Ionically Modified Gelatin Hydrogels Maintain Murine Myogenic Cell Viability and Fusion Capacity.

For tissue engineering of skeletal muscles, there is a need for biomaterials which do not only allow cell attachment, proliferation, and differentiation, but also support the physiological conditions of the tissue. Next to the chemical nature and structure of the biomaterial, its response to the application of biophysical stimuli, such as mechanical deformation or application of electrical pulses, can impact in vitro tissue culture. In this study, gelatin methacryloyl (GelMA) is modified with hydrophilic 2-acryloxyethyltrimethylammonium chloride (AETA) and 3-sulfopropyl acrylate potassium (SPA) ionic comonomers to obtain a piezoionic hydrogel. Rheology, mass swelling, gel fraction, and mechanical characteristics are determined. The piezoionic properties of the SPA and AETA-modified GelMA are confirmed by a significant increase in ionic conductivity and an electrical response as a function of mechanical stress. Murine myoblasts display a viability of >95% after 1 week on the piezoionic hydrogels, confirming their biocompatibility. The GelMA modifications do not influence the fusion capacity of the seeded myoblasts or myotube width after myotube formation. These results describe a novel functionalization providing new possibilities to exploit piezo-effects in the tissue engineering field.

Priming of the murine mammary gland with Staphylococcus chromogenes IM reduces bacterial growth of Streptococcus uberis: a proof-of-concept study.

Streptococcus uberis is a major causative agent of bovine mastitis, an inflammation of the mammary gland with substantial economic consequences. To reduce antibiotic use in animal agriculture, alternative strategies to treat or prevent mastitis are being investigated. Bovine-associated non-aureus staphylococci are proposed in that respect due to their capacity to inhibit the in vitro growth of S. uberis. We demonstrate that priming the murine mammary gland with Staphylococcus chromogenes IM reduces S. uberis growth in comparison with non-primed glands. The innate immune system is activated by increasing IL-8 and LCN2, which may explain this decreased growth.

Novel Quantitative Assay to Describe In Vitro Bovine Mastitis Bacterial Pathogen Inhibition by Non-aureus Staphylococci.

In this paper, we describe a new quantitative method to evaluate and quantify in vitro growth inhibition of mastitis-related bacteria. Colony-forming units of Staphylococcus (S.) aureus (n = 10), Escherichia (E.) coli (n = 10), and Streptococcus (S.) uberis (n = 10) were quantified after their growth on top of layers of trypticase soy agar (TSA) containing six different concentrations (varying from 102 to 107 CFU/mL) of bovine non-aureus staphylococci (NAS), i.e., S. chromogenes (n = 3) and S. simulans (n = 3) isolates. Growth inhibition of the mastitis-related major bacterial pathogens, including E. coli, was confirmed by all NAS, an effect that varied highly among NAS isolates and was not evident from the semiquantitative method with which the new method was compared. By subsequent application of the new method on a larger set of 14 bovine NAS isolates, we observed that S. simulans and NAS originating from teat apices (especially S. epidermidis) required lower concentrations to inhibit both methicillin-sensitive (MSSA) (n = 5) and methicillin-resistant S. aureus (MRSA) isolates (n = 5) originating from milk. Therefore, the new assay is a promising tool to precisely quantify the intra- and inter-species differences in growth inhibition between NAS.

Child-onset thrombotic thrombocytopenic purpura caused by p.R498C and p.G259PfsX133 mutations in ADAMTS13.

INTRODUCTION: Patients suffering from congenital thrombotic thrombocytopenic purpura (cTTP) have a deficiency in ADAMTS13 due to mutations in their ADAMTS13 gene. OBJECTIVE: The aim of this study was to determine ADAMTS13 parameters (activity, antigen, and mutations), to investigate if the propositus suffered from child-onset cTTP, and to study the in vitro effect of the ADAMTS13 mutations. METHODS: ADAMTS13 activity and antigen were determined using the FRETS VWF73 assay and ELISA and ADAMTS13 mutations via sequencing of the exons. Mutant proteins were expressed in Chinese hamster ovary cells, and their expression was studied using fluorescence microscopy and ELISA. Molecular modeling was used to evaluate the effect of the mutations on ADAMTS13 structure and stability. RESULTS: The propositus was diagnosed with cTTP at the age of 20. ADAMTS13 activity was below 10%, and 2 compound heterozygous mutations, the p.R498C point and the p.G259PfsX133 frameshift mutation, were identified. Expression of ADAMTS13 mutants revealed that the p.R498C and the p.G259PfsX133 mutation cause secretion and translation defects in vitro, respectively. Molecular modeling showed that the R498 intra-domain interactions are lacking in the p.R498C mutant, resulting in protein instability. CONCLUSION: The ADAMTS13 mutations result in a severe ADAMTS13 deficiency explaining the patient's phenotype.