Protein Adsorption on Solid Surfaces: Data Mining, Database, Molecular Surface-Derived Properties, and Semiempirical Relationships.

Protein adsorption on solid surfaces is a process relevant to biological, medical, industrial, and environmental applications. Despite this wide interest and advancement in measurement techniques, the complexity of protein adsorption has frustrated its accurate prediction. To address this challenge, here, data regarding protein adsorption reported in the last four decades was collected, checked for completeness and correctness, organized, and archived in an upgraded, freely accessible Biomolecular Adsorption Database, which is equivalent to a large-scale, ad hoc, crowd-sourced multifactorial experiment. The shape and physicochemical properties of the proteins present in the database were quantified on their molecular surfaces using an in-house program (ProMS) operating as an add-on to the PyMol software. Machine learning-based analysis indicated that protein adsorption on hydrophobic and hydrophilic surfaces is modulated by different sets of operational, structural, and molecular surface-based physicochemical parameters. Separately, the adsorption data regarding four "benchmark" proteins, i.e., lysozyme, albumin, IgG, and fibrinogen, was processed by piecewise linear regression with the protein monolayer acting as breakpoint, using the linearization of the Langmuir isotherm formalism, resulting in semiempirical relationships predicting protein adsorption. These relationships, derived separately for hydrophilic and hydrophobic surfaces, described well the protein concentration on the surface as a function of the protein concentration in solution, adsorbing surface contact angle, ionic strength, pH, and temperature of the carrying fluid, and the difference between pH and the isoelectric point of the protein. When applying the semiempirical relationships derived for benchmark proteins to two other "test" proteins with known PDB structure, i.e., ß-lactoglobulin and α-lactalbumin, the errors of this extrapolation were found to be in a linear relationship with the dissimilarity between the benchmark and the test proteins. The work presented here can be used for the estimation of operational parameters modulating protein adsorption for various applications such as diagnostic devices, pharmaceuticals, biomaterials, or the food industry.

Biosensing using antibody-modulated motility of actin filaments on myosin-coated surfaces.

Motor proteins, such as myosin and kinesin, are biological molecular motors involved in force generation and intracellular transport within living cells. The characteristics of molecular motors, i.e., their motility over long distances, their capacity of transporting cargoes, and their very efficient energy consumption, recommend them as potential operational elements of a new class of dynamic nano-devices, with potential applications in biosensing, analyte concentrators, and biocomputation. A possible design of a biosensor based on protein molecular motor comprises a surface with immobilized motors propelling cytoskeletal filaments, which are decorated with antibodies, presented as side-branches. Upon biomolecular recognition of these branches by secondary antibodies, the 'extensions' on the cytoskeletal filaments can achieve considerable lengths (longer than several diameters of the cytoskeletal filament carrier), thus geometrically impairing or halting motility. Because the filaments are several micrometers long, this sensing mechanism converts an event in the nanometer range, i.e., antibody-antigen sizes, into an event in the micrometer range: the visualization of the halting of motility of microns-long cytoskeletal filaments. Here we demonstrate the proof of concept of a sensing system comprising heavy-mero-myosin immobilized on surfaces propelling actin filaments decorated with actin antibodies, whose movement is halted upon the recognition with secondary anti-actin antibodies. Because antibodies to the actin-myosin system are involved in several rare diseases, the first possible application for such a device may be their prognosis and diagnosis. The results also provide insights into guidelines for designing highly sensitive and very fast biosensors powered by motor proteins.

Hydrophobic Recovery of PDMS Surfaces in Contact with Hydrophilic Entities: Relevance to Biomedical Devices.

Polydimethylsiloxane (PDMS), a silicone elastomer, is increasingly being used in health and biomedical fields due to its excellent optical and mechanical properties. Its biocompatibility and resistance to biodegradation led to various applications (e.g., lung on a chip replicating blood flow, medical interventions, and diagnostics). The many advantages of PDMS are, however, partially offset by its inherent hydrophobicity, which makes it unsuitable for applications needing wetting, thus requiring the hydrophilization of its surface by exposure to UV or O2 plasma. Yet, the elastomeric state of PDMS translates in a slow, hours to days, process of reducing its surface hydrophilicity-a process denominated as hydrophobic recovery. Using Fourier transform infrared spectroscopy (FTIR) and atomic force microscopy (AFM), the present study details the dynamics of hydrophobic recovery of PDMS, on flat bare surfaces and on surfaces embedded with hydrophilic beads. It was found that a thin, stiff, hydrophilic, silica film formed on top of the PDMS material, following its hydrophilization by UV radiation. The hydrophobic recovery of bare PDMS material is the result of an overlap of various nano-mechanical, and diffusional processes, each with its own dynamics rate, which were analyzed in parallel. The hydrophobic recovery presents a hysteresis, with surface hydrophobicity recovering only partially due to a thin, but resilient top silica layer. The monitoring of hydrophobic recovery of PDMS embedded with hydrophilic beads revealed that this is delayed, and then totally stalled in the few-micrometer vicinity of the embedded hydrophilic beads. This region where the hydrophobic recovery stalls can be used as a good approximation of the depth of the resilient, moderately hydrophilic top layer on the PDMS material. The complex processes of hydrophilization and subsequent hydrophobic recovery impact the design, fabrication, and operation of PDMS materials and devices used for diagnostics and medical procedures. Consequently, especially considering the emergence of new surgical procedures using elastomers, the impact of hydrophobic recovery on the surface of PDMS warrants more comprehensive studies.

Effect of physicochemical parameters on the stability and activity of garlic alliinase and its use for in-situ allicin synthesis.

Garlic is a well-known example of natural self-defence system consisting of an inactive substrate (alliin) and enzyme (alliinase) which, when combined, produce highly antimicrobial allicin. Increase of alliinase stability and its activity are of paramount importance in various applications relying on its use for in-situ synthesis of allicin or its analogues, e.g., pulmonary drug delivery, treatment of superficial injuries, or urease inhibitors in fertilizers. Here, we discuss the effect of temperature, pH, buffers, salts, and additives, i.e. antioxidants, chelating agents, reducing agents and cosolvents, on the stability and the activity of alliinase extracted from garlic. The effects of the storage temperature and relative humidity on the stability of lyophilized alliinase was demonstrated. A combination of the short half-life, high reactivity and non-specificity to particular proteins are reasons most bacteria cannot deal with allicin's mode of action and develop effective defence mechanism, which could be the key to sustainable drug design addressing serious problems with escalating emergence of multidrug-resistant (MDR) bacterial strains.

Lensless, reflection-based dark-field microscopy (RDFM) on a CMOS chip.

We present for the first time a lens-free, oblique illumination imaging platform for on-sensor dark- field microscopy and shadow-based 3D object measurements. It consists of an LED point source that illuminates a 5-megapixel, 1.4 µm pixel size, back-illuminated CMOS sensor at angles between 0° and 90°. Analytes (polystyrene beads, microorganisms, and cells) were placed and imaged directly onto the sensor. The spatial resolution of this imaging system is limited by the pixel size (∼1.4 µm) over the whole area of the sensor (3.6×2.73 mm). We demonstrated two imaging modalities: (i) shadow imaging for estimation of 3D object dimensions (on polystyrene beads and microorganisms) when the illumination angle is between 0° and 85°, and (ii) dark-field imaging, at >85° illumination angles. In dark-field mode, a 3-4 times drop in background intensity and contrast reversal similar to traditional dark-field imaging was observed, due to larger reflection intensities at those angles. With this modality, we were able to detect and analyze morphological features of bacteria and single-celled algae clusters.

Review on Stress Tolerance in Campylobacter jejuni.

Campylobacter spp. are the leading global cause of bacterial colon infections in humans. Enteropathogens are subjected to several stress conditions in the host colon, food complexes, and the environment. Species of the genus Campylobacter, in collective interactions with certain enteropathogens, can manage and survive such stress conditions. The stress-adaptation mechanisms of Campylobacter spp. diverge from other enteropathogenic bacteria, such as Escherichia coli, Salmonella enterica serovar Typhi, S. enterica ser. Paratyphi, S. enterica ser. Typhimurium, and species of the genera Klebsiella and Shigella. This review summarizes the different mechanisms of various stress-adaptive factors on the basis of species diversity in Campylobacter, including their response to various stress conditions that enhance their ability to survive on different types of food and in adverse environmental conditions. Understanding how these stress adaptation mechanisms in Campylobacter, and other enteric bacteria, are used to overcome various challenging environments facilitates the fight against resistance mechanisms in Campylobacter spp., and aids the development of novel therapeutics to control Campylobacter in both veterinary and human populations.

Single-molecule analysis reveals the mechanism of transcription activation in M. tuberculosis.

The σ subunit of bacterial RNA polymerase (RNAP) controls recognition of the -10 and -35 promoter elements during transcription initiation. Free σ adopts a "closed," or inactive, conformation incompatible with promoter binding. The conventional two-state model of σ activation proposes that binding to core RNAP induces formation of an "open," active, σ conformation, which is optimal for promoter recognition. Using single-molecule Förster resonance energy transfer, we demonstrate that vegetative-type σ subunits exist in open and closed states even after binding to the RNAP core. As an extreme case, RNAP from Mycobacterium tuberculosis preferentially retains σ in the closed conformation, which is converted to the open conformation only upon binding by the activator protein RbpA and interaction with promoter DNA. These findings reveal that the conformational dynamics of the σ subunit in the RNAP holoenzyme is a target for regulation by transcription factors and plays a critical role in promoter recognition.

Mycobacterium RbpA cooperates with the stress-response σB subunit of RNA polymerase in promoter DNA unwinding.

RbpA, a transcriptional activator that is essential for Mycobacterium tuberculosis replication and survival during antibiotic treatment, binds to RNA polymerase (RNAP) in the absence of promoter DNA. It has been hypothesized that RbpA stimulates housekeeping gene expression by promoting assembly of the σ(A) subunit with core RNAP. Here, using a purified in vitro transcription system of M. tuberculosis, we show that RbpA functions in a promoter-dependent manner as a companion of RNAP essential for promoter DNA unwinding and formation of the catalytically active open promoter complex (RPo). Screening for RbpA activity using a full panel of the M. tuberculosis σ subunits demonstrated that RbpA targets σ(A) and stress-response σ(B), but not the alternative σ subunits from the groups 3 and 4. In contrast to σ(A), the σ(B) subunit activity displayed stringent dependency upon RbpA. These results suggest that RbpA-dependent control of RPo formation provides a mechanism for tuning gene expression during the switch between different physiological states, and in the stress response.