Approaches for Mitigating Microbial Biofilm-Related Drug Resistance: A Focus on Micro- and Nanotechnologies

Biofilms play an essential role in chronic and healthcare-associated infections and are more resistant to antimicrobials compared to their planktonic counterparts due to their (1) physiological state, (2) cell density, (3) quorum sensing abilities, (4) presence of extracellular matrix, (5) upregulation of drug efflux pumps, (6) point mutation and overexpression of resistance genes, and (7) presence of persister cells. The genes involved and their implications in antimicrobial resistance are well defined for bacterial biofilms but are understudied in fungal biofilms. Potential therapeutics for biofilm mitigation that have been reported include (1) antimicrobial photodynamic therapy, (2) antimicrobial lock therapy, (3) antimicrobial peptides, (4) electrical methods, and (5) antimicrobial coatings. These approaches exhibit promising characteristics for addressing the impending crisis of antimicrobial resistance (AMR). Recently, advances in the micro- and nanotechnology field have propelled the development of novel biomaterials and approaches to combat biofilms either independently, in combination or as antimicrobial delivery systems. In this review, we will summarize the general principles of clinically important microbial biofilm formation with a focus on fungal biofilms. We will delve into the details of some novel micro- and nanotechnology approaches that have been developed to combat biofilms and the possibility of utilizing them in a clinical setting.     

Improved Optimum Radius for Robust Stability of Schur Polynomials

An approach based on the Rouché theorem was introduced in the literature to compute the optimum radius for robust stability of Schur polynomials. Later an attempt was made to improve the result, but it was shown to be incorrect. The purpose of this note is to show that an improved optimum radius still can be obtained by modifying the proposed method. The result of this note can be easily extended to the multidimensional cases.     

Morphological investigation of polylactide/microfibrillated cellulose composites

Optical microscopy and transmission electron microscopy have been used to investigate the morphology of polylactide (PLA)/microfibrillated cellulose (MFC) composites prepared by: compression molding of wet-comingled MFC and PLA latex or powder, twin-screw extrusion of the wet-comingled compounds, and solvent mixing of PLA with MFC or acetylated MFC. Compression molding of wet-comingled MFC and PLA latex or powder compounds resulted in a cellular MFC network, whereas solvent-cast films showed a more uniform dispersion of MFC fibers. Somewhat lower aggregate diameters observed in the acetylated MFC were assumed to be due to decreased MFC hydrophilicity and improved chemical affinity with the PLA matrix. The MFC networks in the commingled compounds were severely disrupted after twin-screw extrusion. This confirmed the limited deformability of the networks inferred from the extensive syneresis during the initial compression molding step, and accounted for substantial losses in stiffness reinforcement by the MFC after extrusion.     

Large area impingement spray cooling from multiple normal and inclined spray nozzles

An inclined spray chamber with four multiple nozzles to cool a 1 kW 6U electronic test card has been designed and tested in this study. The multiple inclined sprays can cover the same heated surface area as that with the multiple normal sprays but halve the volume of the spray chamber. The spray cooling system used R134a as a working fluid in a modified refrigeration cycle. It is observed that increasing mass flow rate and pressure drop across the nozzles improved the heat transfer coefficient with a maximum enhancement of 117 %, and reduced the maximum temperature difference at the heated surface from 13.8 to 8.4 °C in the inclined spray chamber with a heat flux of 5.25 W/cm², while the heat transfer coefficient of the normal spray increased with a maximum enhancement of 215 % and the maximum temperature difference decreased from 10.8 to 5.4 °C under similar operating conditions. We conclude that the multiple inclined sprays could produce a higher heat transfer coefficient but with an increase in non-uniformity of the surface temperature compared with the multiple normal sprays.     

A novel approach to determine the molecular overlap of polyelectrolyte using an ultrafiltration membrane

The overlapping of charged polymers (e.g., polyelectrolyte) in the liquid phase is one of the key parameters affecting the processing of the macromolecular solutions. This work focused on the development of a novel method for determining the overlap concentration of polyelectrolytes with stirred cell ultrafiltration (UF) techniques. A new, simplified equation that incorporated the resistance-in-series UF model into the osmosis phenomenon caused by polymers was developed, and then applied to estimate the threshold concentration for macromolecular overlap. The overlap concentrations of model polymers, such as poly(dimethylamine-co-epichlorohydrin-co-ethylenediamine) and poly(diallyldimethylammonium chloride), were evaluated with different, initial polymer concentrations and membrane pore sizes. In the correlation between ln R_t/C_r and ln C_r, the concentration where ln R_t/C_r had the minimal value was referred to as the overlap concentration. The gyration radius of polyelectrolytes was calculated using the overlap concentration, which can provide insight into the understanding of the size of polymers and their rejections by membranes. Determining the overlap concentration of polymers with a UF membrane appeared to be viable and practical.     

Engineering of a two-step purification strategy for a panel of monoclonal immunoglobulin M directed against undifferentiated human embryonic stem cells

A two-step purification strategy comprising of polyethylene glycol (PEG) precipitation and anion-exchange chromatography was developed for a panel of monoclonal immunoglobulin M (IgM) (pI 5.5–7.7) produced from hybridoma cultures. PEG precipitation was optimized with regards to concentration, pH and mixing. For anion-exchange chromatography, different resins were screened of which Fractogel EMD, a polymer grafted porous resin had the highest capacity. Despite its significantly slower mass transfer, the binding capacity was still higher compared to a convection driven resin (monolith). This purification strategy was successfully demonstrated for all 9 IgMs in the panel. In small scale most antibodies could be purified to >95% purity with the exception of two which gave a lower final purity (46% and 85%). The yield was dependent on the different antibodies ranging from 28% to 84%. Further improvement of recovery and purity was obtained by the digestion of DNA present in the hybridoma supernatant using an endonuclease, benzonase. So far this strategy has been applied for the purification of up to 2 l hybridoma supernatants.     

Quantitative analysis of human serum leptin using a nanoarray protein chip based on single-molecule sandwich immunoassay

We report a method for the quantitative analysis of human serum leptin, which is a protein hormone associated with obesity, using a nanoarray protein chip based on a single-molecule sandwich immunoassay. The nanoarray patterning of a biotin-probe with a spot diameter of 150 nm on a self-assembled monolayer functionalized by MPTMS on a glass substrate was successfully accomplished using atomic force microscopy (AFM)-based dip-pen nanolithography (DPN). Unlabeled leptin protein molecules in human serum were detected based on the sandwich fluorescence immunoassay by total internal reflection fluorescence microscopy (TIRFM). The linear regression equation for leptin in the range of 100 zM-400 aM was determined to be y = 456.35x + 80,382 (R = 0.9901). The accuracy and sensitivity of the chip assay were clinically validated by comparing the leptin level in adult serum obtained by this method with those measured using the enzyme-linked immunosorbent assay (ELISA) performed with the same leptin standards and serum samples. In contrast to conventional ELISA techniques, the proposed chip methodology exhibited the advantages of ultra-sensitivity, a smaller sample volume and faster analysis time.     

Two-dimensional fluorescence difference gel electrophoresis for comparison of affinity and non-affinity based downstream processing of recombinant monoclonal antibody

Although Staphylococcus Protein A (SpA) affinity chromatography is the state of the art capture step for antibody purification, non-affinity methods are more economical. We used two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) to evaluate the purification of a recombinant IgG₁ antibody from cultured cells, with two different processes: (1) SpA capture followed by cation-exchange chromatography (CEX); and (2) CEX capture, followed by anion exchanger, then hydrophobic interaction chromatography. Efficiencies were similar in sodium dodecylsulphate polyacrylamide gel electrophoresis and size-exclusion chromatography; however, 2-D DIGE revealed higher efficiency with SpA than with CEX capture. Thus, 2-D DIGE is a valuable tool for downstream process development.