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.

A well-accepted method for identification of microorganisms uses matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) coupled to analysis software which identifies and classifies the organism according to its ribosomal protein spectral profile. The method, called MALDI biotyping, is widely used in clinical diagnostics and has partly replaced conventional microbiological techniques such as biochemical identification due to its shorter time to result (minutes for MALDI biotyping versus hours or days for classical phenotypic or genotypic identification). Besides its utility for identifying bacteria, MS-based identification has been shown to be applicable also to yeasts and molds. A limitation to this method, however, is that accurate identification is most reliably achieved on the species level on the basis of reference mass spectra, making further phylogenetic classification unreliable. Here, it is shown that combining tryptic digestion of the acid/organic solvent extracted (classical biotyping preparation) and resolubilized proteins, nano-liquid chromatography (nano-LC), and subsequent identification of the peptides by MALDI-tandem TOF (MALDI-TOF/TOF) mass spectrometry increases the discrimination power to the level of subspecies. As a proof of concept, using this targeted proteomics workflow, we have identified subspeciesspecific biomarker peptides for three Salmonella subspecies, resulting in an extension of the mass range and type of proteins investigated compared to classical MALDI biotyping. This method therefore offers rapid and cost-effective identification and classification of microorganisms at a deeper taxonomic level.

Despite the compelling clinical needs in enhancing bone regeneration and the potential offered by the field of tissue engineering, the adoption of cell-based bone graft substitutes in clinical practice is limited to date. In fact, no study has yet convincingly demonstrated reproducible clinical performance of tissue-engineered implants and at least equivalent cost-effectiveness compared to the current treatment standards. Here, we propose and discuss how tissue engineering strategies could be evolved towards more efficient solutions, depicting three different experimental paradigms: (i) bioreactor-based production; (ii) intraoperative manufacturing, and (iii) developmental engineering. The described approaches reflect the need to streamline graft manufacturing processes while maintaining the potency of osteoprogenitors and recapitulating the sequence of biological steps occurring during bone development, including vascularization. The need to combine the assessment of efficacy of the different strategies with the understanding of their mechanisms of action in the target regenerative processes is highlighted. This will be crucial to identify the necessary and sufficient set of signals that need to be delivered at the injury or defect site and should thus form the basis to define release criteria for reproducibly effective engineered bone graft substitutes.

BackgroundThe relevance of femoral neck fractures (FNFs) increases with the ageing of numerous societies, injury-related decline is observed in many patients. Treatment strategies have evolved towards primary joint replacement, but the impact of different approaches remains a matter of debate. The aim of this trial was to evaluate the benefit of an anterior minimally-invasive (AMIS) compared to a lateral Hardinge (LAT) approach for hemiarthroplasty in these oftentimes frail patients.MethodsFour hundred thirty-nine patients were screened during the 44-months trial, aiming at the evaluation of 150 patients > 60 yrs. of age. Eligible patients were randomised using an online-tool with completely random assignment. As primary endpoint, early mobility, a predictor for long-term outcomes, was evaluated at 3 weeks via the “Timed up and go” test (TUG). Secondary endpoints included the Functional Independence Measure (FIM), pain, complications, one-year mobility and mortality.ResultsA total of 190 patients were randomised; both groups were comparable at baseline, with a predominance for frailty-associated factors in the AMIS-group. At 3 weeks, 146 patients were assessed for the primary outcome. There was a reduction in the median duration of TUG performance of 21.5% (CI [− 41.2,4.7], p = 0.104) in the AMIS-arm (i.e., improved mobility). This reduction was more pronounced in patients with signs of frailty or cognitive impairment. FIM scores increased on average by 6.7 points (CI [0.5–12.8], p = 0.037), pain measured on a 10-point visual analogue scale decreased on average by 0.7 points (CI: [− 1.4,0.0], p = 0.064). The requirement for blood transfusion was lower in the AMIS- group, the rate of complications comparable, with a higher rate of soft tissue complications in the LAT-group. The mortality was higher in the AMIS-group.ConclusionThese results, similar to previous reports, support the concept that in elderly patients at risk of frailty, the AMIS approach for hemiarthroplasty can be beneficial, since early mobilisation and pain reduction potentially reduce deconditioning, morbidity and loss of independence. The results are, however, influenced by a plethora of factors. Only improvements in every aspect of the therapeutic chain can lead to optimisation of treatment and improve outcomes in this growing patient population.Trial registrationwww.clinicaltrials.gov: NCT01408693 (registered August 3rd 2011).Electronic supplementary materialThe online version of this article (10.1186/s12877-018-0898-9) contains supplementary material, which is available to authorized users.