The Toxoplasma gondii active serine hydrolase 4 regulates parasite division and intravacuolar parasite architecture

ABSTRACT Hydrolase are enzymes that regulate diverse biological processes, including posttranslational protein modifications. Recent work identified four active serine hydrolases (ASHs) in Toxoplasma gondii as candidate depalmitoylases. However, only TgPPT1 (ASH1) has been confirmed to remove palmitate from proteins. ASH4 (TgME49_264290) was reported to be refractory to genetic disruption. We demonstrate that recombinant ASH4 is an esterase that processes short acyl esters but not palmitoyl thioesters. Genetic disruption of ASH4 causes defects in cell division and premature scission of parasites from residual bodies. These defects lead to the presence of vacuoles with a disordered intravacuolar architecture, with parasites arranged in pairs around multiple residual bodies. Importantly, we found that the deletion of ASH4 correlates with a defect in radial dispersion from host cells after egress. This defect in dispersion of parasites is a general phenomenon that is observed for disordered vacuoles that occur at low frequency in wild-type parasites, suggesting a possible general link between intravacuolar organization and dispersion after egress. IMPORTANCE This work defines the function of an enzyme in the obligate intracellular parasite Toxoplasma gondii. We show that this previously uncharacterized enzyme is critical for aspects of cellular division by the parasite and that loss of this enzyme leads to parasites with cell division defects and which also are disorganized inside their vacuoles. This leads to defects in the ability of the parasite to disseminate from the site of an infection and may have a significant impact on the parasite's overall infectivity of a host organism

The Drosophila Inhibitor of Apoptosis (IAP) DIAP2 Is Dispensable for Cell Survival, Required for the Innate Immune Response to Gram-negative Bacterial Infection, and Can Be Negatively Regulated by the Reaper/Hid/Grim Family of IAP-binding Apoptosis Inducers

Many inhibitor of apoptosis (IAP) family proteins inhibit apoptosis. IAPs contain N-terminal baculovirus IAP repeat domains and a C-terminal RING ubiquitin ligase domain. Drosophila IAP DIAP1 is essential for the survival of many cells, protecting them from apoptosis by inhibiting active caspases. Apoptosis initiates when proteins such as Reaper, Hid, and Grim bind a surface groove in DIAP1 baculovirus IAP repeat domains via an N-terminal IAP-binding motif. This evolutionarily conserved interaction disrupts DIAP1-caspase interactions, unleashing apoptosis-inducing caspase activity. A second Drosophila IAP, DIAP2, also binds Rpr and Hid and inhibits apoptosis in multiple contexts when overexpressed. However, due to a lack of mutants, little is known about the normal functions of DIAP2. We report the generation of diap2 null mutants. These flies are viable and show no defects in developmental or stress-induced apoptosis. Instead, DIAP2 is required for the innate immune response to Gram-negative bacterial infection. DIAP2 promotes cytoplasmic cleavage and nuclear translocation of the NF-{kappa}B homolog Relish, and this requires the DIAP2 RING domain. Increasing the genetic dose of diap2 results in an increased immune response, whereas expression of Rpr or Hid results in down-regulation of DIAP2 protein levels. Together these observations suggest that DIAP2 can regulate immune signaling in a dose-dependent manner, and this can be regulated by IBM-containing proteins. Therefore, diap2 may identify a point of convergence between apoptosis and immune signaling pathways

Rancang Bangun Aplikasi Pencatatan Transaksi Penjualan Tiket pada PT Gery Anugerah Tour And Travel di Kupang - Nusa Tenggara Timur

PT. Gery Anugerah Tours & Travel is a travel agency company located in the city of Kupang, East Nusa Tenggara. PT. Gery Anugerah Tours & Travel is here to serve the needs of customers for services ticketing domestic and International flights. The management of the company is promoting superior service to customers. In 2013 and 2014 the company experienced a very rapid increase in the sale of airline tickets, with increasing sales then give a positive impact to the company so that the company provides to the customer convenience in transaction using cash or credit. Given the ease of appearing an obstacle in the form of ticket sales related record keeping became sloppy in filing into sales reporting. Ticket sales transaction records that are not neatly arranged this represented a loss of 12% of the records of the sales and the loss of customers who have previously made booking tickets. Any problems that occur, it will be created an information system that will govern the sale of tickets as well as ticket sales reporting company, in order to eliminate the losses incurred earlier. The information system that will be created is the application of transaction records in ticket sales

The glycolytic enzyme phosphofructokinase-1 assembles into filaments.

Despite abundant knowledge of the regulation and biochemistry of glycolytic enzymes, we have limited understanding on how they are spatially organized in the cell. Emerging evidence indicates that nonglycolytic metabolic enzymes regulating diverse pathways can assemble into polymers. We now show tetramer- and substrate-dependent filament assembly by phosphofructokinase-1 (PFK1), which is considered the "gatekeeper" of glycolysis because it catalyzes the step committing glucose to breakdown. Recombinant liver PFK1 (PFKL) isoform, but not platelet PFK1 (PFKP) or muscle PFK1 (PFKM) isoforms, assembles into filaments. Negative-stain electron micrographs reveal that filaments are apolar and made of stacked tetramers oriented with exposed catalytic sites positioned along the edge of the polymer. Electron micrographs and biochemical data with a PFKL/PFKP chimera indicate that the PFKL regulatory domain mediates filament assembly. Quantified live-cell imaging shows dynamic properties of localized PFKL puncta that are enriched at the plasma membrane. These findings reveal a new behavior of a key glycolytic enzyme with insights on spatial organization and isoform-specific glucose metabolism in cells

Evaluation of early implementations of antibiotic stewardship program initiatives in nine Dutch hospitals

Background Antibiotic resistance is a global threat to patient safety and care. In response, hospitals start antibiotic stewardship programs to optimise antibiotic use. Expert-based guidelines recommend strategies to implement such programs, but local implementations may differ per hospital. Earlier published assessments determine maturity of antibiotic stewardship programs based on expert-based structure indicators, but they disregard that there may be valid deviations from these expert-based programs. Aim To analyse the progress and barriers of local implementations of antibiotic stewardship programs with stakeholders in nine Dutch hospitals and to develop a toolkit that guides implementing local antibiotic stewardship programs. Methods An online questionnaire based on published guidelines and recommendations, conducted with seven clinical microbiologists, seven infectious disease physicians and five clinical pharmacists at nine Dutch hospitals. Results Results show local differences in antibiotic stewardship programs and the uptake of interventions in hospitals. Antibiotic guidelines and antibiotic teams are the most extensively implemented interventions. Education, decision support and audit-feedback are deemed important interventions and they are either piloted in implementations at academic hospitals or in preparation for application in non-academic hospitals. Other interventions that are recommended in guidelines - benchmarking, restriction and antibiotic formulary - appear to have a lower priority. Automatic stop-order, pre-authorization, automatic substitution, antibiotic cycling are not deemed to be worthwhile according to respondents. Conclusion There are extensive local differences in the implementation of antibiotic stewardship interventions. These differences suggest a need to further explore the rationale behind the choice of interventions in antibiotic stewardship programs. Rather than reporting this rationale, this study reports where rationale can play a key role in the implementation of antibiotic stewardship programs. A one-size-fits-all solution is unfeasible as there may be barriers or valid reasons for local experts to deviate from expert-based guidelines. Local experts can be supported with a toolkit containing advice based on possible barriers and considerations. These parameters can be used to customise an implementation of antibiotic stewardship programs to local needs (while retaining its expert-based foundation)

Transitions Regulating the Timing of Cytokinesis in Embryonic Cells

AbstractAnaphase, mitotic exit, and cytokinesis proceed in rapid succession, and while mitotic exit is a requirement for cytokinesis in yeast [1, 2], it may not be a direct requirement for furrow initiation in animal cells [3, 4]. In this report, we physically manipulated the proximity of the mitotic apparatus (MA) to the cell cortex in combination with microinjection of effectors of the spindle checkpoint and CDK1 activity to determine how the initiation of cytokinesis is coupled to the onset of anaphase and mitotic exit. Whereas precocious contact between the MA and the cell surface advanced the onset of cytokinesis into early anaphase A, furrowing could not be advanced prior to the metaphase-anaphase transition. Additionally, while cells arrested in anaphase could be induced to initiate cleavage furrows, cells arrested in metaphase could not. Finally, activation of the mitotic checkpoint in one spindle of a binucleate cell failed to arrest cytokinesis induced by the control spindle but did inhibit the formation of furrows between the arrested MA and the control, nonarrested MA. Our experiments suggest that the competence of the mitotic apparatus to initiate cytokinesis is not dependent on cyclin degradation but does require anaphase-promoting complex (APC) activity and, thus, inactivation of the mitotic checkpoint

Improving the Stability of High and Low Bandgap Polymers Organic Photovoltaic Devices Using a Solution Based Titanium Sub-Oxide Interfacial Layer

The improvement in device efficiency has brought organic photovoltaic (OPV) devices closer to commercial viability, highlighting the importance of studying the lifetime and stability of OPV devices. At present, the lifetime and stability of OPV devices is much shorter and poor mainly caused by oxygen, moisture, and light resulting in the oxidation on low work function electrodes and the degradation of the morphology of the photoactive layer. To improve the lifetime and stability of the OPV devices, we used newly developed low bandgap polymer, PCDTBT, as the electron acceptor material and a solution based titanium sub-oxide (TiOx) interfacial layer inserted between the active layer and the cathode. In our experiment, we fabricated unencapsulated bulk heterojunctions OPV devices based on the high and low bandgap polymers of P3HT:PC61BM and PCDTBT:PC71BM, respectively. We synthesized a solution based TiOx by using a sol-gel chemistry method. We performed stability tests on the OPV devices: (1) with and without the TiOx layer (Case (I)) to test the effectiveness of the TiOx layer in protecting the photoactive layer from degradation, (2) with and without a protection cover (a high research grade opaque Al foil) to observe the device performance in a dark/light environment (Case (II)), and (3) in different storage media conditions: (a) air, (b) glove box, (3) ante-chamber of a glove box, and (4) (Case (III)). We spent significant time and effort in optimizing the fabrication processing steps including; the thickness of the active layer, pre-annealing and post-annealing treatments. We fabricated the OPV devices by using the optimal fabrication procedure. We found that the best PCE value of 4.1% achieved for the P3HT:PC61BM OPV cell and 5.1% for the PCDTBT:PC71BM OPV cell. On the air stability test, we found that the OPV cell of P3HT:PC61BM materials showed good air stability performance resulting in the PCE only dropping 26% over a period of 70 days (stored in a glove box). The PCDTBT:PC71BM devices stored in the glove box over a period of 30 days showed relatively good air stability performances; (1) the device with a TiOx, layer and an opaque Al cover the PCE dropped only 16%, (2) the device with the TiOx layer and without an opaque Al cover PCE dropped 34%, and (3) the device without a TiO x, layer and with an Al cover PCE dropped 48%. While the PCDTBT:PC71BM devices stored in the air; (1-2) with a TiOx layer and with/without opaque Al covers the PCE values dropped 92% after 18 days, and (3) without the TiOx, layer and with an opaque Al cover, the PCE dropped 100% after 3 days. These results highlight the effectiveness of the TiOx layer in protecting the active layer from degradation. We concluded that the TiOx, layer effectively improved the stability the OPV devices

The Role of Regulated mRNA Stability in Establishing Bicoid Morphogen Gradient in Drosophila Embryonic Development

The Bicoid morphogen is amongst the earliest triggers of differential spatial pattern of gene expression and subsequent cell fate determination in the embryonic development of Drosophila. This maternally deposited morphogen is thought to diffuse in the embryo, establishing a concentration gradient which is sensed by downstream genes. In most model based analyses of this process, the translation of the bicoid mRNA is thought to take place at a fixed rate from the anterior pole of the embryo and a supply of the resulting protein at a constant rate is assumed. Is this process of morphogen generation a passive one as assumed in the modelling literature so far, or would available data support an alternate hypothesis that the stability of the mRNA is regulated by active processes? We introduce a model in which the stability of the maternal mRNA is regulated by being held constant for a length of time, followed by rapid degradation. With this more realistic model of the source, we have analysed three computational models of spatial morphogen propagation along the anterior-posterior axis: (a) passive diffusion modelled as a deterministic differential equation, (b) diffusion enhanced by a cytoplasmic flow term; and (c) diffusion modelled by stochastic simulation of the corresponding chemical reactions. Parameter estimation on these models by matching to publicly available data on spatio-temporal Bicoid profiles suggests strong support for regulated stability over either a constant supply rate or one where the maternal mRNA is permitted to degrade in a passive manner