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. While lacking physical diffusion barriers, scaffolded systems could concentrate intermediates locally through a mechanism analogous to naturally occurring microdomains. A more direct strategy for compartmentalizing pathway components would be to encapsulate them within protein shells. Several classes of shells have been loaded with exogenous proteins and expressed successfully in industrial hosts. A critical challenge for achieving ideal pathway compartmentalization with protein shells will likely be evolving pores to selectively limit intermediate diffusion. Eventually, these tools should enhance our ability to rationally design metabolic 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. In contrast, a version of NLG-1, which lacks insert B (NLG-1DeltaB), induces rapid presynaptic differentiation, reaching the rate seen at native neuronal synapses. We show that this acceleration is attributed to the removal of the N-linked glycosylation site within insert B. NLG-1DeltaB also increases synaptic density at neuro-neuronal synapses more than does full-length NLG-1. Other postsynaptic adhesion proteins, such as N-cadherin, EphB2, and SynCAM-1, alone or in combination with full-length NLG-1, do not trigger fast differentiation, suggesting that rapid presynaptic differentiation depends on a unique interaction of NLG-1DeltaB with axonal proteins. Indeed, we find that NLG-1DeltaB recruits more axonal alpha-NRX. Our results suggest that the engagement of alpha-NRX is a key to rapid induction of synapses at new sites of axo-dendritic contact.

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. Based on these calculations, silent mutations aimed at removing internal RBSs can effectively reduce truncation products from internal translation. However, a solution for complete elimination of internal translation initiation is not always feasible due to constraints of available coding sequences. Fluorescence assays and Western blot analysis showed that in genes with internal RBSs, increasing the strength of the intended upstream RBS had little influence on the internal translation strength. Another strategy to minimize truncated products from an internal RBS is to increase the relative strength of the upstream RBS with a concomitant reduction in promoter strength to achieve the same protein expression level. Unfortunately, lower transcription levels result in increased noise at the single cell level due to stochasticity in gene expression. At the low expression regimes desired for many synthetic biology applications, this problem becomes particularly pronounced. We found that balancing promoter strengths and upstream RBS strengths to intermediate levels can achieve the target protein concentration while avoiding both excessive noise and truncated protein.

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. Here we report a retrospective analysis of 59 SEMS placed for gastroduodenal and colorectal obstruction in 48 patients at Harbor-UCLA Medical Center during the last 5 years, as well as review the literature published on SEMS placement. Technical and clinical success rates were approximately 92% and 80%, respectively. The majority of patients tolerated oral food intake by 36 hours after SEMS. There were no major complications of perforation, bleeding, or death. Thirteen patients had obstructive symptom recurrence, which in most cases was successfully managed with additional endoscopic interventions. Our data confirm SEMS efficacy in palliation of malignant intestinal obstruction with lower rates of major complications than previously reported.

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. Injection sites were selected based on intrinsic optical imaging. Expression of Flare occurred in the cell bodies and dendritic processes. Neurons imaged in vivo using two-photon scanning microscopy typically revealed the soma best, discernable against the background labeling of the neuropil. Somatic fluorescence changes were correlated with flashed visual stimuli; however, averaging was essential to observe these changes. This study demonstrates that the genetic modification of single neurons to express a fluorescent voltage sensor can be used to assess neuronal activity in vivo.

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. We have now developed a new method to measure single molecule and ensemble distribution of Nrx and Nlg at the contact site between a non-neuronal Nrx-expressing cell and a flat supported glycosylphosphoinositol-neuroligin-1 (GPI-Nlg) lipid bilayer and relate them to adhesion as measured by cell migration and gravity dissociation. We find that within minutes after cell-bilayer contact, Nrx accumulates at the contact site and the contact area is expanded. The strength of cell-bilayer adhesion depends on the morphology of Nrx accumulation, with the focal concentration strengthening adhesion. The results suggest that Nlg-Nrx interaction rapidly establishes a weak, but specific, adhesion between dynamic pre- and postsynaptic processes, which may ultimately require additional molecules for synapse stabilization.

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. Removal of the submucosal tumor was done to prevent future re-bleeding. Histologic and immunohistochemical characterization of the tumor showed an endocrine tumor that expressed a variety of endocrine peptides.