Endocrine cell proliferation fluctuates dramatically in response to signals that communicate hormone demand. The genetic alterations that override these controls in endocrine tumors often are not ...associated with oncogenes common to other tumor types, suggesting that unique pathways govern endocrine proliferation. Within the pancreas, for example, activating mutations of the prototypical oncogene KRAS drive proliferation in all pancreatic ductal adenocarcimomas but are never found in pancreatic endocrine tumors. Therefore, we asked how cellular context impacts K-RAS signaling. We found that K-RAS paradoxically suppressed, rather than promoted, growth in pancreatic endocrine cells. Inhibition of proliferation by K-RAS depended on antiproliferative RAS effector RASSF1A and blockade of the RAS-activated proproliferative RAF/MAPK pathway by tumor suppressor menin. Consistent with this model, a glucagon-like peptide 1 (GLP1) agonist, which stimulates ERK1/2 phosphorylation, did not affect endocrine cell proliferation by itself, but synergistically enhanced proliferation when combined with a menin inhibitor. In contrast, inhibition of MAPK signaling created a synthetic lethal interaction in the setting of menin loss. These insights suggest potential strategies both for regenerating pancreatic β cells for people with diabetes and for targeting menin-sensitive endocrine tumors.
Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical regions, is caused by infection with Plasmodium ...parasites. The development of more effective drugs to combat malaria can be accelerated by improving our understanding of the biology of this complex parasite. Genetic manipulation of these parasites is key to understanding their biology; however, historically the genome of P. falciparum has been difficult to manipulate. Recently, CRISPR/Cas9 genome editing has been utilized in malaria parasites, allowing for easier protein tagging, generation of conditional protein knockdowns, and deletion of genes. CRISPR/Cas9 genome editing has proven to be a powerful tool for advancing the field of malaria research. Here, we describe a CRISPR/Cas9 method for generating glmS-based conditional knockdown mutants in P. falciparum. This method is highly adaptable to other types of genetic manipulations, including protein tagging and gene knockouts.
Malaria remains a major public health issue, infecting nearly 220 million people every year. The spread of drug-resistant strains of
around the world threatens the progress made against this disease. ...Therefore, identifying druggable and essential pathways in
parasites remains a major area of research. One poorly understood area of parasite biology is the formation of disulfide bonds, which is an essential requirement for the folding of numerous proteins. Specialized chaperones with thioredoxin (Trx) domains catalyze the redox functions necessary for breaking incorrect and forming correct disulfide bonds in proteins. Defining the substrates of these redox chaperones is difficult and immunoprecipitation based assays cannot distinguish between substrates and interacting partners. Further, the substrate or client interactions with the redox chaperones are usually transient in nature. Activity based crosslinkers that rely on the nucleophilic cysteines on Trx domains and the disulfide bond forming cysteines on clients provide an easily scalable method to trap and identify the substrates of Trx-domain containing chaperones. The cell permeable crosslinker divinyl sulfone (DVSF) is active only in the presence of nucleophilic cysteines in proteins and, therefore, traps Trx domains with their substrates, as they form mixed disulfide bonds during the course of their catalytic activity. This allows the identification of substrates that rely on Trx activity for their folding, as well as discovering small molecules that interfere with Trx domain activity. Graphic abstract: Identification of thioredoxin domain substrates via divinylsulfone crosslinking and immunoprecipitation-mass spectrometry.
Export of parasite proteins into the host erythrocyte is essential for survival of
during its asexual life cycle. While several studies described key factors within the parasite that are involved in ...protein export, the mechanisms employed to traffic exported proteins within the host cell are currently unknown. Members of the Hsp70 family of chaperones, together with their Hsp40 cochaperones, facilitate protein trafficking in other organisms, and are thus likely used by
in the trafficking of its exported proteins. A large group of Hsp40 proteins is encoded by the parasite and exported to the host cell, but only one Hsp70,
Hsp70x (PfHsp70x), is exported with them. PfHsp70x is absent in most
species and is found only in
and closely related species that infect apes. Herein, we have utilized clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome editing in
to investigate the essentiality of PfHsp70x. We show that parasitic growth was unaffected by knockdown of PfHsp70x using both the dihydrofolate reductase (DHFR)-based destabilization domain and the
ribozyme system. Similarly, a complete gene knockout of PfHsp70x did not affect the ability of
to proceed through its intraerythrocytic life cycle. The effect of PfHsp70x knockdown/knockout on the export of proteins to the host red blood cell (RBC), including the critical virulence factor
erythrocyte membrane protein 1 (PfEMP1), was tested, and we found that this process was unaffected. These data show that although PfHsp70x is the sole exported Hsp70, it is not essential for the asexual development of
.
Half of the world's population lives at risk for malaria. The intraerythrocytic life cycle of
spp. is responsible for clinical manifestations of malaria; therefore, knowledge of the parasite's ability to survive within the erythrocyte is needed to combat the deadliest agent of malaria,
. An outstanding question in the field is how
undertakes the essential process of trafficking its proteins within the host cell. In most organisms, chaperones such as Hsp70 are employed in protein trafficking. Of the
species causing human disease, the chaperone PfHsp70x is unique to
, and it is the only parasite protein of its kind exported to the host (S. Külzer et al., Cell Microbiol 14:1784-1795, 2012). This has placed PfHsp70x as an ideal target to inhibit protein trafficking and kill the parasite. However, we show that PfHsp70x is not required for export of parasite effectors and it is not essential for parasite survival inside the RBC.
The US Government is the world’s de facto provider of space object cataloging data, but it is challenged to maintain pace in an increasingly complex space environment. This work advances a ...multi-disciplinary approach to better understand and evaluate an underexplored solution recommended by national policy in which current collection capabilities are augmented with non-traditional sensors. System architecting techniques and extant literature identified likely needs, performance measures, and potential contributors to a conceptualized Augmented Network (AN). Multiple hypothetical architectures of ground- and space-based telescopes with representative capabilities were modeled and simulated on four separate days throughout the year, then evaluated against performance measures and constraints using Multi-Objective Optimization. Decision analysis and Pareto optimality identified a small, diverse set of high-performing architectures while preserving design flexibility. Should decision-makers adopt the AN approach, this research effort indicates (1) a threefold increase in average capacity, (2) a 55% improvement in coverage, and (3) a 2.5-h decrease in the average maximum time a space object goes unobserved.
Purpose
Patients with systemic lupus erythematosus (SLE) frequently develop lupus nephritis (LN), a complication frequently leading to end stage kidney disease. Immune complex deposition in the ...glomerulus is central to the development of LN. Using a targeted proteomic approach, we tested the hypothesis that autoantibodies targeting glomerular antigens contribute to the development of LN.
Experimental design
Human podocyte and glomerular proteins were separated by SDS‐PAGE and immunoblotted with sera from SLE patients with and without LN. The regions of those gels corresponding to reactive bands observed with sera from LN patients were analyzed using LC‐MS/MS.
Results
LN reactive bands were seen at approximately 50 kDa in podocyte extracts and between 36 and 50 kDa in glomerular extracts. Those bands were analyzed by LC‐MS/MS and 102 overlapping proteins were identified. Bioinformatic analysis determined that 36 of those proteins were membrane associated, including a protein previously suggested to contribute to glomerulonephritis and LN, annexin A2. By ELISA, patients with proliferative LN demonstrated significantly increased antibodies against annexin A2.
Conclusion and clinical relevance
Proteomic approaches identified multiple candidate antigens for autoantibodies in patients with LN. Serum antibodies against annexin A2 were significantly elevated in subjects with proliferative LN, validating those antibodies as potential biomarkers.
Improving space situational awareness (SSA) remains one of the Department of Defense’s (DoD) top priorities. Current research has shown that the modeling of geosynchronous orbit (GEO) SSA ...architectures can help identify optimal combinations of ground- and space-based sensors. This paper extends previous research by expanding design boundaries and refining the methodology. A multi-objective genetic algorithm was used to examine this increased trade-space containing 1022 possible design combinations. The results of the optimizer clearly favor 1.0 m aperture ground telescopes combined with 0.15 m aperture sensors in a 12-satellite geosynchronous polar orbit (GPO) constellation. The GPO regime offers increased access to GEO resident space objects (RSO) since other orbits are restricted by a 40° solar exclusion angle. When performance is held constant, a GPO satellite constellation offers a 22.4% reduction in total system cost when compared to Sun synchronous orbit (SSO), equatorial low earth orbit (LEO), and near-GEO constellations. Parallel high-performance computing provides the possibility of solving an entirely new class of complex problems of interest to the DoD. The results of this research can educate national policy makers on the benefits of proposed upgrades to current and future SSA systems.
The Foundational Data Initiative for Parkinson Disease (FOUNDIN-PD) is an international collaboration producing fundamental resources for Parkinson disease (PD). FOUNDIN-PD generated a multi-layered ...molecular dataset in a cohort of induced pluripotent stem cell (iPSC) lines differentiated to dopaminergic (DA) neurons, a major affected cell type in PD. The lines were derived from the Parkinson’s Progression Markers Initiative study, which included participants with PD carrying monogenic PD variants, variants with intermediate effects, and variants identified by genome-wide association studies and unaffected individuals. We generated genetic, epigenetic, regulatory, transcriptomic, and longitudinal cellular imaging data from iPSC-derived DA neurons to understand molecular relationships between disease-associated genetic variation and proximate molecular events. These data reveal that iPSC-derived DA neurons provide a valuable cellular context and foundational atlas for modeling PD genetic risk. We have integrated these data into a FOUNDIN-PD data browser as a resource for understanding the molecular pathogenesis of PD.
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•Differentiated iPSCs to DA neurons from 95 PPMI participants with varying genetic risks•DA neurons derived from human iPSCs provide valuable cellular context for genetic risk•Data are available at https://www.ppmi-info.org and https://www.foundinpd.org
Bressan et al. report the first data release of the Foundational Data Initiative for Parkinson Disease (FOUNDIN-PD), which includes a multi-layered molecular dataset of 95 induced pluripotent stem cell (iPSC) lines with different genetic risk backgrounds differentiated to dopaminergic (DA) neurons, a major affected cell type in Parkinson disease (PD).
The goal of this project was to create a system that was easy for radiologists to use and that could reliably identify, communicate, and track communication of important but non-urgent radiology ...findings to providers and patients. Prior to 2012, our workflow for communicating important non-urgent diagnostic imaging results was cumbersome, rarely used by our radiologists, and resulted in delays in report turnaround time. In 2012, we developed a new system to communicate important non-urgent findings (the RADiology CATegorization 3 (RADCAT-3) system) that was easy for radiologists to use and documented communication of results in the electronic medical record. To evaluate the performance of the new system, we reviewed our radiology reports before (June 2011–June 2012) and after (June 2012–June 2014) the implementation of the new system to compare utilization by the radiologists and success in communicating these findings. During the 12 months prior to implementation, 250 radiology reports (0.06 % of all reports) entered our workflow for communicating important non-urgent findings. One-hundred percent were successfully communicated. During the 24 months after implementation, 13,158 radiology reports (1.4 % of all reports) entered our new RADCAT-3 workflow (3995 (0.8 % of all reports) during year 1 and 9163 (1.9 % of all reports) during year 2). 99.7 % of those reports were successfully communicated. We created a reliable system to ensure communication of important but non-urgent findings with providers and/or patients and to document that communication in the electronic medical record. The rapid adoption of the new system by radiologists suggests that they found it easy to use and had confidence in its integrity. This system has the potential to improve patient care by improving the likelihood of appropriate follow-up for important non-urgent findings that could become life threatening.
Malaria remains a major public health issue, infecting nearly 220 million people every year. The spread of drug-resistant strains of Plasmodium falciparum around the world threatens the progress made ...against this disease. Therefore, identifying druggable and essential pathways in P. falciparum parasites remains a major area of research. One poorly understood area of parasite biology is the formation of disulfide bonds, which is an essential requirement for the folding of numerous proteins. Specialized chaperones with thioredoxin (Trx) domains catalyze the redox functions necessary for breaking incorrect and forming correct disulfide bonds in proteins. Defining the substrates of these redox chaperones is difficult and immunoprecipitation based assays cannot distinguish between substrates and interacting partners. Further, the substrate or client interactions with the redox chaperones are usually transient in nature. Activity based crosslinkers that rely on the nucleophilic cysteines on Trx domains and the disulfide bond forming cysteines on clients provide an easily scalable method to trap and identify the substrates of Trx-domain containing chaperones. The cell permeable crosslinker divinyl sulfone (DVSF) is active only in the presence of nucleophilic cysteines in proteins and, therefore, traps Trx domains with their substrates, as they form mixed disulfide bonds during the course of their catalytic activity. This allows the identification of substrates that rely on Trx activity for their folding, as well as discovering small molecules that interfere with Trx domain activity.Graphic abstract:Identification of thioredoxin domain substrates via divinylsulfone crosslinking and immunoprecipitation-mass spectrometry.
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