Strategies are needed to improve repopulation of decellularized lung scaffolds with stromal and functional epithelial cells. We demonstrate that decellularized mouse lungs recellularized in a dynamic low fluid shear suspension bioreactor, termed the rotating wall vessel (RWV), contained more cells with decreased apoptosis, increased proliferation and enhanced levels of total RNA compared to static recellularization conditions. These results were observed with two relevant mouse cell types: bone marrow-derived mesenchymal stromal (stem) cells (MSCs) and alveolar type II cells (C10). In addition, MSCs cultured in decellularized lungs under static but not bioreactor conditions formed multilayered aggregates. Gene expression and immunohistochemical analyses suggested differentiation of MSCs into collagen I-producing fibroblast-like cells in the bioreactor, indicating enhanced potential for remodeling of the decellularized scaffold matrix. In conclusion, dynamic suspension culture is promising for enhancing repopulation of decellularized lungs, and could contribute to remodeling the extracellular matrix of the scaffolds with subsequent effects on differentiation and functionality of inoculated cells.

Extra-intestinal pathogenic E. coli (ExPEC), including avian pathogenic E. coli (APEC), pose a considerable threat to both human and animal health, with illness causing substantial economic loss. APEC strain χ7122 (O78∶K80∶H9), containing three large plasmids [pChi7122-1 (IncFIB/FIIA-FIC), pChi7122-2 (IncFII), and pChi7122-3 (IncI₂)]; and a small plasmid pChi7122-4 (ColE2-like), has been used for many years as a model strain to study the molecular mechanisms of ExPEC pathogenicity and zoonotic potential. We previously sequenced and characterized the plasmid pChi7122-1 and determined its importance in systemic APEC infection; however the roles of the other pChi7122 plasmids were still ambiguous. Herein we present the sequence of the remaining pChi7122 plasmids, confirming that pChi7122-2 and pChi7122-3 encode an ABC iron transport system (eitABCD) and a putative type IV fimbriae respectively, whereas pChi7122-4 is a cryptic plasmid. New features were also identified, including a gene cluster on pChi7122-2 that is not present in other E. coli strains but is found in Salmonella serovars and is predicted to encode the sugars catabolic pathways. In vitro evaluation of the APEC χ7122 derivative strains with the three large plasmids, either individually or in combinations, provided new insights into the role of plasmids in biofilm formation, bile and acid tolerance, and the interaction of E. coli strains with 3-D cultures of intestinal epithelial cells. In this study, we show that the nature and combinations of plasmids, as well as the background of the host strains, have an effect on these phenomena. Our data reveal new insights into the role of extra-chromosomal sequences in fitness and diversity of ExPEC in their phenotypes.

Streptococcus pneumoniae still causes severe morbidity and mortality worldwide, especially in young children and the elderly. Much effort has been dedicated to developing protein-based universal vaccines to conquer the current shortcomings of capsular vaccines and capsular conjugate vaccines, such as serotype replacement, limited coverage and high costs. A recombinant live vector vaccine delivering protective antigens is a promising way to achieve this goal. In this review, we discuss the researches using live recombinant vaccines, mainly live attenuated Salmonella and lactic acid bacteria, to deliver pneumococcal antigens. We also discuss both the limitations and the future of these vaccines.

Cyanovirin-N (CV-N) is an antiviral lectin with potent activity against enveloped viruses, including HIV. The mechanism of action involves high affinity binding to mannose-rich glycans that decorate the surface of enveloped viruses. In the case of HIV, antiviral activity of CV-N is postulated to require multivalent interactions with envelope protein gp120, achieved through a pseudo-repeat of sequence that adopts two near-identical glycan-binding sites, and possibly involves a 3D-domain-swapped dimeric form of CV-N. Here, we present a covalent dimer of CV-N that increases the number of active glycan-binding sites, and we characterize its ability to recognize four glycans in solution. A CV-N variant was designed in which two native repeats were separated by the “nested” covalent insertion of two additional repeats of CV-N, resulting in four possible glycan-binding sites. The resulting Nested CV-N folds into a wild-type-like structure as assessed by circular dichroism and NMR spectroscopy, and displays high thermal stability with a T_m of 59 °C, identical to WT. All four glycan-binding domains encompassed by the sequence are functional as demonstrated by isothermal titration calorimetry, which revealed two sets of binding events to dimannose with dissociation constants K_d of 25 μM and 900 μM, assigned to domains B and B’ and domains A and A’ respectively. Nested CV-N displays a slight increase in activity when compared to WT CV-N in both an anti-HIV cellular assay and a fusion assay. This construct conserves the original binding specifityies of domain A and B, thus indicating correct fold of the two CV-N repeats. Thus, rational design can be used to increase multivalency in antiviral lectins in a controlled manner.

Misfolding and aggregation of α-synuclein into toxic soluble oligomeric α-synuclein aggregates has been strongly correlated with the pathogenesis of Parkinson’s disease (PD). Here, we show that two different morphologically distinct oligomeric α-synuclein aggregates are present in human post-mortem PD brain tissue and are responsible for the bulk of α-synuclein induced toxicity in brain homogenates from PD samples. Two antibody fragments that selectively bind the different oligomeric α-synuclein variants block this α-synuclein induced toxicity and are useful tools to probe how various cell models replicate the α-synuclein aggregation pattern of human PD brain. Using these reagents, we show that mammalian cell type strongly influences α-synuclein aggregation, where neuronal cells best replicate the PD brain α-synuclein aggregation profile. Overexpression of α-synuclein in the different cell lines increased protein aggregation but did not alter the morphology of the oligomeric aggregates generated. Differentiation of the neuronal cells into a cholinergic-like or dopaminergic-like phenotype increased the levels of oligomeric α-synuclein where the aggregates were localized in cell neurites and cell bodies.

Myxoma virus (MYXV) is Leporipoxvirus that possesses a specific rabbit‐restricted host tropism but exhibits a much broader cellular host range in cultured cells. MYXV is able to efficiently block all aspects of the type I interferon (IFN)‐induced antiviral state in rabbit cells, partially in human cells and very poorly in mouse cells. The mechanism(s) of this species‐specific inhibition of type I IFN‐induced antiviral state is not well understood. Here we demonstrate that MYXV encoded protein M029, a truncated relative of the vaccinia virus (VACV) E3 double‐stranded RNA (dsRNA) binding protein that inhibits protein kinase R (PKR), can also antagonize the type I IFN‐induced antiviral state in a highly species‐specific manner. In cells pre‐treated with type I IFN prior to infection, MYXV exploits M029 to overcome the induced antiviral state completely in rabbit cells, partially in human cells, but not at all in mouse cells. However, in cells pre‐infected with MYXV, IFN‐induced signaling is fully inhibited even in the absence of M029 in cells from all three species, suggesting that other MYXV protein(s) apart from M029 block IFN signaling in a speciesindependent manner. We also show that the antiviral state induced in rabbit, human or mouse cells by type I IFN can inhibit M029‐knockout MYXV even when PKR is genetically knocked‐out, suggesting that M029 targets other host proteins for this antiviral state inhibition. Thus, the MYXV dsRNA binding protein M029 not only antagonizes PKR from multiple species but also blocks the type I IFN antiviral state independently of PKR in a highly species‐specific fashion.