Gliding Associated Proteins Play Essential Roles during the Formation of the Inner Membrane Complex of Toxoplasma gondii

The inner membrane complex (IMC) of apicomplexan parasites is a specialised structure localised beneath the parasite’s plasma membrane, and is important for parasite stability and intracellular replication. Furthermore, it serves as an anchor for the myosin A motor complex, termed the glideosome. While the role of this protein complex in parasite motility and host cell invasion has been well described, additional roles during the asexual life cycle are unknown. Here, we demonstrate that core elements of the glideosome, the gliding associated proteins GAP40 and GAP50 as well as members of the GAPM family, have critical roles in the biogenesis of the IMC during intracellular replication. Deletion or disruption of these genes resulted in the rapid collapse of developing parasites after initiation of the cell cycle and led to redistribution of other glideosome components.     

The Natural History of Kidney Graft Cortical Microcirculation Determined by Real-Time Contrast-Enhanced Sonography (RT-CES)

Background

Kidney transplantation is the therapy of choice for end-stage kidney disease. Graft’s life span is shorter than expected due in part to the delayed diagnosis of various complications, specifically those related to silent progression. It is recognized that serum creatinine levels and proteinuria are poor markers of mild kidney lesions, which results in delayed clinical information. There are many investigation looking for early markers of graft damage. Decreasing kidney graft cortical microcirculation has been related to poor prognosis in kidney transplantation. Cortical capillary blood flow (CCBF) can be measured by real-time contrast-enhanced sonography (RT-CES). Our aim was to describe the natural history of CCBF over time under diverse conditions of kidney transplantation, to explore the influence of donor conditions and recipient events, and to determine the capacity of CCBF for predicting renal function in medium term.

Patients and Methods

RT-CES was performed in 79 consecutive kidney transplant recipients during the first year under regular clinical practice. Cortical capillary blood flow was measured. Clinical variables were analyzed. The influence of CCBF has been determined by univariate and multivariate analysis using mixed regression models based on sequential measurements for each patient over time. We used a first-order autoregression model as the structure of the covariation between measures. The post-hoc comparisons were considered using the Bonferroni correction.

Results

The CCBF values varied significantly over the study periods and were significantly lower at 48 h and day 7. Brain-death donor age and CCBF levels showed an inverse relationship (r: -0.62, p<0.001). Living donors showed higher mean CCBF levels than brain-death donors at each point in the study. These significant differences persisted at month 12 (54.5 ± 28.2 vs 33.7 ± 30 dB/sec, living vs brain-death donor, respectively, p = 0.004) despite similar serum creatinine levels (1.5 ± 0.3 and 1.5 ± 0.5 mg/dL). A sole rejection episode was associated with lower overall CCBF values over the first year. CCBF defined better than level of serum creatinine the graft function status at medium-term.

Conclusion

RT-CES is a non-invasive tool that can quantify and iteratively estimate cortical microcirculation. We have described the natural history of cortical capillary blood flow under regular clinical conditions.     

Dp71Δ78‐79 dystrophin mutant stimulates neurite outgrowth in PC12 cells via upregulation and phosphorylation of HspB1

PC12 cells acquire a neuronal phenotype in response to nerve growth factor (NGF). However, this phenotype is more efficiently achieved when the Dp71Δ_78‐79 dystrophin mutant is stably expressed in PC12‐C11 cells. To investigate the effect of Dp71Δ_78‐79 overexpression on the protein profile of PC12‐C11 cells, we compared the expression profiles of undifferentiated and NGF‐differentiated PC12‐C11 and PC12 cells by 2DE. In undifferentiated cultures, one protein was downregulated, and five were upregulated. Dp71Δ_78‐79 overexpression had a greater effect on differentiated cultures, with ten proteins downregulated and seven upregulated. The protein with the highest upregulation was HspB1. Changes in HspB1 expression were validated by Western blot and immunofluorescence analyses. Interestingly, the neurite outgrowth in PC12‐C11 cells was affected by a polyclonal antibody against HspB1, and the level of HspB1 and HspB1Ser86 decreased, suggesting an important role for this protein in this cellular process. Our results show that Dp71Δ_78‐79 affects the expression level of some proteins and that the stimulated neurite outgrowth produced by this mutant is mainly through upregulation and phosphorylation of HspB1.     

Non-straight cell edges are important to invasion and engulfment as demonstrated by cell mechanics model

Computational models of cell–cell mechanical interactions typically simulate sorting and certain other motions well, but as demands on these models continue to grow, discrepancies between the cell shapes, contact angles and behaviours they predict and those that occur in real cells have come under increased scrutiny. To investigate whether these discrepancies are a direct result of the straight cell–cell edges generally assumed in these models, we developed a finite element model that approximates cell boundaries using polylines with an arbitrary number of segments. We then compared the predictions of otherwise identical polyline and monoline (straight-edge) models in a variety of scenarios, including annealing, single- and multi-cell engulfment, sorting, and two forms of mixing—invasion and checkerboard pattern formation. Keeping cell–cell edges straight influences cell motion, cell shape, contact angle, and boundary length, especially in cases where one cell type is pulled between or around cells of a different type, as in engulfment or invasion. These differences arise because monoline cells have restricted deformation modes. Polyline cells do not face these restrictions, and with as few as three segments per edge yielded realistic edge shapes and contact angle errors one-tenth of those produced by monoline models, making them considerably more suitable for situations where angles and shapes matter, such as validation of cellular force–inference techniques. The findings suggest that non-straight cell edges are important both in modelling and in nature.     

Orthogonal intercellular signaling for programmed spatial behavior

Bidirectional intercellular signaling is an essential feature of multicellular organisms, and the engineering of complex biological systems will require multiple pathways for intercellular signaling with minimal crosstalk. Natural quorum‐sensing systems provide components for cell communication, but their use is often constrained by signal crosstalk. We have established new orthogonal systems for cell–cell communication using acyl homoserine lactone signaling systems. Quantitative measurements in contexts of differing receiver protein expression allowed us to separate different types of crosstalk between 3‐oxo‐C6‐ and 3‐oxo‐C12‐homoserine lactones, cognate receiver proteins, and DNA promoters. Mutating promoter sequences minimized interactions with heterologous receiver proteins. We used experimental data to parameterize a computational model for signal crosstalk and to estimate the effect of receiver protein levels on signal crosstalk. We used this model to predict optimal expression levels for receiver proteins, to create an effective two‐channel cell communication device. Establishment of a novel spatial assay allowed measurement of interactions between geometrically constrained cell populations via these diffusible signals. We built relay devices capable of long‐range signal propagation mediated by cycles of signal induction, communication and response by discrete cell populations. This work demonstrates the ability to systematically reduce crosstalk within intercellular signaling systems and to use these systems to engineer complex spatiotemporal patterning in cell populations.