Drivers for Change: A Study of Distributed Leadership and Performance Adaptation During Policy Innovation Implementation

Scaling up innovation in the instructional core remains a vexing proposition. Such disruptive innovations require teachers to engage in performance adaptation. Schools vary in their capacity to support changes in teachers’ day-today work. By comparing distributed instructional leadership practices of “odds-beating” schools with those at “typically performing schools,” this study identified four qualities of distributed instructional leadership that drive teacher performance adaptation: collective goal setting, instructional feedback, collective guided learning, and trusting relationships. These findings reiterate the need for policy to go beyond standards and accountability mandates to focus on the right drivers of change: capacity building, and opportunities for collaboration in tandem with pedagogical improvement

The Role of District and School Leaders\u27 Trust and Communications in the Simultaneous Implementation of Innovative Policies

This mixed-method multiple case study investigated nine elementary schools. Six “odds-beating schools, which serve relatively high numbers of economically disadvantaged children, achieved higher than predicted performance on state assessments when compared with three typically performing schools. The overarching research question guiding this study was: What forces, factors, and actors account for odds-beating schools\u27 better outcomes? The trust-communication connection provided one answer. Relational trust in odds-beating schools is an intraorganizational phenomenon, and it is accompanied by interorganizational trust (reciprocal trust). These two kinds of trust are accompanied by intraschool and district office-school communication mechanisms. Trust and communications are mutually constitutive as innovations are implemented. This connection is also an implementation outcome. When today\u27s innovation implementation initiatives reinforce this trust–communication connection, it becomes an organizational resource for future innovation implementation

An E. coli-produced single-chain variable fragment (scFv) targeting hepatitis B virus surface protein potently inhibited virion secretion

Hepatitis B virus (HBV) envelopes as well as empty subviral particles carry in their lipid membranes the small (S), middle (M), and large (L) surface proteins, collectively known as hepatitis B surface antigen (HBsAg). Due to their common S domain all three proteins share a surface-exposed hydrophilic antigenic loop (AGL) with a complex disulfide bridge-dependent structure. The AGL is critical for HBV infectivity and virion secretion, and thus represents a major target for neutralizing antibodies. Previously, a human monoclonal antibody (mAb) targeting a conformational epitope in the AGL, IgG12, exhibited 1000-fold higher neutralizing activity than hepatitis B immune globulin (HBIG). Here we designed a single-chain variable fragment (scFv) homolog of IgG12, G12-scFv, which could be efficiently produced in soluble form in the cytoplasm of E. coli SHuffle cells. Independent in vitro assays verified specific binding of G12-scFv to a conformational S epitope shared with IgG12. Despite 20-fold lower affinity, G12-scFv but not an irrelevant scFv potently neutralized HBV infection of susceptible hepatoma cells (IC50=1.8nM). Strikingly, low concentrations of G12-scFv blocked virion secretion from HBV producing cells (IC50=1.25nM) without disturbing intracellular viral replication, whereas extracellular HBsAg was reduced only at >100-fold higher though still nontoxic concentration. The inhibitory effects correlated with S binding specificity and presumably also G12-scFv internalization into cells. Together these data suggest G12-scFv as a highly specific yet easily accessible novel tool for basic, diagnostic, and possibly future therapeutic applications

Reduced expression of p27 is a novel mechanism of docetaxel resistance in breast cancer cells

INTRODUCTION: Docetaxel is one of the most effective chemotherapeutic agents in the treatment of breast cancer. Breast cancers can have an inherent or acquired resistance to docetaxel but the causes of this resistance remain unclear. However, apoptosis and cell cycle regulation are key mechanisms by which most chemotherapeutic agents exert their cytotoxic effects. METHODS: We created two docetaxel-resistant human breast cancer cell lines (MCF-7 and MDA-MB-231) and performed cDNA microarray analysis to identify candidate genes associated with docetaxel resistance. Gene expression changes were validated at the RNA and protein levels by reverse transcription PCR and western analysis, respectively. RESULTS: Gene expression cDNA microarray analysis demonstrated reduced p27 expression in docetaxel-resistant breast cancer cells. Although p27 mRNA expression was found to be reduced only in MCF-7 docetaxel-resistant sublines (2.47-fold), reduced expression of p27 protein was noted in both MCF-7 and MDA-MB-231 docetaxel-resistant breast cancer cells (2.83-fold and 3.80-fold, respectively). CONCLUSIONS: This study demonstrates that reduced expression of p27 is associated with acquired resistance to docetaxel in breast cancer cells. An understanding of the genes that are involved in resistance to chemotherapy may allow further development in modulating drug resistance, and may permit selection of those patients who are most likely to benefit from such therapies

VgrG and PAAR Proteins Define Distinct Versions of a Functional Type VI Secretion System

The Type VI secretion system (T6SS) is widespread among bacterial pathogens and acts as an effective weapon against competitor bacteria and eukaryotic hosts by delivering toxic effector proteins directly into target cells. The T6SS utilises a bacteriophage-like contractile machinery to expel a puncturing device based on a tube of Hcp topped with a VgrG spike, which can be extended by a final tip from a PAAR domain-containing protein. Effector proteins are believed to be delivered by specifically associating with particular Hcp, VgrG or PAAR proteins, either covalently ('specialised') or non-covalently ('cargo' effectors). Here we used the T6SS of the opportunistic pathogen Serratia marcescens, together with integratecd genetic, proteomic and biochemical approaches, to elucidate the role of specific VgrG and PAAR homologues in T6SS function and effector specificity, revealing new aspects and unexpected subtleties in effector delivery by the T6SS. We identified effectors, both cargo and specialised, absolutely dependent on a particular VgrG for delivery to target cells, and discovered that other cargo effectors can show a preference for a particular VgrG. The presence of at least one PAAR protein was found to be essential for T6SS function, consistent with designation as a 'core' T6SS component. We showed that specific VgrG-PAAR combinations are required to assemble a functional T6SS and that the three distinct VgrG-PAAR assemblies in S. marcescens exhibit distinct effector specificity and efficiency. Unexpectedly, we discovered that two different PAAR-containing Rhs proteins can functionally pair with the same VgrG protein. Showing that accessory EagR proteins are involved in these interactions, native VgrG-Rhs-EagR complexes were isolated and specific interactions between EagR and cognate Rhs proteins identified. This study defines an essential yet flexible role for PAAR proteins in the T6SS and highlights the existence of distinct versions of the machinery with differential effector specificity and efficiency of target cell delivery

An E. coli-produced single-chain variable fragment (scFv) targeting hepatitis B virus surface protein potently inhibited virion secretion

Hepatitis B virus (HBV) envelopes as well as empty subviral particles carry in their lipid membranes the small (S), middle (M), and large (L) surface proteins, collectively known as hepatitis B surface antigen (HBsAg). Due to their common S domain all three proteins share a surface-exposed hydrophilic antigenic loop (AGL) with a complex disulfide bridge-dependent structure. The AGL is critical for HBV infectivity and virion secretion, and thus represents a major target for neutralizing antibodies. Previously, a human monoclonal antibody (mAb) targeting a conformational epitope in the AGL, IgG12, exhibited 1000-fold higher neutralizing activity than hepatitis B immune globulin (HBIG). Here we designed a single-chain variable fragment (scFv) homolog of IgG12, G12-scFv, which could be efficiently produced in soluble form in the cytoplasm of E. coli SHuffle cells. Independent in vitro assays verified specific binding of G12-scFv to a conformational S epitope shared with IgG12. Despite 20-fold lower affinity, G12-scFv but not an irrelevant scFv potently neutralized HBV infection of susceptible hepatoma cells (IC50=1.8nM). Strikingly, low concentrations of G12-scFv blocked virion secretion from HBV producing cells (IC50=1.25nM) without disturbing intracellular viral replication, whereas extracellular HBsAg was reduced only at >100-fold higher though still nontoxic concentration. The inhibitory effects correlated with S binding specificity and presumably also G12-scFv internalization into cells. Together these data suggest G12-scFv as a highly specific yet easily accessible novel tool for basic, diagnostic, and possibly future therapeutic applications

Evolution of apoptosis-like programmed cell death in unicellular protozoan parasites

Apoptosis-like programmed cell death (PCD) has recently been described in multiple taxa of unicellular protists, including the protozoan parasites Plasmodium, Trypanosoma and Leishmania. Apoptosis-like PCD in protozoan parasites shares a number of morphological features with programmed cell death in multicellular organisms. However, both the evolutionary explanations and mechanisms involved in parasite PCD are poorly understood. Explaining why unicellular organisms appear to undergo 'suicide' is a challenge for evolutionary biology and uncovering death executors and pathways is a challenge for molecular and cell biology. Bioinformatics has the potential to integrate these approaches by revealing homologies in the PCD machinery of diverse taxa and evaluating their evolutionary trajectories. As the molecular mechanisms of apoptosis in model organisms are well characterised, and recent data suggest similar mechanisms operate in protozoan parasites, key questions can now be addressed. These questions include: which elements of apoptosis machinery appear to be shared between protozoan parasites and multicellular taxa and, have these mechanisms arisen through convergent or divergent evolution? We use bioinformatics to address these questions and our analyses suggest that apoptosis mechanisms in protozoan parasites and other taxa have diverged during their evolution, that some apoptosis factors are shared across taxa whilst others have been replaced by proteins with similar biochemical activities

Mechanism of Disruption of the Amt-GlnK Complex by PII-Mediated Sensing of 2-Oxoglutarate

GlnK proteins regulate the active uptake of ammonium by Amt transport proteins by inserting their regulatory T-loops into the transport channels of the Amt trimer and physically blocking substrate passage. They sense the cellular nitrogen status through 2-oxoglutarate, and the energy level of the cell by binding both ATP and ADP with different affinities. The hyperthermophilic euryarchaeon Archaeoglobus fulgidus possesses three Amt proteins, each encoded in an operon with a GlnK ortholog. One of these proteins, GlnK2 was recently found to be incapable of binding 2-OG, and in order to understand the implications of this finding we conducted a detailed structural and functional analysis of a second GlnK protein from A. fulgidus, GlnK3. Contrary to Af-GlnK2 this protein was able to bind both ATP/2-OG and ADP to yield inactive and functional states, respectively. Due to the thermostable nature of the protein we could observe the exact positioning of the notoriously flexible T-loops and explain the binding behavior of GlnK proteins to their interaction partner, the Amt proteins. A thermodynamic analysis of these binding events using microcalorimetry evaluated by microstate modeling revealed significant differences in binding cooperativity compared to other characterized PII proteins, underlining the diversity and adaptability of this class of regulatory signaling proteins