Crystallographic characterization of two novel crystal forms of human insulin induced by chaotropic agents and a shift in pH

Background: Insulin is a therapeutic protein that is widely used for the treatment of diabetes. Its biological function was discovered more than 80 years ago and it has since then been characterized extensively. Crystallization of the insulin molecule has always been a key activity since the protein is often administered by subcutaneous injections of crystalline insulin formulations. Over the years, insulin has been crystallized and characterized in a number of crystal systems. Results: Interestingly, we have now discovered two new crystal forms of human insulin. The crystals were obtained when the two chaotropic agents, urea and thiocyanate were present in the crystallization experiments, and their structures were determined by X-ray crystallography. The crystals belong to the orthorhombic and monoclinic crystal systems, with space groups C222(I) and C2 respectively. The orthorhombic crystals were obtained at pH 6.5 and contained three insulin hexamers in R-6 conformation in the asymmetric unit whilst the monoclinic C2 crystals were obtained at pH 7.0 and contained one R6 hexamer in the asymmetric unit. Common for the two new crystals is a hexamer-hexamer interaction that has not been found in any of the previous crystal forms of insulin. The contacts involve a tight glutamate-glutamate interaction with a distance of 2.3 angstrom between groups. The short distance suggests a low barrier hydrogen bond. In addition, two tyrosine-tyrosine interactions occupying a known phenol binding pocket contribute to the stabilization of the contacts. Within the crystals, distinct binding sites for urea were found, adding further to the discussion on the role of urea in protein denaturation. Conclusion: The change in space group from C222(I) to C2 was primarily caused by an increase in pH. The fewer number of hexamer-hexamer interactions comprising the short hydrogen bond in the C2 space group suggest that pH is the driving force. In addition, the distance between the two glutamates increases from 2.32 angstrom in the C222(I) crystals to 2.4 angstrom in the C2 crystals. However, in both cases the low barrier hydrogen bond and the tyrosine-tyrosine interaction should contribute to the stability of the crystals which is crucial when used in pharmaceutical formulations

Developing adaptive capacity in times of climate change in central rural Vietnam: exploring smallholders’ learning and governance

Climate change already affects Vietnam in virtually all sectors. Agriculture in small communities is particularly vulnerable to current and projected climate change impacts. Many of the smallholder farmers in Vietnam have limited adaptive capacity to deal with these impacts. Increasingly social learning is proposed as an important mechanism to build the adaptive capacity of local farming communities. However, little is known about the interplay between social learning and adaptive capacity and how adaptive capacity could be increased in a complex hierarchical governance setting that is typical in a country like Vietnam. The dissertation therefore aims to elicit and explore the ways through which social learning can increase the adaptive capacity of smallholder farmers in central Vietnam to respond to climate change impacts. Four research questions are addressed: (i) what insights does the existing body of climate change adaptation literature provide into the interplay between social learning and adaptive capacity?; (ii) what do smallholder farmers in Vietnam perceive as their current adaptive capacity and what enables or constrains them in increasing it?; (iii) how can social learning configurations strengthen the adaptive capacity of farming communities?; and (iv) how do different levels of government enable and constrain the process of building adaptive capacity and social learning of smallholder farmers to respond to impacts of climate change in Vietnam? Overall, the dissertation shows that social learning offers many possibilities to help farmers adapt to climate change, but that climate change adaptation in developing countries creates specific contextual conditions that require an adaptive capacity-focused perspective. An adequate learning configuration that can successfully help farmers build their adaptive capacity, considers responsive design, facilitation, monitoring, and evaluation steps. Furthermore, efforts of increasing adaptive capacity should not only focus on technical, social and human dimensions, but also on market conditions. The critical importance in creating an environment that enables social learning is the role of government across different levels. In order for the Vietnamese government to be more actively involved in building adaptive capacity through social learning, investments in transparent legal institutions, efficient use of limited available resources, and enhancing capacity of local policy actors will be critical in helping smallholder farmers learn how to adapt to climate change impacts.</p

O papel dos EUA e da URSS na reconstrução do Estado alemão na República de Weimar

O Tratado de Versalhes, assinado após o fim da Primeira Guerra Mundial, impôs duras restrições ao Estado Alemão, no que diz respeito ao seu poder econômico e militar. As perdas territoriais e os pagamentos em espécie das reparações impostas pelo Tratado de Versalhes faz com que a República de Weimar se inicie enfrentado uma Balança de Pagamentos deficitária que constituiu o principal fator para um período inflacionário que vai de 1918 a 1923. Além da dificuldade econômica, a Alemanha também sofreu com as restrições impostas ao seu exército. Tais restrições deixaram o exército alemão em posição inferior, em relação aos demais países europeus e, portanto, incapaz de garantir a segurança e a integridade territorial da Alemanha. A superação de tais dificuldades era condição essencial para que a Alemanha reconstruísse seu poder econômico e militar para, assim, voltar a ter papel relevante no cenário internacional. Para tal, contou com o auxílio de dois países importantes: EUA e URSS. A participação dos EUA foi de importância fundamental para a reconstrução do poder econômico do Estado Alemão. Através do capital norte americano, tornou-se possível encerrar o período inflacionário iniciando, portanto uma era de prosperidade que perdurou até a crise econômica de 1929. A importância da URSS ficou demonstrada a partir da análise do pacto militar germano-soviético, que desempenhou um papel importante, tanto na manutenção da integridade territorial alemã como na preparação militar de seu exército ao proporcionar os meios que permitiram à Alemanha se esquivar das cláusulas militares do Tratado de Versalhes

Food protein-induced enterocolitis syndrome &ndash; a review of the literature with focus on clinical management

Food protein-induced enterocolitis syndrome (FPIES) is a potentially severe presentation of non-IgE-mediated gastrointestinal food allergy (non-IgE-GI-FA) with heterogeneous clinical manifestations. Acute FPIES is typically characterized by profuse vomiting and lethargy, occurring classically 1-4 hours after ingestion of the offending food. When continuously exposed to the incriminated food, a chronic form has been described with persistent vomiting, diarrhea, and/or failure to thrive. Although affecting mainly infants, FPIES has also been described in adults. Although FPIES is actually one of the most actively studied non-IgE-GI-FAs, epidemiologic data are lacking, and estimation of the prevalence is based on a limited number of prospective studies. The exact pathomechanisms of FPIES remain not well defined, but recent data suggest involvement of neutrophils and mast cells, in addition to T cells. There is a wide range of food allergens that can cause FPIES with some geographical variations. The most frequently incriminated foods are cow milk, soy, and grains in Europe and USA. Furthermore, FPIES can be induced by foods usually considered as hypoallergenic, such as chicken, potatoes or rice. The diagnosis relies currently on typical clinical manifestations, resolving after the elimination of the offending food from the infant's/child's diet and/or an oral food challenge (OFC). The prognosis is usually favorable, with the vast majority of the case resolving before 5 years of age. Usually, assessment of tolerance acquisition by OFC is proposed every 12-18 months. Of note, a switch to an IgE-mediated FA is possible and has been suggested to be associated with a more severe phenotype. Avoiding the offending food requires education of the family of the affected child. A multidisciplinary approach including ideally allergists, gastroenterologists, dieticians, specialized nurses, and caregivers is often useful to optimize the management of these patients, that might be difficult

The Significance of Hemosiderin Deposition in the Lungs and Organs of the Mononucleated Macrophage Resorption System in Infants and Children

Hemosiderin deposition is not often recognized on routine examination with hematoxylin and eosin staining; however, iron stains may be helpful in the evaluation of hemosiderin deposition in infant autopsies. This report describes the data obtained from autopsy of 86 infants and children whose deaths were investigated at the Forensic Medicine Council Bursa Morgue Department from January 2000 to January 2003. A histochemical technique was used to identify hemosiderin in lung, liver and spleen specimens, which was correlated with other descriptive variables such as the reported cause of death, postmortem interval, trauma history, gender, and age. There was a weakly positive but significant correlation between lung and liver hemosiderin scores (Spearman's rank correlation coefficient, rho=0.348, p=0.001); i.e., given an increase in lung hemosiderin scores, an increase in liver hemosiderin scores was also observed. Similarly, a marked positive correlation between spleen and liver hemosiderin scores (Spearman's rank correlation coefficient, rho=0.335, p=0.002) was observed. The probability of spleen hemosiderin-positive cases belonging to the age group under 6 months was found to be 4.3 times greater than those who were hemosiderin-negative (95% confidence interval, 1.6-11.8). After the major differential diagnoses were ruled out, this study demonstrated, that depending on the statistically assessed morphometric grounds, the presence of hemosiderin deposits in the liver and spleen were significantly higher in the age group under 6 months

Insights into the hyperthermostability and unusual region-specificity of archaeal Pyrococcus abyssi tRNA m1A57/58 methyltransferase

The S-adenosyl-l-methionine dependent methylation of adenine 58 in the T-loop of tRNAs is essential for cell growth in yeast or for adaptation to high temperatures in thermophilic organisms. In contrast to bacterial and eukaryotic tRNA m1A58 methyltransferases that are site-specific, the homologous archaeal enzyme from Pyrococcus abyssi catalyzes the formation of m1A also at the adjacent position 57, m1A57 being a precursor of 1-methylinosine. We report here the crystal structure of P. abyssi tRNA m1A57/58 methyltransferase (PabTrmI), in complex with S-adenosyl-l-methionine or S-adenosyl-l-homocysteine in three different space groups. The fold of the monomer and the tetrameric architecture are similar to those of the bacterial enzymes. However, the inter-monomer contacts exhibit unique features. In particular, four disulfide bonds contribute to the hyperthermostability of the archaeal enzyme since their mutation lowers the melting temperature by 16.5°C. His78 in conserved motif X, which is present only in TrmIs from the Thermococcocales order, lies near the active site and displays two alternative conformations. Mutagenesis indicates His78 is important for catalytic efficiency of PabTrmI. When A59 is absent in tRNAAsp, only A57 is modified. Identification of the methylated positions in tRNAAsp by mass spectrometry confirms that PabTrmI methylates the first adenine of an AA sequence

Reality Hackers: The Next Wave of Media Revolutionaries

Just as the printing press gave rise to the nation-state, emerging technologies are reshaping collective identities and challenging our understanding of what it means to be human. Should citizens have the right to be truly anonymous on-line? Should we be concerned about the fact that so many people are choosing to migrate to virtual worlds? Are injectible microscopic radio-frequency ID chips a blessing or a curse? Is the use of cognitive enhancing nootropics a human right or an unforgivable transgression? Should genomic data about human beings be hidden away with commercial patents or open-sourced like software? Should hobbyists known as biohackers be allowed to experiment with genetic engineering in their home laboratories? The time-frame for acting on such questions is relatively short, and these decisions are too important to be left up to a small handful of scientists and policymakers. If democracy is to continue as a viable alternative to technocracy, the average citizen must become more involved in these debates. To borrow a line from the computer visionary Ted Nelson, all of us can -- and must -- understand technology now. Challenging the popular stereotype of hackers as ciminal sociopaths, reality hackers uphold the basic tenets of what Steven Levy (1984) terms the hacker ethic. These core principles include a commitment to: sharing, openness, decentralization, public access to information, and the use of new technologies to make the world a better place.https://digitalcommons.trinity.edu/mono/1000/thumbnail.jp