Rapid Formation of Non-canonical Phospholipid Membranes by Chemoselective Amide-Forming Ligations with Hydroxylamines.

There has been increasing interest in methods to generate synthetic lipid membranes as key constituents of artificial cells or to develop new tools for remodeling membranes in living cells. However, the biosynthesis of phospholipids involves elaborate enzymatic pathways that are challenging to reconstitute in vitro. An alternative approach is to use chemical reactions to non-enzymatically generate natural or non-canonical phospholipids de novo. Previous reports have shown that synthetic lipid membranes can be formed in situ using various ligation chemistries, but these methods lack biocompatibility and/or suffer from slow kinetics at physiological pH. Thus, it would be valuable to develop chemoselective strategies for synthesizing phospholipids from water-soluble precursors that are compatible with synthetic or living cells Here, we demonstrate that amide-forming ligations between lipid precursors bearing hydroxylamines and α-ketoacids (KAs) or potassium acyltrifluoroborates (KATs) can be used to prepare non-canonical phospholipids at physiological pH conditions. The generated amide-linked phospholipids spontaneously self-assemble into cell-like micron-sized vesicles similar to natural phospholipid membranes. We show that lipid synthesis using KAT ligation proceeds extremely rapidly, and the high selectivity and biocompatibility of the approach facilitates the in situ synthesis of phospholipids and associated membranes in living cells.

Expression of Fatty Acyl-CoA Ligase Drives One-Pot De Novo Synthesis of Membrane-Bound Vesicles in a Cell-Free Transcription-Translation System.

Despite the central importance of lipid membranes in cellular organization, it is challenging to reconstitute their formation de novo from minimal chemical and biological elements. Here, we describe a chemoenzymatic route to membrane-forming noncanonical phospholipids in which cysteine-modified lysolipids undergo spontaneous coupling with fatty acyl-CoA thioesters generated enzymatically by a fatty acyl-CoA ligase. Due to the high efficiency of the reaction, we were able to optimize phospholipid formation in a cell-free transcription-translation (TX-TL) system. Combining DNA encoding the fatty acyl-CoA ligase with suitable lipid precursors enabled one-pot de novo synthesis of membrane-bound vesicles. Noncanonical sphingolipid synthesis was also possible by using a cysteine-modified lysosphingomyelin as a precursor. When the sphingomyelin-interacting protein lysenin was coexpressed alongside the acyl-CoA ligase, the in situ assembled membranes were spontaneously decorated with protein. Our strategy of coupling gene expression with membrane lipid synthesis in a one-pot fashion could facilitate the generation of proteoliposomes and brings us closer to the bottom-up generation of synthetic cells using recombinant synthetic biology platforms.

Cooperativity and Steep Voltage Dependence in a Bacterial Channel.

This paper reports on the discovery of a novel three-membrane channel unit exhibiting very steep voltage dependence and strong cooperative behavior. It was reconstituted into planar phospholipid membranes formed by the monolayer method and studied under voltage-clamp conditions. The behavior of the novel channel-former, isolated from Escherichia coli, is consistent with a linearly organized three-channel unit displaying steep voltage-gating (a minimum of 14 charges in the voltage sensor) that rivals that of channels in mammalian excitable membranes. The channels also display strong cooperativity in that closure of the first channel permits the second to close and closure of the second channel permits closure of the third. All three have virtually the same conductance and selectivity, and yet the first and third close at positive potentials whereas the second closes at negative potentials. Thus, is it likely that the second channel-former is oriented in the membrane in a direction opposite to that of the other two. This novel structure is named "triplin." The extraordinary behavior of triplin indicates that it must have important and as yet undefined physiological roles.

Deleterious Germline Mutations in Patients With Apparently Sporadic Pancreatic Adenocarcinoma.

Purpose Deleterious germline mutations contribute to pancreatic cancer susceptibility and are well documented in families in which multiple members have had pancreatic cancer. Methods To define the prevalence of these germline mutations in patients with apparently sporadic pancreatic cancer, we sequenced 32 genes, including known pancreatic cancer susceptibility genes, in DNA prepared from normal tissue obtained from 854 patients with pancreatic ductal adenocarcinoma, 288 patients with other pancreatic and periampullary neoplasms, and 51 patients with non-neoplastic diseases who underwent pancreatic resection at Johns Hopkins Hospital between 2000 and 2015. Results Thirty-three (3.9%; 95% CI, 3.0% to 5.8%) of 854 patients with pancreatic cancer had a deleterious germline mutation, 31 (3.5%) of which affected known familial pancreatic cancer susceptibility genes: BRCA2 (12 patients), ATM (10 patients), BRCA1 (3 patients), PALB2 (2 patients), MLH1 (2 patients), CDKN2A (1 patient), and TP53 (1 patient). Patients with these germline mutations were younger than those without (mean ± SD, 60.8 ± 10.6 v 65.1 ± 10.5 years; P = .03). Deleterious germline mutations were also found in BUB1B (1) and BUB3 (1). Only three of these 33 patients had reported a family history of pancreatic cancer, and most did not have a cancer family history to suggest an inherited cancer syndrome. Five (1.7%) of 288 patients with other periampullary neoplasms also had a deleterious germline mutation. Conclusion Germline mutations in pancreatic cancer susceptibility genes are commonly identified in patients with pancreatic cancer without a significant family history of cancer. These deleterious pancreatic cancer susceptibility gene mutations, some of which are therapeutically targetable, will be missed if current family history guidelines are the main criteria used to determine the appropriateness of gene testing.

Duodenal Involvement is an Independent Prognostic Factor for Patients with Surgically Resected Pancreatic Ductal Adenocarcinoma.

BACKGROUND: The current staging system for pancreatic ductal adenocarcinoma (PDAC) includes information about size and local extension of the primary tumor (T stage). The value of incorporating any local tumor extension into pancreatic staging systems has been questioned because it often is difficult to evaluate tumor extension to the peri-pancreatic soft tissues and because most carcinomas of the head of the pancreas infiltrate the intra-pancreatic common bile duct. This study sought to evaluate the prognostic implications of having PDAC with local tumor extension. METHODS: A single-institution, prospectively collected database of 1128 patients who underwent surgical resection for PDAC was queried to examine the prognostic significance of extra-pancreatic tumor involvement ("no involvement," "duodenal involvement," and "extensive involvement"; e.g., gastric, colon or major vein involvement). RESULTS: The median overall survival for the patients without extra-pancreatic involvement was 26 months versus 19 months for the patients with duodenal involvement and 16 months for the patients with extensive involvement (p < 0.001). In the multivariable analysis, duodenal and extensive involvement independently predicted increased risk of death compared with no involvement (hazard ratio [HR] 1.30; 95% confidence interval [CI] 1.08-1.57 and 1.78; 95% CI 1.25-2.55, respectively). A multivariable model combining duodenal and extensive extra-pancreatic involvement, tumor grade, lymph node ratio, and other prognostic features had the highest c-index (0.67). CONCLUSIONS: Inclusion of duodenal involvement in the staging of PDAC adds independent prognostic information.

Bax channel triplet: co-operativity and voltage gating.

Bax, despite being a cytosolic protein, has the distinct ability to form channels in the mitochondrial outer membrane, which are capable of releasing proteins that initiate the execution phase of apoptosis. When studied in a planar phospholipid membrane system, full-length activated Bax can form conducting entities consistent with linearly organized three-channel units displaying steep voltage-gating (n=14) that rivals that of channels in excitable membranes. In addition, the channels display strong positive co-operativity possibly arising from the charge distribution of the voltage sensors. On the basis of functional behaviour, one of the channels in this functional triplet is oriented in the opposite direction to the others often resulting in conflicts between the effects of the electric field and the positive co-operativity of adjacent channels. The closure of the first channel occurs at positive potentials and this permits the second to close, but at negative potentials. The closure of the second channel in turn permits closure of the third, but at positive potentials. Positive co-operativity manifests itself in a number of ways including the second and the third channels opening virtually simultaneously. This extraordinary behaviour must have important, although as yet undefined, physiological roles.