeeping has annabelle the countries of the world to meet common threats to peace and security and share the burden under the UN flag.

over the past 7 years more than 1 million peacekeeping women and men soldiers police and civilians from the countries across the world have responded to Abha strangers of conflict and peace keeping has adapted constantly to meet the demands..

in mail and Central African Republic I saw the important work the blue helmet to every day not only keeping the peace about supporting the delivery of human tourism ad protecting civilians.

I have also late too many rate for filling peacekeeper.
we have enacted new measure to address the rise in fatalities and have commissioned independent strategic review of each peacekeeping operations.

Expectations of peacekeeping fastly outstripped both support and resources.

Friday, October 12, 2018

Textile processing

Weaving machines identified as either Patel or Saturdays explaining they are filling process.

Textile processing

The first step in the textile process is spinning.

This is paid transform fibre into yarn..

Raw fibre like cotton and oil mask the span for making textile products..
Years ago this process used to be performed by hand but development in technology and machinery have create a machinery control process.

The iron produced by this machine is classified as either filament who is a long continuous strand or staple.

Filaments hot short length of iBall filament yarn spinning is an easier process then is staple yarn production and also cheaper.

The various component of the spinning process include

Carding removes impurities from the fibres.

Combined straighter the yarn.

Drawing create awareness in the yarn.

Roving inserts twist into the yarn to hold the fibre together.

Shading separating the warp into two layers.

Picking inserting more and while the shed is open.

Beating pushing the feeling yarn into the oven fabric.

Weaving machine or identified as Idol on Saturdays which explain their filling process..

The moment we make Vai places safe for women mast involve yrs to all

In what has been called India's me to home and the social media I think with women coming for 3 days to reach the 6th August harassment.

In the interview in the End of September of harassment at the hands of Actor Nana Patekar on the comfort A decade ago and have been speaking of their Experience At The trauma mostly on the Twitter and Facebook.

The testimonial on sofa the mostly concerned him all the word and the mainstream media David bothra classes in the private spaces from the story of the soul dance training and testing.

In the best majority of cases the naming of the result of the features of the receiving gauge of women from the systems and signal that is no longer possible for such behaviour scooby Bracelet and necklace because the following the boy the main twisted pointed out the current year here I go to this even come out there after the another of the first converter gallons of Sheldon.

In the Outsourcing of India to wear on disturbing picture is amazing it is not only this many of the celebration that extremely Crave for internship against Christianity exposed aggregate about the left journalism from government to become this minister of the state of external affairs for group discussion.

Wednesday, October 10, 2018

The factors that help who forgive

What are the factors that may help us to stay calm at work what is there to let it down when you forgive and forget your letting go of the past and what is happen I forgive the person for what has happened then I am going to forget about it one technique to prepare ourselves to be forgiving so we can stay call at work is meditation the problems of life will not in how can we can focus our intention and peace so we can realise about life difficulty meditation place .

How to remain calm at workplaces

As we go about our life who is seldom think about the world we speak or the action we perform.

Who is yet to happen without giving a thought of possible consequences.
Samoan does something at work I do not like so Vilas out at them.

Haan bol resentment for our dates quick month and sometimes Years. At work consider how many time people think angry thoughts about others. Consider how many time who is say something Housefull 2 someone else.

If we had been and had tempered and we had other most likely they will react in a similar fashion.

So we became the object of our Angar.
It is easy to fall victim not only to others who may hurt or mystreet us but also to our own anger.
It takes strength to overcome anger.
Apex Systems actually second link to emotion out of weakness. It takes one who is brave at hard to resist anger.
At work when faced with people are going or fighting with us we can choose to retaliate and heart them back we can forgive them or just stay cool instead of holding a grudge and increasing our Lord of the word and deed of retaliation.

When we forget we also help Aircel physically and mentally.

There is physical Haal we do to our sale when we all caught up in anger hatred and vengeans.
the solution for protecting the body against harmful hormone released by Hitler angle Vengeance simple forgiveness.
It is only soap for fairness that we can calm down and.
A majority of time Airtel job responding to minor problem with the same intensity as your life written.
we can learn how to stay at work through.

Monday, October 8, 2018

Improvement of Mental Health

People should be open to listen they do not have to solve the problem but this should not be dismissed it of someone who is trying to access their problem and refer them to a specialist.

Anxiety is when a person feel that the world is overwhelming and depression is when a person feel underwhelmed.

In both cases however people often do not seek help.

The most common disorders for anxiety and depression.

It is very common among Toma victim especially victim of sexual violence to stay bottled up for long as the land to feel guilty NCR and stigma associated with it.

Global is 70 person have suffered some kind of Soma in their life and 4 person suffer from a post traumatic stress disorder.

Sex and afraid that stigmatization is global and not just a challenges for India it is not going away because our society does not accept mental illness or like physical illness and need same type of care.

Our society label them as terrible red and dangerous.

Less than 10 person of people who have mental illness globally get any kind of treatment and even in developed countries only 50% of those patients are treated.

If someone says that they feel like killing them since it should be considered as a medical emergency.

Depression is treatable.
To identify the sign one has to be observant.

The red flags that one should look for include frequent mood swings daily routine getting affected not sleeping adequately and change it in Qatar for more than 2 week.

you

Techling mental illness begins by landing and ear.

The first and waste away to deal with mental illness is listen to people if they can buy an expired say that section on mental health.

Dr David C I N Wilson professor and Associate share of the psychiatry and behavioral science standard University School of medicine and doctor Sekhar Saxena visiting professor harvest Chan School of Public Health spoke on mental health how to start conversation.

people should be open to listening they do not have to solve the problem but they should not be dismissive of someone who is trying to access their problems and refer them to a specialist.

The most common mental disorder and anxiety and depres when a person feel that the world is overwhelming and depression is find a person feel underwe

Central command to keep Hawk eye on Civic services

Help at end by March 19 the command and control centre at NDMC headquarter at Palika Kendra will help in better coordination real time monitoring and fixing of public grievance.

President of call playing had Logistic complaint with a New Delhi Municipal Council about damages road in his neighborhood.
The next day he comes to know that the Civic agency head resolve the problem on Paypal but nothing had actually improved on ground.
The next day he had to a file access complain and start from the scratches which was a time consuming process.
But chance is at hand and no complain maybe have to go through his experiences by the market next year as the city currency is in the process of setting a command and control centre and its headquarter in the Palika Kendra for the better coordination real time monitoring and fixing a public grievance.

The initiatives first any Municipal agency in Delhi is under the Smart City project.

Council chairman Naresh Kumar said that system would provide live update of all 35 until the Civic service by various department and help keep a busy on the movement of all IT services vehicle has a garbage lifting traction machine mechanical people complaint request received for specific utility services.
The centre is likely to be operational in by March 2019.

Explaining how the system will work a senior official NDMC Information Technology department faith we have already installed Global positioning system on the girl that the garbages are used for electricity for road repair and other Maintenance Services.
detailed part 2 monitors in real time for movement of services may get there is faith the route before law and duration of their halt at any specific location.

In case they tryna divert from the ER marked wrote the controller at the centre will immediately send an alert to the operator and ask about the reason.

The Civic agency has decided to integrate the theosophical with the radio frequency identification system installed at residential dwelling.

The RFID plate installed outside houses will read the tag and sticker past in front of vehicle and plans for the information to command centre.

this will help us to find out the exact time the place duration for which the vehicle had visited the house.

After attending a complaint the staff will men with really need to upload real-time photograph of the place on NDMC 311 app as group of.

The central will record daily information of the number of people visiting inoculation Centre death and birth registration Centre property tax collection centre.

The information will be forwarded to the department of identification of grade areas.

For example if mole percent solicited the inoculation centre in the month of November then arrangement will be made for accidental kids at the time of the year.

Similarly the finance department will be able to do comparative study of property tax collected from the various residential and commercial areas.

The second part with included close monitoring of Civic services with the help of 2,500 closed circuit camera installed at NDMC parking lot smart a lot market Complex cell service Thane Law School offices complex as Park public places etc.

This official will watch video and issue instruction in the field stuff.

Advance ruling of GST

Scope of advance ruling expanded substantially under GST

Advance ruling can be sought in respect of classification time and value of supply registration admissibility of ITC.

GST lodge provide for the facility of appeal against the advance ruling.

Advance ruling can be obtained easily as an advance ruling authority has been constituted for all the states.

E-Commerce operator means any person who own operate or managers digital electronics facility for platform for eCommerce.

Search person need to be registered under GST irrespective of the value of suppliers.

No threshold exception is available.

E-Commerce operator or required to collect tax at sources at a rate of one person of net value of taxable suppliers confidence is collected by them.
Thank you very much for collecting and addressing such a beautiful topic about GST.
Goods and simple tax

Kejriwal 6 support of non BJP CM against changes in power act

Aam Aadmi Party kondana and Delhi Chief Minister Arvind Kejriwal on Saturday road to Chief Minister of 11 states that are not ruled by the Bharatiya Janata Party seeking their support in his standing against the proposed amendment to the Electricity Act 2003.

Kejriwal said he will also travel to the state to build a confessions to install the amendment.

The electricity Amendment Bill 2014 was introduced in the Lok Sabha in December 2014.

It was then referred to the standing committee on energy and after it is recommendations consultations held with the all states. The central head pain the proposed amendment to the states for their feedback on September 7.
According to the draught amendment the central one to give consumer provider us by promoting the competition in the power distribution factor and addressing the contracting is suit with a.

It will allow the consumer to pay electricity from a portal to their choice.

Imaging that the chances would lead to an immediate and steep hike in electricity tariff Kejriwal text the cm to keep the document proposal wide publicity to make people are of how it will hit them.

The proposed amendment to the Electricity Act 2003 or a matter of serious concern as they concentrate on power in the electricity sector in the hands of Central government to the exclusion of the state government and it will lead to an immediate is traffic in the electricity tariff.
Draught of proposed amendment to the Electricity Act 2003 has also been circulated among the CM of 11 States for the proposal.

The little has been sent to Mamata Banerjee West Bengal and Chandrababu Naidu Andhra Pradesh winner bigyan Kerala HD Kumaraswamy Karnataka Pondicherry Tamil Nadu Orissa and Sikkim.

It appears that the motive of the central government is to import and husband privatisation of electricity all over the country and to have her a few companies by concentrating call power with itself the centre will be able to do that.

Kitchen has been critical of electricity Hanumaan been calling it Jacqueline because it according to him it would put an end to cross subsidy with US President helps to keep electricity career blower for the small medium and Agriculture consumer by imposing higher rate on Industrial and commercial consumer.

We all need to get together to ensure the central government does not submit to getting this Amendment Bill passed in the parliament.

Responding to he delegation the BJP switched it to the national democratic Alliance government at Central said the proposed amendment to the act the centre will know about parrot.

At a public meeting in the Rohini neighborhood Kejriwal told gathering he will travel to the state and made the chief minister to secure the hell backing in the flight against the proposed amendment.

If the Modi government passed the amendment power tariff in Delhi will become 7.5 per unit for all consumers in every category.

In Delhi Bharthari for consumers who used upto 400 unit who is comprised 85% of electricity connection in the city will have to hiked by over 5 times he was said earlier.

In the national capital the average cost of power is 7.4 per unit.

Nobel Laureate Nadia Murad story is that of Survival and hope

Nadia Murad from Iraq and benefits from Democratic Republic of Congo on the Nobel Peace prices fall 2018 on Friday for their efforts.

The Nobel Peace Prize is awarded for  efforts to end the use of sexual violence as a weapon of War and arms conflict.

I meet Na Diya at goalkeepers Global Gold Award ceremony in New York on September 25 this year while he given the change maker award for using her own story to put the spotlight on were crying including raped in Iraq and across areas of conflict across the world.

She refused all interview but we said hello she does not speak but understands a smattering of English so the conversation was shot a nice period.

But I have noticed her in a crowded hall March before I realized who he was her eyes speak of the an imaginary level horror she witnessed and ended fall months as a bro of Islamic state militants.

The 24 year old was either he is a survivor of IAS enthe cleaning in their finger in North Iran Iraq in August 2014 for the message card mm of from their ancestral land.

Nadia lost 18 of her family in genocide including 6 mother and her mother was killed soon after.
They were all damps in mass Graves.

Nadiya was allowed to live because he was young and could be used as a sapire who are raped by 10 and sold several times.

This was the first time I had the world savai or 6 lamp applied to me.

Like other attractive girls and women Nadiya was beaten state on with Pal band with cigarettes raped and told repeatedly.

See escaped captivity in January 2015 with the help of a Muslim family in mosul and since then he became the voice of the people including 1380 women Steel enslaved by the IS.

Deciding to be honest was one of the hardest decision I have ever met and also the most important it never gets easier to tell your story if time you speak it you really read it when I tell someone about the checkpoint where the man with me or the feeling of Haji Salman the man who first brought her wife on the blanket as I lay under it or the darkening Sky while searching the neighbours for some kind of help.

My story told honestly and matter of factly is that the best with them I gave against tourism and I plan on using it until those tourist or put on trial.

She has founded the Nadiad initiative with support women and minorities through the redevelopment and stabilization of community in Crisis.

It is not easy to call out and really is sexual assault and balances pastor prevent as evidence from Christine Bradley thought destimoney against builders flat favourite who is it to the complicated us Supreme Court justice on Saturday decided I'm ending acquisition she is like Nadiya for everyone who believes in human value.

A focus on elections in Madhya Pradesh Rajasthan and Chhattisgarh

Rahul Gandhi gave stewardship of Rajasthan early enough in February 2014 to Sachin Pilot to retrieve the state for Congress.

Pilot got down to the task in earnest shifted his days to Jaipur kilometre the state in the last 4 years to rebuild the organisation and did not let go of any opportunity to Vasundhara Raje.

This is now beginning to show results.

III sector and this is going to be the real challenges for Congress is the extent to which young and old can work together.

Ashok Gehlot is still popular in rural Rajasthan and many want him to CM again if the party images Big trees what is the icon of the young and his elevation will give an impetus to the futuristic politics of Rahul Gandhi.

Both aur condom Dal for Mrs and no matter what they feel about each other they have so restrain and maturity and complain all together.

This was not done in Madhya Pradesh or for that matter in Chhattisgarh where the party suffers from a leadership crisis or even in Telangana where Congress could have destroyed IT self Sooner.

4 years now MP Congress has been elected by anal dispensaries the story might have been different had different type of government.

Mayawati the toughest beginners in India politics today is obviously trying to drive a hard bargain for the Lok Sabha election or she would not have stayed the Gandhi and directed her instead at Digvijay Singh who is on the margins of Congress today they are by keeping a door open on them.
Hard decision to go it alone in Rajasthan Madhya Pradesh and Alliance with Congress and rebul Ajit Jogi in Chhattisgarh help Vijay see but then she does not have has stopped in this state elections.

This would not be in the case in national policy would like to emerge as the state strap with the largest number of seats under her belt.

To do that she will have to Alliance with the opposition BJP will not let it sit for her in Uttar Pradesh with Akhilesh Yadav is prepared to do 9 is it likely to offer CMC of UP to win her to its side.

Going it alone could still finish for her politics with new and younger Dholak player amazing like Chandrashekhar Azad in UP.

It goes without saying that a Congress victory in the heartland states will strengthen the parties bargaining position with Regional parties for big battle that lives in 2019.

But it would be happy to describe the five state police and seminal for 2019.

Even if Congress oil to win all three Hindi heartland States YouTube not necessarily lead to its emergence as the single largest party in 2019.

The question that banks there's an inside it will 2019 to be an aggregate of state elections a sum total of 543 constituencies?

This is what opposition leaders are hoping for as they speak of alliances in a state like u p Bihar Maharashtra Jharkhand Karnataka and Andhra Pradesh.

Congress hope that if it can win just announced States poll that would translate into games at the national level.

But will people vote in a national election the same away.

Even as BJP had lost ground among guest delete Pharma traders minorities unemployed youth Modi remains its waste date event today.

The other side does not have his equivalent.

No matter what the results are in the five states it will still be too early to predict National election.

Youth Congress losses MP and Chhattisgarh despite the anti incumbency that has built up over 3 ton against Shivraj Singh Chouhan and Raman Singh no a small period then even if it will Rajasthan it will appear to be a weak player in 2019.

Forthcoming assembly election in 5 states or more crucial for Congress then for BJP

The die is cast with announcement of election 25 state assemble and your oil and truly entering pole season now.

Clearly and the it takes or higher for Congress then for BJP this is for the simple reason that Congress hope of rejuvenation will depend on how it there in the three heartland states Rajasthan Madhya Pradesh and Chhattisgarh it is fitted directly against BJP is equal a party on the comeback trail.

If Congress Lodges MP and Chhattisgarh despite the anti incumbency that has ruled over 3 ton against Shivraj Singh Chouhan and Raman Singh know a small period then even if it wins Rajasthan it will appear to be a week player in 2019.

After all they crisis have hit this to government farmer distress is purchased in Madhya Pradesh and Vyapam scam look is school and Raman Singh continue to face challenges from left extremist groups in Chhattisgarh.

Which is why it is only state for the election commission has ordered the election to be held in two phase.

The trouble is that Congress which appears to be doing well in Rajasthan.

Russian shield S400 acquisitions will boast India's Defence and diplomacy can avoid us sanctions

Russia and India on Friday entered into a contract for surface to air missile which will significantly and hands in the air defence capabilities.

Over the next 54 months will fail v s 400 Trump missiles Quick Heal cost of 5.48 Delhi on US dollar.

The conclusion of the deal during Russian President Vladimir Putin visit to Delhi despite the threat of us sanctions on countries entering into commercial contract with Russia main that India has decided to prioritise its national security interest.

This acquisition according to Indian Air Force Chief BS dhanoa will be a booster shot.

Separately public sector oil refinery or reported to have finalized contract to purchase more oil from Iran.
Event this mob runs the risk of us sanctions but this financial year Iran has been the second largest Oil supplier to India.

At a time when India's macroeconomic parameters are being tested by a shark in crease in the price of oil. We can ill afford to start over a major suppliers.

India has dealt with prior us sanctions on trade relations with Iran by avoiding dollarization transaction and routine payment through Indian banks with all family domestic in business orientation.

It is a testing time for India's policy makers.
Unilateral us sanctions on countries with which India has overlapping interest recover nimble footedness.

But it is not as if India does not have never rates.

In September the two countries signed the commercial Communications compatibility and security agreement one of four fundamental agreement us science with its alliances to facilitate Intel operatively between military.

This agreement also opens up additional commercial opportunities for us India must use the prospect of Sardaar designing of tires to persuade President Donald Trump to offer India A for the S400 deal.

The relevant us law empowered Trump to web sanctions on individual transactions to help alliances.

In the current global scenario India had to play it card carefully.
Even ads it is dependent on a strategic relationship with us it has two rings and its sides with countries such as Iran and Russia from Collateral Damage triggered by us foreign policy.

This requires skillful diplomacy and the need to make a compelling case to the American.

The benefit of doing this is that it will allow India to expand its relationship simultaneously with my life partner without compromising on its strategic interest.

External affairs minister has its work cut out. So it is very crucial to Indian defence and protecting power to raise a bilateral meeting and agreement between India and Russia on the acceptance of Rafael A 400 the Warriors air missile.

Saturday, October 6, 2018

The Royal Swedish Academy of Sciences has decided to award the Bank of Sweden Prize in Economic Sciences in Memory of Alfred Nobel, 1994, jointly to

for their pioneering analysis of equilibria in the theory of non-cooperative games.

Games as the Foundation for Understanding Complex Economic Issues
Game theory emanates from studies of games such as chess or poker. Everyone knows that in these games, players have to think ahead – devise a strategy based on expected countermoves from the other player(s). Such strategic interaction also characterizes many economic situations, and game theory has therefore proved to be very useful in economic analysis.
The foundations for using game theory in economics were introduced in a monumental study by John von Neumann and Oskar Morgenstern entitled Theory of Games and Economic Behavior(1944). Today, 50 years later, game theory has become a dominant tool for analyzing economic issues. In particular, non-cooperative game theory, i.e., the branch of game theory which excludes binding agreements, has had great impact on economic research. The principal aspect of this theory is the concept of equilibrium, which is used to make predictions about the outcome of strategic interaction. John F. Nash, Reinhard Selten and John C. Harsanyi are three researchers who have made eminent contributions to this type of equilibrium analysis.
John F. Nash introduced the distinction between cooperative games, in which binding agreements can be made, and non-cooperative games, where binding agreements are not feasible. Nash developed an equilibrium concept for non-cooperative games that later came to be called Nash equilibrium.
Reinhard Selten was the first to refine the Nash equilibrium concept for analyzing dynamic strategic interaction. He has also applied these refined concepts to analyses of competition with only a few sellers.
John C. Harsanyi showed how games of incomplete information can be analyzed, thereby providing a theoretical foundation for a lively field of research – the economics of information – which focuses on strategic situations where different agents do not know each others’ objectives.
Strategic Interaction
Game theory is a mathematical method for analyzing strategic interaction. Many classical analyses in economics presuppose such a large number of agents that each of them can disregard the others’ reactions to their own decision. In many cases, this assumption is a good description of reality, but in other cases it is misleading. When a few firms dominate a market, when countries have to make an agreement on trade policy or environmental policy, when parties on the labor market negotiate about wages, and when a government deregulates a market, privatizes companies or pursues economic policy, each agent in question has to consider other agents’ reactions and expectations regarding their own decisions, i.e., strategic interaction.
As far back as the early nineteenth century, beginning with Auguste Cournot in 1838, economists have developed methods for studying strategic interaction. But these methods focused on specific situations and, for a long time, no overall method existed. The game-theoretic approach now offers a general toolbox for analyzing strategic interaction.
Game Theory
Whereas mathematical probability theory ensued from the study of pure gambling without strategic interaction, games such as chess, cards, etc. became the basis of game theory. The latter are characterized by strategic interaction in the sense that the players are individuals who think rationally. In the early 1900s, mathematicians such as Zermelo, Borel and von Neumann had already begun to study mathematical formulations of games. It was not until the economist Oskar Morgenstern met the mathematician John von Neumann in 1939 that a plan originated to develop game theory so that it could be used in economic analysis.
The most important ideas set forth by von Neumann and Morgenstern in the present context may be found in their analysis of two-person zero-sum games. In a zero-sum game, the gains of one player are equal to the losses of the other player. As early as 1928, von Neumann introduced the minimax solution for a two-person zero-sum game. According to the minimax solution, each player tries to maximize his gain in the outcome which is most disadvantageous to him (where the worst outcome is determined by his opponent’s choice of strategy). By means of such a strategy, each player can guarantee himself a minimum gain. Of course, it is not certain that the players’ choices of strategy will be consistent with each other. von Neumann was able to show, however, that there is always a minimax solution, i.e., a consistent solution, if so-called mixed strategies are introduced. A mixed strategy is a probability distribution of a player’s available strategies, whereby a player is assumed to choose a certain “pure” strategy with some probability.
John F. NashJohn Nash arrived at Princeton University in 1948 as a young doctoral student in mathematics. The results of his studies are reported in his doctoral dissertation entitled Non-cooperative Games(1950). The thesis gave rise to Equilibrium Points in n-person Games (Proceedings of the National Academy of Sciences of the USA 1950), and to an article entitled Non-cooperative Games, (Annals of Mathematics 1951).
In his dissertation, Nash introduced the distinction between cooperative and non-cooperative games. His most important contribution to the theory of non-cooperative games was to formulate a universal solution concept with an arbitrary number of players and arbitrary preferences, i.e., not solely for two-person zero-sum games. This solution concept later came to be called Nash equilibrium. In a Nash equilibrium, all of the players’ expectations are fulfilled and their chosen strategies are optimal. Nash proposed two interpretations of the equilibrium concept: one based on rationality and the other on statistical populations. According to the rationalistic interpretation, the players are perceived as rational and they have complete information about the structure of the game, including all of the players’ preferences regarding possible outcomes, where this information is common knowledge. Since all players have complete information about each others’ strategic alternatives and preferences, they can also compute each others’ optimal choice of strategy for each set of expectations. If all of the players expect the same Nash equilibrium, then there are no incentives for anyone to change his strategy. Nash’s second interpretation – in terms of statistical populations – is useful in so-called evolutionary games. This type of game has also been developed in biology in order to understand how the principles of natural selection operate in strategic interaction within and among species. Moreover, Nash showed that for every game with a finite number of players, there exists an equilibrium in mixed strategies.
Many interesting economic issues, such as the analysis of oligopoly, originate in non-cooperative games. In general, firms cannot enter into binding contracts regarding restrictive trade practices because such agreements are contrary to trade legislation. Correspondingly, the interaction among a government, special interest groups and the general public concerning, for instance, the design of tax policy is regarded as a non-cooperative game. Nash equilibrium has become a standard tool in almost all areas of economic theory. The most obvious is perhaps the study of competition between firms in the theory of industrial organization. But the concept has also been used in macroeconomic theory for economic policy, environmental and resource economics, foreign trade theory, the economics of information, etc. in order to improve our understanding of complex strategic interactions. Non-cooperative game theory has also generated new research areas. For example, in combination with the theory of repeated games, non-cooperative equilibrium concepts have been used successfully to explain the development of institutions and social norms. Despite its usefulness, there are problems associated with the concept of Nash equilibrium. If a game has several Nash equilibria, the equilibrium criterion cannot be used immediately to predict the outcome of the game. This has brought about the development of so-called refinements of the Nash equilibrium concept. Another problem is that when interpreted in terms of rationality, the equilibrium concept presupposes that each player has complete information about the other players’ situation. It was precisely these two problems that Selten and Harsanyi undertook to solve in their contributions.
Reinhard SeltenThe problem of numerous non-cooperative equilibria has generated a research program aimed at eliminating “uninteresting” Nash equilibria. The principal idea has been to use stronger conditions not only to reduce the number of possible equilibria, but also to avoid equilibria which are unreasonable in economic terms. By introducing the concept of subgame perfection, Selten provided the foundation for a systematic endeavor in Spieltheoretische Behandlung eines Oligopolmodells mit Nachfrageträgheit, (Zeitschrift für die Gesamte Staatswissenschaft 121, 301-24 and 667-89, 1965).
An example might help to explain this concept. Imagine a monopoly market where a potential competitor is deterred by threats of a price war. This may well be a Nash equilibrium – if the competitor takes the threat seriously, then it is optimal to stay out of the market – and the threat is of no cost to the monopolist because it is not carried out. But the threat is not credible if the monopolist faces high costs in a price war. A potential competitor who realizes this will establish himself on the market and the monopolist, confronted with fait accompli, will not start a price war. This is also a Nash equilibrium. In addition, however, it fulfills Selten’s requirement of subgame perfection, which thus implies systematic formalization of the requirement that only credible threats should be taken into account.
Selten’s subgame perfection has direct significance in discussions of credibility in economic policy, the analysis of oligopoly, the economics of information, etc. It is the most fundamental refinement of Nash equilibrium. Nevertheless, there are situations where not even the requirement of subgame perfection is sufficient. This prompted Selten to introduce a further refinement, usually called the “trembling-hand” equilibrium, in Reexamination of the Perfectness Concept for Equilibrium Points in Extensive Games (International Journal of Game Theory 4, 25-55, 1975). The analysis assumes that each player presupposes a small probability that a mistake will occur, that someone’s hand will tremble. A Nash equilibrium in a game is “trembling-hand perfect” if it is robust with respect to small probabilities of such mistakes. This and closely related concepts, such as sequential equilibrium (Kreps and Wilson, 1982), have turned out to be very fruitful in several areas, including the theory of industrial organization and macroeconomic theory for economic policy.
John C. HarsanyiIn games with complete information, all of the players know the other players’ preferences, whereas they wholly or partially lack this knowledge in games with incomplete information. Since the rationalistic interpretation of Nash equilibrium is based on the assumption that the players know each others’ preferences, no methods had been available for analyzing games with incomplete information, despite the fact that such games best reflect many strategic interactions in the real world.
This situation changed radically in 1967-68 when John Harsanyi published three articles entitled Games with Incomplete Information Played by Bayesian Players, (Management Science 14, 159-82, 320-34 and 486-502). Harsanyi’s approach to games with incomplete information may be viewed as the foundation for nearly all economic analysis involving information, regardless of whether it is asymmetric, completely private or public.
Harsanyi postulated that every player is one of several “types”, where each type corresponds to a set of possible preferences for the player and a (subjective) probability distribution over the other players’ types. Every player in a game with incomplete information chooses a strategy for each of his types. Under a consistency requirement on the players’ probability distributions, Harsanyi showed that for every game with incomplete information, there is an equivalent game with complete information. In the jargon of game theory, he thereby transformed games with incomplete information into games with imperfect information. Such games can be handled with standard methods.
An example of a situation with incomplete information is when private firms and financial markets do not exactly know the preferences of the central bank regarding the tradeoff between inflation and unemployment. The central bank’s policy for future interest rates is therefore unknown. The interactions between the formation of expectations and the policy of the central bank can be analyzed using the technique introduced by Harsanyi. In the most simple case, the central bank can be of two types, with adherent probabilities: Either it is oriented towards fighting inflation and thus prepared to pursue a restrictive policy with high rates, or it will try to combat unemployment by means of lower rates. Another example where similar methods can be applied is regulation of a monopoly firm. What regulatory or contractual solution will produce a desirable outcome when the regulator does not have perfect knowledge about the firm’s costs?
Other Contributions of the Laureates
In addition to his contributions to non-cooperative game theory, John Nash has developed a basic solution for cooperative games, usually referred to as Nash’s bargaining solution, which has been applied extensively in different branches of economic theory. He also initiated a project that subsequently came to be called the Nash program, a research program designed to base cooperative game theory on results from non-cooperative game theory. In addition to his prizewinning achievements, Reinhard Selten has contributed powerful new insights regarding evolutionary games and experimental game theory. John Harsanyi has also made significant contributions to the foundations of welfare economics and to the area on the boundary between economics and moral philosophy. Harsanyi and Selten have worked closely together for more than 20 years, sometimes in direct collaboration.

Through their contributions to equilibrium analysis in non-cooperative game theory, the three laureates constitute a natural combination: Nash provided the foundations for the analysis, while Selten developed it with respect to dynamics, and Harsanyi with respect to incomplete information.

Amartya Sen and his contribution to Welfare economics

Amartya Sen Nobel Prize winning economist of Indian Origin who has made by their contribution to the field of development economics.

One of his majesty Mahal contribution to the social choice theory but his contribution extend the Welfare economics and starting the problem of poverty as well.

By identifying house at police is manifest themselves in society recommended civil construction methods which basic welfares .

The Norwegian Nobel committee has decided about the 2018 Nobel Prize in peace

For the effort to end the use of sexual violence as a weapon of War and armed conflict.

The Nobel Prize in Physiology or medicine 2018 was awarded jointly

The new discovery of Cancer therapy by inhibition of negative immune regulation.

Friday, October 5, 2018

Advancement and applications of peptide phage display technology in biomedical science

Combinatorial phage library is a powerful research tool for high-throughput screening of protein interactions. Of all available molecular display techniques, phage display has proven to be the most popular approach. Screening phage-displayed random peptide libraries is an effective means of identifying peptides that can bind target molecules and regulate their function. Phage-displayed peptide libraries can be used for (i) B-cell and T-cell epitope mapping, (ii) selection of bioactive peptides bound to receptors or proteins, disease-specific antigen mimics, peptides bound to non-protein targets, cell-specific peptides, or organ-specific peptides, and (iii) development of peptide-mediated drug delivery systems and other applications. Targeting peptides identified using phage display technology may be useful for basic research and translational medicine. In this review article, we summarize the latest technological advancements in the application of phage-displayed peptide libraries to applied biomedical sciences.
Keywords: Phage display, Peptides, Epitope mapping, Antigen mimics, Drug delivery..

Phage display is a selection technique in which a peptide or protein is fused with a bacteriophage coat protein and displayed on the surface of a virion. This technology was first described by George P. Smith in 1985, when he demonstrated the display of peptides on filamentous phage by fusing the peptide of interest to gene III of filamentous phage []. Phage-displayed random peptide libraries enable functional access to the peptides and provide a physical link between phenotype (the displayed peptide) and genotype (the encoding DNA); these libraries lend themselves to a screening process in which binding clones are separated from nonbinding clones by affinity purification.
Peptides binding to individual targets can be identified by affinity selection (called biopanning). For biopanning, a display library is incubated with an immobilized target, followed by extensive washing to remove nonreacting phages. Binders are usually eluted using acid or high salt and are enriched by amplification in the appropriate host cells. Three to five rounds of biopanning are usually performed in order to obtain targets that bind with high affinity (Fig. 1). The primary structure of the peptide can then be determined by sequencing the DNA of individual clones. Using this approach, it is easy to identify peptides that bind specifically to target molecules.

Phage-displayed peptide library can be used in B-cell and T-cell epitope mapping, selection of bioactive peptides bound to receptors or proteins, selection of disease-specific antigen mimics, selection of peptides bound to non-protein targets, selection of cell-specific peptides, selection of organ-specific peptides, and development of peptide-mediated drug delivery systems and other applications. Targeting peptides identified using phage-displayed peptide libraries have potential use in basic research and translational medicine. In this review paper, we discuss in detail each of the applications of the phage-displayed technology platform in the biomedical sciences.

B-cell and T-cell epitope mapping


Upon encountering antigen, host humoral immunity activates and triggers production of antibodies directed against foreign protein epitopes. Knowledge of these protein epitopes is pivotal in understanding the pathogenesis of pathogen infections and in developing diagnostic reagents, therapeutic antibodies, and effective vaccines. An epitope (known as an antigenic determinant) is recognized by components of the immune system, including antibodies, B cells, and T cells. The epitopes of antigens, which are dependent on their structural properties, can be either linear or conformational []. Linear epitopes have some continuous amino acid sequences of antigens, which correspond with their primary structure. In contrast, conformational epitopes contain discontinuous amino acid sequences of antigens, which are based on their protein tertiary structure. There are several experimental methods with which to identify B-cell epitopes, such as Pepscan [], co-crystallization [], nuclear magnetic resonance (NMR) [], computational docking [], and site-directed mutagenesis []. The phage display method provides an economical and rapid approach with which to map B-cell epitopes []. Previous studies have used various phage-displayed random peptide libraries to identify B-cell epitopes [] or neutralizing epitopes [] from monoclonal antibodies (mAbs). In addition to mAbs, polyclonal antibodies (e.g. special sera from patients or immunized mice) can also be captured on solid disks or magnetic beads, and then reacted with a comprehensive library of random peptides. Peptides have been selected by biopanning with antibodies from complex sera of patients with various diseases, including severe acute respiratory syndrome (SARS) [] and infection with human papillomavirus (HPV) [] and avian influenza viruses (AIV) []. Based on information on B-cell epitopes from polyclonal antibodies, certain peptide-based antigens are useful for serological diagnosis, and some are suitable for development of vaccines []. The selected disease-specific epitopes may prove to be invaluable for the identification of the etiological agent [] (Fig. 2).

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Phage-displayed peptide library can be used in B-cell and T-cell epitope mapping, selection of bioactive peptides bound to receptors or proteins, selection of disease-specific antigen mimics, selection of peptides bound to non-protein targets, selection of cell-specific peptides, selection of organ-specific peptides, and development of peptide-mediated drug delivery systems and other applications. Targeting peptides identified using phage-displayed peptide libraries have potential use in basic research and translational medicine. In this review paper, we discuss in detail each of the applications of the phage-displayed technology platform in the biomedical sciences.

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Selection of disease-specific antigen mimics

The considerable potential of phage display peptide libraries lies in their capacity to identify some peptide molecules that mimic epitopes (named mimotopes). Mimotopes have fewer similarities to primary amino acids of antigens and always show discontinuous sequences, but they can elicit an identical or highly similar antibody response to that of the native epitope. The development of diagnostic or preventive reagents requires screening phage-displayed peptide libraries for disease-specific epitopes/mimotopes in serum or cerebrospinal fluid samples obtained from patients with viral infections [], rheumatoid arthritis [], multiple sclerosis [], autoimmune thrombocytopenic purpura [], and neurocysticercosis []. Peptides have been selected by biopanning serum samples from patients infected with HPV [], SARS [], and AIV []. Mimotope-based detection is suitable for broad spectrum antibodies against avian H5N1 influenza virus [], and has been shown to improve serological detection of SARS [] and rheumatoid arthritis []. Another example is recently identified cholera toxin-binding peptides. Cholera toxin, which is secreted by Vibrio cholerae, can enter host cells by binding to GM1, a monosialoganglioside, and would result in acute diarrhea. Biopanning with cholera toxin B (CTB) subunit to select CTB-binding peptides that structurally mimic GM1 could serve as novel agents to block CTB binding on epithelial cells and prevent the ensuing physiological effects of cholera toxin [].
Mimotopes can also be used to characterize unknown initiating events and provide clues toward disease pathogenesis. Biopanning with antibodies from systemic sclerosis patients was used to screen an immunopositive peptide that had a high degree of similarity to autoantigens, including heterogenous ribonucleoproteins (hnRNP), cytochrome c, fibrillarin, and late protein UL94 of human cytomegalovirus (CMV) []. Immunopositive peptide-based affinity purified antibodies from sera of systemic sclerosis patients reacted with a surface component of endothelial cells and induced apoptosis. A dengue anti-NS1 antibody B-cell epitope is cross-reactive to astrocyte elevated gene-1 (AEG-1), a human protein on human endothelial cells that may cause some dengue patients to suffer from hemorrhagic fever (DHF) or dengue shock syndrome (DSS) []. Recently, an antigen peptide mimicking alpha-2–Heremans–Schmid glycoprotein, also known as fetuin-A, was identified from serum antibodies of prostate cancer patients []. Using this antigen mimic peptide, the authors demonstrated increased serum antibody reactivity to fetuin-A during disease progression in the index patient, and strong serum reactivity in a large cohort of metastatic prostate cancer patients []. As mentioned above, study of the disease-specific antigen mimics by phage display helps us to understand the etiology of diseases (Fig. 2).

Selection of bioactive peptides bound to receptors or proteins

For receptors

Membrane receptors are pivotal for cell-cell biochemical and electrical signaling in essential physiological functions. Therefore, the pharmaceutical industries tend to focus on developing drugs targeting membrane receptors []. Novel receptor ligands discovered using phage-displayed random peptide libraries [] may be either agonists or antagonists (Fig. 1). Two typical examples of agonist peptides selected by phage display are peptides targeting erythropoietin receptor (EpoR) [] and thrombopoietin receptor (TpoR) []. After treatment with Epo mimetic peptide, conformational changes in the extracellular domain of EpoR trigger intracellular signal transduction []. This discovery may form the basis for the design of small molecule mimetics of Epo. On the other hand, the selected small peptides targeting TpoR can compete for the binding of the natural ligand, thrombopoietin, and stimulate the proliferation of a TPO-responsive cell line []. Antagonists of membrane receptors from phage display have also been reported []. For example, vascular endothelial growth factor (VEGF) plays an important role in angiogenesis through binding to the kinase domain receptor (KDR/FLK1 or VEGFR2). Peptides as antagonists that block VEGF-mediated angiogenesis have been obtained by phage display []. Human CXC ligand 8 (hCXCL8) is a type of chemoattractant that binds to hCXCR1 and hCXCR2, which are involved in inflammation. Inhibition of hCXCL8 binding to hCXCR1 and hCXCR2 by antagonistic peptides has been investigated, and used to develop new therapeutics for treatment of inflammatory diseases [].
Phage display is also used to isolate receptor-independent peptide modulators, such as G protein-coupled receptors. Some selected peptides from G proteins can interact with the Gαi subunit, leading to an elevated sensitivity of guanine nucleotides to bind to A1 adenosine receptors [] and to Gβγ subunit in order to enhance the dissociation of G protein heterotrimers in vitro, and activate G protein signaling in intact cells [].

For enzyme inhibitors

The pathogenesis of some diseases occurs through the expression of abnormal enzymes, which can serve as potential targets for developing inhibitors as new drugs to block the activities of these enzymes. Phage display has been used to identify the peptide substrate inhibitors that modulate enzyme activities [] (Fig. 1). Because filamentous phages are resistant to a wide range of proteases, they have been used to identify substrates and generate protease inhibitors []. Bahudhanapati et al. identified selective inhibitors of collagenase-1 (metalloproteinase 1, MMP-1) by screening variants of tissue inhibitor of metalloproteinases-2 (TIMP-2) using phage display. TIMP-2 is a broad range inhibitor of matrix metalloproteinases (MMPs) []. In addition to protease inhibitors, peptide-based inhibitors against various enzymes, such as human HMG-CoA reductase [], ubiquitin ligases [], and tyrosinase [], have all been identified by phage-displayed random peptide libraries.

Protein-protein interactions

In cells, protein-protein interactions regulate the mechanisms of several important normal physiological functions. Phage display is a potent and versatile method for studying protein-protein interaction []. It can be applied to a wide range of protein interaction partners and used in a number of applications, especially in mapping intracellular interactions of the distinct protein domain. Good examples of protein interaction partners include Src homology (SH) 3 domains, which are highly conserved protein interaction modules comprised of 50 to 70 amino acids. SH3 domains are also found in a variety of functionally unrelated proteins. Kärkkäinen et al. generated a library in which all human SH3 domains are expressed on the surface of M13 bacteriophage, thereby enabling analysis of human SH3 domain binding to target proteins of interest, including human immunodeficiency virus-1 Nef, p21-activated kinase (PAK)2, and ADAM15 []. Voss et al. also established similar libraries to define the SH3 domain that reacts with the intracellular region of Fas ligand. In addition to the known SH3 domains, the authors also identified a number of additional SH3 domains that may also be associated with FasL []. CDBP2 is a cellular adapter protein that contains a GYF domain in its C-terminal fragment. Kofler et al. identified a new conserved motif, PPG (W/F/Y/M/L), in the GYF domain of CDBP2, using phage display. Alignment of these consensus motifs to protein databases, in combination with yeast display or NMR methods, has allowed scientists to rapidly identify novel interaction partners of GYF domain [].

Selection of peptides bound to non-protein targets

Many different types of bacteria trigger protective immune responses through cell surface non-protein antigens, such as polysaccharides. Many tumor antigens are carbohydrates. Epitopes mimicking low immunogenic polysaccharides or carbohydrate antigens can be screened and identified by phage-displayed random peptide libraries [] (Fig. 1). These isolated peptide mimotopes coupled with carrier proteins can be used as potential vaccine candidates to stimulate stronger antibody responses [].
The phage display technique has also allowed us to identify novel peptides against RNA of interest by screening random peptide libraries []. Selective peptides targeting helix 31 of bacterial 16S RNA can be used to inhibit cell-free translation []. Bose et al. used phage display to find a selective peptide binding to pre-miR-21, which blocks Dicer processing and decreases miR-21 expression [].
A few studies have successfully employed peptide phage display to select binders of small molecules, such as fluorophores [], microcystin-LR [], and paclitaxel (Taxol) []. Recently, Liu et al. performed phage display to screen peptide ligands recognizing an insecticide, imidacloprid, for environmental monitoring in water and soil []. The biopanning strategy of phage display can be used to select specific peptides against nanomaterials. These phage-displayed nanomaterial binding peptides are broadly useful in the field of nanotechnology []. Whaley et al. showed the power of using combinatorial phage-display libraries to select highly specific peptides that bind to a range of crystalline semiconductor structures, such as GaAs, depending on the nanocrystal orientation []. The application of specific peptides has been extended to recognize other substrates of inorganic nanocrystals, including ZnS, CdS, TiO2, and SiO2, as well as magnetic materials such as Fe2O3 and Fe3O4 []. Peptides derived from phage-displayed libraries can be specifically bound to a conducting polymer for use as a biomaterial to functionalize the surface of conductive polymers, thereby enabling various electronic and biomedical applications []. Moreover, the Au-, Ag-, Ti-, Pt-, and Pd-binding peptides were all acquired using the combinatorial phage display peptide library []. Such metal and semiconductor-targeting peptides have been genetically engineered for use in nano- and biotechnology, particularly in the molecular biomimetics field [].

Selection of cell-specific peptides

Peptide phage display through whole-cell panning offers a high-throughput approach for identifying peptides that bind specifically to a single cell type. Johnston and co-workers were the first to describe the use of peptide-displayed phage to identify peptide binding to several different cell types []. The most common screening method is a process called “biopanning”, shown in Fig. 3, in which the binding affinities of the targeted phage clones are enriched. It can be performed in vitro against various types of cells, including cultured cell lines, primary cells isolated from animal models or human patients, or processed cells (fixed cells, activated cells etc.). Within the past ten years, several studies have focused on the in vitro biopanning of phage displayed peptide libraries using various cancer cell types [] to identify cell-specific ligands [].

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Depending on the applications of the ligand, selection can be performed with adherent or fixed cells. The experimental approach can be modified to isolate phages, which bind to the cell surface or peptides, thereby triggering the cellular uptake of the peptides. Peptide-displayed phage libraries are incubated with the cells for a defined period of time. The cells are subsequently washed to remove non-specific and weakly bound phage. In order to reduce the cross-reactivity of the peptide or the phage, blocking agents such as BSA are occasionally used. Removing unbound phage is required to obtain phage clones with strong binding to the desired target, and remove non-specific binding from the background. In general, the washing processes are relatively gentle; however, more stringent washes may increase the affinity of selected phage clones. In some cases, negative selection is performed to avoid the aforementioned problem. In general negative selection is not essential.
Phage bound to the target is recovered using several elution strategies, including the use of acidic buffers, Dithiothreitol, and high ionic strength, which tend to decrease the interaction between the peptide and the target. Most commonly, acidic buffer is sufficient for the elution of target bound phage. However, in the case of strong peptide-target interactions, these elution procedures may only partially break peptide-target interactions, thereby resulting in loss of the high-affinity phage clones. To circumvent this problem, Strukelj and co-workers used a modified method, in which ultrasound was applied during acidic buffer elution to release target-bound phage and enable the selection of high-affinity phage clones []. In cases where ligands of a particular target are known and available, competitive elution is the preferred method of isolating the target molecule. This method can specifically elute desired target-bound phage clones while avoiding elution of background-bound phage. Alternatively phage can also be eluted competitively but nonspecifically by using the free target molecule, such as an eluant, or by adding bacterial host directly to the target-bound phage.
Using whole cells instead of purified proteins as target for in vitro biopanning has several advantages. The cellular receptors expressed on live cells can retain their native states (correct protein folding, quaternary structure, expression level, and association with neighboring proteins), and their biological functions and activities. Biopanning with modified protocols can be used for the isolation of peptides that mediate specific cellular functions. For example, selection can be aimed at isolating surface-bound or internalized peptides. Direction elution of phage enables isolation of surface-bound phage. If surface-bound phages are removed by low-pH washes or through treatment with a protease, phage with internalizing characteristics can be isolated. In addition, the use of whole cells for biopanning enables the identification of cell surface molecules with unknown biological functions. This can be used to characterize cell surface profiles and provide information on molecular changes (such as expression level and protein localization) between normal and disease cells.
Although numerous cell-binding peptides have been successfully isolated using in vitro panning against cultured cells, several challenges still remain []. In particular, systematic experimental approaches for target identification are lacking []. This is a key problem because accurate identification of peptide-targeted molecules is important for basic and clinical research. Conventional receptor identification focuses on membrane protein extraction and affinity purification, followed by mass spectrometric identification of the purified protein. However, the problems associated with this approach arise from the difficulty in maintaining the native interaction between targeting peptide and isolated whole membrane receptor []. Furthermore, the binding affinities of targeting peptides are, in general, too low to enable purification by affinity-based methods. Wu and co-workers aimed to overcome the problems outlined above by using biotinylated peptides to directly bind intact cells, and subsequent fixation of ligand-receptor complexes by cross-linking with 3,3′-dithiobis[sulfosuccinimidyl propionate] (DTSSP). After affinity trapping and LC-MS/MS analysis, the unknown target protein on the plasma membrane of the cells could be identified []. It is important to note that advances in peptide identification and subsequent receptor identification can lead to the discovery of important cellular targets that were previously unknown. This not only improves our understanding of the molecules expressed in the pathological state, but may also provide useful information on molecules that are not expressed under normal physiological conditions.

Selection of organ-specific peptides

Organ-specific peptides can be isolated from phage display peptide libraries by performing the selection in a living animal. In vivo phage display technology was first described by Ruoslahti and co-workers in 1996 []. They aimed to discover brain vasculature targeting peptides using phage-displayed peptide libraries. Typically, a random peptide phage library is introduced by intravenous injection into the circulation of animal. After a brief circulation time, the animals are sacrificed, and the unbound phage clones are washed off through perfusion of the left ventricle with saline. Based on the half-life of the phage in previous studies, the circulation time of a peptide-displayed phage is generally estimated to be in the 5 to 15 min time range []. There is evidence that the displayed exogenous peptide or protein can exhibit circulation half-lives as short as 1.5 min to as long as 4.5 h []. Molenaar and co-workers reported that degradation of phage occurs as rapidly as 30 min after injection []. A circulation time of over 30 min may be caused by lysosomal degradation of phage after uptake by the target tissue.
After removing the unbound phage in circulation, the desired organs are collected and homogenized. The organ-associated phage is recovered from the homogenized tissue. A fraction of the organ lysates are used to infect bacteria for phage amplification and for subsequent rounds of selection in another animal. Another fraction is used for phage titering to measure the amount of recovered phage. After 3–5 rounds of in vivo biopanning, the phage titer recovered from the target tissue should increase []. Several peptide motifs are typically identified for a given organ (Fig. 4). After biopanning, the specificity of the isolated peptide needs to be further confirmed. In general, wild type phage without insert or phage with scrambled peptide is used as a negative control. The ability of the isolated peptide to home to the target organ can be confirmed either by comparative analysis using a scrambled peptide, or by competitive studies using a combination of synthetic peptide and selected phage clone with identical peptide sequence. Other approaches, such as immunostaining of homing phage, or fluorescent or radioactive labeling of either phage or synthetic peptide, can be used to directly determine the organ tissue distribution.
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Use of phage display libraries to identify peptides that bind to a specific organ. a The in vivo process is based on panning the library of peptides against the target organs. The phage peptide library is injected into the mouse’s tail vein to allow it to circulate throughout the body. After a few minutes in circulation, phage molecules that bind to organs and tissues of interest are isolated. The isolated phage molecules are amplified and subjected to further rounds of selection to enrich organ-specific phage. Finally, DNA from the phage is sequenced to identify the encoded peptide, and the peptide can be used for the development of organ-specific therapeutics and diagnostics. b The specificity of selected phage clones can be further confirmed using the phage homing assay. Mice are injected with the selected phage clones through the tail vein. Phage clones are allowed to circulate, and the nonspecific binders are washed out. The organs are subsequently recovered, and the titers of phage in various organs are determined
This approach has been applied to a number of different organs, including brain, kidney, lungs, liver, uterus, muscle, pancreas, thymus, and mammary gland []. Various types of tumor and malignant tissue vasculature have been targeted using phage-displayed random peptide libraries []. In addition to tumor blood vessels, many tumors induce the growth of new lymphatic vessels and change characteristics during tumor development. Several researchers have used the in vivo phage display technology to map tumor-specific differences in the lymphatic vasculature, and have identified peptides that specifically home to tumor lymphatics [].
Using in vivo phage display technology to identify specific homing peptides is not without pitfalls. Peptides isolated from animal models using this method may not translate to human because of differences in the vasculature and peptide binding between species. Such limitations can be overcome by performing biopanning in humans. The phage library can be injected into terminal human patients and retrieved from human tissues to identify organ specific peptides []. Another study reported the use of phage-display libraries in cancer patients to identify tumor-targeting ligands []. In vivo phage display has been shown to be an effective and powerful technique for the isolation of peptides that specifically bind to an organ with high affinity and specificity. However, optimization of these peptides is needed to enhance the clinical applicability of in vivo phage display research.

Development of peptide-mediated drug delivery systems

The delivery of anti-cancer drugs to solid tumors is limited by physical transport barriers within tumors, and such restrictions directly contribute to decreased therapeutic index and the emergence of drug resistance. Drug delivery systems designed to precise spatiotemporal control have demonstrated potential to enhance drug delivery in animal models. Many nanoparticle delivery systems for anticancer drugs have entered the clinic, where they have been shown to exert anticancer effects by improving the pharmacokinetic and pharmacodynamic properties of their associated drugs []. Liposomes are the most advanced form of particulate drug carriers. Liposomal systems, like RES-avoiding and long-circulating systems, can confer stable formulation, improved pharmacokinetics, and “passive” targeting of tumor tissue. The newest generation of drug carriers utilizes multicapable nanotechnology-oriented strategies aimed at greater specificity and efficiency. They feature direct molecular targeting of cancer cells via ligand-mediated interactions. Molecular targeted drug delivery can be achieved using targeting liposomes created by linking liposomal drugs to specific ligands. The development of targeting liposomes has been made possible by advances in liposomal systems. These two technologies can in principle be fully integrated, thus combining the specificity of ligands with the drug delivery capabilities of liposomes (Fig. 5

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Peptide-mediated liposomes include three main components: an anticancer drug, a liposome carrier, and targeting ligands (Fig. 5). Remote loading methods, such as the ammonium sulfate method [] and the pH gradient method [], can encapsulate weak bases, such as doxorubicin or vinorelbine, into the liposomes with more than 95 % efficiency []. Schedule-dependent drugs, such as vinca alkaloids and topotecan, are reasonable candidates for liposomal delivery because of their effectiveness at enhancing the exposure time of cancer cells to therapeutic drug levels. The use of peptide-mediated liposomes also avoids exposing normal tissue to cytotoxic drugs, and prevents adverse side effects. High tumor interstitial fluid pressure (IFP) is a barrier for efficient drug delivery []. Increased IFP contributes to decreased transcapillary transport in tumors, which reduces uptake of drugs. This approach sidesteps the problem of high tumor IFP [] and improves treatment effectiveness, thereby reducing incomplete tumor response, rapid disease relapse, and development of drug resistance due to suboptimal doses, which are often seen when using conventional chemotherapies.
The use of peptides as targeting ligands offers several advantages, including ease of synthesis, structural simplicity, low cost, low immunogenicity, small size, ready diffusion, and simple targeted formulation assembly, as compared to larger biomolecules, such as antibodies. Previous studies showed that using larger biomolecules as targeting ligands may increase the clearance of antibody-modified nanoparticles from the blood []. This may be due to non-specific binding and uptake of nanoparticles by the RES []. Earlier studies with other targeted delivery systems have suggested that targeting moieties increase drug accumulation in tumor tissues []. However, targeted nanoparticles have not always caused a significant increase in overall tumor accumulation as compared to the non-targeted drugs. The use of macromolecule-targeting ligands, such as antibodies [] or transferrin [], has a negligible impact on tumor accumulation and biodistribution. The differential effects of nanoparticles modified with macromolecules and those modified with small molecules may be due to differences in molecular size, affinity, and penetrability of the targeting ligand []. It is possible that targeting moieties with high affinity would be subject to greater internalization and degradation by perivascular tumor cells, thereby limiting their penetration of tumors and reducing their tumor retention [].
Colorectal cancer is one of the most commonly diagnosed cancers and a leading cause of cancer death worldwide. Traditional chemotherapy only has limited therapeutic efficacy due to non-specific delivery to tumor and non-tumor cells, and the development of drug resistance by cancer cells. Therefore, there is an urgent need to develop more tumor-specific targeted drug delivery systems that can more accurately and effectively deliver the anti-cancer chemotherapeutic drugs to the tumor sites. In a recent study, Wu et al. used phage display technology to identify three peptides that could bind to colorectal cancer cells with high specificity and binding affinity []. The authors successfully developed a targeted drug by conjugating these peptides to liposomes. This novel targeted liposomal drug combination specifically delivers chemotherapeutics to tumors, resulting in a much higher dose of drugs being accumulated at the tumor site. This significantly increased the tumor inhibition abilities of these two types of chemotherapeutic drugs, and effectively eliminated cancer without inducing any side effects for 150 days, with no trace of recurrence []. Thus, this study demonstrated that drugs can be accurately delivered to tumor sites by combining the liposomal drugs with peptides generated using phage display technology, resulting in a next-generation targeted anti-cancer drug delivery system.
Advances in nanotechnology have facilitated a multidisciplinary approach toward the development of an ideal “smart nanodrug delivery system,” which can be decorated with a targeting ligand for diagnostic or imaging uses, in addition to therapeutic agents of interest (Fig. 5). The availability of such “smart nanodrug delivery systems” in the near future will allow us to detect, diagnose, target, modulate delivery of, and track the progression of therapy both remotely and noninvasively.

Discussion

Combinatorial phage-displayed random peptide libraries are very valuable tools for studying the interaction between peptides and other substances (or materials). In the past, scientists have been focused on identifying B/T cell epitopes [], disease-specific antigen mimics [], receptor agonists/antagonists [], enzyme inhibitors [] and protein partners []. In recent years, there is an increasing number of researchers who apply this technique to new areas of studies, such as chemistry [] and materials [] science. Although phage display is a powerful technique, it still has some disadvantages. Choosing suitable phage display libraries (i. e. the number of phage-displayed amino acid residues), using applicable selection condition, ensuring the stability and the quality of phage display libraries and following the appropriate screening protocols are all important factors that could impact the quality and desirability of the ligand peptides generated. If these key parameters can be established, functional target peptides are more likely to be obtained. Furthermore, combination of peptide information from phage display with bioinformatics resources may improve the quality of peptides. Tian et al. [] and Sandman et al. [] incorporated genetically-encoded non-natural amino acids into phage-displayed libraries and paved the way for wider chemical diversities. Woiwode et al. [] also combined a new phage-displayed hybrid system with synthetic chemistry through one-compound-one-clone principle (each compound was encoded by a unique nucleotide sequence inserted in a non-coding region of the phage genome). These new techniques help to further advance the possible applications and potentials of phage display going forward.
Cancer cells, different from normal cells, are often found to overexpress certain antigens. Molecules which can recognize these tumor antigens with high specificity are suitable candidates as potential agents for guiding cancer chemotherapy to target tumor sites. The therapeutic monoclonal antibody, antibody-drug conjugates (ADCs), peptide-drug conjugates (PDCs) and peptide-mediated drug delivery systems have a major impact on the field of cancer therapy. Therapeutic monoclonal antibody based therapies have achieved remarkable clinical success and become one of the most important strategies for treating patients with hematological malignancies and solid tumors. The direct action of antibodies by receptor blockade or agonist activity may have limited therapeutic activity. Antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) have been demonstrated to have a major role in antibody efficacy []. Unfortunately, antibody treatment of patients with malignant tumors may not achieve therapeutic effect due to immune suppression, immune escape, complement inhibition etc. ADCs offer the possibility to overcome this issue by enhancing cytotoxicity to cancer cells, thereby reducing undesired side effects. Although ADCs have already been approved as anticancer therapy, there remain several limitations to this type of therapeutics, such as tumor penetration, high manufacturing costs, and challenging conjugation chemistry [].
PDCs have similar promise as ADCs but they differ in their pharmacological outcomes. Compared to ADCs, PDCs have additional advantages of having smaller molecular mass, higher tissue penetration, enhanced flexibility and well-defined conjugation chemistry, and faster and easier synthesis process when prepared in a homogenous form []. Several peptides included RGD motif peptides [], cell penetrating peptides [], and tumor cell specific peptides [] are used for synthesizing PDCs for cancer therapy. After more than decades of research, lots of PDCs have been discovered, and some have been clinically evaluated, although none has yet received regulatory approval. One of the PDCs that has been clinically evaluated and carry most promising is GRN1005. GRN1005 is a LRP-1-targeted peptide-drug conjugate that covalently links paclitaxel to the proprietary 19 amino acid peptide angiopep-2, in a 3:1 ratio. The results from the phase I trial of GRN1005 showed the drug candidate to have acceptable safety profile. GRN1005 is currently undergoing Phase II trials to demonstrate the efficacy, safety, and tolerability of GRN1005 in patients with brain metastases from non-small cell lung cancer (NCT01497665). In addition, GRN1005 in combination with Trastuzumab (Herceptin®) has advanced to Phase II studies as a therapeutic agent for HER2 positive metastatic breast cancer (NCT01480583) []. However, one major drawback of PDCs that limits their clinical uses is their stability. PDCs are highly proteolytic unstable, which results in their short half-life in circulation and poor pharmacokinetics. In addition, the relative small molecular weight of PDCs would be rapidly eliminated through renal excretory system.
Neither ADCs nor PDCs allows for high drug payloads on the conjugates molecule. The major challenges facing most ADCs and PDCs are their relatively low capacity for drugs. Hence, more effective payload strategies using different carriers are urgently needed. In addition to ADCs and PDCs, nanoparticles modified with targeting ligand are more effective payload strategies. Nanoparticles as potential vehicle for encapsulating chemotherapeutics can achieve higher drug-loading efficiency. Nanoparticles have been shown to increase the drug-loading capacity by approximately a thousand-fold compared to ADCs and PDCs []. Much enhanced drug loading capacity and higher tumor cell specificity render nanoparticles as potentially more promising targeted drugs as a result of their higher efficacy and lower toxicity []. ADCs, PDCs and peptide-mediated drug delivery systems have shown considerable promise in improving the delivery of drug to tumor and limiting off-target toxicities simultaneously. These targeting therapeutics represent promising new frontier in cancer treatment.
Despite having the advantages of increased therapeutic efficacy, many challenges still remain for nanoparticles, including potential for off-target effects, ligand stability, immune responses triggering and drugs delivery efficiency in the cytoplasm of target cells. In particular, the rapid clearance of these nanodrugs by the cells of the reticuloendothelial system (RES)/mononuclear phagocyte system (MPS), especially liver and spleen, can lead to increased toxicity to the off-target organs and reduced therapeutic efficacy. However, a recent publication suggested that such off-target effects can be reduced by Intralipid 20 % (a FDA-approved fat emulsion used as parenteral nutrition source), which could decrease the toxicities in liver and spleen and increase the bioavailability of dichloro (1, 2-diaminocyclohexane) platinum (II)-loaded and hyaluronic acid polymer-coated nanoparticle (DACHPt/HANP) by possibly inhibiting peritoneal clearance and impairing the phagocytic activity of Kupffer cells []. Whether such reduced off target effects can also be observed in liposomal based drug delivery system is worthwhile further investigating.
Drug delivery using antibodies or ligands that bind to specific receptor molecules on tumor target cells allow increased drug accumulation at the target tumor site, however, the actual percentage of drug accumulated at the tumor site was often only a few percent of the total dose administered. Identification of effective ligand-receptor interaction may help to improve the effectiveness of active targeting. In the field of targeted drug delivery, scientists still have a long way to go, but this may change within the next couple of years. Over time, the number of targeted drug-delivery nanoparticles approved by the FDA is expected to increase.

Conclusions

Although cancer drugs continue to be discovered, most of these drugs have only limited efficacy against cancer, with less than ideal ability to prolong the lives of cancer patients. Small molecule drugs have the advantage of having higher tissue penetration abilities, but they are non-specific to tumors and have a relatively short half-life. Protein drugs are highly tumor-specific; however, they have lower tumor penetration abilities due to their larger molecular sizes. Peptide-mediated drug delivery systems combine the potent small molecule drugs with high specificity of peptides, thus leveraging the benefits of the two therapeutic regimens while reducing their disadvantages. An ideal drug delivery system should achieve a high level of clinical efficacy and minimize the adverse effects. The development of targeting liposomes can improve drug delivery to the target tissue and reduce drug distribution to nontarget tissues, resulting in increased therapeutic activity with minimal side effects. Peptide-mediated liposomes that target tumor cells and vasculature represent a new generation of chemotherapeutic delivery systems. They offer superior pharmacokinetics, controlled biodistribution, greater efficacy, and enhanced safety profiles, and simultaneously improve eradication of disease and reduce common side effects. This has been a long sought-after goal in the development of chemotherapeutic drugs. The modular organization of targeting liposome technology makes it possible to combine peptides with a series of liposomal drugs to yield next-generation targeted agents, such as peptide-mediated targeting liposomes. However, to date, no peptide-drug conjugates or peptide-modified nanoparticles have successfully reached the market. However, certain hurdles must be overcome before peptides can be widely used as targeting moieties, including development of the appropriate ligand for the targeted receptor, understanding the mechanisms of ligand-receptor uptake, disposition, trafficking, and recycling, and compliance with Chemistry, Manufacturing, and Control (CMC) requirements.



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