Introducing original methods for integrating sociocultural and discourse studies into science and engineering education, this book provides a much-needed framework for how to conduct qualitative ...research in this field. The three dimensions of learning identified in the Next Generation Science Standards (NGSS) create a need for research methods that examine the sociocultural components of science education. With cutting-edge studies and examples consistent with the NGSS, this book offers comprehensive research methods for integrating discourse and sociocultural practices in science and engineering education and provides key tools for applying this framework for students, pre-service teachers, scholars, and researchers.
The asexual blood stages of Plasmodium falciparum cause the most lethal form of human malaria. During growth within an infected red blood cell, parasite multiplication and formation of invasive ...merozoites is called schizogony. Here, we present a detailed analysis of the phosphoproteome of P. falciparum schizonts revealing 2541 unique phosphorylation sites, including 871 novel sites. Prominent roles for cAMP-dependent protein kinase A- and phosphatidylinositol-signaling were identified following analysis by functional enrichment, phosphoprotein interaction network clustering and phospho-motif identification tools. We observed that most key enzymes in the inositol pathway are phosphorylated, which strongly suggests additional levels of regulation and crosstalk with other protein kinases that coregulate different biological processes. A distinct pattern of phosphorylation of proteins involved in merozoite egress and red blood cell invasion was noted. The analyses also revealed that cAMP-PKA signaling is implicated in a wide variety of processes including motility. We verified this finding experimentally using an in vitro kinase assay and identified three novel PKA substrates associated with the glideosome motor complex: myosin A, GAP45 and CDPK1. Therefore, in addition to an established role for CDPK1 in the motor complex, this study reveals the coinvolvement of PKA, further implicating cAMP as an important regulator of host cell invasion.
Ubiquitylation is a common post translational modification of eukaryotic proteins and in the human malaria parasite, Plasmodium falciparum (Pf) overall ubiquitylation increases in the transition from ...intracellular schizont to extracellular merozoite stages in the asexual blood stage cycle. Here, we identify specific ubiquitylation sites of protein substrates in three intraerythrocytic parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified (data available via ProteomeXchange with identifier PXD014998). 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, suggesting a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the apparent extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We created a genetically modified parasite with a rapamycin-inducible functional deletion of uba1; addition of either MLN7243 or rapamycin to the recombinant parasite line resulted in the same phenotype, with parasite development blocked at the schizont stage. Nuclear division and formation of intracellular structures was interrupted. These results indicate that the intracellular target of MLN7243 is UBA1, and this activity is essential for the final differentiation of schizonts to merozoites.
ABSTRACT
INTRODUCTION
The U.S. faces a critical gap between residency training and clinical practice that affects the recruitment and preparation of internal medicine residents for primary care ...careers. The patient-centered medical home (PCMH) represents a new clinical microsystem that is being widely promoted and implemented to improve access, quality, and sustainability in primary care practice.
AIM
We address two key questions regarding the training of internal medicine residents for practice in PCMHs. First, what are the educational implications of practice transformations to primary care home models? Second, what must we do differently to prepare internal medicine residents for their futures in PCMHs?
PROGRAM DESCRIPTION
The 2011 Society of General Internal Medicine (SGIM) PCMH Education Summit established seven work groups to address the following topics: resident workplace competencies, teamwork, continuity of care, assessment, faculty development, ‘medical home builder’ tools, and policy. The output from the competency work group was foundational for the work of other groups. The work group considered several educational frameworks, including developmental milestones, competencies, and entrustable professional activities (EPAs).
RESULTS
The competency work group defined 25 internal medicine resident PCMH EPAs. The 2011 National Committee for Quality Assurance (NCQA) PCMH standards served as an organizing framework for EPAs.
DISCUSSION
The list of PCMH EPAs has the potential to begin to transform the education of internal medicine residents for practice and leadership in the PCMH. It will guide curriculum development, learner assessment, and clinical practice redesign for academic health centers.
Pathology of the most lethal form of malaria is caused by Plasmodium falciparum asexual blood stages and initiated by merozoite invasion of erythrocytes. We present a phosphoproteome analysis of ...extracellular merozoites revealing 1765 unique phosphorylation sites including 785 sites not previously detected in schizonts. All MS data have been deposited in the ProteomeXchange with identifier PXD001684 (http://proteomecentral.proteomexchange.org/dataset/PXD001684). The observed differential phosphorylation between extra and intraerythrocytic life‐cycle stages was confirmed using both phospho‐site and phospho‐motif specific antibodies and is consistent with the core motif K/RxxpS/pT being highly represented in merozoite phosphoproteins. Comparative bioinformatic analyses highlighted protein sets and pathways with established roles in invasion. Within the merozoite phosphoprotein interaction network a subnetwork of 119 proteins with potential roles in cellular movement and invasion was identified and suggested that it is coregulated by a further small subnetwork of protein kinase A (PKA), two calcium‐dependent protein kinases (CDPKs), a phosphatidyl inositol kinase (PI3K), and a GCN2‐like elF2‐kinase with a predicted role in translational arrest and associated changes in the ubquitinome. To test this notion experimentally, we examined the overall ubiquitination level in intracellular schizonts versus extracellular merozoites and found it highly upregulated in merozoites. We propose that alterations in the phosphoproteome and ubiquitinome reflect a starvation‐induced translational arrest as intracellular schizonts transform into extracellular merozoites.
Insights from cell cycle research have led to the hypothesis that tumors may be selectively sensitized to DNA-damaging agents
resulting in improved antitumor activity and a wider therapeutic margin. ...The theory relies on the observation that the majority
of tumors are deficient in the G 1 -DNA damage checkpoint pathway resulting in reliance on S and G 2 checkpoints for DNA repair and cell survival. The S and G 2 checkpoints are regulated by checkpoint kinase 1, a serine/threonine kinase that is activated in response to DNA damage;
thus, inhibition of checkpoint kinase 1 signaling impairs DNA repair and increases tumor cell death. Normal tissues, however,
have a functioning G 1 checkpoint signaling pathway allowing for DNA repair and cell survival. Here, we describe the preclinical profile of AZD7762,
a potent ATP-competitive checkpoint kinase inhibitor in clinical trials. AZD7762 has been profiled extensively in vitro and in vivo in combination with DNA-damaging agents and has been shown to potentiate response in several different settings where inhibition
of checkpoint kinase results in the abrogation of DNA damage-induced cell cycle arrest. Dose-dependent potentiation of antitumor
activity, when AZD7762 is administered in combination with DNA-damaging agents, has been observed in multiple xenograft models
with several DNA-damaging agents, further supporting the potential of checkpoint kinase inhibitors to enhance the efficacy
of both conventional chemotherapy and radiotherapy and increase patient response rates in a variety of settings. Mol Cancer
Ther 2008;7(9):2955–66
Abstract
Background
The resistance of
Plasmodium falciparum
to artemisinin-based (ART) drugs, the front-line drug family used in artemisinin-based combination therapy (ACT) for treatment of malaria, ...is of great concern. Mutations in the
kelch13
(
k13
) gene (for example, those resulting in the Cys580Tyr C580Y variant) were identified as genetic markers for ART-resistant parasites, which suggests they are associated with resistance mechanisms. However, not all resistant parasites contain a
k13
mutation, and clearly greater understanding of resistance mechanisms is required. A genome-wide association study (GWAS) found single nucleotide polymorphisms associated with ART-resistance in
fd
(ferredoxin),
arps10
(apicoplast ribosomal protein S10),
mdr2
(multidrug resistance protein 2), and
crt
(chloroquine resistance transporter), in addition to
k13
gene mutations, suggesting that these alleles contribute to the resistance phenotype. The importance of the FD and ARPS10 variants in ART resistance was then studied since both proteins likely function in the apicoplast, which is a location distinct from that of K13.
Methods
The reported mutations were introduced, together with a mutation to produce the
k13
-C580Y variant into the ART-sensitive 3D7 parasite line and the effect on ART-susceptibility using the 0−3 h ring survival assay (RSA
0−3 h
) was investigated.
Results and conclusion
Introducing both
fd
-D193Y and
arps10
-V127M into a
k13
-C580Y-containing parasite, but not a wild-type
k13
parasite, increased survival of the parasite in the RSA
0−3 h
. The results suggest epistasis of
arps10
and
k13
, with
arps10
-V127M a modifier of ART susceptibility in different
k13
allele backgrounds.
The inducible Di-Cre system was used to delete the putative ubiquitin-conjugating enzyme 13 gene (ubc13) of Plasmodium falciparum to study its role in ubiquitylation and the functional consequence ...during the parasite asexual blood stage. Deletion resulted in a significant reduction of parasite growth in vitro, reduced ubiquitylation of the Lys63 residue of ubiquitin attached to protein substrates, and an increased sensitivity of the parasite to both the mutagen, methyl methanesulfonate and the antimalarial drug dihydroartemisinin (DHA), but not chloroquine. The parasite was also sensitive to the UBC13 inhibitor NSC697923. The data suggest that this gene does code for an ubiquitin conjugating enzyme responsible for K63 ubiquitylation, which is important in DNA repair pathways as was previously demonstrated in other organisms. The increased parasite sensitivity to DHA in the absence of ubc13 function indicates that DHA may act primarily through this pathway and that inhibitors of UBC13 may both enhance the efficacy of this antimalarial drug and directly inhibit parasite growth.
Age‐related changes in resting‐state (RS) neural rhythms in typically developing children (TDC) but not children with autism spectrum disorder (ASD) suggest that RS measures may be of clinical use in ...ASD only for certain ages. The study examined this issue via assessing RS peak alpha frequency (PAF), a measure previous studies, have indicated as abnormal in ASD. RS magnetoencephalographic (MEG) data were obtained from 141 TDC (6.13–17.70 years) and 204 ASD (6.07–17.93 years). A source model with 15 regional sources projected the raw MEG surface data into brain source space. PAF was identified in each participant from the source showing the largest amplitude alpha activity (7‐13 Hz). Given sex differences in PAF in TDC (females > males) and relatively few females in both groups, group comparisons were conducted examining only male TDC (N = 121) and ASD (N = 183). Regressions showed significant group slope differences, with an age‐related increase in PAF in TDC (R2 = 0.32) but not ASD (R2 = 0.01). Analyses examining male children below or above 10‐years‐old (median split) indicated group effects only in the younger TDC (8.90 Hz) and ASD (9.84 Hz; Cohen's d = 1.05). In the older ASD, a higher nonverbal IQ was associated with a higher PAF. In the younger TDC, a faster speed of processing was associated with a higher PAF. PAF as a marker for ASD depends on age, with a RS alpha marker of more interest in younger versus older children with ASD. Associations between PAF and cognitive ability were also found to be age and group specific.
Our understanding of the key phosphorylation-dependent signalling pathways in the human malaria parasite, Plasmodium falciparum, remains rudimentary. Here we address this issue for the essential ...cGMP-dependent protein kinase, PfPKG. By employing chemical and genetic tools in combination with quantitative global phosphoproteomics, we identify the phosphorylation sites on 69 proteins that are direct or indirect cellular targets for PfPKG. These PfPKG targets include proteins involved in cell signalling, proteolysis, gene regulation, protein export and ion and protein transport, indicating that cGMP/PfPKG acts as a signalling hub that plays a central role in a number of core parasite processes. We also show that PfPKG activity is required for parasite invasion. This correlates with the finding that the calcium-dependent protein kinase, PfCDPK1, is phosphorylated by PfPKG, as are components of the actomyosin complex, providing mechanistic insight into the essential role of PfPKG in parasite egress and invasion.
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