Engineering highly active nuclease enzymes with machine learning and high-throughput screening.

Optimizing enzymes to function in novel chemical environments is a central goal of synthetic biology, but optimization is often hindered by a rugged fitness landscape and costly experiments. In this work, we present TeleProt, a machine learning (ML) framework that blends evolutionary and experimental data to design diverse protein libraries, and employ it to improve the catalytic activity of a nuclease enzyme that degrades biofilms that accumulate on chronic wounds. After multiple rounds of high-throughput experiments, TeleProt found a significantly better top-performing enzyme than directed evolution (DE), had a better hit rate at finding diverse, high-activity variants, and was even able to design a high-performance initial library using no prior experimental data.

Extrusion-Based Printing of Myoblast-Loaded Fibrin Microthreads to Induce Myogenesis.

Large skeletal muscle injuries such as volumetric muscle loss (VML) disrupt native tissue structures, including biophysical and biochemical signaling cues that promote the regeneration of functional skeletal muscle. Various biofabrication strategies have been developed to create engineered skeletal muscle constructs that mimic native matrix and cellular microenvironments to enhance muscle regeneration; however, there remains a need to create scalable engineered tissues that provide mechanical stability as well as structural and spatiotemporal signaling cues to promote cell-mediated regeneration of contractile skeletal muscle. We describe a novel strategy for bioprinting multifunctional myoblast-loaded fibrin microthreads (myothreads) that recapitulate the cellular microniches to drive myogenesis and aligned myotube formation.

Avoidance of truncated proteins from unintended ribosome binding sites within heterologous protein coding sequences.

Genetic sequences ported into non-native hosts for synthetic biology applications can gain unexpected properties. In this study, we explored sequences functioning as ribosome binding sites (RBSs) within protein coding DNA sequences (CDSs) that cause internal translation, resulting in truncated proteins. Genome-wide prediction of bacterial RBSs, based on biophysical calculations employed by the RBS calculator, suggests a selection against internal RBSs within CDSs in Escherichia coli, but not those in Saccharomyces cerevisiae.

Two-photon scanning microscopy of in vivo sensory responses of cortical neurons genetically encoded with a fluorescent voltage sensor in rat.

A fluorescent voltage sensor protein "Flare" was created from a Kv1.4 potassium channel with YFP situated to report voltage-induced conformational changes in vivo. The RNA virus Sindbis introduced Flare into neurons in the binocular region of visual cortex in rat.

Spatial organization of enzymes for metabolic engineering.

As synthetic pathways built from exogenous enzymes become more complicated, the probability of encountering undesired interactions with host organisms increases, thereby lowering product titer. An emerging strategy to combat this problem is to spatially organize pathway enzymes into multi-protein complexes, where high local concentrations of enzymes and metabolites may enhance flux and limit problematic interactions with the cellular milieu. Co-localizing enzymes using synthetic scaffolds has improved titers for multiple pathways.

Alternative splicing of neuroligin regulates the rate of presynaptic differentiation.

Neuroligins (NLGs) and Neurexins (NRXs) are important adhesion molecules that promote synapse formation. Multiple splice variants of NLG and NRX exist, but their specific functions are unclear. Here we report that a surrogate postsynaptic cell expressing full-length NLG-1 triggers slow presynaptic differentiation in a contacting axon.

Neuronal synapse interaction reconstituted between live cells and supported lipid bilayers.

In the nervous system, homophilic and heterophilic adhesion molecules participate in the induction and differentiation of presynaptic transmitter release sites. We focus on the heterophilic interaction between postsynaptic neuroligin-1 (Nlg) and presynaptic beta-neurexin (Nrx). Nlg has previously been shown to trigger presynaptic differentiation in a Nrx-expressing axon even when presented on a non-neuronal cell or on beads coated with lipid bilayers.

Islet cell tumor arising from a heterotopic pancreas in the duodenal wall with ulceration.

A rare case of symptomatic islet cell tumor arising from heterotopic pancreas in the duodenum with ulceration is described. Gastrointestinal bleeding was the only sign observed in this patient. Tagged red blood cell scan, upper endoscopy, and computed tomography scan showed active bleeding ulcer from a periampullary mass.

Self-expandable metal stents in palliation of malignant gastrointestinal obstruction: review of the current literature data and 5-year experience at Harbor-UCLA Medical Center.

Management of malignant gastrointestinal obstruction presents a significant challenge. Most patients are in a profoundly decompensated state due to underlying malignancy and are not ideal candidates for invasive surgical procedures. In recent years, self-expandable metal stents (SEMS) have emerged as an effective and safe, less invasive alternative for the treatment of malignant intestinal obstruction.