Glycoprotein Interactions in the Assembly of Hantaviruses

Hantaviruses are one of the five genera of the vector-borne virus family Bunyaviridae. While other members of the family are transmitted via arthropods, hantaviruses are carried and transmitted by rodents and insectivores. Occasional transmission to humans occurs via inhalation of aerosolized rodent excreta. When transmitted to man hantaviruses cause hemorrhagic fever with renal syndrome (HFRS, in Eurasia, mortality ~10%) and hantavirus cardiopulmonary syndrome (HCPS, in the Americas, mortality ~40%). The single-stranded, negative-sense RNA genome of hantaviruses is in segments S, M and L that respectively encode for nucleocapsid (N), glycoproteins Gn and Gc, and RNA-dependent RNA-polymerase (RdRp or L protein). The genome segments, encapsidated by N protein to form ribonucleoprotein (RNP), are enclosed inside a lipid envelope decorated by spikes formed of Gn and Gc. The focus of this study was to understand the mechanisms and interactions through which the virion is formed and maintained. We observed that when extracted from virions both Gn and Gc favor homo- over hetero-oligomerization. The minimal glycoprotein complexes extracted from virion by detergent were observed, by using ultracentrifugation and gel filtration, to be tetrameric Gn and homodimeric Gc. These results led us to suggest a model where tetrameric Gn complexes are interconnected through homodimeric Gc units to form the grid-like surface architecture described for hantaviruses. This model was found to correlate with the three-dimensional (3D) reconstruction of virion surface created using cryo-electron tomography (cryo-ET). The 3D-density map showed the spike complex formed of Gn and Gc to be 10 nm high and to display a four-fold symmetry with dimensions of 15 nm times 15 nm. This unique square-shaped complex on a roughly round virion creates a hitch for the assembly, since a sphere cannot be broken into rectangles. Thus additional interactions are likely required for the virion assembly. In cryo-ET we observed that the RNP makes occasional contacts to the viral membrane, suggesting an interaction between the spike and RNP. We were able to demonstrate this interaction using various techniques, and showed that both Gn and Gc contribute to the interaction. This led us to suggest that in addition to the interactions between Gn and Gc, also the interaction between spike and RNP is required for assembly. We found galectin-3 binding protein (referred to as 90K) to co-purify with the virions and showed an interaction between 90K and the virion. Analysis of plasma samples taken from patients hospitalized for Puumala virus infection showed increased concentrations of 90K in the acute phase and the increased 90K level was found to correlate with several parameters that reflect the severity of acute HFRS. The results of these studies confirmed, but also challenged some of the dogmas on the structure and assembly of hantaviruses. We confirmed that Gn and RNP do interact, as long assumed. On the other hand we demonstrated that the glycoproteins Gn and Gc exist as homo-oligomers or appear in large hetero-oligomeric complexes, rather than form primarily heterodimers as was previously assumed. This work provided new insight into the structure and assembly of hantaviruses.

Effects of gender, and SLCO1B1 and CYP7A1 polymorphisms on plasma bile acids in humans and the pharmacokinetics of therapeutic ursodeoxycholic acid

Bile acids are important steroid-derived molecules essential for fat absorption in the small intestine. They are produced in the liver and secreted into the bile. Bile acids are transported by bile flow to the small intestine, where they aid the digestion of lipids. Most bile acids are reabsorbed in the small intestine and return to the liver through the portal vein. The whole recycling process is referred to as the enterohepatic circulation, during which only a small amount of bile acids are removed from the body via faeces. The enterohepatic circulation of bile acids involves the delicate coordination of a number of bile acid transporters expressed in the liver and the small intestine. Organic anion transporting polypeptide 1B1 (OATP1B1), encoded by the solute carrier organic anion transporter family, member 1B1 (SLCO1B1) gene, mediates the sodium independent hepatocellular uptake of bile acids. Two common SNPs in the SLCO1B1 gene are well known to affect the transport activity of OATP1B1. Moreover, bile acid synthesis is an important elimination route for cholesterol. Cholesterol 7α-hydroxylase (CYP7A1) is the rate-limiting enzyme of bile acid production. The aim of this thesis was to investigate the effects of SLCO1B1 polymorphism on the fasting plasma levels of individual endogenous bile acids and a bile acid synthesis marker, and the pharmacokinetics of exogenously administered ursodeoxycholic acid (UDCA). Furthermore, the effects of CYP7A1 genetic polymorphism and gender on the fasting plasma concentrations of individual endogenous bile acids and the bile acid synthesis marker were evaluated. Firstly, a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the determination of bile acids was developed (Study I). A retrospective study examined the effects of SLCO1B1 genetic polymorphism on the fasting plasma concentrations of individual bile acids and a bile acid synthesis marker in 65 healthy subjects (Study II). In another retrospective study with 143 healthy individuals, the effects of CYP7A1 genetic polymorphism and gender as well as SLCO1B1 polymorphism on the fasting plasma levels of individual bile acids and the bile acid synthesis marker were investigated (Study III). The effects of SLCO1B1 polymorphism on the pharmacokinetics of exogenously administered UDCA were evaluated in a prospective genotype panel study including 27 healthy volunteers (Study IV). A robust, sensitive and simple HPLC-MS/MS method was developed for the simultaneous determination of 16 individual bile acids in human plasma. The method validation parameters for all the analytes met the requirements of the FDA (Food and Drug Administration) bioanalytical guidelines. This HPLC-MS/MS method was applied in Studies II-IV. In Study II, the fasting plasma concentrations of several bile acids and the bile acid synthesis marker seemed to be affected by SLCO1B1 genetic polymorphism, but these findings were not replicated in Study III with a larger sample size. Moreover, SLCO1B1 polymorphism had no effect on the pharmacokinetic parameters of exogenously administered UDCA. Furthermore, no consistent association was observed between CYP7A1 genetic polymorphism and the fasting plasma concentrations of individual bile acids or the bile acid synthesis marker. In contrast, gender had a major effect on the fasting plasma concentrations of several bile acids and also total bile acids. In conclusion, gender, but not SLCO1B1 or CYP7A1 polymorphisms, has a major effect on the fasting plasma concentrations of individual bile acids. Moreover, the common genetic polymorphism of CYP7A1 is unlikely to influence the activity of CYP7A1 under normal physiological conditions. OATP1B1 does not play an important role in the in vivo disposition of exogenously administered UDCA.

Divergencies of Perception : The Possibilities of Merleau-Pontian Phenomenology in Analyses of Contemporary Art

Maurice Merleau-Ponty (1908-1961) has been known as the philosopher of painting. His interest in the theory of perception intertwined with the questions concerning the artist s perception, the experience of an artwork and the possible interpretations of the artwork. For him, aesthetics was not a sub-field of philosophy, and art was not simply a subject matter for the aesthetic experience, but a form of thinking. This study proposes an opening for a dialogue between Merleau-Pontian phenomenology and contemporary art. The thesis examines his phenomenology through certain works of contemporary art and presents readings of these artworks through his phenomenology. The thesis both shows the potentiality of a method, but also engages in the critical task of finding the possible limitations of his approach. The first part lays out the methodological and conceptual points of departure of Merleau-Ponty s phenomenological approach to perception as well as the features that determined his discussion on encountering art. Merleau-Ponty referred to the experience of perceiving art using the notion of seeing with (voir selon). He stressed a correlative reciprocity described in Eye and Mind (1961) as the switching of the roles of the visible and the painter. The choice of artworks is motivated by certain restrictions in the phenomenological readings of visual arts. The examined works include paintings by Tiina Mielonen, a photographic work by Christian Mayer, a film by Douglas Gordon and Philippe Parreno, and an installation by Monika Sosnowska. These works resonate with, and challenge, his phenomenological approach. The chapters with case studies take up different themes that are central to Merleau-Ponty s phenomenology: space, movement, time, and touch. All of the themes are interlinked with the examined artworks. There are also topics that reappear in the thesis, such as the notion of écart and the question of encountering the other. As Merleau-Ponty argued, the sphere of art has a particular capability to address our being in the world. The thesis presents an interpretation that emphasises the notion of écart, which refers to an experience of divergence or dispossession. The sudden dissociation, surprise or rupture that is needed in order for a meeting between the spectator and the artwork, or between two persons, to be possible. Further, the thesis suggests that through artworks it is possible to take into consideration the écart, the divergence, that defines our subjectivity.

Study of Pulsed Plasma in a Crossed Flow Dielectric Barrier Discharge Reactor for Improvement of NOx Removal in Raw Diesel Engine Exhaust

Improved performance of plasma in raw engine exhaust treatment is reported. A new type of reactor referred to as of cross-flow dielectric barrier discharge (DBD) was used, in which the gas flow is perpendicular to the corona electrode. In raw exhaust environment, the cross-flow (radial-flow) reactor exhibits a superior performance with regard to NOX removal when compared to that with axial flow of gas. Experiments were conducted at different flow rates ranging from 2 L/min to 25 L/min. The plasma assisted barrier discharge reactor has shown encouraging results in NOx removal at high flow rates.

Molecular and serological studies on the recent seal virus epizootics in Europe and Siberia

The virus epizootics which occurred in seals in both Europe and Siberia during 1987/1988 were caused by two different morbillivirus, referred to as phocid distemper virus (PDV) 1 and 2, respectively. Molecular and serological studies have shown that the European virus is quite distinct from canine distemper virus (CDV), its closest relative in the morbillivirus group. Analysis of tissues obtained from infected seals from a wide geographical distrubution over Northern Europe showed that the infectious agent (PDV 1) was identical in all cases. Nucleotide sequence analysis of one of the virus genes suggested that this virus has evolved away from CDV over a long time period and is most probably an enzootic virus of marine mammals. In contrast, the virus (PDV 2) which caused the deaths of many Siberian seals was indistinguishable, both serologically and at the molecular level, from CDV and must have originated from a land source.

Locating and sizing plants for bottling propane in south India

Shri Shakti LPG Ltd. (SSLPG) imports and markets propane (referred to as liquefied petroleum gas (LPG) in India) in south India. It sells LPG in packed (cylinder) form to domestic customers and commercial establishments through a network of dealers. Dealers replenish their stocks of filled cylinders from bottling plants, which in turn receive LPG in bulk from the cheaper of SSLPG's two import-and-storage facilities that are located on the Indian coast. We implemented integer programming to help SSLPG decide on the locations and long-run sizes of its bottling plants. We estimate that our recommended configuration of bottling plants is about $1 million cheaper annually than the one that SSLPG had initially planned.

Crucible Steel in South India-Preliminary Investigations on Crucibles from Some Newly Identified Sites

European accounts from the 17th century onwards have referred to the repute and manufacture of “wootz’, a traditional crucible steel made especially in parts of southern India in the former provinces of Golconda, Mysore and Salem. Pliny's Natural History mentions the import of iron and steel from the Seres which have been thought to refer to the ancient southern Indian kingdom of the Cheras. As yet the scale of excavations and surface surveys is too limited to link the literary accounts to archaeometallurgical evidence, although pioneering exploratory investigations have been made by scholars, especially on the pre-industrial production sites of Konasamudram and Gatihosahalli discussed in 18th-19th century European accounts. In 1991–2 during preliminary surveys of ancient base metal mining sites, Srinivasan came across unreported dumps with crucible fragments at Mel-Siruvalur in Tamil Nadu, and Tintini and Machnur in Karnataka and she collected surface specimens from these sites as well as from the known site of Gatihosahalli. She was also given crucible fragments by the Tamil University, Tanjavur, from an excavated megalithic site at Kodumanal, dated to ca 2nd c. Bc, mentioned in Tamil Sangam literature (ca 3rd c. BC-3rd c. AD), and very near Karur, the ancient capital of the Sangam Cheras. Analyses of crucible fragments from the surface collection at Mel-Siruvalur showed several iron prills with a uniform pearlitic structure of high-carbon hypereutectoid steel (∼1–1.5% C) suggesting that the end product was uniformly a high-carbon steel of a structure consistent with those of high-carbon steels used successfully to experimentally replicate the watered steel patterns on ‘Damascus’ swords. Investigations indicate that the process was of carburisation of molten low carbon iron (m.p. 1400° C) in crucibles packed with carbonaceous matter. The fabric of crucibles from all the above mentioned sites appears similar. Preliminary investigations on these crucibles are thus reported to establish their relationship to crucible production of carbon steel and to thereby extend the known horizons of this technology further.

The Archaeometallurgical Implications of New Findings of Traditional Crafts of Making High Tin ‘Delta’ Bronze Mirrors and ‘Beta’ Bronze Vessels in Kerala State of South India

New metallurgical and ethnographic observations of the traditional manufacture of specular high-tin bronze mirrors in Kerala state of southern India are discussed, which is an exceptional example of a surviving craft practice of metal mirror-making in the world. The manufacturing process has been reconstructed from analytical investigations made by Srinivasan following a visit late in 1991 to a mirror making workshop and from her technical studies of equipment acquired by Glover in March 1992 from another group of mirror makers from Pathanamthita at an exhibition held at Crafts Museum, Delhi. Finished and unfinished mirror from two workshops were of a binary, copper-tin alloy of 33% tin which is close to the composition of pure delta phase, so that these mirrors are referred to here as ‘delta’ bronzes. For the first time, metallurgical and field observations were made by Srinivasan in 1991 of the manufacture of high-tin ‘beta’ bonze vessels from Palghat district, Kerala, i‥e of wrought and quenched 23% tin bronze. This has provided the first metallurgical record for a surviving craft of high-tin bronze bowl making which can be directly related to archaeological finds of high-tin bronze vessels from the Indian subcontinent and Southeast Asia. New analytical investigations are presented of high-tin beta bronzes from the Indian subcontinent which are some of the earliest reported worldwide. These coupled with the archaeometallurgical evidence suggests that these high-tin bronze techniques are part of a long, continuing, and probably indigenous tradition of the use of high-tin bronzes in the Indian subcontinent with finds reported even from Indus Valley sites. While the source of tin has been problematic, new evidence on bronze smelting slags and literary evidence suggests there may have been some sources of tin in South India.

Task Dependent Boundary Mannequins in Statistical DHM

The primary objective of the paper is to make use of statistical digital human model to better understand the nature of reach probability of points in the taskspace. The concept of task-dependent boundary manikin is introduced to geometrically characterize the extreme individuals in the given population who would accomplish the task. For a given point of interest and task, the map of the acceptable variation in anthropometric parameters is superimposed with the distribution of the same parameters in the given population to identify the extreme individuals. To illustrate the concept, the task space mapping is done for the reach probability of human arms. Unlike the boundary manikins, who are completely defined by the population, the dimensions of these manikins will vary with task, say, a point to be reached, as in the present case. Hence they are referred to here as the task-dependent boundary manikins. Simulations with these manikins would help designers to visualize how differently the extreme individuals would perform the task. Reach probability at the points in a 3D grid in the operational space is computed; for objects overlaid in this grid, approximate probabilities are derived from the grid for rendering them with colors indicating the reach probability. The method may also help in providing a rational basis for selection of personnel for a given task.

A tropical oviparous lizard, Calotes versicolor, exhibiting a potentially novel FMFM pattern of temperature-dependent sex determination

Among squamate reptiles, lizards exhibit an impressive array of sex-determining modes viz. genotypic sex determination, temperature-dependent sex determination, co-occurrence of both these and those that reproduce parthenogenetically. The oviparous lizard, Calotes versicolor, lacks heteromorphic sex chromosomes and there are no reports on homomorphic chromosomes. Earlier studies on this species presented little evidence to the sex-determining mechanism. Here we provide evidences for the potential role played by incubation temperature that has a significant effect (P<0.01) on gonadal sex and sex ratio. The eggs were incubated at 14 different incubation temperatures. Interestingly, 100% males were produced at low (25.5 +/- 0.5 degrees C) as well as high (34 +/- 0.5 degrees C) incubation temperatures and 100% females were produced at low (23.5 +/- 0.5 degrees C) and high (31.5 +/- 0.5 degrees C) temperatures, clearly indicating the occurrence of TSD in this species. Sex ratios of individual clutches did not vary at any of the critical male-producing or female-producing temperatures within as well as across the seasons. However, clutch sex ratios were female- or male-biased at intermediate temperatures. Thermosensitive period occurred during the embryonic stages 3033. Three pivotal temperatures operate producing 1:1 sex ratio. Histology of gonad and accessory reproductive structures provide additional evidence for TSD. The sex-determining pattern, observed for the first time in this species, that neither compares to Pattern I [Ia (MF) and Ib (FM)] nor to Pattern II (FMF), is being referred to as FMFM pattern of TSD. This novel FMFM pattern of sex ratio exhibited by C. versicolor may have an adaptive significance in maintaining sex ratio. J. Exp. Zool. 317:3246, 2012. (c) 2011 Wiley Periodicals, Inc.

Fast algorithms for frequent episode discovery in event sequences

In this paper we consider the process of discovering frequent episodes in event sequences. The most computationally intensive part of this process is that of counting the frequencies of a set of candidate episodes. We present two new frequency counting algorithms for speeding up this part. These, referred to as non-overlapping and non-inteleaved frequency counts, are based on directly counting suitable subsets of the occurrences of an episode. Hence they are different from the frequency counts of Mannila et al [1], where they count the number of windows in which the episode occurs. Our new frequency counts offer a speed-up factor of 7 or more on real and synthetic datasets. We also show how the new frequency counts can be used when the events in episodes have time-durations as well.

How Does the Spacer Length of Cationic Gemini Lipids Influence the Lipoplex Formation with Plasmid DNA? Physicochemical and Biochemical Characterizations and their Relevance in Gene Therapy

Lipoplexes formed by the pEGFP-C3 plasmid DNA (pDNA) and lipid mixtures containing cationic gemini surfactant of the 1,2-bis(hexadecyl dimethyl ammonium) Acmes family referred to as C16CnC16, where n = 2 3, 5, or 12, and the zwitterionic helper lipid, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) have been studied from a wide variety of physical, chemical, and biological standpoints. The study has been carried out using several experimental methods, such as zeta potential, gel electrophoresis, small-angle X-ray scattering (SAXS), cryo-TEM, gene transfection, cell viability/cytotoxicity, and confocal fluorescence microscopy. As reported recently in a communication (J. Am. Chem. Soc. 2011, 133, 18014), the detailed physicochemical and biological studies confirm that, in the presence of the studied series lipid mixtures, plasmid DNA is compacted with a large number of its associated Na+ counterions. This in turn yields a much lower effective negative charge, q(pDNA)(-), a value that has been experimentally obtained for each mixed lipid mixture. Consequently, the cationic lipid (CL) complexes prepared with pDNA and CL/DOPE mixtures to be used in gene transfection require significantly less amount of CL than the one estimated assuming a value of q(DNA)(-) = -2. This drives to a considerably lower cytotoxicity of the gene vector. Depending on the CL molar composition, alpha, of the lipid mixture, and the effective charge ratio of the lipoplex, rho(eff), the reported SAXS data indicate the presence of two or three structures in the same lipoplex, one in the DOPE-rich region, other in the CL-rich region, and another one present at any CL composition. Cryo-TEM and SAXS studies with C16CnC16/DOPE-pDNA lipoplexes indicate that pDNA is localized between the mixed lipid bilayers of lamellar structures within a monolayer of similar to 2 nm. This is consistent with a highly compacted supercoiled pDNA conformation compared with that of linear DNA. Transfection studies were carried out with HEK293T, HeLa, CHO, U343, and H460 cells. The alpha and rho(eff) values for each lipid mixture were optimized on HEK293T cells for transfection, and using these values, the remaining cells were also transfected in absence (-FBS-FBS) and presence (-FBS+FBS) of serum. The transfection efficiency was higher with the CLs of shorter gemini spacers (n = 2 or 3). Each formulation expressed GFP on pDNA transfection and confocal fluorescence microscopy corroborated the results. C16C2C16/DOPE mixtures were the most efficient toward transfection among all the lipid mixtures and, in presence of serum, even better than the Lipofectamine2000, a commercial transfecting agent Each lipid combination was safe and did not show any significant levels of toxicity. Probably, the presence of two coexisting lamellar structures in lipoplexes synergizes the transfection efficiency of the lipid mixtures which are plentiful in the lipoplexes formed by CLs with short spacer (n = 2, 3) than those with the long spacer (n = 5, 12).

Structural insights into the role of Bacillus subtilis YwfH (BacG) in tetrahydrotyrosine synthesis

The synthesis of the dipeptide antibiotic bacilysin involves the sequential action of multiple enzymes in the bac operon. YwfH (also referred to as BacG) catalyzes the stereoselective reduction of dihydro-hydroxyphenylpyruvate (H2HPP) to tetrahydro-hydroxyphenylpyruvate (H4HPP) in this biosynthetic pathway. YwfH is an NADPH-dependent reductase that facilitates the conjugate addition of a hydride at the C4 olefin terminus of H2HPP. Here, the structure of YwfH is described at three conformational steps: the apo form, an apo-like conformation and the NADPH complex. YwfH is structurally similar to other characterized short-chain dehydrogenase/reductases despite having marginal sequence similarity. The structures of YwfH in different conformational states provide a rationale for the ping-pong reaction mechanism. The identification and role of the residues in the catalytic tetrad (Lys113Tyr117Ser155Asn158) in proton transfer were examined by mutational analysis. Together, the structures and biochemical features revealed synchronized conformational changes that facilitate cofactor specificity and catalysis of H4HPP formation en route to tetrahydrotyrosine synthesis.

Tethering preferences of domain families co-occurring in multi-domain proteins

Genomic data of several organisms have revealed the presence of a vast repertoire of multi-domain proteins. The role played by individual domains in a multi-domain protein has a profound influence on the overall function of the protein. In the present analysis an attempt has been made to better understand the tethering preferences of domain families that occur in multi-domain proteins. The analysis has been carried out on an exhaustive dataset of 2 961 898 sequences of proteins from 930 organisms, where 741 274 proteins are comprised of at least two domain families. For every domain family, the number of other domain families with which it co-occurs within a protein in this dataset has been enumerated and is referred to as the tethering number of the domain family. It was found that, in the general dataset, the AAA ATPase family and the family of Ser/Thr kinases have the highest tethering numbers of 450 and 444 respectively. Further analysis reveals significant correlation between the number of members in a family and its tethering number. Positive correlation was also observed for the extent of a sequence and functional diversity within a family and the tethering numbers of domain families. Domain families that are present ubiquitously in diverse organisms tend to have large tethering numbers, while organism/kingdom-specific families have low tethering numbers. Thus, the analysis uncovers how domain families recombine and evolve to give rise to multi-domain proteins.

Classification, representation, and automatic extraction of deformation features in sheet metal parts

This paper presents classification, representation and extraction of deformation features in sheet-metal parts. The thickness is constant for these shape features and hence these are also referred to as constant thickness features. The deformation feature is represented as a set of faces with a characteristic arrangement among the faces. Deformation of the base-sheet or forming of material creates Bends and Walls with respect to a base-sheet or a reference plane. These are referred to as Basic Deformation Features (BDFs). Compound deformation features having two or more BDFs are defined as characteristic combinations of Bends and Walls and represented as a graph called Basic Deformation Features Graph (BDFG). The graph, therefore, represents a compound deformation feature uniquely. The characteristic arrangement of the faces and type of bends belonging to the feature decide the type and nature of the deformation feature. Algorithms have been developed to extract and identify deformation features from a CAD model of sheet-metal parts. The proposed algorithm does not require folding and unfolding of the part as intermediate steps to recognize deformation features. Representations of typical features are illustrated and results of extracting these deformation features from typical sheet metal parts are presented and discussed. (C) 2013 Elsevier Ltd. All rights reserved.