Disaccharide uptake and priming in animal cells: inhibition of sialyl Lewis X by acetylated Gal beta 1-->4GlcNAc beta-O-naphthalenemethanol.

Inhibitors of glycosylation provide a tool for studying the biology of glycoconjugates. One class of inhibitors consists of glycosides that block glycoconjugate synthesis by acting as primers of free oligosaccharide chains. A typical primer contains one sugar linked to a hydrophobic aglycone. In this report, we describe a way to use disaccharides as primers. Chinese hamster ovary cells readily take up glycosides containing a pentose linked to naphthol, but they take up hexosides less efficiently and disaccharides not at all. Linking phenanthrol to a hexose improves its uptake dramatically but has no effect on disaccharides. To circumvent this problem, analogs of Xyl beta 1-->6Gal beta-O-2-naphthol were tested as primers of glycosaminoglycan chains. The unmodified disaccharide did not prime, but methylated derivatives had activity in the order Xyl beta 1-->6Gal(Me)3-beta-O-2-naphthol > Xyl beta 1-->6Gal (Me)2 beta-O-2-naphthol >> Xyl beta 1-->6Gal(Me)beta-O-2-naphthol. Acetylated Xyl beta 1-->6Gal beta-O-2-naphthol also primed glycosaminoglycans efficiently, suggesting that the terminal xylose residue was exposed by removing the acetyl groups. The general utility of using acetyl groups to create disaccharide primers was shown by the priming of oligosaccharides on peracetylated Gal beta 1-->4GlcNAc beta-O-naphthalenemethanol. This disaccharide inhibited sialyl Lewis X expression on HL-60 cells.

Self-standing curved crystals for X and Gamma rays focusing

The efficiency of a Laue lens for X and Gamma ray focusing in the energy range 60 ÷ 600 keV is closely linked to the diffraction efficiency of the single crystals composing the lens. A powerful focusing system is crucial for applications like medical imaging and X ray astronomy where wide beams must be focused. Mosaic crystals with a high density, such as Cu or Au, and bent crystals with curved diffracting planes (CDP) are considered for the realization of a focusing system for X rays, owing to their high diffraction efficiency. In this work, a comparison of the efficiency of CDP crystals and mosaic crystals was performed on the basis of the theory of X-ray diffraction. Si, GaAs and Ge CDP crystals with optimized thicknesses and moderate radii of curvature of several tens of metres demonstrate comparable or superior performance with respect to the higher atomic number mosaic crystals generally used. A simplified approach for calculating the integrated reflectivity of the crystals is applied. A bending technique used during this work to realize CDP crystals consists in a controlled surface damaging induced by a mechanical lapping process. A compressive strained layer of few micrometres in thickness is generated and causes the convex curvature of the damaged side of the crystal. Another new bending technique is developed and the main results are shown. The process consists on a film deposition of a selected bi-component epoxy resin on one side of crystal, made uniform in thickness by mean of a spin-coater. Choosing the speed of spin-coating, so changing the thickness of the film, a control of radius of curvature can be obtained. Moreover the possibility to combine the two bending technique to obtain CDP crystal with a stronger curvature in rather thick crystals was demonstrated. Detailed characterization of Si, and GaAs CDP crystals at low and high x-ray energies are performed on flat and bent crystals obtained with the damaging and the resin deposition technique. As expected an increase of diffraction efficiency in asymmetrical diffraction geometry in CDP crystals with respect to the flat ones is observed. On the other hand an unexpected increase of the integrated intensity in symmetrical geometry, not predicted by the theory, is observed in all the measurements performed with different set up. The experimental trend of the integrated reflectivity as a function of the radius of curvature is in a good agreement with that predicted by the theory of bent perfect crystals, so it is possible to conclude that the surface damage has a limited effect on the crystal reflectivity. A study of the integrated reflectivity in the energy range of interest (100÷350 keV) in CDP crystals realized with damaging and resin deposition technique at symmetrical and asymmetrical geometries was performed at ILL Institute. Also at these energies the diffraction efficiency of bent crystals was much larger (a 12 time increase is observed for bent crystals in asymmetrical 111 geometry) than that measured in flat crystals. The diffraction efficiency of CDP crystals realized with both techniques tends to coincide with that of flat crystals at very high energies (> 200 keV). This suggesting that also real flat perfect crystals can be considered as strongly bent or mosaic crystals at very high X ray energies.

Reciprocal N ((NH4+)-N-15 or (NO3-)-N-15) transfer between nonN(2)-fixing Eucalyptus maculata and N-2-fixing Casuarina cunninghamiana linked by the ectomycorrhizal fungus Pisolithus sp.

Two-way N transfers mediated by Pisolithus sp. were examined by excluding root contact and supplying (NH4+)-N-15 or (NO3-)-N-15 to 6-month-old Eucalyptus maculata or Casuarina cunninghamiana grown in two-chambered-pots separated by 37 m screens. Mycorrhizal colonization was 35% in Eucalyptus and 66% in Casuarina (c. 29% N-2-fixation). Using an environmental scanning electron microscope, living hyphae were observed to interconnect Eucalyptus and Casuarina. Biomass and N accumulation was greatest in nodulated mycorrhizal Casuarina/mycorrhizal Eucalyptus pairs, less in nonnodulated mycorrhizal Casuarina/mycorrhizal Eucalyptus pairs, and least in nonnodulated nonmycorrhizal Casuarina/nonmycorrhizal Eucalyptus pairs. In nonnodulated mycorrhizal pairs, N transfers to Eucalyptus or to Casuarina were similar (2.4-4.1 mg per plant in either direction) and were 2.6-4.0 times greater than in nonnodulated nonmycorrhizal pairs. In nodulated mycorrhizal pairs, N transfers were greater to Eucalyptus (5-7 times) and to Casuarina (12-18 times) than in nonnodulated mycorrhizal pairs. Net transfer to Eucalyptus or to Casuarina was low in both nonnodulated nonmycorrhizal (< 0.7 mg per plant) and nonnodulated mycorrhizal pairs (< 1.1 mg per plant). In nodulated mycorrhizal pairs, net transfer to Casuarina was 26.0 mg per plant. The amount and direction of two-way mycorrhiza-mediated N transfer was increased by the presence of Pisolithus sp. and Frankia, resulting in a net N transfer from low-N-demanding Eucalyptus to high-N-demanding Casuarina.

On dendrites in Down syndrome and DS murine models: a spiny way to learn

Since the discovery in the 1970s that dendritic abnormalities in cortical pyramidal neurons are the most consistent pathologic correlate of mental retardation, research has focused on how dendritic alterations are related to reduced intellectual ability. Due in part to obvious ethical problems and in part to the lack of fruitful methods to study neuronal circuitry in the human cortex, there is little data about the microanatomical contribution to mental retardation. The recent identification of the genetic bases of some mental retardation associated alterations, coupled with the technology to create transgenic animal models and the introduction of powerful sophisticated tools in the field of microanatomy, has led to a growth in the studies of the alterations of pyramidal cell morphology in these disorders. Studies of individuals with Down syndrome, the most frequent genetic disorder leading to mental retardation, allow the analysis of the relationships between cognition, genotype and brain microanatomy. In Down syndrome the crucial question is to define the mechanisms by which an excess of normal gene products, in interaction with the environment, directs and constrains neural maturation, and how this abnormal development translates into cognition and behaviour. In the present article we discuss mainly Down syndrome-associated dendritic abnormalities and plasticity and the role of animal models in these studies. We believe that through the further development of such approaches, the study of the microanatomical substrates of mental retardation will contribute significantly to our understanding of the mechanisms underlying human brain disorders associated with mental retardation. (C) 2004 Elsevier Ltd. All rights reserved.

Response of crayfish, Procambarus clarkii, haemocytes infected by white spot syndrome virus

White spot syndrome virus ( WSSV) is a serious pathogen of aquatic crustaceans. Little is known about its transmission in vivo and the immune reaction of its hosts. In this study, the circulating haemocytes of crayfish, Procambarus clarkii, infected by WSSV, and primary haemocyte cultures inoculated with WSSV, were collected and observed by transmission electron microscopy and light microscopy following in situ hybridization. In ultrathin sections of infected haemocytes, the enveloped virions were seen to be phagocytosed in the cytoplasm and no viral particles were observed in the nuclei. In situ hybridization with WSSV-specific probes also demonstrated that there were no specific positive signals present in the haemocytes. Conversely, strong specific positive signals showed that WSSV replicated in the nuclei of gill cells. As a control, the lymphoid organ of shrimp, Penaeus monodon, infected by WSSV was examined by in situ hybridization which showed that WSSV did not replicate within the tubules of the lymphoid organ. In contrast to previous studies, it is concluded that neither shrimp nor crayfish haemocytes support WSSV replication.White spot syndrome virus (WSSV) is a serious pathogen of aquatic crustaceans. Little is known about its transmission in vivo and the immune reaction of its hosts. In this study, the circulating haemocytes of crayfish, Procambarus clarkii, infected by WSSV, and primary haemocyte cultures inoculated with WSSV, were collected and observed by transmission electron microscopy and light microscopy following in situ hybridization. In ultra-thin sections of infected haemocytes, the enveloped virions were seen to be phagocytosed in the cytoplasm and no viral particles were observed in the nuclei. In situ hybridization with WSSV-specific probes also demonstrated that there were no specific positive signals present in the haemocytes. Conversely, strong specific positive signals showed that WSSV replicated in the nuclei of gill cells. As a control, the lymphoid organ of shrimp, Penaeus monodon, infected by WSSV was examined by in situ hybridization which showed that WSSV did not replicate within the tubules of the lymphoid organ. In contrast to previous studies, it is concluded that neither shrimp nor crayfish haemocytes support WSSV replication.

Dietary patterns and metabolic syndrome - a review of epidemiologic evidence

Metabolic syndrome is associated with increased risk of coronary heart disease and type 2 diabetes, and appears to be widely prevalent in both developed and developing countries. While lifestyle modification is recommended for management of the syndrome, the dietary pattern most beneficial for patients is yet to be ascertained. Original research papers from the Medline database were examined for dietary patterns that may be associated with the syndrome. Three large-scale epidemiological studies were found fitting our criteria. Dietary patterns high in fruit and vegetable content were generally found to be associated with lower prevalence of metabolic syndrome. Diet patterns with high meat intake were frequently associated with components of metabolic syndrome, particularly impaired glucose tolerance. High dairy intake was generally associated with reduced risk for components of metabolic syndrome with some inconsistency in the literature regarding risk of obesity. Minimally processed cereals appeared to be associated with decreased risk of metabolic syndrome, while highly processed cereals with high glycaemic index are associated with higher risk. Fried foods were noticeably absent from any dietary pattern associated with decreased prevalence of metabolic syndrome. The conclusion of this review is that no individual dietary component could be considered wholly responsible for the association of diet with metabolic syndrome. Rather it is the overall quality of the diet that appears to offer protection against lifestyle disease such as metabolic syndrome. Further research is required into conditions, such as overweight and obesity, which may influence the effect of diet on the development of metabolic syndrome.

Radioimmunoassay of insulin and glucagon with studies on the obese hyperglycaemic syndrome in mice

Sensitive and precise radioimmunoassays for insulin and glucagon have been established. Although it was possible to employ similar precepts to the development of both hormone assays, the establishment of a reliable glucagon radioimmunoassay was complicated by the poor immunogenicity and instability of the peptide. Thus, unlike insulin antisera which were prepared by monthly injection of guinea pigs with crystalline insulin emulsified in adjuvant, the successful production of glucagon antisera was accomplished by immunisation of rabbits and guinea pigs with glucagon covalently linked to bovine plasma albumin. The conventional chloramine-T iodination with purification by gel chromatography was only suitable for the production of labelled insulin. Quality tracer for use in the glucagon radioimmunoassay was prepared by trace iodination, with subsequent purification of monoiodinated glucagon by anion exchange chromatography. Separation of free and antibody bound moieties by coated charcoal was applicable to both hormone assays, and a computerised data processing system, relying on logit-log transformation, was used to analyse all assay results. The assays were employed to evaluate the regulation of endocrine pancreatic function and the role of insulin and glucagon in the pathogenesis of the obese hyperglycaemic syndrome in mice. In the homozygous (ob/ob) condition, mice of the Birmingham strain were characterised by numerous abnormalities of glucose homeostasis, several of which were detected in heterozygous (ob/+) mice. Obese mice exhibited pancreatic alpha cell dysfunction and hyperglucagonaemia. Investigation of this defect revealed a marked insensitivity of an insulin dependent glucose sensing mechanism that inhibited glucagon secretion. Although circulating glucagon was of minor importance in the maintenance of hyperinsulinaemia, lack of suppression of alpha cell function by glucose and insulin contributed significantly to both the insulin insensitivity and the hyperglycaemia of obese mice.

Under the influence? The construction of foetal alcohol syndrome in UK newspapers

Today, alongside many other proscriptions, women are expected to abstain or at least limit their alcohol consumption during pregnancy. This advice is reinforced through warning labels on bottles and cans of alcoholic drinks. In most (but not all) official policies, this is linked to a risk of Foetal Alcohol Syndrome (FAS) or one of its associated conditions. However, given that there is little medical evidence that low levels of alcohol consumption have an adverse impact on the foetus, we need to examine broader societal ideas to explain why this has now become a policy concern. This paper presents a quantitative and qualitative assessment of analysis of the media in this context. By analysing the frames over time, this paper will trace the emergence of concerns about alcohol consumption during pregnancy. It will argue that contemporary concerns about FAS are framed around a number of pre-existing discourses including alcohol consumption as a social problem, heightened concerns about children at risk and shifts in ideas about the responsibility of motherhood including during the pre-conception and pregnancy periods. Whilst the newspapers regularly carried critiques of the abstinence position now advocated, these challenges focused did little to refute current parenting cultures.

An X-ray micro-fluorescence study to investigate the distribution of Al, Si, P and Ca ions in the surrounding soft tissue after implantation of a calcium phosphate-mullite ceramic composite in a rabbit animal model

Synthetic calcium phosphates, despite their bioactivity, are brittle. Calcium phosphate-mullite composites have been suggested as potential dental and bone replacement materials which exhibit increased toughness. Aluminium, present in mullite, has however been linked to bone demineralisation and neurotoxicity: it is therefore important to characterise the materials fully in order to understand their in vivo behaviour. The present work reports the compositional mapping of the interfacial region of a calcium phosphate-20 wt% mullite biocomposite/soft tissue interface, obtained from the samples implanted into the long bones of healthy rabbits according to standard protocols (ISO-10993) for up to 12 weeks. X-ray micro-fluorescence was used to map simultaneously the distribution of Al, P, Si and Ca across the ceramic-soft tissue interface. A well defined and sharp interface region was present between the ceramic and the surrounding soft tissue for each time period examined. The concentration of Al in the surrounding tissue was found to fall by two orders of magnitude, to the background level, within similar to 35 mu m of the implanted ceramic.

A case of bilateral perisylvian syndrome with reading disability

Bilateral Perisylvian Syndrome (BPS) often presents with epilepsy and significant behavioral impairments that can include mental retardation, dysarthria, delayed speech development, and delayed fine motor development (Graff-Radford et al., 1986 and Kuzniecky et al., 1993). While a small subset of BPS cases have been described as having relatively isolated language delays (Leventer et al., 2010), BPS is not expected in children with dyslexia. As part of a Medical University of South Carolina, IRB approved multi-site study involving retrospective and de-identified dyslexia data, we unexpectedly identified a 14.05 year old male with evidence of BPS whose father had been diagnosed with dyslexia and dysgraphia. This child had been recruited for a neuroimaging study on dyslexia from a school specializing in educating children with dyslexia. The T1-weighted MRI scan from this child demonstrated a highly unusual perisylvian sulcal/gyral patterning that is a defining feature of BPS (Fig. 1). BPS cases exhibit bilateral dysgenesis of the Sylvian fissure and surrounding gyri, which appears to occur because of a limited or absent arcuate fasciculus (Kilinc, Ekinci, Demirkol, & Agan, 2015). This BPS case also had a relatively enlarged atrium of the lateral ventricle that is consistent with the BPS anatomical presentation and reduction of parietal white matter (Graff-Radford et al., 1986, Kilinc et al., 2015 and Toldo et al., 2011).

Raw X-ray fluorescence (XRF) scannings and radiocarbon age of sediment cores GeoB8331-4, and GeoB8322-2

Variations in the sediment input to the Namaqualand mudbelt during the Holocene are assessed using an integrative terrestrial to marine, source to sink approach. Geochemical and Sr and Nd isotopic signatures are used to distinguish fluvial sediment source areas. Relative to the sediments of the Olifants River, craton outcrops in the northern Orange River catchment have a more radiogenic Sr and a more unradiogenic Nd isotopic signature. Furthermore, upper Orange River sediments are rich in heavier elements such as Ti and Fe derived from the chemical weathering of Drakensberg flood basalt. Suspension load signatures change along the Orange River's westward transit as northern catchments contribute physical weathering products from the Fish and Molopo River catchment area. Marine cores offshore of the Olifants (GeoB8323-2) and Orange (GeoB8331-4) River mouths show pulses of increased contribution of Olifants River and upper Orange River input, respectively. These pulses coincide with intervals of increased terrestrial organic matter flux and increased paleo-production at the respective core sites. We attribute this to an increase in fluvial activity and vegetation cover in the adjacent catchments during more humid climate conditions. The contrast in the timing of these wet phases in the catchment areas reflects the bipolar behavior of the South African summer and winter rainfall zones. While rainfall in the Orange River catchment is related to southward shifts in the ICTZ, rainfall in the Olifants catchment is linked to northward shifts in Southern Hemisphere Westerly storm tracks. The later may also have increased southern Benguela upwelling in the past by reducing the shedding of Agulhas eddies into the Atlantic. The high-resolution records of latitudinal shifts in these atmospheric circulation systems correspond to late Holocene centennial-millennial scale climate variability evident in Antarctic ice core records. The mudbelt cores indicate that phases of high summer rainfall zone and low winter rainfall zone humidity (at ca. 2.8 and 1 ka BP) may be synchronous with Antarctic warming events. On the other hand, dry conditions in the summer rainfall zone along with wet conditions in the winter rainfall zone (at ca 3.3, 2 and 0.5 ka BP) may be associated with Antarctic cooling events.

Mutation of SLC35D3 causes metabolic syndrome by impairing dopamine signaling in striatal D1 neurons

Funding: This work was partially supported by grants from National Basic Research Program of China (2013CB530605; 2014CB942803), from National Natural Science Foundation of China 1230046; 31071252; 81101182) and from Chinese Academy of Sciences (KSCX2-EW-R-05, KJZD-EW-L08). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Arterial entrapment syndrome in the cubital fossa: a rare cause of acute stress-related arterial thrombosis in a patient with brachial artery duplication

Arterial entrapment syndrome (AES) at elbow level is very rare and to our knowledge no case of AES by lacertus fibrosus in the cubital fossa in presence of brachial artery duplication has been described to date. We describe a rare case of acute arterial thrombosis of one of two brachial arteries highlighted in the cubital fossa which developed after strenuous right elbow flexor muscle activity and hyper-extensions presumably related to AES by lacertus fibrosus at elbow level. A 43-year-old right-handed woman, experienced paleness, coldness and numbness of the right hand, after 8 consecutive hours of gardening. As she worked, her ipsilateral flexor elbow muscles remained in prolonged and inappropriate tension. Clinical examination evidenced the absence of radial artery pulse in the wrist and mild hypothermia in the second and third finger. During surgical exploration two anastomosed brachial arteries were detected in the cubital fossa under the lacertus fibrosus. The lateral superficial brachial artery was occluded. Intraoperative arteriography evidenced brachial artery duplication at the third superior of the arm and normal vascular pattern at the forearm level. In cases of unexplained atypical intermittent upper extremity claudication or acute ischemic symptoms an AES should always be ruled out, particularly when symptoms are exacerbated by strenuous upper extremity activity or when upper limb muscular hypertrophy is evident. In these cases a thorough dynamic clinical and instrumental examination is mandatory to confirm a diagnosis of AES and to avoid possible future ischemic complications.

Microfluidic platforms for the characterization of in meso membrane protein crystallization

Membrane proteins, which reside in the membranes of cells, play a critical role in many important biological processes including cellular signaling, immune response, and material and energy transduction. Because of their key role in maintaining the environment within cells and facilitating intercellular interactions, understanding the function of these proteins is of tremendous medical and biochemical significance. Indeed, the malfunction of membrane proteins has been linked to numerous diseases including diabetes, cirrhosis of the liver, cystic fibrosis, cancer, Alzheimer's disease, hypertension, epilepsy, cataracts, tubulopathy, leukodystrophy, Leigh syndrome, anemia, sensorineural deafness, and hypertrophic cardiomyopathy.1-3 However, the structure of many of these proteins and the changes in their structure that lead to disease-related malfunctions are not well understood. Additionally, at least 60% of the pharmaceuticals currently available are thought to target membrane proteins, despite the fact that their exact mode of operation is not known.4-6 Developing a detailed understanding of the function of a protein is achieved by coupling biochemical experiments with knowledge of the structure of the protein. Currently the most common method for obtaining three-dimensional structure information is X-ray crystallography. However, no a priori methods are currently available to predict crystallization conditions for a given protein.7-14 This limitation is currently overcome by screening a large number of possible combinations of precipitants, buffer, salt, and pH conditions to identify conditions that are conducive to crystal nucleation and growth.7,9,11,15-24 Unfortunately, these screening efforts are often limited by difficulties associated with quantity and purity of available protein samples. While the two most significant bottlenecks for protein structure determination in general are the (i) obtaining sufficient quantities of high quality protein samples and (ii) growing high quality protein crystals that are suitable for X-ray structure determination,7,20,21,23,25-47 membrane proteins present additional challenges. For crystallization it is necessary to extract the membrane proteins from the cellular membrane. However, this process often leads to denaturation. In fact, membrane proteins have proven to be so difficult to crystallize that of the more than 66,000 structures deposited in the Protein Data Bank,48 less than 1% are for membrane proteins, with even fewer present at high resolution (< 2Å)4,6,49 and only a handful are human membrane proteins.49 A variety of strategies including detergent solubilization50-53 and the use of artificial membrane-like environments have been developed to circumvent this challenge.43,53-55 In recent years, the use of a lipidic mesophase as a medium for crystallizing membrane proteins has been demonstrated to increase success for a wide range of membrane proteins, including human receptor proteins.54,56-62 This in meso method for membrane protein crystallization, however, is still by no means routine due to challenges related to sample preparation at sub-microliter volumes and to crystal harvesting and X-ray data collection. This dissertation presents various aspects of the development of a microfluidic platform to enable high throughput in meso membrane protein crystallization at a level beyond the capabilities of current technologies. Microfluidic platforms for protein crystallization and other lab-on-a-chip applications have been well demonstrated.9,63-66 These integrated chips provide fine control over transport phenomena and the ability to perform high throughput analyses via highly integrated fluid networks. However, the development of microfluidic platforms for in meso protein crystallization required the development of strategies to cope with extremely viscous and non-Newtonian fluids. A theoretical treatment of highly viscous fluids in microfluidic devices is presented in Chapter 3, followed by the application of these strategies for the development of a microfluidic mixer capable of preparing a mesophase sample for in meso crystallization at a scale of less than 20 nL in Chapter 4. This approach was validated with the successful on chip in meso crystallization of the membrane protein bacteriorhodopsin. In summary, this is the first report of a microfluidic platform capable of performing in meso crystallization on-chip, representing a 1000x reduction in the scale at which mesophase trials can be prepared. Once protein crystals have formed, they are typically harvested from the droplet they were grown in and mounted for crystallographic analysis. Despite the high throughput automation present in nearly all other aspects of protein structure determination, the harvesting and mounting of crystals is still largely a manual process. Furthermore, during mounting the fragile protein crystals can potentially be damaged, both from physical and environmental shock. To circumvent these challenges an X-ray transparent microfluidic device architecture was developed to couple the benefits of scale, integration, and precise fluid control with the ability to perform in situ X-ray analysis (Chapter 5). This approach was validated successfully by crystallization and subsequent on-chip analysis of the soluble proteins lysozyme, thaumatin, and ribonuclease A and will be extended to microfluidic platforms for in meso membrane protein crystallization. The ability to perform in situ X-ray analysis was shown to provide extremely high quality diffraction data, in part as a result of not being affected by damage due to physical handling of the crystals. As part of the work described in this thesis, a variety of data collection strategies for in situ data analysis were also tested, including merging of small slices of data from a large number of crystals grown on a single chip, to allow for diffraction analysis at biologically relevant temperatures. While such strategies have been applied previously,57,59,61,67 they are potentially challenging when applied via traditional methods due to the need to grow and then mount a large number of crystals with minimal crystal-to-crystal variability. The integrated nature of microfluidic platforms easily enables the generation of a large number of reproducible crystallization trials. This, coupled with in situ analysis capabilities has the potential of being able to acquire high resolution structural data of proteins at biologically relevant conditions for which only small crystals, or crystals which are adversely affected by standard cryocooling techniques, could be obtained (Chapters 5 and 6). While the main focus of protein crystallography is to obtain three-dimensional protein structures, the results of typical experiments provide only a static picture of the protein. The use of polychromatic or Laue X-ray diffraction methods enables the collection of time resolved structural information. These experiments are very sensitive to crystal quality, however, and often suffer from severe radiation damage due to the intense polychromatic X-ray beams. Here, as before, the ability to perform in situ X-ray analysis on many small protein crystals within a microfluidic crystallization platform has the potential to overcome these challenges. An automated method for collecting a "single-shot" of data from a large number of crystals was developed in collaboration with the BioCARS team at the Advanced Photon Source at Argonne National Laboratory (Chapter 6). The work described in this thesis shows that, even more so than for traditional structure determination efforts, the ability to grow and analyze a large number of high quality crystals is critical to enable time resolved structural studies of novel proteins. In addition to enabling X-ray crystallography experiments, the development of X-ray transparent microfluidic platforms also has tremendous potential to answer other scientific questions, such as unraveling the mechanism of in meso crystallization. For instance, the lipidic mesophases utilized during in meso membrane protein crystallization can be characterized by small angle X-ray diffraction analysis. Coupling in situ analysis with microfluidic platforms capable of preparing these difficult mesophase samples at very small volumes has tremendous potential to enable the high throughput analysis of these systems on a scale that is not reasonably achievable using conventional sample preparation strategies (Chapter 7). In collaboration with the LS-CAT team at the Advanced Photon Source, an experimental station for small angle X-ray analysis coupled with the high quality visualization capabilities needed to target specific microfluidic samples on a highly integrated chip is under development. Characterizing the phase behavior of these mesophase systems and the effects of various additives present in crystallization trials is key for developing an understanding of how in meso crystallization occurs. A long term goal of these studies is to enable the rational design of in meso crystallization experiments so as to avoid or limit the need for high throughput screening efforts. In summary, this thesis describes the development of microfluidic platforms for protein crystallization with in situ analysis capabilities. Coupling the ability to perform in situ analysis with the small scale, fine control, and the high throughput nature of microfluidic platforms has tremendous potential to enable a new generation of crystallographic studies and facilitate the structure determination of important biological targets. The development of platforms for in meso membrane protein crystallization is particularly significant because they enable the preparation of highly viscous mixtures at a previously unachievable scale. Work in these areas is ongoing and has tremendous potential to improve not only current the methods of protein crystallization and crystallography, but also to enhance our knowledge of the structure and function of proteins which could have a significant scientific and medical impact on society as a whole. The microfluidic technology described in this thesis has the potential to significantly advance our understanding of the structure and function of membrane proteins, thereby aiding the elucidation of human biology, the development of pharmaceuticals with fewer side effects for a wide range of diseases. References (1) Quick, M.; Javitch, J. A. P Natl Acad Sci USA 2007, 104, 3603. (2) Trubetskoy, V. S.; Burke, T. J. Am Lab 2005, 37, 19. (3) Pecina, P.; Houstkova, H.; Hansikova, H.; Zeman, J.; Houstek, J. Physiol Res 2004, 53, S213. (4) Arinaminpathy, Y.; Khurana, E.; Engelman, D. M.; Gerstein, M. B. Drug Discovery Today 2009, 14, 1130. (5) Overington, J. P.; Al-Lazikani, B.; Hopkins, A. L. Nat Rev Drug Discov 2006, 5, 993. (6) Dauter, Z.; Lamzin, V. S.; Wilson, K. S. Current Opinion in Structural Biology 1997, 7, 681. (7) Hansen, C.; Quake, S. R. Current Opinion in Structural Biology 2003, 13, 538. (8) Govada, L.; Carpenter, L.; da Fonseca, P. C. A.; Helliwell, J. R.; Rizkallah, P.; Flashman, E.; Chayen, N. E.; Redwood, C.; Squire, J. M. J Mol Biol 2008, 378, 387. (9) Hansen, C. L.; Skordalakes, E.; Berger, J. M.; Quake, S. R. P Natl Acad Sci USA 2002, 99, 16531. (10) Leng, J.; Salmon, J.-B. Lab Chip 2009, 9, 24. (11) Zheng, B.; Gerdts, C. J.; Ismagilov, R. F. Current Opinion in Structural Biology 2005, 15, 548. (12) Lorber, B.; Delucas, L. J.; Bishop, J. B. J Cryst Growth 1991, 110, 103. (13) Talreja, S.; Perry, S. L.; Guha, S.; Bhamidi, V.; Zukoski, C. F.; Kenis, P. J. A. The Journal of Physical Chemistry B 2010, 114, 4432. (14) Chayen, N. E. Current Opinion in Structural Biology 2004, 14, 577. (15) He, G. W.; Bhamidi, V.; Tan, R. B. H.; Kenis, P. J. A.; Zukoski, C. F. Cryst Growth Des 2006, 6, 1175. (16) Zheng, B.; Tice, J. D.; Roach, L. S.; Ismagilov, R. F. Angew Chem Int Edit 2004, 43, 2508. (17) Li, L.; Mustafi, D.; Fu, Q.; Tereshko, V.; Chen, D. L. L.; Tice, J. D.; Ismagilov, R. F. P Natl Acad Sci USA 2006, 103, 19243. (18) Song, H.; Chen, D. L.; Ismagilov, R. F. Angew Chem Int Edit 2006, 45, 7336. (19) van der Woerd, M.; Ferree, D.; Pusey, M. Journal of Structural Biology 2003, 142, 180. (20) Ng, J. D.; Gavira, J. A.; Garcia-Ruiz, J. M. Journal of Structural Biology 2003, 142, 218. (21) Talreja, S.; Kenis, P. J. A.; Zukoski, C. F. Langmuir 2007, 23, 4516. (22) Hansen, C. L.; Quake, S. R.; Berger, J. M. US, 2007. (23) Newman, J.; Fazio, V. J.; Lawson, B.; Peat, T. S. Cryst Growth Des 2010, 10, 2785. (24) Newman, J.; Xu, J.; Willis, M. C. Acta Crystallographica Section D 2007, 63, 826. (25) Collingsworth, P. D.; Bray, T. L.; Christopher, G. K. J Cryst Growth 2000, 219, 283. (26) Durbin, S. D.; Feher, G. Annu Rev Phys Chem 1996, 47, 171. (27) Talreja, S.; Kim, D. Y.; Mirarefi, A. Y.; Zukoski, C. F.; Kenis, P. J. A. J Appl Crystallogr 2005, 38, 988. (28) Yoshizaki, I.; Nakamura, H.; Sato, T.; Igarashi, N.; Komatsu, H.; Yoda, S. J Cryst Growth 2002, 237, 295. (29) Anderson, M. J.; Hansen, C. L.; Quake, S. R. P Natl Acad Sci USA 2006, 103, 16746. (30) Hansen, C. L.; Sommer, M. O. A.; Quake, S. R. P Natl Acad Sci USA 2004, 101, 14431. (31) Lounaci, M.; Rigolet, P.; Abraham, C.; Le Berre, M.; Chen, Y. Microelectron Eng 2007, 84, 1758. (32) Zheng, B.; Roach, L. S.; Ismagilov, R. F. J Am Chem Soc 2003, 125, 11170. (33) Zhou, X.; Lau, L.; Lam, W. W. L.; Au, S. W. N.; Zheng, B. Anal. Chem. 2007. (34) Cherezov, V.; Caffrey, M. J Appl Crystallogr 2003, 36, 1372. (35) Qutub, Y.; Reviakine, I.; Maxwell, C.; Navarro, J.; Landau, E. M.; Vekilov, P. G. J Mol Biol 2004, 343, 1243. (36) Rummel, G.; Hardmeyer, A.; Widmer, C.; Chiu, M. L.; Nollert, P.; Locher, K. P.; Pedruzzi, I.; Landau, E. M.; Rosenbusch, J. P. Journal of Structural Biology 1998, 121, 82. (37) Gavira, J. A.; Toh, D.; Lopez-Jaramillo, J.; Garcia-Ruiz, J. M.; Ng, J. D. Acta Crystallogr D 2002, 58, 1147. (38) Stevens, R. C. Current Opinion in Structural Biology 2000, 10, 558. (39) Baker, M. Nat Methods 2010, 7, 429. (40) McPherson, A. In Current Topics in Membranes, Volume 63; Volume 63 ed.; DeLucas, L., Ed.; Academic Press: 2009, p 5. (41) Gabrielsen, M.; Gardiner, A. T.; Fromme, P.; Cogdell, R. J. In Current Topics in Membranes, Volume 63; Volume 63 ed.; DeLucas, L., Ed.; Academic Press: 2009, p 127. (42) Page, R. In Methods in Molecular Biology: Structural Proteomics - High Throughput Methods; Kobe, B., Guss, M., Huber, T., Eds.; Humana Press: Totowa, NJ, 2008; Vol. 426, p 345. (43) Caffrey, M. Ann Rev Biophys 2009, 38, 29. (44) Doerr, A. Nat Methods 2006, 3, 244. (45) Brostromer, E.; Nan, J.; Li, L.-F.; Su, X.-D. Biochemical and Biophysical Research Communications 2009, 386, 634. (46) Li, G.; Chen, Q.; Li, J.; Hu, X.; Zhao, J. Anal Chem 2010, 82, 4362. (47) Jia, Y.; Liu, X.-Y. The Journal of Physical Chemistry B 2006, 110, 6949. (48) RCSB Protein Data Bank. http://www.rcsb.org/ (July 11, 2010). (49) Membrane Proteins of Known 3D Structure. http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html (July 11, 2010). (50) Michel, H. Trends Biochem Sci 1983, 8, 56. (51) Rosenbusch, J. P. Journal of Structural Biology 1990, 104, 134. (52) Garavito, R. M.; Picot, D. Methods 1990, 1, 57. (53) Kulkarni, C. V. 2010; Vol. 12, p 237. (54) Landau, E. M.; Rosenbusch, J. P. P Natl Acad Sci USA 1996, 93, 14532. (55) Pebay-Peyroula, E.; Rummel, G.; Rosenbusch, J. P.; Landau, E. M. Science 1997, 277, 1676. (56) Cherezov, V.; Liu, W.; Derrick, J. P.; Luan, B.; Aksimentiev, A.; Katritch, V.; Caffrey, M. Proteins: Structure, Function, and Bioinformatics 2008, 71, 24. (57) Cherezov, V.; Rosenbaum, D. M.; Hanson, M. A.; Rasmussen, S. G. F.; Thian, F. S.; Kobilka, T. S.; Choi, H. J.; Kuhn, P.; Weis, W. I.; Kobilka, B. K.; Stevens, R. C. Science 2007, 318, 1258. (58) Cherezov, V.; Yamashita, E.; Liu, W.; Zhalnina, M.; Cramer, W. A.; Caffrey, M. J Mol Biol 2006, 364, 716. (59) Jaakola, V. P.; Griffith, M. T.; Hanson, M. A.; Cherezov, V.; Chien, E. Y. T.; Lane, J. R.; IJzerman, A. P.; Stevens, R. C. Science 2008, 322, 1211. (60) Rosenbaum, D. M.; Cherezov, V.; Hanson, M. A.; Rasmussen, S. G. F.; Thian, F. S.; Kobilka, T. S.; Choi, H. J.; Yao, X. J.; Weis, W. I.; Stevens, R. C.; Kobilka, B. K. Science 2007, 318, 1266. (61) Wacker, D.; Fenalti, G.; Brown, M. A.; Katritch, V.; Abagyan, R.; Cherezov, V.; Stevens, R. C. J Am Chem Soc 2010, 132, 11443. (62) Höfer, N.; Aragão, D.; Caffrey, M. Biophys J 2010, 99, L23. (63) Li, L.; Ismagilov, R. F. Ann Rev Biophys 2010. (64) Pal, R.; Yang, M.; Lin, R.; Johnson, B. N.; Srivastava, N.; Razzacki, S. Z.; Chomistek, K. J.; Heldsinger, D. C.; Haque, R. M.; Ugaz, V. M.; Thwar, P. K.; Chen, Z.; Alfano, K.; Yim, M. B.; Krishnan, M.; Fuller, A. O.; Larson, R. G.; Burke, D. T.; Burns, M. A. Lab Chip 2005, 5, 1024. (65) Jayashree, R. S.; Gancs, L.; Choban, E. R.; Primak, A.; Natarajan, D.; Markoski, L. J.; Kenis, P. J. A. J Am Chem Soc 2005, 127, 16758. (66) Wootton, R. C. R.; deMello, A. J. Chem Commun 2004, 266. (67) McPherson, A. J Appl Crystallogr 2000, 33, 397.

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This article belongs to the Special Issue Diet and Metabolic Dysfunction