Dracocephalum moldavica L. and Melissa officinalis L. : Chemistry and Bioactivities Relevant in Alzheimer’s Disease Therapy

Oksidatiivisen stressin eli liiallisen reaktiivisten happiyhdisteiden määrän soluissa on jo pitkään arveltu olevan tärkeä Alzheimerin taudin kehittymiseen ja etenemiseen vaikuttava tekijä. Tämän vuoksi kiinnostus erilaisten antioksidanttien (yhdisteitä, jotka neutraloivat näitä happiradikaaleja soluissa) mahdollisia terapeuttisia ominaisuuksia Alzheimerin taudin hoidossa on tutkittu laajalti. Tähän mennessä ei kuitenkaan ole vielä onnistuttu löytämään antioksidanttia, joka olisi hyödyksi Alzheimerin taudin hoidossa. Tämän vuoksi on tärkeää pyrkiä löytämään uusia antioksidanttien lähteitä sekä tutkia niistä löytyviä aktiivisia yhdisteitä. Kiinnostus luonnon antioksidantteja kohtaan on kasvanut voimakkaasti viime aikoina. Huomio on kiinnittynyt erityisesti aromaattisista sekä lääkekasveista löytyviin antioksidantteihin. Lamiaceae- perheeseen kuuluvia tuoksuampiaisyrttiä (Dracocephalum moldavica L.) ja sitruunamelissaa (Melissa officinalis L.) on käytetty Iranissa pitkään sekä ruoanlaitossa että lääkinnässä, minkä vuoksi näiden kasvien uutteiden antioksidanttisisältöä päätettiin analysoida käyttäen useaa erilaista in vitro- menetelmää. Näissä kokeissa ilmeni, että uutteilla oli useita antioksidanttisia vaikutuksia. Näistä antioksidanttisista vaikutuksista vastaavia yhdisteitä pyrittiin tunnistamaan käyttäen HPLC-PDA- tekniikkaa, minkä seurauksena niiden havaittiin sisältävän erilaisia polyfenoleita, kuten hydroksyloituneita bentsoeeni- ja cinnamamidihapon johdannaisia sekä flavonoideja. Kummankin kasvin uutteissa runsaimmin esiintynyt yhdiste oli rosmariinihappo. Sitruunamelissaa (M. officinalis) on käytetty antiikin ajoista alkaen kognitiivisten toimintojen häiriöiden hoidossa. Perustuen tietoon kasvin käytöstä perinteisessä lääkinnässä, sen tehoa Alzheimerin taudin hoidossa on tutkittu viime aikoina kliinisin kokein. Sitruunamelissan todettiinkin olevan hyödyksi lievää ja keskivaikeaa Alzheimeimerin tautia sairastavien potilaiden hoidossa. Väitöskirjan osanan olevasta kooste-artikkelista käy ilmi, että tutkimalla lääkekasvien ominaisuuksia voidaan saada arvokkaita suuntaa-antavia vihjeitä Alzheimerin taudin lääkehoidon kehittämiseen. Tämän perusteella päätettiinkin testatata myös sitruunamelissauutteen kykyä estää asetyylikoliiniesteraasin (AChE) toimintaa, koska tämän entsyymin toiminna estämisen tiedetään olevan hyödyksi Alzheimerin taudin hoidossa. Uute kykeni estämään AChE:n toimintaa, minkä vuoksi uutteen sisältämiä komponentteja päätettiin tutkia terkemmin. Uute jaettiin erilaisiin fraktioihin käyttäen HPLC-menetelmää, minkä jälkeen testattiin jokaisen fraktion kykyä inhiboida AchE. Suurin osa fraktioista kykeni inhiboimaan AChE:n toimintaa selkeästi tehokkaammin, kuin raakauute. Kaikista tehokkainta fraktiota analysoitiin tarkemmin sen aktiivisten yhdisteiden tunnistamiseksi, minkä seurauksena sen sisältämät yhdisteet tunnistettiin cis ja trans-rosmariinihapoiksi. Tässä tutkimuksessa tunnistettujen yhdisteiden hyödyllisyyttä Alzheimerin taudin hoidossa tulisi seuraavaksi tutkia erilaisissa in vivo-malleissa. Lisäksi jäljellä olevien fraktioiden kemiallinen koostumus tulisi selvittää sekä antioksidanttiaktiivisuuden ja AChE:n toiminnan inhiboinnin välistä mahdollista yhteyttä tulisi tutkia tarkemmin. Tämä tutkimus osoittaa tuoksuampiasyrtin (D. moldavica) sekä sitruunamelissan (M. officinalis) sisältävän monenlaisia aktiivisia antioksidantteja. Lisäksi sitruunamelissan sisältämät yhdisteet kykenivät estämään asetyylikoliiniesteraasin (AchE) toimintaa. Nämä tulokset tukevat osaltaan väitöskirjan osana olevan kooste-artikkelin johtopäätöksiä, joiden mukaan etnofarmakologinen kasvitutkimus voi osoittautua erittäin hyödylliseksi kehitettäessä uutta lääkehoitoa Alzheimerin tautiin. Lisäksi tässä väitöskirjassa kuvattu tutkimus osoittaakin, että perinteisesti lääkekasvina käytettyä sitruunamelissaa voidaan mahdollisesti hyödyntää uusien Alzheimerin taudin hoitoon käytettävien lääkkeiden kehityksessä.

Domestication to crop improvement: Genetic resources for Sorghum and Saccharum (Andropogoneae).

Background: Both sorghum (Sorghum bicolor) and sugarcane (Saccharum officinarum) are members of the Andropogoneae tribe in the Poaceae and are each other's closest relatives amongst cultivated plants. Both are relatively recent domesticates and comparatively little of the genetic potential of these taxa and their wild relatives has been captured by breeding programmes to date. This review assesses the genetic gains made by plant breeders since domestication and the progress in the characterization of genetic resources and their utilization in crop improvement for these two related species. Genetic Resources: The genome of sorghum has recently been sequenced providing a great boost to our knowledge of the evolution of grass genomes and the wealth of diversity within S. bicolor taxa. Molecular analysis of the Sorghum genus has identified close relatives of S. bicolor with novel traits, endosperm structure and composition that may be used to expand the cultivated gene pool. Mutant populations (including TILLING populations) provide a useful addition to genetic resources for this species. Sugarcane is a complex polyploid with a large and variable number of copies of each gene. The wild relatives of sugarcane represent a reservoir of genetic diversity for use in sugarcane improvement. Techniques for quantitative molecular analysis of gene or allele copy number in this genetically complex crop have been developed. SNP discovery and mapping in sugarcane has been advanced by the development of high-throughput techniques for ecoTILLING in sugarcane. Genetic linkage maps of the sugarcane genome are being improved for use in breeding selection. The improvement of both sorghum and sugarcane will be accelerated by the incorporation of more diverse germplasm into the domesticated gene pools using molecular tools and the improved knowledge of these genomes.

Status, challenges and tools for identification and diagnosis of Peronosclerospora and Sclerophthora of regulatory concern for graminicolous crops

Graminicolous Downy Mildew (GDM) diseases caused by the genera Peronosclerospora (13 spp.) and Sclerophthora (6 spp. and 1 variety) are poorly studied but destructive diseases of major crops such as corn, sorghum, sugarcane and other graminoids. Eight of the 13 described Peronosclerospora spp. are able to infect corn. In particular, P. philippinensis (= P. sacchari), P. maydis, P. heteropogonis, and S. rayssiae var. zeae cause major losses in corn yields in tropical Asia. In 2012 a new species, P. australiensis, was described based on isolates previously identified as P. maydis in Australia; this species is now a pathogen of major concern. Despite the strong impact of GDM diseases, there are presently no reliable molecular methods available for their detection. GDM pathogens are among the most difficult Oomycetes to identify using molecular tools, as their taxonomy is very challenging, and little genetic sequence data are available for development of molecular tools to detect GDM pathogens to species level. For example, from over 15 genes used in identification, diagnostics or phylogeny of Phytophthora, only ITS1 and cox2 show promise for use with GDM pathogens. Multiplex/multigene conventional and qPCR assays are currently under evaluation for the detection of economically important GDM spp. Scientists from the USA, Germany, Canada, Australia, and the Philippines are collaborating on the development and testing of diagnostic tools for these pathogens of concern.

Turing and the Trans-Pacific Partnership: Intellectual Property, Public Health, and Access to Essential Medicines

A recent controversy in the United States over drug pricing by Turing Pharmaceuticals AG has raised larger issues in respect of intellectual property, access to medicines, and the Trans-Pacific Partnership (TPP). In August 2015, Turing Pharmaceuticals AG – a private biopharmaceutical company with offices in New York, the United States, and Zug, Switzerland - acquired the exclusive marketing rights to Daraprim in the United States from Impax Laboratories Incorporated. Martin Shkreli, Turing’s Founder and Chief Executive Officer, maintained: “The acquisition of Daraprim and our toxoplasmosis research program are significant steps along Turing’s path of bringing novel medications to patients with serious disorders, some of whom often go undiagnosed and untreated.” He emphasised: “We intend to invest in the development of new drug candidates that we hope will yield an even better clinical profile, and also plan to launch an educational effort to help raise awareness and improve diagnosis for patients with toxoplasmosis.” In September 2015, there was much public controversy over the decision of Martin Shkreli to raise the price of a 62 year old drug, Daraprim, from $US13.50 to $US750 a pill. The drug is particularly useful in respect to the treatment and prevention of malaria, and in the treatment of infections in individuals with HIV/AIDS. Daraprim is listed on the World Health Organization’s (WHO) List of Essential Medicines. In the face of much criticism, Martin Shkreli has said that he will reduce the price of Daraprim. He observed: “We've agreed to lower the price on Daraprim to a point that is more affordable and is able to allow the company to make a profit, but a very small profit.” He maintained: “We think these changes will be welcomed.” However, he has been vague and ambiguous about the nature of the commitment. Notably, the lobby group, Pharmaceutical Research and Manufacturers of America (PhARMA), disassociated itself from the claims of Turing Pharmaceuticals. The group said: “PhRMA members have a long history of drug discovery and innovation that has led to increased longevity and improved lives for millions of patients.” The group noted: “Turing Pharmaceutical is not a member of PhRMA and we do not embrace either their recent actions or the conduct of their CEO.” The biotechnology peak body Biotechnology Industry Organization also sought to distance itself from Turing Pharmaceuticals. A hot topic: United States political debate about access to affordable medicines This controversy over Daraprim is unusual – given the age of drug concerned. Daraprim is not subject to patent protection. Nonetheless, there remains a monopoly in respect of the marketplace. Drug pricing is not an isolated problem. There have been many concerns about drug pricing – particularly in respect of essential medicines for HIV/AIDS, tuberculosis, and malaria. This recent controversy is part of a larger debate about access to affordable medicines. The dispute raises larger issues about healthcare, consumer rights, competition policy, and trade. The Daraprim controversy has provided impetus for law reform in the US. US Presidential Candidate Hillary Clinton commented: “Price gouging like this in this specialty drug market is outrageous.” In response to her comments, the Nasdaq Biotechnology Index fell sharply. Hillary Clinton has announced a prescription drug reform plan to protect consumers and promote innovation – while putting an end to profiteering. On her campaign site, she has emphasised that “affordable healthcare is a basic human right.” Her rival progressive candidate, Bernie Sanders, was also concerned about the price hike. He wrote a letter to Martin Shkreli, complaining about the price increase for the drug Daraprim. Sanders said: “The enormous, overnight price increase for Daraprim is just the latest in a long list of skyrocketing price increases for certain critical medications.” He has pushed for reforms to intellectual property to make medicines affordable. The TPP and intellectual property The Daraprim controversy and political debate raises further issues about the design of the TPP. The dispute highlights the dangers of extending the rights of pharmaceutical drug companies under intellectual property, investor-state dispute settlement, and drug administration. Recently, the civil society group Knowledge Ecology International published a leaked draft of the Intellectual Property Chapter of the TPP. Knowledge Ecology International Director, James Love, was concerned the text revealed that the US “continues to be the most aggressive supporter of expanded intellectual property rights for drug companies.” He was concerned that “the proposals contained in the TPP will harm consumers and in some cases block innovation.” James Love feared: “In countless ways, the Obama Administration has sought to expand and extend drug monopolies and raise drug prices.” He maintained: “The astonishing collection of proposals pandering to big drug companies make more difficult the task of ensuring access to drugs for the treatment of cancer and other diseases and conditions.” Love called for a different approach to intellectual property and trade: “Rather than focusing on more intellectual property rights for drug companies, and a death-inducing spiral of higher prices and access barriers, the trade agreement could seek new norms to expand the funding of medical research and development (R&D) as a public good, an area where the US has an admirable track record, such as the public funding of research at the National Institutes of Health (NIH) and other federal agencies.” In addition, there has been much concern about the Investment Chapter of the TPP. The investor-state dispute settlement regime would enable foreign investors to challenge government policy making, which affected their investments. In the context of healthcare, there is a worry that pharmaceutical drug companies will deploy their investor rights to challenge public health measures – such as, for instance, initiatives to curb drug pricing and profiteering. Such concerns are not merely theoretical. Eli Lilly has brought an investor action against the Canadian Government over the rejection of its drug patents under the investor-state dispute settlement regime of the North American Free Trade Agreement (NAFTA). The Health Annex to the TPP also raises worries that pharmaceutical drug companies will able to object to regulatory procedures in respect of healthcare. It is disappointing that the TPP – in the leaks that we have seen – has only limited recognition of the importance of access to essential medicines. There is a need to ensure that there are proper safeguards to provide access to essential medicines – particularly in respect of HIV/AIDs, malaria, and tuberculosis. Moreover, there must be protection against drug profiteering and price gouging in any trade agreement. There should be strong measures against the abuse of intellectual property rights. The dispute over Turing Pharmaceuticals AG and Daraprim is an important cautionary warning in respect of some of the dangers present in the secret negotiations in respect of the TPP. There is a need to preserve consumer rights, competition policy, and public health in trade negotiations over an agreement covering the Pacific Rim.

Greenwashing the Trans-Pacific Partnership: Fossil fuels, the environment, and climate change

There has been much controversy over the Trans-Pacific Partnership (TPP) – a plurilateral trade agreement involving a dozen nations from throughout the Pacific Rim – and its impact upon the environment, biodiversity, and climate change. The secretive treaty negotiations involve Australia and New Zealand; countries from South East Asia such as Brunei Darussalam, Malaysia, Singapore, Vietnam, and Japan; the South American nations of Peru and Chile; and the members of the 1994 North American Free Trade Agreement (NAFTA), Canada, Mexico and the United States. There was an agreement reached between the parties in October 2015. The participants asserted: ‘We expect this historic agreement to promote economic growth, support higher-paying jobs; enhance innovation, productivity and competitiveness; raise living standards; reduce poverty in our countries; and to promote transparency, good governance, and strong labor and environmental protections.’ The final texts of the agreement were published in November 2015. There has been discussion as to whether other countries – such as Indonesia, the Philippines, and South Korea – will join the deal. There has been much debate about the impact of this proposed treaty upon intellectual property, the environment, biodiversity and climate change. There have been similar concerns about the Trans-Atlantic Trade and Investment Partnership (TTIP) – a proposed trade agreement between the United States and the European Union. In 2011, the United States Trade Representative developed a Green Paper on trade, conservation, and the environment in the context of the TPP. In its rhetoric, the United States Trade Representative has maintained that it has been pushing for strong, enforceable environmental standards in the TPP. In a key statement in 2014, the United States Trade Representative Mike Froman insisted: ‘The United States’ position on the environment in the Trans-Pacific Partnership negotiations is this: environmental stewardship is a core American value, and we will insist on a robust, fully enforceable environment chapter in the TPP or we will not come to agreement.’ The United States Trade Representative maintained: ‘Our proposals in the TPP are centered around the enforcement of environmental laws, including those implementing multilateral environmental agreements (MEAs) in TPP partner countries, and also around trailblazing, first-ever conservation proposals that will raise standards across the region’. Moreover, the United States Trade Representative asserted: ‘Furthermore, our proposals would enhance international cooperation and create new opportunities for public participation in environmental governance and enforcement.’ The United States Trade Representative has provided this public outline of the Environment Chapter of the TPP: A meaningful outcome on environment will ensure that the agreement appropriately addresses important trade and environment challenges and enhances the mutual supportiveness of trade and environment. The Trans-Pacific Partnership countries share the view that the environment text should include effective provisions on trade-related issues that would help to reinforce environmental protection and are discussing an effective institutional arrangement to oversee implementation and a specific cooperation framework for addressing capacity building needs. They also are discussing proposals on new issues, such as marine fisheries and other conservation issues, biodiversity, invasive alien species, climate change, and environmental goods and services. Mark Linscott, an assistant Trade Representative testified: ‘An environment chapter in the TPP should strengthen country commitments to enforce their environmental laws and regulations, including in areas related to ocean and fisheries governance, through the effective enforcement obligation subject to dispute settlement.’ Inside US Trade has commented: ‘While not initially expected to be among the most difficult areas, the environment chapter has emerged as a formidable challenge, partly due to disagreement over the United States proposal to make environmental obligations binding under the TPP dispute settlement mechanism’. Joshua Meltzer from the Brookings Institute contended that the trade agreement could be a boon for the protection of the environment in the Pacific Rim: Whether it is depleting fisheries, declining biodiversity or reduced space in the atmosphere for Greenhouse Gas emissions, the underlying issue is resource scarcity. And in a world where an additional 3 billion people are expected to enter the middle class over the next 15 years, countries need to find new and creative ways to cooperate in order to satisfy the legitimate needs of their population for growth and opportunity while using resources in a manner that is sustainable for current and future generations. The TPP parties already represent a diverse range of developed and developing countries. Should the TPP become a free trade agreement of the Asia-Pacific region, it will include the main developed and developing countries and will be a strong basis for building a global consensus on these trade and environmental issues. The TPP has been promoted by its proponents as a boon to the environment. The United States Trade Representative has maintained that the TPP will protect the environment: ‘The United States’ position on the environment in the TPP negotiations is this: environmental stewardship is a core American value, and we will insist on a robust, fully enforceable environment chapter in the TPP or we will not come to agreement.’ The United States Trade Representative discussed ‘Trade for a Greener World’ on World Environment Day. Andrew Robb, at the time the Australian Trade and Investment Minister, vowed that the TPP will contain safeguards for the protection of the environment. In November 2015, after the release of the TPP text, Rohan Patel, the Special Assistant to the President and Deputy Director of Intergovernmental Affairs, sought to defend the environmental credentials of the TPP. He contended that the deal had been supported by the Nature Conservancy, the International Fund for Animal Welfare, the Joint Ocean Commission Initiative, the World Wildlife Fund, and World Animal Protection. The United States Congress, though, has been conflicted by the United States Trade Representative’s arguments about the TPP and the environment. In 2012, members of the United States Congress - including Senator Ron Wyden (D-OR), Olympia Snowe (R-ME), and John Kerry (D-MA) – wrote a letter, arguing that the trade agreement needs to provide strong protection for the environment: ‘We believe that a '21st century agreement' must have an environment chapter that guarantees ongoing sustainable trade and creates jobs, and this is what American businesses and consumers want and expect also.’ The group stressed that ‘A binding and enforceable TPP environment chapter that stands up for American interests is critical to our support of the TPP’. The Congressional leaders maintained: ‘We believe the 2007 bipartisan congressional consensus on environmental provisions included in recent trade agreements should serve as the framework for the environment chapter of the TPP.’ In 2013, senior members of the Democratic leadership expressed their opposition to granting President Barack Obama a fast-track authority in respect of the TPP House of Representatives Minority Leader Nancy Pelosi said: ‘No on fast-track – Camp-Baucus – out of the question.’ Senator Majority leader Harry Reid commented: ‘I’m against Fast-Track: Everyone would be well-advised to push this right now.’ Senator Elizabeth Warren has been particularly critical of the process and the substance of the negotiations in the TPP: From what I hear, Wall Street, pharmaceuticals, telecom, big polluters and outsourcers are all salivating at the chance to rig the deal in the upcoming trade talks. So the question is, Why are the trade talks secret? You’ll love this answer. Boy, the things you learn on Capitol Hill. I actually have had supporters of the deal say to me ‘They have to be secret, because if the American people knew what was actually in them, they would be opposed. Think about that. Real people, people whose jobs are at stake, small-business owners who don’t want to compete with overseas companies that dump their waste in rivers and hire workers for a dollar a day—those people, people without an army of lobbyists—they would be opposed. I believe if people across this country would be opposed to a particular trade agreement, then maybe that trade agreement should not happen. The Finance Committee in the United States Congress deliberated over the Trans-Pacific Partnership negotiations in 2014. The new chair Ron Wyden has argued that there needs to be greater transparency in trade. Nonetheless, he has mooted the possibility of a ‘smart-track’ to reconcile the competing demands of the Obama Administration, and United States Congress. Wyden insisted: ‘The new breed of trade challenges spawned over the last generation must be addressed in imaginative new policies and locked into enforceable, ambitious, job-generating trade agreements.’ He emphasized that such agreements ‘must reflect the need for a free and open Internet, strong labor rights and environmental protections.’ Elder Democrat Sander Levin warned that the TPP failed to provide proper protection for the environment: The TPP parties are considering a different structure to protect the environment than the one adopted in the May 10 Agreement, which directly incorporated seven multilateral environmental agreements into the text of past trade agreements. While the form is less important than the substance, the TPP must provide an overall level of environmental protection that upholds and builds upon the May 10 standard, including fully enforceable obligations. But many of our trading partners are actively seeking to weaken the text to the point of falling short of that standard, including on key issues like conservation. Nonetheless, 2015, President Barack Obama was able to secure the overall support of the United States Congress for his ‘fast-track’ authority. This was made possible by the Republicans and dissident Democrats. Notably, Oregon Senator Ron Wyden switched sides, and was transformed from a critic of the TPP to an apologist for the TPP. For their part, green political parties and civil society organisations have been concerned about the secretive nature of the negotiations; and the substantive implications of the treaty for the environment. Environmental groups and climate advocates have been sceptical of the environmental claims made by the White House for the TPP. The Green Party of Aotearoa New Zealand, the Australian Greens and the Green Party of Canada have released a joint declaration on the TPP observing: ‘More than just another trade agreement, the TPP provisions could hinder access to safe, affordable medicines, weaken local content rules for media, stifle high-tech innovation, and even restrict the ability of future governments to legislate for the good of public health and the environment’. In the United States, civil society groups such as the Sierra Club, Public Citizen, WWF, the Friends of the Earth, the Rainforest Action Network and 350.org have raised concerns about the TPP and the environment. Allison Chin, President of the Sierra Club, complained about the lack of transparency, due process, and public participation in the TPP talks: ‘This is a stealth affront to the principles of our democracy.’ Maude Barlow’s The Council of Canadians has also been concerned about the TPP and environmental justice. New Zealand Sustainability Council executive director Simon Terry said the agreement showed ‘minimal real gains for nature’. A number of organisations have joined a grand coalition of civil society organisations, which are opposed to the grant of a fast-track. On the 15th January 2013, WikiLeaks released the draft Environment Chapter of the TPP - along with a report by the Chairs of the Environmental Working Group. Julian Assange, WikiLeaks' publisher, stated: ‘Today's WikiLeaks release shows that the public sweetener in the TPP is just media sugar water.’ He observed: ‘The fabled TPP environmental chapter turns out to be a toothless public relations exercise with no enforcement mechanism.’ This article provides a critical examination of the draft Environment Chapter of the TPP. The overall argument of the article is that the Environment Chapter of the TPP is an exercise in greenwashing – it is a public relations exercise by the United States Trade Representative, rather than a substantive regime for the protection of the environment in the Pacific Rim. Greenwashing has long been a problem in commerce, in which companies making misleading and deceptive claims about the environment. In his 2012 book, Greenwash: Big Brands and Carbon Scams, Guy Pearse considers the rise of green marketing and greenwashing. Government greenwashing is also a significant issue. In his book Storms of My Grandchildren, the climate scientist James Hansen raises his concerns about government greenwashing. Such a problem is apparent with the TPP – in which there was a gap between the assertions of the United States Government, and the reality of the agreement. This article contends that the TPP fails to meet the expectations created by President Barack Obama, the White House, and the United States Trade Representative about the environmental value of the agreement. First, this piece considers the relationship of the TPP to multilateral environmental treaties. Second, it explores whether the provisions in respect of the environment are enforceable. Third, this article examines the treatment of trade and biodiversity in the TPP. Fourth, this study considers the question of marine capture fisheries. Fifth, there is an evaluation of the cursory text in the TPP on conservation. Sixth, the article considers trade in environmental services under the TPP. Seventh, this article highlights the tensions between the TPP and substantive international climate action. It is submitted that the TPP undermines effective and meaningful government action and regulation in respect of climate change. The conclusion also highlights that a number of other chapters of the TPP will impact upon the protection of the environment – including the Investment Chapter, the Intellectual Property Chapter, the Technical Barriers to Trade Chapter, and the text on public procurement.

Development and reproducibility of a short questionnaire to measure use and usability of custom-made orthopaedic shoes

OBJECTIVE To develop a short and easy to use questionnaire to measure use and usability of custom-made orthopaedic shoes, and to investigate its reproducibility. DESIGN Development of the questionnaire (Monitor Orthopaedic Shoes) was based on a literature search, expert interviews, 2 expert meetings, and exploration and testing of reproducibility. The questionnaire comprises 2 parts: a pre part, measuring expectations; and a post part, measuring experiences. Patients The pre part of the final version was completed twice by 37 first-time users before delivery of their orthopaedic shoes. The post part of the final version was completed twice by 39 first-time users who had worn their orthopaedic shoes for 2–4 months. RESULTS High reproducibility scores (Cohen’s kappa > 0.60 or intra class correlation > 0.70) were found in all but one question of both parts of the final version of the Monitor Orthopaedic Shoes questionnaire. The smallest real difference on a visual analogue scale (100 mm) ranged from 21 to 50 mm. It took patients approximately 15 minutes to complete one part. CONCLUSION Monitor Orthopaedic Shoes is a practical and reproducible questionnaire that can measure relevant aspects of use and usability of orthopaedic shoes from a patient’s perspective.

Bending the Rules of Cell Protrusions : Molecular Mechanisms and Biological Roles of Inverse-BAR Proteins in Cell Morphogenesis

Plasma membrane adopts myriad of different shapes to carry out essential cellular processes such as nutrient uptake, immunological defence mechanisms and cell migration. Therefore, the details how different plasma membrane structures are made and remodelled are of the upmost importance. Bending of plasma membrane into different shapes requires substantial amount of force, which can be provided by the actin cytoskeleton, however, the molecules that regulate the interplay between the actin cytoskeleton and plasma membrane have remained elusive. Recent findings have placed new types of effectors at sites of plasma membrane remodelling, including BAR proteins, which can directly bind and deform plasma membrane into different shapes. In addition to their membrane-bending abilities, BAR proteins also harbor protein domains that intimately link them to the actin cytoskeleton. The ancient BAR domain fold has evolved into at least three structurally and functionally different sub-groups: the BAR, F-BAR and I-BAR domains. This thesis work describes the discovery and functional characterization of the Inverse-BAR domains (I-BARs). Using synthetic model membranes, we have shown that I-BAR domains bind and deform membranes into tubular structures through a binding-surface composed of positively charged amino acids. Importantly, the membrane-binding surface of I-BAR domains displays an inverse geometry to that of the BAR and F-BAR domains, and these structural differences explain why I-BAR domains induce cell protrusions whereas BAR and most F-BAR domains induce cell invaginations. In addition, our results indicate that the binding of I-BAR domains to membranes can alter the spatial organization of phosphoinositides within membranes. Intriguingly, we also found that some I-BAR domains can insert helical motifs into the membrane bilayer, which has important consequences for their membrane binding/bending functions. In mammals there are five I-BAR domain containing proteins. Cell biological studies on ABBA revealed that it is highly expressed in radial glial cells during the development of the central nervous system and plays an important role in the extension process of radial glia-like C6R cells by regulating lamellipodial dynamics through its I-BAR domain. To reveal the role of these proteins in the context of animals, we analyzed MIM knockout mice and found that MIM is required for proper renal functions in adult mice. MIM deficient mice displayed a severe urine concentration defect due to defective intercellular junctions of the kidney epithelia. Consistently, MIM localized to adherens junctions in cultured kidney epithelial cells, where it promoted actin assembly through its I-BAR andWH2 domains. In summary, this thesis describes the mechanism how I-BAR proteins deform membranes and provides information about the biological role of these proteins, which to our knowledge are the first proteins that have been shown to directly deform plasma membrane to make cell protrusions.

Malaria burden and costs of intensified control in Bhutan, 2006-2014: A situational analysis

- Introduction Malaria cases have dwindled in Bhutan with aim of malaria elimination by 2016. The aims of this study are to determine the trends and burden of malaria, the costs of intensified control activities, the main donors of the control activities and the costs of different preventive measures in the pre-elimination phase (2006-2014). - Methods A descriptive analysis of malaria surveillance data from 2006-2014 was carried out, using data from the Vector-borne Disease Control Programme (VDCP), Bhutan. Malaria morbidity and mortality among local Bhutanese and foreign nationals were analysed. The cost of different control and preventive measures, and estimation of the average numbers of long-lasting insecticidal nests (LLINs) per person were calculated. - Findings There were 5,491 confirmed malaria cases from 2006 to 2014. By 2013, there was an average of one LLIN for every 1·51 individuals. The Global Fund was the main international donor accounting for > 80% of the total funds. The cost of procuring LLINs accounted for > 90% of the total cost of prevention measures. - Interpretation The malaria burden reduced significantly over the study period with high coverage of LLINs in Bhutan. This foreseeable challenges that require national attention to maintain malaria-free status after elimination are importation of malaria, particularly from India; continued protection of the population in endemic districts through complete coverage with LLINs and IRS; and exploration of local funding modalities post elimination in the event there is a reduction in international funding.

Computational systems approach for drug target discovery

Importance of the field: The shift in focus from ligand based design approaches to target based discovery over the last two to three decades has been a major milestone in drug discovery research. Currently, it is witnessing another major paradigm shift by leaning towards the holistic systems based approaches rather the reductionist single molecule based methods. The effect of this new trend is likely to be felt strongly in terms of new strategies for therapeutic intervention, new targets individually and in combinations, and design of specific and safer drugs. Computational modeling and simulation form important constituents of new-age biology because they are essential to comprehend the large-scale data generated by high-throughput experiments and to generate hypotheses, which are typically iterated with experimental validation. Areas covered in this review: This review focuses on the repertoire of systems-level computational approaches currently available for target identification. The review starts with a discussion on levels of abstraction of biological systems and describes different modeling methodologies that are available for this purpose. The review then focuses on how such modeling and simulations can be applied for drug target discovery. Finally, it discusses methods for studying other important issues such as understanding targetability, identifying target combinations and predicting drug resistance, and considering them during the target identification stage itself. What the reader will gain: The reader will get an account of the various approaches for target discovery and the need for systems approaches, followed by an overview of the different modeling and simulation approaches that have been developed. An idea of the promise and limitations of the various approaches and perspectives for future development will also be obtained. Take home message: Systems thinking has now come of age enabling a `bird's eye view' of the biological systems under study, at the same time allowing us to `zoom in', where necessary, for a detailed description of individual components. A number of different methods available for computational modeling and simulation of biological systems can be used effectively for drug target discovery.

The mesenchymal-to-epithelial reverting transition is enriched in metastatic castration resistant prostate cancer and correlates with poor survival

Despite recent recognition that the epithelial-mesenchymal transition (EMT) program acts in a dynamic manner (termed Epithelial to Mesenchymal Plasticity or EMP) during carcinoma metastasis, it has largely been ignored in the discovery and development of EMT-targeted therapies. In part, this has stemmed from a lack of preclinical models that can mimic the full dynamic nature of EMP and the perception that the EMT-reverting transition [or mesenchymal-epithelial reverting transition; (MErT)] is a mere antithesis of EMT. The objective of this study was to develop the first PCa model capable of recapitulating the dynamic nature of EMP.

Lectin Microarrays for Glycomic Analysis

Glycomics is the study of comprehensive structural elucidation and characterization of all glycoforms found in nature and their dynamic spatiotemporal changes that are associated with biological processes. Glycocalyx of mammalian cells actively participate in cell-cell, cell-matrix, and cell-pathogen interactions, which impact embryogenesis, growth and development, homeostasis, infection and immunity, signaling, malignancy, and metabolic disorders. Relative to genomics and proteomics, glycomics is just growing out of infancy with great potential in biomedicine for biomarker discovery, diagnosis, and treatment. However, the immense diversity and complexity of glycan structures and their multiple modes of interactions with proteins pose great challenges for development of analytical tools for delineating structure function relationships and understanding glycocode. Several tools are being developed for glycan profiling based on chromatography,m mass spectrometry, glycan microarrays, and glyco-informatics. Lectins, which have long been used in glyco-immunology, printed on a microarray provide a versatile platform for rapid high throughput analysis of glycoforms of biological samples. Herein, we summarize technological advances in lectin microarrays and critically review their impact on glycomics analysis. Challenges remain in terms of expansion to include nonplant derived lectins, standardization for routine clinical use, development of recombinant lectins, and exploration of plant kingdom for discovery of novel lectins.

Assessment of In-House Natural Product and Synthetic Compound Libraries Based on In vitro Inhibition of Cholinesterases

The first line medication for mild to moderate Alzheimer s disease (AD) is based on cholinesterase inhibitors which prolong the effect of the neurotransmitter acetylcholine in cholinergic nerve synapses which relieves the symptoms of the disease. Implications of cholinesterases involvement in disease modifying processes has increased interest in this research area. The drug discovery and development process is a long and expensive process that takes on average 13.5 years and costs approximately 0.9 billion US dollars. Drug attritions in the clinical phases are common due to several reasons, e.g., poor bioavailability of compounds leading to low efficacy or toxic effects. Thus, improvements in the early drug discovery process are needed to create highly potent non-toxic compounds with predicted drug-like properties. Nature has been a good source for the discovery of new medicines accounting for around half of the new drugs approved to market during the last three decades. These compounds are direct isolates from the nature, their synthetic derivatives or natural mimics. Synthetic chemistry is an alternative way to produce compounds for drug discovery purposes. Both sources have pros and cons. The screening of new bioactive compounds in vitro is based on assaying compound libraries against targets. Assay set-up has to be adapted and validated for each screen to produce high quality data. Depending on the size of the library, miniaturization and automation are often requirements to reduce solvent and compound amounts and fasten the process. In this contribution, natural extract, natural pure compound and synthetic compound libraries were assessed as sources for new bioactive compounds. The libraries were screened primarily for acetylcholinesterase inhibitory effect and secondarily for butyrylcholinesterase inhibitory effect. To be able to screen the libraries, two assays were evaluated as screening tools and adapted to be compatible with special features of each library. The assays were validated to create high quality data. Cholinesterase inhibitors with various potencies and selectivity were found in natural product and synthetic compound libraries which indicates that the two sources complement each other. It is acknowledged that natural compounds differ structurally from compounds in synthetic compound libraries which further support the view of complementation especially if a high diversity of structures is the criterion for selection of compounds in a library.

Network approaches to drug discovery

Introduction: Advances in genomics technologies are providing a very large amount of data on genome-wide gene expression profiles, protein molecules and their interactions with other macromolecules and metabolites. Molecular interaction networks provide a useful way to capture this complex data and comprehend it. Networks are beginning to be used in drug discovery, in many steps of the modern discovery pipeline, with large-scale molecular networks being particularly useful for the understanding of the molecular basis of the disease. Areas covered: The authors discuss network approaches used for drug target discovery and lead identification in the drug discovery pipeline. By reconstructing networks of targets, drugs and drug candidates as well as gene expression profiles under normal and disease conditions, the paper illustrates how it is possible to find relationships between different diseases, find biomarkers, explore drug repurposing and study emergence of drug resistance. Furthermore, the authors also look at networks which address particular important aspects such as off-target effects, combination-targets, mechanism of drug action and drug safety. Expert opinion: The network approach represents another paradigm shift in drug discovery science. A network approach provides a fresh perspective of understanding important proteins in the context of their cellular environments, providing a rational basis for deriving useful strategies in drug design. Besides drug target identification and inferring mechanism of action, networks enable us to address new ideas that could prove to be extremely useful for new drug discovery, such as drug repositioning, drug synergy, polypharmacology and personalized medicine.

A game theoretic approach for feature clustering and its application to feature selection

In this paper, we develop a game theoretic approach for clustering features in a learning problem. Feature clustering can serve as an important preprocessing step in many problems such as feature selection, dimensionality reduction, etc. In this approach, we view features as rational players of a coalitional game where they form coalitions (or clusters) among themselves in order to maximize their individual payoffs. We show how Nash Stable Partition (NSP), a well known concept in the coalitional game theory, provides a natural way of clustering features. Through this approach, one can obtain some desirable properties of the clusters by choosing appropriate payoff functions. For a small number of features, the NSP based clustering can be found by solving an integer linear program (ILP). However, for large number of features, the ILP based approach does not scale well and hence we propose a hierarchical approach. Interestingly, a key result that we prove on the equivalence between a k-size NSP of a coalitional game and minimum k-cut of an appropriately constructed graph comes in handy for large scale problems. In this paper, we use feature selection problem (in a classification setting) as a running example to illustrate our approach. We conduct experiments to illustrate the efficacy of our approach.

Biodegradation of sodium isopropyl xanthate by Paenibacillus polymyxa and Pseudomonas putida

Biodegradation of sodium isopropyl xanthate using two types of bacterial strains, Paenibacillus polymyxa and Pseudomonas putida, is demonstrated. At concentrations higher than 50 mg/L, the presence of xanthate in the growth medium resulted in bacterial toxicity, retarding growth kinetics. Adaptation through serial subculturing in the presence of higher xanthate concentrations resulted in the development of xanthate-tolerant bacterial strains. Stress proteins secreted by bacterial cells grown in the presence of xanthate were isolated. Bacterial cells could utilize xanthate as a growth substrate, degrading xanthate species in the process. Acidic metabolic products generated by bacterial metabolism promoted efficient xanthate decomposition. Probable mechanisms for the biodegradation of isopropyl xanthate are illustrated.