Sorting live stem cells based on sox2 mRNA expression.

While cell sorting usually relies on cell-surface protein markers, molecular beacons (MBs) offer the potential to sort cells based on the presence of any expressed mRNA and in principle could be extremely useful to sort rare cell populations from primary isolates. We show here how stem cells can be purified from mixed cell populations by sorting based on MBs. Specifically, we designed molecular beacons targeting Sox2, a well-known stem cell marker for murine embryonic (mES) and neural stem cells (NSC). One of our designed molecular beacons displayed an increase in fluorescence compared to a nonspecific molecular beacon both in vitro and in vivo when tested in mES and NSCs. We sorted Sox2-MB(+)SSEA1(+) cells from a mixed population of 4-day retinoic acid-treated mES cells and effectively isolated live undifferentiated stem cells. Additionally, Sox2-MB(+) cells isolated from primary mouse brains were sorted and generated neurospheres with higher efficiency than Sox2-MB(-) cells. These results demonstrate the utility of MBs for stem cell sorting in an mRNA-specific manner.

Old world arenaviruses enter the host cell via the multivesicular body and depend on the endosomal sorting complex required for transport.

The highly pathogenic Old World arenavirus Lassa virus (LASV) and the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) use α-dystroglycan as a cellular receptor and enter the host cell by an unusual endocytotic pathway independent of clathrin, caveolin, dynamin, and actin. Upon internalization, the viruses are delivered to acidified endosomes in a Rab5-independent manner bypassing classical routes of incoming vesicular trafficking. Here we sought to identify cellular factors involved in the unusual and largely unknown entry pathway of LASV and LCMV. Cell entry of LASV and LCMV required microtubular transport to late endosomes, consistent with the low fusion pH of the viral envelope glycoproteins. Productive infection with recombinant LCMV expressing LASV envelope glycoprotein (rLCMV-LASVGP) and LCMV depended on phosphatidyl inositol 3-kinase (PI3K) as well as lysobisphosphatidic acid (LBPA), an unusual phospholipid that is involved in the formation of intraluminal vesicles (ILV) of the multivesicular body (MVB) of the late endosome. We provide evidence for a role of the endosomal sorting complex required for transport (ESCRT) in LASV and LCMV cell entry, in particular the ESCRT components Hrs, Tsg101, Vps22, and Vps24, as well as the ESCRT-associated ATPase Vps4 involved in fission of ILV. Productive infection with rLCMV-LASVGP and LCMV also critically depended on the ESCRT-associated protein Alix, which is implicated in membrane dynamics of the MVB/late endosomes. Our study identifies crucial cellular factors implicated in Old World arenavirus cell entry and indicates that LASV and LCMV invade the host cell passing via the MVB/late endosome. Our data further suggest that the virus-receptor complexes undergo sorting into ILV of the MVB mediated by the ESCRT, possibly using a pathway that may be linked to the cellular trafficking and degradation of the cellular receptor.

Characterization of the expression, regulation and function of Sorting Nexin 9 and Sorting Nexin 18

Summary : Sorting nexin (SNX) family members play important roles in intracellular protein and membrane trafficking, The membrane-tubulating SNX9 protein has been shown to interact with multiple components of the endocytic machinery and to participate in clathrin-mediated endocytosis of cell surface receptors. It has not been investigated if SNX9 may also participate in other protein sorting pathways that involve vesicular transport, specifically the biogenesis of lysosome-related organelles (LROs). Closely related to SNX9 is SNXl8, whose function is largely unknown. In this work, we have characterized the expression of SNX9 and SNXl8 in LRO-containing cells and investigated their role in protein trafficking during the formation of LROs. Our results indicate that SNX9 and SNXl8 are not essential for the formation of LROs, nor for the sorting of melanosomal proteins. We investigated how the level of intracellular SNX9 protein is regulated and found that it is a substrate of the ubiquitin ligase Itch, a member of the NEDD4 family of E3 ubiquitin ligases. Itch ubiquitylates SNX9 and regulates SNX9 levels by enhancing its degradation. Using ? truncated proteins we found that the interaction with SNX9 is mediated by the proline-rich domain of Itch, a domain distinct from the conventional WW recognition domain, and the SH3 domain of SNX9. Interaction with the PRD of Itch is essential for SNX9 ubiquitylation and degradation. We further showed that Itch binding is not affected by tyrosine phosphorylation of SNX9. Using lentivector-mediated siRNA techniques, we found that Itch regulates the level of melanosomal proteins, while knock-down of SNX9 does not alter their level. Interestingly, we revealed that silencing of SNXIS affects the amount of the melanosomal protein Melan-A, but also of SNX9, and that SNXl8 can interact with SNX9. Taken together, our results highlight that the pool of substrates of NEDD4 family E3 ligases extends to proteins containing SH3 domains and provide insight into the potential functions of SNXI8. Résumé : Les membres de la famille des Sorting Nexins (SNX) jouent des rôles importants dans le trafic intracellulaire de protéines et membranes. Il a été démontré que la protéine SNX9, qui génère les tubules membranaires, interagit avec plusieurs composants de la machinerie d'endocytose et participe à l'endocytose des récepteurs de surface mediée par la clathrine. Aucune étude n'a investigué si SNX9 pourrait aussi participer à d'autres voies de trafic de protéines tel que le transport vésiculaire, et plus particulièrement la biogenèse des organites lysosomaux ("lysosome-related organelles", LR©s). SNXl8 est similaire à SNX9, mais sa fonction est largement inconnue. Dans ce travail, nous avons caractérisé l'expression de SNX9 et SNX18 dans des cellules contenants des LROs et investigué leur rôle dans le trafic de protéines pendant la formation des LROS. Nos résultats indiquent que SNX9 et SNXI8 ne sont essentiels ni pour la formation des LR©s, ni pour le trafic de protéines mélanosomales. Nous avons examiné la régulation du niveau intracellulaire de la protéine SNX9 et avons trouvé qu'elle est un substrat de l'ubiquitine ligase Itch, un membre de la famille NEDD4 des ubiquitine ligases E3. Itch ubiquitine SNX9 et régule les niveaux de SNX9 en augmentant sa dégradation. En utilisant des protéines mutées nous avons découvert que l'interaction avec SNX9 est médiée par le domaine riche en proline de Itch, qui est différent du domaine conventionnel de reconnaissance WW, et par le domaine SH3 de SNX9. L'interaction avec le domaine riche en proline de Itch est essentielle pour l'ubiquitination et la dégradation de SNX9. De plus, nous avons montré que cette liaison n'est pas affectée par la phosphorylation des résidus tyrosine de SNX9. En utilisant des vecteurs lentiviraux exprimant des siARN, nous avons trouvé que Itch régule les niveaux de protéines mélanosomales, alors que l'extinction de l'expression de SNX9 ne change pas leurs niveaux. En autre, nous avons révélé que la diminution de SNXl8 affecte le niveau de la protéine mélanosomale Melan-A et de SNX9, et aussi que SNXl8 peut interagir avec SNX9. En résumé, nos résultats démontrent que l'ensemble des substrats de la famille NEDD4 des ubiquitine ligases E3 s'élargit aux protéines contenant des domaines SH3 et ouvrent des perspectives sur les fonctions potentielles de SNXl8.

Sorting on periodic surfaces

Particles moving on crystalline surfaces and driven by external forces or flow fields can acquire velocities along directions that deviate from that of the external force. This effect depends upon the characteristics of the particles, most notably particle size or particle index of refraction, and can therefore be (and has been) used to sort different particles. We introduce a simple model for particles subject to thermal fluctuations and moving in appropriate potential landscapes. Numerical results are compared to recent experiments on landscapes produced with holographic optical tweezers and microfabricated technology. Our approach clarifies the relevance of different parameters, the direction and magnitude of the external force, particle size, and temperature.

An analytical approach to sorting in periodic and random potentials

There has been a recent revolution in the ability to manipulate micrometer-sized objects on surfaces patterned by traps or obstacles of controllable configurations and shapes. One application of this technology is to separate particles driven across such a surface by an external force according to some particle characteristic such as size or index of refraction. The surface features cause the trajectories of particles driven across the surface to deviate from the direction of the force by an amount that depends on the particular characteristic, thus leading to sorting. While models of this behavior have provided a good understanding of these observations, the solutions have so far been primarily numerical. In this paper we provide analytic predictions for the dependence of the angle between the direction of motion and the external force on a number of model parameters for periodic as well as random surfaces. We test these predictions against exact numerical simulations.

Consumer Advisory Bulletin, Work-at-Home Scams:So You Didn’t Order That? Protecting yourself from unauthorized credit card charges, August 2012

The Attorney General’s Consumer Protection Division receives hundreds of calls and consumer complaints every year. Follow these tips to avoid unexpected expense and disappointments. This record is about: So You Didn’t Order That? Protecting yourself from unauthorized credit card charges

Consumer Advisory Bulletin, Credit Card Offers: They’re Not All the Same, October 2012

The Attorney General’s Consumer Protection Division receives hundreds of calls and consumer complaints every year. Follow these tips to avoid unexpected expense and disappointments. This record is about: Credit Card Offers: They’re Not All the Same

Fluorescence-activated cell sorting and cloning of bona fide CD8+ CTL with reversible MHC-peptide and antibody Fab' conjugates.

The isolation of subsets of Ag-specific T cells for in vitro and in vivo studies by FACS is compromised by the fact that the soluble MHC-peptide complexes and Abs used for staining, especially when combined, induce unwanted T cell activation and eventually apoptosis. This is especially a problem for CD8+ CTL, which are susceptible to activation-dependent cell death. In this study, we show that reversible MHC-peptide complexes (tetramers) can be prepared by conjugating MHC-peptide monomers with desthiobiotin (DTB; also called dethiobiotin) and multimerization by reaction with fluorescent streptavidin. While in the cold these reagents are stable and allow good staining, they rapidly dissociate in monomers at elevated temperatures, especially in the presence of free biotin. FACS cloning of Melan-A (MART-1)-specific CTL from a melanoma-infiltrated lymph node with reversible HLA-A2 Melan-A26-35 multimers yielded over two times more clones than when using the conventional biotin-containing multimers. CTL clones obtained by means of reversible multimers killed Melan-A-positive tumor cells more efficiently as compared with clones obtained with the stable multimers. Among the CTL obtained with the reversible multimers, but much less among those obtained with the stable multimers, a high proportion of clones exhibited high functional and physical avidity and died upon incubation with soluble MHC-peptide complexes. Finally, we show that Fab' of an anti-CD8 Ab can be converted in reversible DTB streptavidin conjugates the same way. These DTB reagents efficiently and reversibly stained murine and human CTL without affecting their viability.

The State Report Card for No Child Left Behind Iowa Department of Education, December 2011

The No Child Left Behind Act (NCLB) of 2001 requires each state to provide an annual report card to inform stakeholders and the public about the progress of students and schools on indicators of student achievement and other information that relates to student success. The State Report Card provides state level data to serve as a comparison for schools and districts as they consider and implement improvement efforts to increase the success for all Iowa students.

The State Report Card for No Child Left Behind Iowa Department of Education, September 2014

The No Child Left Behind Act (NCLB) of 2001 requires each state to provide an annual report card to inform stakeholders and the public about the progress of students and schools on indicators of student achievement and other information that relates to student success. The State Report Card provides state level data to serve as a comparison for schools and districts as they consider and implement improvement efforts to increase the success for all Iowa students.

The State Report Card for No Child Left Behind Iowa Department of Education, August 2004

The No Child Left Behind Act (NCLB) of 2001 requires each state to provide an annual report card to inform stakeholders and the public about the progress of students and schools on indicators of student achievement and other information that relates to student success. The State Report Card provides state level data to serve as a comparison for schools and districts as they consider and implement improvement efforts to increase the success for all Iowa students.

The State Report Card for No Child Left Behind Iowa Department of Education, September 2009

The No Child Left Behind Act (NCLB) of 2001 requires each state to provide an annual report card to inform stakeholders and the public about the progress of students and schools on indicators of student achievement and other information that relates to student success. The State Report Card provides state level data to serve as a comparison for schools and districts as they consider and implement improvement efforts to increase the success for all Iowa students.

The State Report Card for No Child Left Behind Iowa Department of Education, August 2005

The No Child Left Behind Act (NCLB) of 2001 requires each state to provide an annual report card to inform stakeholders and the public about the progress of students and schools on indicators of student achievement and other information that relates to student success. The State Report Card provides state level data to serve as a comparison for schools and districts as they consider and implement improvement efforts to increase the success for all Iowa students.

The State Report Card for No Child Left Behind Iowa Department of Education, September 2012

The No Child Left Behind Act (NCLB) of 2001 requires each state to provide an annual report card to inform stakeholders and the public about the progress of students and schools on indicators of student achievement and other information that relates to student success. The State Report Card provides state level data to serve as a comparison for schools and districts as they consider and implement improvement efforts to increase the success for all Iowa students.

The State Report Card for No Child Left Behind Iowa Department of Education, September 2013

The No Child Left Behind Act (NCLB) of 2001 requires each state to provide an annual report card to inform stakeholders and the public about the progress of students and schools on indicators of student achievement and other information that relates to student success. The State Report Card provides state level data to serve as a comparison for schools and districts as they consider and implement improvement efforts to increase the success for all Iowa students.