Engineered bacteriophages provide powerful tools for biotechnology, diagnostics, pathogen control, and therapy. However, current techniques for phage editing are experimentally challenging and limited to few phages and host organisms. Viruses that target Gram-positive bacteria are particularly difficult to modify. Here, we present a platform technology that enables rapid, accurate, and selection-free construction of synthetic, tailor-made phages that infect Gram-positive bacteria. To this end, custom-designed, synthetic phage genomes were assembled in vitro from smaller DNA fragments. We show that replicating, cell wall-deficient L-form bacteria can reboot synthetic phage genomes upon transfection, i.e., produce virus particles from naked, synthetic DNA. Surprisingly, L-form cells not only support rebooting of native and synthetic phage genomes but also enable cross-genus reactivation of and phages from their DNA, thereby broadening the approach to phages that infect other important Gram-positive pathogens. We then used this platform to generate virulent phages by targeted modification of temperate phage genomes and demonstrated their superior killing efficacy. These synthetic, virulent phages were further armed by incorporation of enzybiotics into their genomes as a genetic payload, which allowed targeting of phage-resistant bystander cells. In conclusion, this straightforward and robust synthetic biology approach redefines the possibilities for the development of improved and completely new phage applications, including phage therapy.

L-forms are cell wall-deficient bacteria that divide through unusual mechanisms, involving dynamic perturbations of the cellular shape and generation of vesicles, independently of the cell-division protein FtsZ. Here we describe FtsZ-independent mechanisms, involving internal and external vesicles, by which Listeria monocytogenes L-forms proliferate. Using micromanipulation of single cells and vesicles, we show that small vesicles are formed by invagination within larger intracellular vesicles, receive cytoplasmic content, and represent viable progeny. In addition, the L-forms can reproduce by pearling, that is, generation of extracellular vesicles that remain transiently linked to their mother cell via elastic membranous tubes. Using photobleaching and fluorescence recovery, we demonstrate cytoplasmic continuity and transfer through these membranous tubes. Our findings indicate that L-forms' polyploidy and extended interconnectivity through membranous tubes contribute to the generation of viable progeny independently of dedicated division machinery, and further support L-forms as models for studies of potential multiplication mechanisms of hypothetical primitive cells.

A care pathway for elderly hip fracture patients allowed decreased LoS without affecting mortality or change of residential status 1 year after fracture compared to prefracture baseline.

Pubic rami fractures are frequently associated with concomitant posterior pelvic ring injuries, making these injuries more unstable than generally assumed. Based on this fact and the long duration of hospital stay, more aggressive management of these injuries may be considered. The principle aims in this patient population are satisfying pain management, early mobilisation, conservation of independence and return to previous place of residence.

Stromal Vascular Fraction (SVF) cells freshly isolated from adipose tissue include osteogenic- and vascular-progenitors, yet their relevance in bone fracture healing is currently unknown. Here, we investigated whether human SVF cells directly contribute to the repair of experimental fractures in nude rats, and explored the feasibility/safety of their clinical use for augmentation of upper arm fractures in elderly individuals. Human SVF cells were loaded onto ceramic granules within fibrin gel and implanted in critical nude rat femoral fractures after locking-plate osteosynthesis, with cell-free grafts as control. After 8 weeks, only SVF-treated fractures did not fail mechanically and displayed formation of ossicles at the repair site, with vascular and bone structures formed by human cells. The same materials combined with autologous SVF cells were then used to treat low-energy proximal humeral fractures in 8 patients (64-84 years old) along with standard open reduction and internal fixation. Graft manufacturing and implantation were compatible with intraoperative settings and led to no adverse reactions, thereby verifying feasibility/safety. Biopsies of the repair tissue after up to 12 months, upon plate revision or removal, demonstrated formation of bone ossicles, structurally disconnected and morphologically distinct from osteoconducted bone, suggesting the osteogenic nature of implanted SVF cells. We demonstrate that SVF cells, without expansion or exogenous priming, can spontaneously form bone tissue and vessel structures within a fracture-microenvironment. The gained clinical insights into the biological functionality of the grafts, combined with their facile, intra-operative manufacturing modality, warrant further tests of effectiveness in larger, controlled trials. Stem Cells 2016;34:2956-2966.