We report an automated microfluidic platform for ‘digitally’ screening the composition space of droplets containing cocktails of small molecules and demonstrate the features of this system by studying epistatic interactions between antibiotics and Escherichia coli ATCC 25922. This system has several key characteristics: (i) it uses small (<100 μL) samples of liquids and suspensions of bacteria that are introduced directly into the chip; (ii) it generates a sequence of droplets with compositions, including reagents and bacterial cell suspensions that are programmed by the user; (iii) it exports the sequence of droplets to an external segment of tubing that is subsequently disconnected for incubation and storage; and (iv) after incubation of bacteria in droplets, the droplets are injected into a second device equipped with an in-line fiber optic spectrophotometer that measures cell growth. The system generates and fuses droplets with precise (<1% in standard deviation) control of liquid volumes and of the concentrations of input substrates. We demonstrate the application of this technology in determining the minimum inhibitory concentration and pair-wise interactions of ampicillin, tetracycline, and chloramphenicol against E. coli. The experiments consumed small volumes of reagents and required minutes to create the droplets and several hours for their incubation and analysis.

Objectives: The antibacterial activity of antimicrobial peptides is influenced by various factors such as salt content, pH and the presence of proteins. In this study, we explored the antibacterial action of the human cathelicidin LL-37 in physiologically relevant conditions, i.e. various human wound fluids, human plasma fractions and serum. Methods: Radial diffusion assays using Staphylococcus aureus and Escherichia coli were employed for the study of antibacterial effects of LL-37 in the presence of 12 different wound fluids, citrate-, heparin- or EDTA-plasma, or human serum. Glycosaminoglycan content of wound fluids was determined by an Alcian Blue-binding assay. Protein content of wound fluids was measured by the Bradford method. A slot-binding assay was used to study the effects of inhibitors on the interaction between LL-37 and glycosaminoglycans. Results: Five of twelve wound fluids derived from acute wounds showed marked inhibitory effects on the antibacterial action of LL-37. The inhibition was significantly correlated with high glycosaminoglycan content in wound fluid. Analogous to these findings, heparin-plasma strongly inhibited the antibacterial effect of LL-37. The interaction between LL-37 and glycosaminoglycans was abrogated by the cationic polymers DEAE-dextran and chitosan, yielding increased activity of LL-37. Conclusions: Glycosaminoglycan-rich biological fluids inhibit the antibacterial effects of LL-37. Furthermore, polycations that bind to glycosaminoglycans increase the antibacterial activities of endogenous antimicrobial peptides in glycosaminoglycan-containing biological fluids.

An increasing number of multidrug-resistant pathogens is a serious problem of modern medicine and new antibiotics are highly demanded. In this study, different n-alkyl acids (C₂-C₁₄) and aromatic acids (benzoic and trans-cinnamic) were conjugated to the N-terminus of KR12 amide. The effect of this modification on antimicrobial activity (ESKAPE bacteria and biofilm of Staphylococcus aureus) and cytotoxicity (human red blood cells and HaCaT cell line) was examined. The effect of lipophilic modifications on helicity was studied by CD spectroscopy, whereas peptide self-assembly was studied by surface tension measurements and NMR spectroscopy. As shown, conjugation of the KR12-NH₂ peptide with C₄-C₁₄ fatty acid chains enhanced the antimicrobial activity with an optimum demonstrated by C₈-KR12-NH₂ (MIC 1–4 μg/mL against ESKAPE strains; MBEC of S. aureus 4–16 μg/mL). Correlation between antimicrobial activity and self-assembly behavior of C₁₄-KR12-NH₂ and C₈-KR12-NH₂ has shown that the former self-assembled into larger aggregated structures, which reduced its antimicrobial activity. In conclusion, N-terminal modification can enhance antimicrobial activity of KR12-NH₂; however, at the same time, the cytotoxicity increases. It seems that the selectivity against pathogens over human cells can be achieved through conjugation of peptide N-terminus with appropriate n-alkyl fatty and aromatic acids.

Hyaluronic acid (HA), as one of the main components of the extracellular matrix (ECM), plays an important role in the process of wound-healing and tissue-repair processes due to its unique properties and different physiological functions. HA has an ability to maintain a moist environment that promotes healing, the stimulation of growth factors and cellular constituents, and the migration of various cells essential for healing. This paper offers a review of HA use in the process of wound healing, with emphasis on hard-to-heal wounds, and examines its various applications in ophthalmology and otorhinolaryngology. It proves HA to be a versatile agent which finds its use in various fields of medicine for its antioxidant, anti-inflammatory, antibacterial properties and accelerated wound healing.

Background: Neonatal lupus erythematosus is an autoimmune disease acquired during fetal life as a result of transplacental passage of maternal anti-Sjögren’s-syndrome-related antigen A (anti-SSA/Ro), anti-Sjögren’s-syndrome-related antigen B (anti-SSB/La) or anti-U1 ribonucleoprotein (anti-U1-RNP) antinuclear autoantibodies. Contents: Clinical manifestations include skin lesions, congenital heart block, hepatobiliary involvement and cytopenias. Most of the disorders disappear spontaneously after clearance of maternal antibodies. Cardiac symptoms, however, are not self-resolving and often pacemaker implantation is required. Diagnosis is based on clinical presentation and the presence of typical antibodies in the mother’s or infant’s serum. Outlook: Neonatal lupus erythematosus may develop in children born to anti-SSA/Ro or anti-SSB/La women with various systemic connective tissue diseases. However, in half of the cases, the mother is asymptomatic, which may delay the diagnosis and have negative impact on the child’s prognosis. Testing for antinuclear antibodies should be considered in every pregnant woman since early treatment with hydroxychloroquine or intravenous immunoglobulin (IVIG) has proven to be effective in preventing congenital heart block.