A Framework for Formal Modeling and Analysis of Organizations

A new, formal, role-based, framework for modeling and analyzing both real world and artificial organizations is introduced. It exploits static and dynamic properties of the organizational model and includes the (frequently ignored) environment. The transition is described from a generic framework of an organization to its deployed model and to the actual agent allocation. For verification and validation of the proposed model, a set of dedicated techniques is introduced. Moreover, where most computational models can handle only two or three layered organizational structures, our framework can handle any arbitrary number of organizational layers. Henceforth, real-world organizations can be modeled and analyzed, as illustrated by a case study, within the DEAL project line. © Springer Science+Business Media, LLC 2007

Bestrophin 1 is indispensable for volume regulation in human retinal pigment epithelium cells

In response to cell swelling, volume-regulated anion channels (VRACs) participate in a process known as regulatory volume decrease (RVD). Only recently, first insight into the molecular identity of mammalian VRACs was obtained by the discovery of the leucine-rich repeats containing 8A (LRRC8A) gene. Here, we show that bestrophin 1 (BEST1) but not LRRC8A is crucial for volume regulation in human retinal pigment epithelium (RPE) cells. Whole-cell patch-clamp recordings in RPE derived from human-induced pluripotent stem cells (hiPSC) exhibit an outwardly rectifying chloride current with characteristic functional properties of VRACs. This current is severely reduced in hiPSC-RPE cells derived from macular dystrophy patients with pathologic BEST1 mutations. Disruption of the orthologous mouse gene (Best1−/−) does not result in obvious retinal pathology but leads to a severe subfertility phenotype in agreement with minor endogenous expression of Best1 in murine RPE but highly abundant expression in mouse testis. Sperm from Best1−/− mice showed reduced motility and abnormal sperm morphology, indicating an inability in RVD. Together, our data suggest that the molecular identity of VRACs is more complex—that is, instead of a single ubiquitous channel, VRACs could be formed by cell type- or tissue-specific subunit composition. Our findings provide the basis to further examine VRAC diversity in normal and diseased cell physiology, which is key to exploring novel therapeutic approaches in VRAC-associated pathologies

Genome-wide expression profiling of the retinoschisin-deficient retina in early postnatal mouse development

purpose. The Rs1h knockout mouse displays retinal features typical for X-linked juvenile retinoschisis (RS). Consequently, this mouse line represents an excellent model to study early molecular events in RS. methods. Whole genome expression profiling using DNA-microarrays was performed on total RNA extracts from retinoschisin-deficient and wild-type murine retinas from postnatal days 7, 9, 11, and 14. Quantitative real-time RT-PCR (qRT-PCR) analysis of additional time points facilitated the refinement of the temporal expression profile of differentially regulated transcripts. Differential protein expression was confirmed by Western blot analysis. results. Based on biostatistic and knowledge-based DNA-microarray analyses we have identified differentially regulated retinal genes in early postnatal stages of the Rs1h-deficient mouse defining key molecular pathways including adhesion, cytoskeleton, vesicular trafficking, and immune response. A significant upregulation of Egr1 at P11 and several microglia/glia-related transcripts starting at P11 with a peak at P14 were identified in the diseased retina. The results provided evidence that macrophage/microglia activation precedes apoptotic photoreceptor cell death. Finally, the role of Egr1 in the pathogenesis of Rs1h-deficiency was investigated, and the results indicated that activation of the MAPK Erk1/2 pathway occurs as early as P7. Analyses of Rs1h −/Y /Egr1 −/− double-knockout mice suggest that Egr1 upregulation is not a prerequisite for macrophage/microglia activation or apoptosis. conclusions. The findings imply that microglia/glia activation may be triggering events in the photoreceptor degeneration of retinoschisin-deficient mice. Furthermore, the data point to a role of Erk1/2-Egr1 pathway activation in RS pathogenesis

Addressing robustness in time-critical, distributed, task allocation algorithms.

The aim of this work is to produce and test a robustness module (ROB-M) that can be generally applied to distributed, multi-agent task allocation algorithms, as robust versions of these are scarce and not well-documented in the literature. ROB-M is developed using the Performance Impact (PI) algorithm, as this has previously shown good results in deterministic trials. Different candidate versions of the module are thus bolted on to the PI algorithm and tested using two different task allocation problems under simulated uncertain conditions, and results are compared with baseline PI. It is shown that the baseline does not handle uncertainty well; the task-allocation success rate tends to decrease linearly as degree of uncertainty increases. However, when PI is run with one of the candidate robustness modules, the failure rate becomes very low for both problems, even under high simulated uncertainty, and so its architecture is adopted for ROB-M and also applied to MIT’s baseline Consensus Based Bundle Algorithm (CBBA) to demonstrate its flexibility. Strong evidence is provided to show that ROB-M can work effectively with CBBA to improve performance under simulated uncertain conditions, as long as the deterministic versions of the problems can be solved with baseline CBBA. Furthermore, the use of ROB-M does not appear to increase mean task completion time in either algorithm, and only 100 Monte Carlo samples are required compared to 10,000 in MIT’s robust version of the CBBA algorithm. PI with ROB-M is also tested directly against MIT’s robust algorithm and demonstrates clear superiority in terms of mean numbers of solved tasks.N/

Whole-genome SNP association analysis of reproduction traits in the Finnish Landrace pig breed

<p>Abstract</p> <p>Background</p> <p>Good genetic progress for pig reproduction traits has been achieved using a quantitative genetics-based multi-trait BLUP evaluation system. At present, whole-genome single nucleotide polymorphisms (SNP) panels provide a new tool for pig selection. The purpose of this study was to identify SNP associated with reproduction traits in the Finnish Landrace pig breed using the Illumina PorcineSNP60 BeadChip.</p> <p>Methods</p> <p>Association of each SNP with different traits was tested with a weighted linear model, using SNP genotype as a covariate and animal as a random variable. Deregressed estimated breeding values of the progeny tested boars were used as the dependent variable and weights were based on their reliabilities. Statistical significance of the associations was based on Bonferroni-corrected <it>P</it>-values.</p> <p>Results</p> <p>Deregressed estimated breeding values were available for 328 genotyped boars. Of the 62 163 SNP in the chip, 57 868 SNP had a call rate > 0.9 and 7 632 SNP were monomorphic. Statistically significant results (<it>P</it>-value < 2.0E-06) were obtained for total number of piglets born in first and later parities and piglet mortality between birth and weaning in later parity, and suggestive associations (<it>P</it>-value < 4.0E-06) for piglet mortality between birth and weaning in first parity, number of stillborn piglets in later parity, first farrowing interval and second farrowing interval. Two of the statistically significant regions for total number of piglets born in first and later parities are located on chromosome 9 around 95 and 79 Mb. The estimated SNP effect in these regions was approximately one piglet between the two homozygote classes. By combining the two most significant SNP in these regions, favourable double homozygote animals are expected to have 1.3 piglets (<it>P</it>-value = 1.69E-08) more than unfavourable double homozygote animals. A region on chromosome 9 (66 Mb) was statistically significant for piglet mortality between birth and weaning in later parity (0.44 piglets between homozygotes, <it>P</it>-value = 6.94E-08).</p> <p>Conclusions</p> <p>Three separate regions on chromosome 9 gave significant results for litter size and pig mortality. The frequencies of favourable alleles of the significant SNP are moderate in the Finnish Landrace population and these SNP are thus valuable candidates for possible marker-assisted selection.</p

Genetic Characterization of Hantaviruses Transmitted by the Korean Field Mouse (Apodemus peninsulae), Far East Russia

In an epizootiologic survey of 122 rodents captured in Vladivostok, Russia, antibodies positive for hantavirus were found in Apodemus peninsulae (4/70), A. agrarius (1/39), and Clethrionomys rufocanus (1/8). The hantavirus sequences identified in two seropositive A. peninsulae and two patients with hemorrhagic fever with renal syndrome (HFRS) from the Primorye region of Far East Russia were designated as Solovey and Primorye, respectively. The nucleotide sequences of the Solovey, Primorye, and Amur (obtained through GenBank) sequences were closely related (>92% identity). Solovey and Primorye sequences shared 84% nucleotide identity with the prototype Hantaan 76-118. Phylogenetic analysis also indicated a close relationship between Solovey, Primorye, Amur, and other viruses identified in Russia, China, and Korea. Our findings suggest that the Korean field mouse (A. peninsulae) is the reservoir for a hantavirus that causes HFRS over a vast area of east Asia, including Far East Russia

Regulation of Bestrophins by Ca2+: A Theoretical and Experimental Study

Bestrophins are a recently discovered family of Cl− channels, for which no structural information is available. Some family members are activated by increased intracellular Ca2+ concentration. Bestrophins feature a well conserved Asp-rich tract in their COOH terminus (Asp-rich domain), which is homologous to Ca2+-binding motifs in human thrombospondins and in human big-conductance Ca2+- and voltage-gated K+ channels (BKCa). Consequently, the Asp-rich domain is also a candidate for Ca2+ binding in bestrophins. Based on these considerations, we constructed homology models of human bestrophin-1 (Best1) Asp-rich domain using human thrombospondin-1 X-ray structure as a template. Molecular dynamics simulations were used to identify Asp and Glu residues binding Ca2+ and to predict the effects of their mutations to alanine. We then proceeded to test selected mutations in the Asp-rich domain of the highly homologous mouse bestrophin-2. The mutants expressed in HEK-293 cells were investigated by electrophysiological experiments using the whole-cell voltage-clamp technique. Based on our molecular modeling results, we predicted that Asp-rich domain has two defined binding sites and that D301A and D304A mutations may impact the binding of the metal ions. The experiments confirmed that these mutations do actually affect the function of the protein causing a large decrease in the Ca2+-activated Cl− current, fully consistent with our predictions. In addition, other studied mutations (E306A, D312A) did not decrease Ca2+-activated Cl− current in agreement with modeling results

Regulation of Bestrophins by Ca2+: A Theoretical and Experimental Study

Bestrophins are a recently discovered family of Cl− channels, for which no structural information is available. Some family members are activated by increased intracellular Ca2+ concentration. Bestrophins feature a well conserved Asp-rich tract in their COOH terminus (Asp-rich domain), which is homologous to Ca2+-binding motifs in human thrombospondins and in human big-conductance Ca2+- and voltage-gated K+ channels (BKCa). Consequently, the Asp-rich domain is also a candidate for Ca2+ binding in bestrophins. Based on these considerations, we constructed homology models of human bestrophin-1 (Best1) Asp-rich domain using human thrombospondin-1 X-ray structure as a template. Molecular dynamics simulations were used to identify Asp and Glu residues binding Ca2+ and to predict the effects of their mutations to alanine. We then proceeded to test selected mutations in the Asp-rich domain of the highly homologous mouse bestrophin-2. The mutants expressed in HEK-293 cells were investigated by electrophysiological experiments using the whole-cell voltage-clamp technique. Based on our molecular modeling results, we predicted that Asp-rich domain has two defined binding sites and that D301A and D304A mutations may impact the binding of the metal ions. The experiments confirmed that these mutations do actually affect the function of the protein causing a large decrease in the Ca2+-activated Cl− current, fully consistent with our predictions. In addition, other studied mutations (E306A, D312A) did not decrease Ca2+-activated Cl− current in agreement with modeling results

Minimal Essential Human Factor VIII Alterations Enhance Secretion and Gene Therapy Efficiency

One important limitation for achieving therapeutic expression of human factor VIII (FVIII) in hemophilia A gene therapy is inefficient secretion of the FVIII protein. Substitution of five amino acids in the A1 domain of human FVIII with the corresponding porcine FVIII residues generated a secretion-enhanced human FVIII variant termed B-domain-deleted (BDD)-FVIII-X5 that resulted in 8-fold higher FVIII activity levels in the supernatant of an in vitro cell-based assay system than seen with unmodified human BDD-FVIII. Analysis of purified recombinant BDD-FVIII-X5 and BDD-FVIII revealed similar specific activities for both proteins, indicating that the effect of the X5 alteration is confined to increased FVIII secretion. Intravenous delivery in FVIII-deficient mice of liver-targeted adeno-associated virus (AAV) vectors designed to express BDD-FVIII-X5 or BDD-FVIII achieved substantially higher plasma FVIII activity levels for BDD-FVIII-X5, even when highly efficient codon-optimized F8 nucleotide sequences were employed. A comprehensive immunogenicity assessment using in vitro stimulation assays and various in vivo preclinical models of hemophilia A demonstrated that the BDD-FVIII-X5 variant does not exhibit an increased immunogenicity risk compared to BDD-FVIII. In conclusion, BDD-FVIII-X5 is an effective FVIII variant molecule that can be further developed for use in gene- and protein-based therapeutics for patients with hemophilia A