London Education and Inclusion Project (LEIP): Exploring Negative and Null Effects of a Cluster-Randomised School-Intervention to Reduce School Exclusion—Findings from Protocol-Based Subgroup Analyses

This paper presents subgroup analyses from the London Education and Inclusion Project (LEIP). LEIP was a cluster-randomised controlled trial of an intervention called Engage in Education-London (EiE-L) which aimed to reduce school exclusions in those at greatest risk of exclusion. Pupils in the control schools attended an hour-long employability seminar. Minimisation was used to randomly assign schools to treatment and control following baseline data collection. The study involved 36 schools (17 in treatment—373 pupils; 19 in control—369 pupils) with >28% free school meal eligibility across London and utilised on pupil self-reports, teacher reports as well as official records to assess the effectiveness of EiE-L. Due to multiple data sources, sample sizes varied according to analysis. Analyses of pre-specified subgroups revealed null and negative effects on school exclusion following the intervention. Our findings suggest that the design and implementation of EiE-L may have contributed to the negative outcomes for pupils in the treatment schools when compared to those in the control schools. These findings call into question the effectiveness of bolt-on short-term interventions with pupils, particularly those at the highest risk of school exclusion and when they are faced with multiple problems. This is especially pertinent given the possibility of negative outcomes.Trial Registration: Controlled Trials: ISRCTN23244695     

Identification of Kaposi's sarcoma-associated herpesvirus (KSHV)-specific cytotoxic T-lymphocyte epitopes and evaluation of reconstitution of KSHV-specific responses in human immunodeficiency virus type 1-Infected patients receiving highly active antiretroviral therapy

Following the introduction of highly active antiretroviral therapy (HAART), the incidence of Kaposi's sarcoma (KS) has significantly declined in human immunodeficiency virus type 1 (HIV-1)-positive (HIV-1(+)) individuals and clinical remission is often observed. We hypothesize that these effects are partly due to anti-KS-associated herpesvirus (KSHV) immune restoration. Here, 15-mer overlapping peptides from proteins K12 and K8.1 were used to identify novel KSHV-specific cytotoxic T-lymphocyte epitopes. Three immunogenic peptides, two lytic and one latent, were subsequently used to monitor the anti-KSHV CD8(+) T-cell responses in a cohort of 19 HIV-1(+) KSHV(+/-) KS(+/-) individuals during 52 weeks of HAART. KSHV and HIV-1 loads, KSHV antibody titers, and both CD4(+) and CD8(+) T-lymphocyte counts were enumerated. Prior to HAART, the total number of spot-forming cells (SFC) for all three peptides correlated with both CD4(+) and CD8(+) T-lymphocyte counts (P < or = 0.05) in the KSHV-positive KS-positive cohort (n = 11). Following 52 weeks of HAART, significant decreases in HIV-1 and KSHV loads were associated with significant increases in CD4(+) T-lymphocyte counts and number of SFC for the three KSHV-specific peptides. Although these increases were modest in comparison to the number of SFC observed with the HIV-1 gag peptide SLYNTVATL, they represented a fourfold increase from the baseline, continuing an upward trend to week 52.     

Negative regulation of yeast Eps15-like Arp2/3 complex activator, Pan1p, by the Hip1R-related protein, Sla2p, during endocytosis

Control of actin assembly nucleated by the Arp2/3 complex plays a crucial role during budding yeast endocytosis. The yeast Eps15-related Arp2/3 complex activator, Pan1p, is essential for endocytic internalization and proper actin organization. Pan1p activity is negatively regulated by Prk1 kinase phosphorylation after endocytic internalization. Phosphorylated Pan1p is probably then dephosphorylated in the cytosol. Pan1p is recruited to endocytic sites approximately 25 s before initiation of actin polymerization, suggesting that its Arp2/3 complex activation activity is kept inactive during early stages of endocytosis by a yet-to-be-identified mechanism. However, how Pan1p is maintained in an inactive state is not clear. Using tandem affinity purification-tagged Pan1p, we identified End3p as a stoichiometric component of the Pan1p complex, and Sla2p, a yeast Hip1R-related protein, as a novel binding partner of Pan1p. Interestingly, Sla2p specifically inhibited Pan1p Arp2/3 complex activation activity in vitro. The coiled-coil region of Sla2p was important for Pan1p inhibition, and a pan1 partial loss-of-function mutant suppressed the temperature sensitivity, endocytic phenotypes, and actin phenotypes observed in sla2DeltaCC mutant cells that lack the coiled-coil region. Overall, our results establish that Sla2p's regulation of Pan1p plays an important role in controlling Pan1p-stimulated actin polymerization during endocytosis.     

The UspA1 protein and a second type of UspA2 protein mediate adherence of Moraxella catarrhalis to human epithelial cells in vitro

The UspA1 and UspA2 proteins of Moraxella catarrhalis are structurally related, are exposed on the bacterial cell surface, and migrate as very high-molecular-weight complexes in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Previous analysis of uspA1 and uspA2 mutants of M. catarrhalis strain 035E indicated that UspA1 was involved in adherence of this organism to Chang conjunctival epithelial cells in vitro and that expression of UspA2 was essential for resistance of this strain to killing by normal human serum (C. Aebi, E. R. Lafontaine, L. D. Cope, J. L. Latimer, S. R. Lumbley, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 66:3113-3119, 1998). In the present study, isogenic uspA1, uspA2, and uspA1 uspA2 mutations were constructed in three additional M. catarrhalis strains: 012E, TTA37, and 046E. The uspA1 mutant of strain 012E had a decreased ability to attach to Chang cells. However, inactivation of the uspA1 gene in both strain TTA37 and strain 046E did not cause a significant decrease in attachment ability. Inactivation of the uspA2 gene of strain TTA37 did result in a loss of attachment ability. Nucleotide sequence analysis revealed that the predicted protein encoded by the uspA2 genes of both strains TTA37 and 046E had a N-terminal half that resembled the N-terminal half of UspA1 proteins, whereas the C-terminal half of this protein was nearly identical to those of previously characterized UspA2 proteins. The gene encoding this "hybrid" protein was designated uspA2H. PCR-based analysis revealed that approximately 20% of M. catarrhalis strains apparently possess a uspA2H gene instead of a uspA2 gene. The M. catarrhalis uspA1, uspA2, and uspA2H genes were cloned and expressed in Haemophilus influenzae cells, which were used to prove that both the UspA1 and UspA2H proteins can function as adhesins in vitro.     

Orderly recruitment among motoneurons supplying different muscles.

Virtually all movements involve the recruitment of motor units from multiple muscles. Given the functional diversity of motor units (motoneurons and the muscle fibers they supply), the effective production of specific movements undoubtedly depends upon some principle(s) to organize the ensemble of active motor units. The principle acting to organize the recruitment of motor units within muscles is the size principle, whereby the first motor units to be recruited have the smallest values for axonal conduction velocity and contractile force, and are the slowest to contract and fatigue. Here we consider the possibility that the size principle applies in the recruitment of motor units across muscles, i.e., that regardless of their muscles of origin, active motor units are recruited in rank order, for example, from low to high conduction velocity. The benefits of orderly recruitment across muscles could be similar to the acknowledged advantages of orderly recruitment within muscles. One benefit is that the neural process involved in organizing active motor units would be simplified. In a muscle-based scheme, the size principle would organize only those motor units within individual muscles, leaving the nervous system with the additional task of coordinating the relative activities of motor units from different muscles. By contrast, in an ensemble-based scheme, orderly recruitment of all motor units according to the size principle would automatically coordinate motor units both within and across motor nuclei. Another potential benefit is the provision for movements with smooth trajectory, the result of interleaving the divergent torque contributions made by motor units from muscles that differ in their orientations about joints. Otherwise, if order were restricted within muscles, the torque trajectory of a joint would change unevenly as participating muscles begin contracting at different times and grade activity at different rates. These considerations support speculation that motor units recruited from co-contracting muscles are collectively recruited according to the size principle.     

Regulation of a wheat actin-depolymerizing factor during cold acclimation

We have previously shown that the wheat (Triticum aestivum) TaADF gene expression level is correlated with the plants capacity to tolerate freezing. Sequence analysis revealed that this gene encodes a protein homologous to members of the actin-depolymerizing factor (ADF)/cofilin family. We report here on the characterization of the recombinant TaADF protein. Assays for ADF activity showed that TaADF is capable of sequestering actin, preventing nucleotide exchange, and inducing actin depolymerization. In vitro phosphorylation studies showed that TaADF is a substrate for a wheat 52-kD kinase. The activity of this kinase is modulated by low temperature during the acclimation period. Western-blot analyses revealed that TaADF is expressed only in cold-acclimated Gramineae species and that the accumulation level is much higher in the freezing-tolerant wheat cultivars compared with the less tolerant ones. This accumulation was found to be regulated by a factor(s) encoded by a gene(s) located on chromosome 5A, the chromosome most often found to be associated with cold hardiness. The induction of an active ADF during cold acclimation and the correlation with an increased freezing tolerance suggest that the protein may be required for the cytoskeletal rearrangements that may occur upon low temperature exposure. These remodelings might be important for the enhancement of freezing tolerance.     

Expression profiling of lymphocyte plasma membrane proteins

The physicochemical properties of plasma membrane proteins of mammalian cells render them refractory to systematic analysis by two-dimensional electrophoresis. We have therefore used in vivo cell surface labeling with a water-soluble biotinylation reagent, followed by cell lysis and membrane purification, prior to affinity capture of biotinylated proteins. Purified membrane proteins were then separated by solution-phase isoelectric focusing and SDS-PAGE and identified by high-pressure liquid chromatography electrospray/tandem mass spectrometry. Using this approach, we identified 42 plasma membrane proteins from a murine T cell hybridoma and 46 from unfractionated primary murine splenocytes. These included three unexpected proteins; nicastrin, osteoclast inhibitory lectin, and a transmembrane domain-containing hypothetical protein of 11.4 kDa. Following stimulation of murine splenocytes with phorbol ester and calcium ionophore, we observed differences in expression of CD69, major histocompatibility complex class II molecules, the glucocorticoid-induced TNF receptor family-related gene product, and surface immunoglobulin M and D that were subsequently confirmed by Western blot or flow cytometric analysis. This approach offers a generic and powerful strategy for investigating differential expression of surface proteins in many cell types under varying environmental and pathophysiological conditions.     

Calculation and measurement of semiconduction activation energy and electron mobility in cytochrome oxidase,with evidence that charge carriers are polarons,which may couple oxidation to phosphorylation

It is shown that an electron transport reaction which is rate-limited by electron conduction 4 across a solid biological particle or membrane in accord with Ohm's law should have a first order rate constant approximately proportional to exp (E _a/KT ), whereT is absolute temperature,k is the Boltzmann constant andE _a is the activation energy for semiconduction in the solid particle, where resistance in the semiconductor is proportional to exp (E _a /KT). For two different preparations of cytochrome oxidase, this method yields an average value ofE _a =0.27 ev, which agrees well with direct conductivity measurements on dry solid enzyme, which provide an average value ofE _a =0.26 ev. Electron mobility in dry cytochrome oxidase is estimated to be approximately \gm=10^\t-5 cm² volt^\t-1 sec^\t-1. Elovich decay of current in dry cytochrome oxidase was observed, which parallels the Elovich kinetics of cytochrome oxidase activity in yeast observed previously by M\:uhlig (1966). Finally, the solid state kinetic theory is used to deduce that conduction of polarons may be involved in cytochrome oxidase activity (1 polaron=1 electron + 1 phonon), which provides a link with the solid state phonon phosphorylation theory of Straub.