A universal microfluidic approach for quantitative study of bacterial biofilms

Bacteria usually live in densely packed communities called biofilms, where interactions between the bacteria give rise to complex properties. Quantitative analysis is indispensable in understanding those properties. However, current biofilm culturing approaches impose various limitations to these types of analysis. Here, we developed a microfluidic approach for quantitative study of biofilms, which is universal and can be used to culture biofilms of various bacterial species. To demonstrate the advantages of this approach, we present two examples, both of which revealed new biological insights. In the first example, we explored the response of Escherichia coli biofilms to exogenous hydrogen peroxide; We found the biofilms gained resistance to H2O2, but their growth was slowed down due to the metabolic cost of maintaining the resistance; However, under oxygen limitation, H2O2 can anti-intuitively boost biofilm growth. In the second example, we explored resource retention by Pseudomonas aeruginosa biofilms; We observed a fluorescent substance within the biofilm and identified it as the siderophore pyoverdine; We further showed that the extracellular matrix component Psl acted as a retention barrier for pyoverdine, minimizing its loss into the environment and therefore potentially promoting sharing of pyoverdine within the biofilm.

A Novel Leptospira interrogans Protein LIC13086 Inhibits Fibrin Clot Formation and Interacts With Host Components

Leptospirosis is a neglected zoonosis, caused by pathogenic spirochetes bacteria of the genus Leptospira. The molecular mechanisms of leptospirosis infection are complex, and it is becoming clear that leptospires express several functionally redundant proteins to invade, disseminate, and escape the host’s immune response. Here, we describe a novel leptospiral protein encoded by the gene LIC13086 as an outer membrane protein. The recombinant protein LIC13086 can interact with the extracellular matrix component laminin and bind plasminogen, thus possibly participating during the adhesion process and dissemination. Also, by interacting with fibrinogen and plasma fibronectin, the protein LIC13086 probably has an inhibitory effect in the fibrin clot formation during the infection process. The newly characterized protein can also bind molecules of the complement system and the regulator C4BP and, thus, might have a role in the evasion mechanism of Leptospira. Taken together, our results suggest that the protein LIC13086 may have a multifunctional role in leptospiral pathogenesis, participating in host invasion, dissemination, and immune evasion processes.

Comparison of Decellularization Protocols to Generate Peripheral Nerve Grafts: A Study on Rat Sciatic Nerves

In critical nerve gap repair, decellularized nerve allografts are considered a promising tissue engineering strategy that can provide superior regeneration results compared to nerve conduits. Decellularized nerves offer a well-conserved extracellular matrix component that has proven to play an important role in supporting axonal guiding and peripheral nerve regeneration. Up to now, the known decellularized techniques are time and effort consuming. The present study, performed on rat sciatic nerves, aims at investigating a novel nerve decellularization protocol able to combine an effective decellularization in short time with a good preservation of the extracellular matrix component. To do this, a decellularization protocol proven to be efficient for tendons (DN-P1) was compared with a decellularization protocol specifically developed for nerves (DN-P2). The outcomes of both the decellularization protocols were assessed by a series of in vitro evaluations, including qualitative and quantitative histological and immunohistochemical analyses, DNA quantification, SEM and TEM ultrastructural analyses, mechanical testing, and viability assay. The overall results showed that DN-P1 could provide promising results if tested in vivo, as the in vitro characterization demonstrated that DN-P1 conserved a better ultrastructure and ECM components compared to DN-P2. Most importantly, DN-P1 was shown to be highly biocompatible, supporting a greater number of viable metabolically active cells.

Evidence of Swim secretion and association with extracellular matrix in the Drosophila embryo

Secreted wingless-interacting protein (Swim) is the Drosophila ortholog gene of the mammalian Tubulointerstitial Nephritis Antigen Like 1 (TINAGL1), known also as lipocalin-7 (LCN7), or adrenocortical zonation factor 1 (AZ-1). Swim and TINAGL1 proteins share a significant homology, including the somatomedin B and the predictive inactive C1 cysteine peptidase domains. In mammals, both TINAGL1 and its closely related homolog TINAG have been identified in basement membranes, where they may function as modulators of integrin-mediated adhesion. In Drosophila, Swim was initially identified in the eggshell matrix and subsequently was detected in the culture medium of S2 cells. Further biochemical analysis indicated that Swim binds to wingless (wg) in a lipid-dependent manner. This observation together with RNAiknockdown studies suggested that Swim is an essential cofactor of wg-signalling. However, recent elegant genetic studies ruled out the possibility that Swim is required alone to facilitate wgsignalling in Drosophila, because flies without Swim are viable and fertile. Here, we use the UAS/Gal4 expression system together with confocal imaging to analyze the in vivo localization of a chimeric Swim-GFP in the developing Drosophila embryo. Our data fully support the notion that Swim is an extracellular matrix component that upon ectopic expression is secreted and preferentially associates with the basement membranes of various organs and with the specialized tendon matrix at the muscle attachment sites (MAS). Interestingly, the accumulation of Swim at the MAS does not require integrins. In conclusion, Swim is an extracellular matrix component, and it is possible that Swim exhibits overlapping functions in concert with other undefined components.

Inhibition of Fibroblast Activation by Components of Rhizoma Curcumae and Rhizoma Sparganii in A Rat Model of Uterine Leiomyoma

Background: Traditional Chinese medicine (TCM) often uses Rhizoma Curcumae and Rhizoma Sparganii (RCRS) ，the natural herbs for the treatment of UL. RCRS has been shown to be effective in the treatment of UL in our previous study. This study was to investigate the molecular mechanism by which RCRS inhibits fibroblast activation protein (FAP) activation and prevents uterine leiomyoma in rats. Methods: The SD rat model of uterine leiomyoma was established by estrogen and progesterone load combined with external stimulation. Subsequently, histological analyses, enzyme-Linked immunosorbent assays, western blotting were performed to evaluate the effect of the drug on uterine leiomyoma and its mechanism. Results: Our data showed that treatment of rats with RCRS significantly reduced the expression of FAP, TGF-β (the FAP activating factor), and significantly decreased the phosphorylation of cell proliferation pathway-related signaling factors AKT/MEK/ERK, as well as the expression of the extracellular matrix component collagen. Conclusions: Our results showed that RCRS is very effective in prevention and treatment of uterine leiomyoma in rats, and RCRS may exert its actions by inhibiting the activation of tumor-associated fibroblasts, inhibiting the cell proliferation, and improving tumor extracellular matrix.

Advances in the Research of Bioinks Based on Natural Collagen, Polysaccharide and Their Derivatives for Skin 3D Bioprinting

The skin plays an important role in protecting the human body, and wound healing must be set in motion immediately following injury or trauma to restore the normal structure and function of skin. The extracellular matrix component of the skin mainly consists of collagen, glycosaminoglycan (GAG), elastin and hyaluronic acid (HA). Recently, natural collagen, polysaccharide and their derivatives such as collagen, gelatin, alginate, chitosan and pectin have been selected as the matrix materials of bioink to construct a functional artificial skin due to their biocompatible and biodegradable properties by 3D bioprinting, which is a revolutionary technology with the potential to transform both research and medical therapeutics. In this review, we outline the current skin bioprinting technologies and the bioink components for skin bioprinting. We also summarize the bioink products practiced in research recently and current challenges to guide future research to develop in a promising direction. While there are challenges regarding currently available skin bioprinting, addressing these issues will facilitate the rapid advancement of 3D skin bioprinting and its ability to mimic the native anatomy and physiology of skin and surrounding tissues in the future.