Chemotactic cell trapping in controlled alternating gradient fields.

Directed cell migration toward spatio-temporally varying chemotactic stimuli requires rapid cytoskeletal reorganization. Numerous studies provide evidence that actin reorganization is controlled by intracellular redistribution of signaling molecules, such as the PI4,5P2/PI3,4,5P3 gradient. However, exploring underlying mechanisms is difficult and requires careful spatio-temporal control of external chemotactic stimuli. We designed a microfluidic setup to generate alternating chemotactic gradient fields for simultaneous multicell exposure, greatly facilitating statistical analysis. For a quantitative description of intracellular response dynamics, we apply alternating time sequences of spatially homogeneous concentration gradients across 300 µm, reorienting on timescales down to a few seconds. Dictyostelium discoideum amoebae respond to gradient switching rates below 0.02 Hz by readapting their migration direction. For faster switching, cellular repolarization ceases and is completely stalled at 0.1 Hz. In this "chemotactically trapped" cell state, external stimuli alternate faster than intracellular feedback is capable to respond by onset of directed migration. To investigate intracellular actin cortex rearrangement during gradient switching, we correlate migratory cell response with actin repolymerization dynamics, quantified by a fluorescence distribution moment of the GFP fusion protein LimEΔcc. We find two fundamentally different cell polarization types and we could reveal the role of PI3-Kinase for cellular repolarization. In the early aggregation phase, PI3-Kinase enhances the capability of D. discoideum cells to readjust their polarity in response to spatially alternating gradient fields, whereas in aggregation competent cells the effect of PI3-Kinase perturbation becomes less relevant.

A 24-year-old patient with testicular benign capillary hemangioma: A rare case report.

Key Clinical Message: The importance of urologists and pathologists being knowledgable about primary testicular hemangiomas and other benign adult testicular neoplasms, though rare, is crucial. Ensuring these professionals are well-versed in these conditions is vital in medicine. Testicular sparing surgery, especially when tumor markers are negative, is a common approach for patients with small or uncertain testicular masses. Abstract: The cause of the uncommon benign testicular tumor known as testicular capillary hemangioma is currently unclear. Children are shown to have a greater incidence than adults. Histopathological examination reveals a vascular tumor with a well-formed capillary lumina. The lesions are bordered by flattened endothelium and have lobulated clusters of closely spaced capillaries with an abundance of vascular gaps. These capillaries have anaplastic characteristics and lack mitotic activity. The patient, who was 24-year-old, came to the urology department complaining of severe testicular pain. He had no significant medical history. Clinical examination found a mass at the upper pole of the testicle. An ultrasonography Doppler study found a left varicocele that measured 3.5 mm in diameter, as well as a mass at the upper part of the left testicle. Pathologic examination confirmed the presence of a benign hemangioma with. Although rare, it is important for urologists and pathologists to be aware of primary testicular hemangiomas and other benign adult testicular neoplasms. Testicular sparing surgery has always been considered in patients with small or indeterminate testicular masses with negative tumor markers.

A 64-year-old male with primary diffuse renal large B-cell non-Hodgkin lymphoma: A rare case report.

Key Clinical Message: In the context of lymphoma, it is of paramount importance to perform subsequent Positron Emission Tomography-Computed Tomography (PET-CT) scans to ensure the comprehensive eradication of neoplasms. Abstract: Primary renal diffuse tumors constitute less than 1% of all renal neoplasms. Among these, diffuse renal large B-cell lymphoma is an exceedingly rare extranodal lymphoma. A 64-year-old male presented to the Department of Urology with complaints of persistent left flank discomfort for a duration of 2 weeks. Additionally, he reported generalized weakness, fatigue, and symptoms indicative of lower urinary tract obstruction, such as discomfort in the left testicle and dysuria. Ultrasound imaging revealed an echogenic structure with thickened, reactive walls and a turbid fluid core, located in the left flank, proximal to the lower pole of the kidney. This structure was subsequently identified as diffuse renal large B-cell lymphoma. For the diagnosis of large B-cell lymphomas, it is imperative that a proficient hematopathologist performs a comprehensive examination of the tumor tissue, preferably utilizing an excisional biopsy. The categorization of lymphoma requires specialized tests such as immunohistochemistry, flow cytometry, fluorescence in situ hybridization (FISH), and molecular testing. In instances where a renal mass is detected, healthcare professionals should consider performing a biopsy. In lymphoma cases, follow-up Positron Emission Tomography-Computed Tomography (PET-CT) scans are crucial to confirm the complete eradication of the tumor.

Visual DSD: a design and analysis tool for DNA strand displacement systems.

SUMMARY: The Visual DSD (DNA Strand Displacement) tool allows rapid prototyping and analysis of computational devices implemented using DNA strand displacement, in a convenient web-based graphical interface. It is an implementation of the DSD programming language and compiler described by Lakin et al. (2011) with additional features such as support for polymers of unbounded length. It also supports stochastic and deterministic simulation, construction of continuous-time Markov chains and various export formats which allow models to be analysed using third-party tools.

Predictive modeling of non-viral gene transfer.

In non-viral gene delivery, the variance of transgenic expression stems from the low number of plasmids successfully transferred. Here, we experimentally determine Lipofectamine- and PEI-mediated exogenous gene expression distributions from single cell time-lapse analysis. Broad Poisson-like distributions of steady state expression are observed for both transfection agents, when used with synchronized cell lines. At the same time, co-transfection analysis with YFP- and CFP-coding plasmids shows that multiple plasmids are simultaneously expressed, suggesting that plasmids are delivered in correlated units (complexes). We present a mathematical model of transfection, where a stochastic, two-step process is assumed, with the first being the low-probability entry step of complexes into the nucleus, followed by the subsequent release and activation of a small number of plasmids from a delivered complex. This conceptually simple model consistently predicts the observed fraction of transfected cells, the cotransfection ratio and the expression level distribution. It yields the number of efficient plasmids per complex and elucidates the origin of the associated noise, consequently providing a platform for evaluating and improving non-viral vectors.

Abstractions for DNA circuit design.

DNA strand displacement techniques have been used to implement a broad range of information processing devices, from logic gates, to chemical reaction networks, to architectures for universal computation. Strand displacement techniques enable computational devices to be implemented in DNA without the need for additional components, allowing computation to be programmed solely in terms of nucleotide sequences. A major challenge in the design of strand displacement devices has been to enable rapid analysis of high-level designs while also supporting detailed simulations that include known forms of interference. Another challenge has been to design devices capable of sustaining precise reaction kinetics over long periods, without relying on complex experimental equipment to continually replenish depleted species over time. In this paper, we present a programming language for designing DNA strand displacement devices, which supports progressively increasing levels of molecular detail. The language allows device designs to be programmed using a common syntax and then analysed at varying levels of detail, with or without interference, without needing to modify the program. This allows a trade-off to be made between the level of molecular detail and the computational cost of analysis. We use the language to design a buffered architecture for DNA devices, capable of maintaining precise reaction kinetics for a potentially unbounded period. We test the effectiveness of buffered gates to support long-running computation by designing a DNA strand displacement system capable of sustained oscillations.

Automated tracking in live-cell time-lapse movies.

The development of high-throughput live cell imaging is currently limited by the capabilities of image analysis. Software is required to generate single cell time courses from large data sets of time-lapse movies and to follow properties of individual cells. Automated cell tracking faces notorious problems associated with cell division, high cell density and cell mobility. In particular, a large number of cell traces are discarded in experiments with extended observation times due to image analysis ambiguities. Here we develop an algorithm for robust tracking based on cost matrices from multiple cell parameters such as object size, position or texture. Singularities in cost indicate tracking conflicts, which can be categorized into event classes such as cell division, lysis or overlap of cells. We demonstrate that multiple parameter tracking (MPT) generates single cell fluorescence time traces more reliably than algorithms based on position tracking only. Context-sensitive automatic evaluation and event management increase the yield of continuous and correctly assigned time traces by 27%.

Actin-cytoskeleton dynamics in non-monotonic cell spreading.

The spreading of motile cells on a substrate surface is accompanied by reorganization of their actin network. We show that spreading in the highly motile cells of Dictyostelium is non-monotonic, and thus differs from the passage of spreading cells through a regular series of stages. Quantification of the gain and loss of contact area revealed fluctuating forces of protrusion and retraction that dominate the interaction of Dictyostelium cells with a substrate. The molecular basis of these fluctuations is elucidated by dual-fluorescence labeling of filamentous actin together with proteins that highlight specific activities in the actin system. Front-to-tail polarity is established by the sorting out of myosin-II from regions where dense actin assemblies are accumulating. Myosin-IB identifies protruding front regions, and the Arp2/3 complex localizes to lamellipodia protruded from the fronts. Coronin is used as a sensitive indicator of actin disassembly to visualize the delicate balance of polymerization and depolymerization in spreading cells. Short-lived actin patches that co-localize with clathrin suggest that membrane internalization occurs even when the substrate-attached cell surface expands. We conclude that non-monotonic cell spreading is characterized by spatiotemporal patterns formed by motor proteins together with regulatory proteins that either promote or terminate actin polymerization on the scale of seconds.