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Bacterial membrane vesicles (MVs) are produced by all bacteria and contribute to numerous bacterial functions due to their ability to package and transfer bacterial cargo. In doing so, MVs have been shown to facilitate horizontal gene transfer, mediate antimicrobial activity, and promote biofilm formation. Uropathogenic Escherichia coli is a pathogenic Gram-negative organism that persists in the urinary tract of its host due to its ability to form persistent, antibiotic-resistant biofilms. The formation of these biofilms is dependent upon proteins such as Antigen 43 (Ag43), which belongs to the widespread Autotransporter group of bacterial surface proteins. In E. coli, the autotransporter Ag43 has been shown to contribute to bacterial cell aggregation and biofilm formation via self-association of Ag43 between neighboring Ag43-expressing bacteria. As MVs package bacterial proteins, we investigated whether MVs produced by E. coli contained Ag43, and the ability of Ag43-expressing MVs to facilitate cell aggregation and biofilm formation. We showed that Ag43 expressing E. coli produced MVs that contained Ag43 on their surface and had an enhanced ability to bind to E. coli bacteria. Furthermore, we demonstrated that the addition of Ag43-containing MVs to Ag43-expressing E. coli significantly enhanced biofilm formation. These findings reveal the contribution of MVs harboring autotransporters in promoting bacterial aggregation and enhancing biofilm formation, highlighting the impact of MVs and their specific composition to bacterial adaptation and pathogenesis.IMPORTANCEAutotransporter proteins are the largest family of outer membrane and secreted proteins in Gram-negative bacteria which contribute to pathogenesis by promoting aggregation, biofilm formation, persistence, and cytotoxicity. Although the roles of bacterial autotransporters are well known, the ability of bacterial membrane vesicles (MVs) naturally released from the surface of bacteria to contain autotransporters and their role in promoting virulence remains less investigated. Our findings reveal that MVs produced by E. coli contain the autotransporter protein Ag43. Furthermore, we show that Ag43-containing MVs function to enhance bacterial cell interactions and biofilm formation. By demonstrating the ability of MVs to carry functional autotransporter adhesins, this work highlights the importance of MVs in disseminating autotransporters beyond the bacterial cell membrane to ultimately promote cellular interactions and enhance biofilm development. Overall, these findings have significant implications in furthering our understanding of the numerous ways in which MVs can facilitate bacterial persistence and pathogenesis.

Antigen 43 (Ag43) is a cell-surface exposed protein of Escherichia coli secreted by the Type V, subtype a, secretion system (T5aSS) and belonging to the family of self-associating autotransporters (SAATs). These modular proteins, comprising a cleavable N-terminal signal peptide, a surface-exposed central passenger and an outer membrane C-terminal translocator, self-recognise in a Velcro-like handshake mechanism. A phylogenetic network analysis focusing on the passenger revealed for the first time that they actually distribute into four distinct classes, namely C1, C2, C3 and C4. Structural alignment and modelling analyses demonstrated these classes arose from shuffling of two different subdomains within the Ag43 passengers. Functional analyses revealed that homotypic interactions occur for all Ag43 classes but significant differences in the sedimentation kinetics and aggregation state were present when Ag43 C3 was expressed. In contrast, heterotypic interaction occurred in a very limited number of cases. Single cell-force spectroscopy demonstrated the importance of specific as well as nonspecific interactions in mediating Ag43-Ag43 recognition. We propose that structural differences in the subdomains of the Ag43 classes account for different autoaggregation dynamics and propensities to co-interact.

Vertebral compression fractures (VCFs) represent a significant cause of disability and primarily result from either underlying vertebral body neoplasms or osteoporosis. Vertebroplasty (VP) is a procedure commonly utilized to repair pathologic VCFs in order to manage pain and reinstate vertebral body height. However, there is a paucity of literature on how to manage painful multilevel VCFs with concomitant bilateral pedicle fractures. We describe a patient with a primary prostatic carcinoma and VCFs of the third and fourth lumbar vertebrae (L3 and L4, respectively) with concomitant bilateral pedicle fractures secondary to metastatic disease. Due to the degree of damage to the L3 and L4 vertebral bodies and pedicles, a VP performed via a percutaneous approach was deemed to be too high risk. VP for L3 and L4 was instead performed by utilizing stereotactic spine navigation and an intraoperative O-arm (Medtronic Corporation, Minneapolis, Minnesota). Our result indicates a potential role for stereotactic spine navigation in vertebroplasty for complex pathologic VCFs., Competing Interests: The authors have declared that no competing interests exist.

Prior to the development of en bloc techniques, vertebral invasion by non-small cell lung cancer (NSCLC) had been considered a relative contraindication to surgical intervention. However, reports in the literature have demonstrated increased progression-free survival with the use of neoadjuvant chemotherapy followed by anterior en bloc resection of the residual tumor. Stereotactic spine navigation has been shown to improve accuracy during complex vertebral osteotomies, improving patient outcomes. We report a 53-year-old woman with an NSCLC in the left upper lobe, a periosteum attachment of the second and third thoracic vertebrae (T2 and T3, respectively), and an infiltration of the corresponding nerve roots. We describe a surgical approach for the resection of NSCLC with vertebral infiltration utilizing stereotactic spine navigation and intraoperative computed tomography (CT) (O-Arm, Medtronic, Minneapolis, Minnesota, US) for a posterior approach laminectomy, osteotomy, and partial vertebrectomy, followed by trans-thoracic en bloc resection of a superior pulmonary sulcus tumor with nerve root infiltration. Posterior approach vertebral osteotomy and en bloc resection for superior sulcus NSCLC infiltrating the vertebrae utilizing stereotactic spine navigation and intraoperative CT (O-Arm) is a viable alternative to the traditional anterior approach., Competing Interests: The authors have declared that no competing interests exist.

Although the effects of ethanol on protein receptors and lipid membranes have been studied extensively, ethanol's effect on vesicles fusing to lipid bilayers is not known. To determine the effect of alcohols on fusion rates, we utilized the nystatin/ergosterol fusion assay to measure fusion of liposomes to a planar lipid bilayer (BLM). The addition of ethanol excited fusion when applied on the cis (vesicle) side, and inhibited fusion on the trans side. Other short-chain alcohols followed a similar pattern. In general, the inhibitory effect of alcohols (trans) occurs at lower doses than the excitatory (cis) effect, with a decrease of 29% in fusion rates at the legal driving limit of 0.08% (w/v) ethanol (IC 50  = 0.2% v/v, 34 mM). Similar inhibitory effects were observed with methanol, propanol, and butanol, with ethanol being the most potent. Significant variability was observed with different alcohols when applied to the cis side. Ethanol and propanol enhanced fusion, butanol also enhanced fusion but was less potent, and low doses of methanol mildly inhibited fusion. The inhibition by trans addition of alcohols implies that they alter the planar membrane structure and thereby increase the activation energy required for fusion, likely through an increase in membrane fluidity. The cis data are likely a combination of the above effect and a proportionally greater lowering of the vesicle lysis tension and hydration repulsive pressure that combine to enhance fusion. Alternate hypotheses are also discussed. The inhibitory effect of ethanol on liposome-membrane fusion is large enough to provide a possible biophysical explanation of compromised neuronal behavior., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)