Limiting the Broadcast Range of a Secreting Cell during Intercellular Signaling Using Protease-Mediated Degradation.

Synthetic biology is revolutionizing our approaches to biocomputing, diagnostics, and environmental monitoring through the use of designed genetic circuits that perform a function within a single cell. More complex functions can be performed by multiple cells that coordinate as they perform different subtasks. Cell-cell communication using molecular signals is particularly suited for aiding in this communication, but the number of molecules that can be used in different communication channels is limited. Here we investigate how proteases can limit the broadcast range of communicating cells. We find that adding barrierpepsin to Saccharomyces cerevisiae cells in two-dimensional multicellular networks that use α-factor signaling prevents cells beyond a specific radius from responding to α-factor signals. Such limiting of the broadcast range of cells could allow multiple cells to use the same signaling molecules to direct different communication processes and functions, provided that they are far enough from one another. These results suggest a means by which complex synthetic cellular networks using only a few signals for communication could be created by structuring a community of cells to create distinct broadcast environments.

Multi-domain automated patterning of DNA-functionalized hydrogels.

DNA-functionalized hydrogels are capable of sensing oligonucleotides, proteins, and small molecules, and specific DNA sequences sensed in the hydrogels' environment can induce changes in these hydrogels' shape and fluorescence. Fabricating DNA-functionalized hydrogel architectures with multiple domains could make it possible to sense multiple molecules and undergo more complicated macroscopic changes, such as changing fluorescence or changing the shapes of regions of the hydrogel architecture. However, automatically fabricating multi-domain DNA-functionalized hydrogel architectures, capable of enabling the construction of hydrogel architectures with tens to hundreds of different domains, presents a significant challenge. We describe a platform for fabricating multi-domain DNA-functionalized hydrogels automatically at the micron scale, where reaction and diffusion processes can be coupled to program material behavior. Using this platform, the hydrogels' material properties, such as shape and fluorescence, can be programmed, and the fabricated hydrogels can sense their environment. DNA-functionalized hydrogel architectures with domain sizes as small as 10 microns and with up to 4 different types of domains can be automatically fabricated using ink volumes as low as 50 µL. We also demonstrate that hydrogels fabricated using this platform exhibit responses similar to those of DNA-functionalized hydrogels fabricated using other methods by demonstrating that DNA sequences can hybridize within them and that they can undergo DNA sequence-induced shape change.

Ophthalmic Microsurgery Lab for Medical Students: Enhancing Learner Intrinsic Motivation and Comfort with Microsurgery.

Objective This study aimed to evaluate the impact of an ophthalmic microsurgery laboratory on medical students' intrinsic motivation, explicit interest in ophthalmology, and comfort with microsurgical skills. Design In this noncontrolled trial, medical students attended a Zoom-based lecture on corneal suturing, watched an instructional video on operating microscopes, and attended a wet laboratory on corneal suturing. Participants completed pre- and posttest surveys assessing comfort with microsurgical skills and explicit interest in ophthalmology. Additionally, the posttest survey included items from the Intrinsic Motivation Inventory (IMI). Setting This study was conducted at a single academic medical center. Participants A total of 20 students enrolled in the MD program at the University of California, San Francisco School of Medicine. Results Pre- and posttest response rates were 100% ( n = 20) and 90% ( n = 18), respectively. Comfort with microsurgical skills increased significantly between pre- and posttest surveys with large effect sizes (95% confidence interval [CI]; p -value): loading a needle, 1.67 (1.04-2.29; p < 0.001); passing a suture, 1.72 (1.04-2.40; p < 0.001); knot tying, 1.05 (0.34-1.76; p = 0.004); using a microscope, 0.83 (0.04-1.63; p = 0.040); and suturing under a microscope, 1.44 (0.88-2.00; p < 0.001). Comparing pre- and posttest surveys, students reporting moderate to extreme interest in ophthalmology increased from 44 to 61%. Intrinsic motivation was high, indicated by the mean IMI Interest score reaching 93% of the maximum score. Multiple linear regression analyses predicted that IMI Interest scores increased with higher scores of familiarity ( p = 0.002), explicit interest in ophthalmology ( p = 0.042), and comfort with microscopes ( p = 0.005), knot tying ( p = 0.026), and performing surgical maneuvers under a microscope ( p = 0.032). Conclusion Ophthalmic microsurgery laboratories may increase medical students' explicit interest in ophthalmology, comfort with microsurgical skills, and intrinsic motivation. Future studies are needed to evaluate the impact of microsurgical electives on students' objective skills and specialty selection.

A flow-based microfluidic device for spatially quantifying intracellular calcium ion activity during cellular electrotaxis.

How a cell senses, responds, and moves toward, or away from an external cue is central to many biological and medical phenomena including morphogenesis, immune response, and cancer metastasis. Many eukaryotic cells have internal sensory mechanisms that allow them to sense these cues, often in the form of gradients of chemoattractant, voltage, or mechanical stress, and bias their motion in a specific direction. In this study, a new method for using microfluidics to study the electrotactic migration of cells is presented. Electrotaxis (also known as galvanotaxis) is the phenomenon by which cells bias their motion directionally in response to an externally applied electrical field. In this work, we present a new flow-based, salt bridge-free microfluidic device for imaging and quantifying cell motility and intracellular ion activity during electrotaxis. To eliminate salt bridges, we used a low nanoliter flow rate to slowly drive Faradaic waste products away from and out of the electrotaxis zone. This cell migration zone consisted of an array of fluidic confinement channels approximately 2 µm in thickness. This confined height served to insulate the migrating cells from the electric field at the top and bottom of the cell, such that only the two-dimensional perimeter of the cells interacted with the electrical source. We demonstrate the ability to quantify the electrotactic velocity of migrating Dictyostelium discoideum cells and show how this confined design facilitates the imaging and quantification of the ion activity of electrotaxing cells. Finally, by spatially imaging the calcium concentration within these cells, we demonstrate that intracellular calcium preferentially translocates to the leading edge of migrating Dictyostelium cells during electrotaxis but does not exhibit this behavior during migration by chemotaxis in a gradient of cyclic adenosine 3',5'-monophosphate or when cells freely migrate in the absence of an external cue.

Outcomes of Endoscopic and Surgical Pancreatic Necrosectomy: A Single Institution Experience.

Pancreatic necrosis can be managed conservatively; however, infection of pancreatic necrosis usually dictates more aggressive management. Our study aimed to assess the outcomes of open pancreatic necrosectomy (OPN) and endoscopic pancreatic necrosectomy (EPN) in a single center. Data from patients undergoing pancreatic necrosectomy at the Geisinger Medical Center from January 1, 2007, to April 25, 2016, were collected and retrospectively analyzed. Cohorts were composed of EPN (n = 22) and OPN (n = 34) groups. The prevalence of preoperative respiratory failure, septic shock, and multiorgan dysfunction syndrome was higher in the OPN group. The OPN group presented with a higher Bedside Index Severity in Acute Pancreatitis score. Postoperative abscess, persistent kidney dysfunction, and death were more frequent in the OPN group. The EPN group had a higher readmission rate. The results of the univariate analysis for complication and mortality demonstrated that higher mortality and persistent kidney dysfunction were associated with the procedure type, specifically OPN and with a higher Bedside Index Severity in Acute Pancreatitis score. Patients who presented with higher severity of disease underwent an OPN, whereas EPN often was performed successfully in a more benign clinical setting. However, patients with infected necrosis are served best in a tertiary medical facility where multiple treatment modalities are available.