Microscale Assays Research Articles

Antioxidants Derived from Natural Products Reduce Radiative Damage in Cultured Retinal Glia to Prevent Oxidative Stress

Retinal pathologies have been heavily studied in response to radiation and microgravity, including spaceflight-associated neuro-ocular syndrome (SANS), which is commonly developed in space flight. SANS has been characterized in clinical studies of astronauts returning to Earth and includes a range of symptoms, such as globe flattening, optic-disc edema, retinal folds, and retinal ischemia. In cases of retinal insult, Müller glia (MG) cells respond via neuroprotective gliotic responses that may become destructive to produce glial scarring and vison loss over time. Retinal pathology is further impacted by the production of excessive reactive oxygen species (ROS) that stimulate retinal inflammation and furthers the gliosis of MG. Neuroprotectants derived from natural products (NPs) able to scavenge excess ROS and mitigate long-term, gliotic responses have garnered recent interest, especially among mature and aging adults. The natural antioxidants aloin and ginkgolide A flavonoids, derived from Aloe vera and Ginkgo biloba species, respectively, have been of particular interest due to their recent use in other nervous-system studies. The current study examined MG behaviors in response to different doses of aloin and ginkgolide A over time by measuring changes in morphology, survival, and ROS production within microscale assays. The study was further enhanced by using galactic cosmic rays (GCR) at the Brookhaven NASA Space Radiation Laboratory to simulate ionizing radiation in low- and high-radiation parameters. Changes in the survival and ROS production of radiation-treated MG were then measured in response to varying dosage of NPs. Our study used in vitro systems to evaluate the potential of NPs to reduce oxidative stress in the retina, highlighting the underexplored interplay between NP antioxidants and MG endogenous responses both in space and terrestrially.

Surveillance utilizes multi-omics in cardiovascular disease: Diet and its potentiality in Preventive index

Cardiovascular Disease (CVD) is characterized by multidimensional risks including drug, diet, lifestyle, stress, and metabolomics diseases which cause mortality and morbidity depending on age and status of chronic diseases. However, emerging evidence indicated it is preventable health complications that depend on risk management along with lifestyle change, and personalized medication that include alternative measures like Diet use following molecular diagnostic and imaging analysis. CVD is mainly attributed to the narrowing of blood vessels through atherosclerotic lesions and/or thrombosis. Hypertension, obesity, and hyperlipidemia are major risk factors for the development of CVD and treating these diseases is essential in slowing down progression of CVD. Inflammation appears to play a pivotal role in CVD and can be measured through a simple blood assay (CRP). Multi-omics approaches have been essential in the development of treatments for CVD, in the prevention of CVD, and in the diagnosis of CVD. There are many outcomes available to help with diagnosing CVD and omics platforms have helped scientists and clinician develop these diagnostic tools. Radiomics has played a key part in the diagnosis of CVD as being able to view the diseased heart is essential in determining CVD progression and the treatment options suitable for that secondary disease related. Nutrigenomics is emerging as the future of medicine such as utilizing treatment strategy innovation instead of medications, but it is still in its infancy. Nutrigenomics will open the doors to different therapeutic drug targets and allow us the ability to be more specific in our treatment options. There are only a few gene-diet interactions documented that increase a person’s chances of developing CVD. Curating an individual diet and treatment plan based on somebody’s genetic disposition or skewed immune responses following personalized diagnosis will be essential in the survival of these severe CVD patients. Key issues referring to risk surveillance and prevention is a distant approach which reflects several factors: for example, what type of tools can be used to conduct diagnosis, molecular diagnostic tools detect what type of biomarkers are present prior to prescribing the personalized diet and to ensure diagnostic accuracy. Recently, increasing findings emphasize dual aspects of diet such as immune enhancers and modulators in which gut microbiota has been proven to play a major factor in development of CVD. The future direction of omics studies will foster the ability to test the impact of gut microbiome of a patient with CVD following diet driven organ protection as well as prescribe essential components of the diet that can be adjusted with proper probiotic medication. Proper diet adjustments can correct the organ dysfunction that occurred due to interaction between molecular mismatch and cellular damage following stress-mediated damage or chronic disease. Further micro-scale assays and molecular diagnostic techniques following nutrigenomics application to the patient could be beneficial to allow patient’ care shift from physician driven and clinic based to self-management with knowledge based at home treatment programs that work by envisioning molecular reprogramming and rejuvenation of damaged organ. These at home treatments can be utilized with development of radiological data with innovation of software. The aim of the short review is to visualize the current role of nutrigenomics and diet formulation for integrative care (e.g., diagnosis, prevention, and treatment of CVD) which would take advantage of earlier prevention synchronized with current medical tests, imaging techniques. Health economy like management can reduce medical cost with disease prevention disease and could modulate the following: enhance knowledge-based interaction between body and diet, discuss cognitive enhancement how sensing with molecular behavior under image-management platform, monitor drug surveillance of current treatment options in CVD and the pitfalls of current omics application and data transformation needs for patient care in the future.

Microfluidic and Microscale Assays to Examine Regenerative Strategies in the Neuro Retina.

Bioengineering systems have transformed scientific knowledge of cellular behaviors in the nervous system (NS) and pioneered innovative, regenerative therapies to treat adult neural disorders. Microscale systems with characteristic lengths of single to hundreds of microns have examined the development and specialized behaviors of numerous neuromuscular and neurosensory components of the NS. The visual system is comprised of the eye sensory organ and its connecting pathways to the visual cortex. Significant vision loss arises from dysfunction in the retina, the photosensitive tissue at the eye posterior that achieves phototransduction of light to form images in the brain. Retinal regenerative medicine has embraced microfluidic technologies to manipulate stem-like cells for transplantation therapies, where de/differentiated cells are introduced within adult tissue to replace dysfunctional or damaged neurons. Microfluidic systems coupled with stem cell biology and biomaterials have produced exciting advances to restore vision. The current article reviews contemporary microfluidic technologies and microfluidics-enhanced bioassays, developed to interrogate cellular responses to adult retinal cues. The focus is on applications of microfluidics and microscale assays within mammalian sensory retina, or neuro retina, comprised of five types of retinal neurons (photoreceptors, horizontal, bipolar, amacrine, retinal ganglion) and one neuroglia (Müller), but excludes the non-sensory, retinal pigmented epithelium.

Comprehensive validation of T- and B-cell deficiency in rag1-null zebrafish: Implication for the robust innate defense mechanisms of teleosts

rag1−/− zebrafish have been employed in immunological research as a useful immunodeficient vertebrate model, but with only fragmentary evidence for the lack of functional adaptive immunity. rag1-null zebrafish exhibit differences from their human and murine counterparts in that they can be maintained without any specific pathogen-free conditions. To define the immunodeficient status of rag1−/− zebrafish, we obtained further functional evidence on T- and B-cell deficiency in the fish at the protein, cellular, and organism levels. Our developed microscale assays provided evidence that rag1−/− fish do not possess serum IgM protein, that they do not achieve specific protection even after vaccination, and that they cannot induce antigen-specific CTL activity. The mortality rate in non-vaccinated fish suggests that rag1−/− fish possess innate protection equivalent to that of rag1+/− fish. Furthermore, poly(I:C)-induced immune responses revealed that the organ that controls anti-viral immunity is shifted from the spleen to the hepatopancreas due to the absence of T- and B-cell function, implying that immune homeostasis may change to an underside mode in rag-null fish. These findings suggest that the teleost relies heavily on innate immunity. Thus, this model could better highlight innate immunity in animals that lack adaptive immunity than mouse models.

An Automated Miniaturized Method to Perform and Analyze Antimicrobial Drug Synergy Assays.

In the light of emerging antibiotic resistance mechanisms found in bacteria throughout the world, discovery of drugs that potentiate the effect of currently available antibiotics remains an important aspect of pharmaceutical research in the 21st century. Well-established clinical tests exist to determine synergy in vitro, but these are only optimal for low-throughput experimentation while leaving analysis of results and interpretation of high-throughput microscale assays poorly standardized. Here, we describe a miniaturized broth microdilution checkerboard assay and data analysis method in 384-well plate format that conforms to the Clinical Laboratory and Standards Institute (CLSI) methods. This method has been automated and developed to rapidly determine the synergism of current antibiotics with various beta-lactamase inhibitors emerging from our antimicrobial research efforts. This technique increases test throughput and integrity of results, and saves test compound and labor. We facilitated the interpretation of results with an automated analysis tool allowing us to rapidly qualify inter- and intraplate robustness, determine efficacy of multiple antibiotics at the same time, and standardize the results of synergy interpretation. This procedure should enhance high-throughput antimicrobial drug discovery and supersedes former techniques.

Managing evaporation for more robust microscale assays. Part 1. Volume loss in high throughput assays.

Evaporation is a well known issue when handling small liquid volumes. Here we present a review of microscale assays prone to evaporation and methods to make them more robust. Applications for these assays span from combinatorial chemistry to cell-biology where the stability of concentrations and osmolarity can be critical. A dimensionless evaporation number Ev is presented and used to characterize volume loss in short term and long term microscale assays. Ev can be used both as a design tool and as an analysis parameter. The advantageous use of evaporation in some applications is also discussed.

Managing evaporation for more robust microscale assays. Part 2. Characterization of convection and diffusion for cell biology.

Cell based microassays allow the screening of a multitude of culture conditions in parallel, which can be used for various applications from drug screening to fundamental cell biology research. Tubeless microfluidic devices based on passive pumping are a step towards accessible high throughput microassays, however they are vulnerable to evaporation. In addition to volume loss, evaporation can lead to the generation of small flows. Here, we focus on issues of convection and diffusion for cell culture in microchannels and particularly the transport of soluble factors secreted by cells. We find that even for humidity levels as high as 95%, convection in a passive pumping channel can significantly alter distributions of these factors and that appropriate system design can prevent convection.

Microassays for a set of enzymes in individual small marine copepods

Fluorogenic assays for a set of five hydrolytic enzymes involved in digestion and food utilization (alanine and arginine aminopeptidase, lipase/esterase, chitobiase, and beta-glucosidase) were optimized to measure activities of these enzymes in the same extracts of individual small North Sea copepods. The enzyme activities of Acartia clausi, Centropages typicus, Corycaeus anglicus, Paracalanus parvus, and Temora longicornis showed distinct species specific activity patterns, but also high intra-specific variability. Protein, lipids, carbon and nitrogen (C, N) were determined with micro-scale assays in individual copepods or in batches of 10 to 50 animals. Water soluble protein contents ranged from 16% to 38%, and lipid contents from 2.4% to 5.5% of dry mass. The molar C/N ratios were between 4.1 and 4.5. The presented microassays provide suitable tools for studying physiological reactions of copepods and other small pelagic crustaceans in response to variable environmental conditions.

A Rapid Method for Determining the Antimicrobial Activity of Novel Natural Molecules

Preservatives used in the Agro-food industries may be of natural origin or obtained chemically. Because of the increasing interest of consumers in food products that contain only natural ingredients, studies on preservative molecules of natural origin, such as organic acids or peptides, have been reported in the past several years. Such studies, which require numerous assays, may be limited by the large amount of molecules required. Microscale assays provide an opportunity for testing natural components available in low quantity. This study examined a rapid method that used microplates for the evaluation of antimicrobial substances. The method was validated using five foodborne pathogens. It required a low amount of product and was convenient for the determination of correlations between the bacterial growth inhibition and concentration of the antimicrobial substance.

Expression of functional recombinant mussel adhesive protein Mgfp-5 in Escherichia coli.

Mussel adhesive proteins have been suggested as a basis for environmentally friendly adhesives for use in aqueous conditions and in medicine. However, attempts to produce functional and economical recombinant mussel adhesive proteins (mainly foot protein type 1) in several systems have failed. Here, the cDNA coding for Mytilus galloprovincialis foot protein type 5 (Mgfp-5) was isolated for the first time. Using this cDNA, we produced a recombinant Mgfp-5 fused with a hexahistidine affinity ligand, which was expressed in a soluble form in Escherichia coli and was highly purified using affinity chromatography. The adhesive properties of purified recombinant Mgfp-5 were compared with the commercial extracted mussel adhesive Cell-Tak by investigating adhesion force using atomic force microscopy, material surface coating, and quartz crystal microbalance. Even though further macroscale assays are needed, these microscale assays showed that recombinant Mgfp-5 has significant adhesive ability and may be useful as a bioadhesive in medical or underwater environments.

Investigating the Adequacy of Selected Micro-Scale Bioassays to Predict the Toxic Potential of Freshwater Sediments through a Tier Process

Abstract A battery of 20 bioassays was applied to assess test and endpoint performance in detecting the toxic potential of 15 freshwater sediment samples collected in the St. Lawrence River/Great Lakes systems. The bioassays included 18 micro-scale assays performed either on whole sediment, pore water or sediment organic extracts, as well as two standardized whole sediment bioassays undertaken with macroinvertebrates (Chironomus riparius and Hyalella azteca). Physical and chemical parameters of selected sediments were also analyzed to aid in the interpretation of results. Several qualitative and quantitative criteria were established for evaluating the reliability and usefulness of the micro-scale bioassays in predicting sediment toxicity based on a two-tier system. In Tier 1, micro-scale testing results were compared for general concordance with those of the benthic bioassays, benthic community structure index and with sediment contaminant levels. Micro-assays which complied with Tier 1 standards were further evaluated via a Tier 2 process consisting of selected (quantitative and qualitative) scientific and practical criteria. Of the 20 bioassays investigated, the two benthic assays and seven micro-scale assays successfully passed the double-tiered assessment. Micro-scale tests whose results most closely matched those of the benthic tests for appraisal of whole sediment toxicity, sediment contamination level and benthic community structure index included three bacterial assays (ATP assay, a micro-plate adaptation of the Microtox® assay, SOS Chromotest™), one algal assay (algal solid-phase test) and three micro-invertebrate assays (Thamnotoxkit™ F, Daphtoxkit™ F and Hydra assay). Two selected batteries, composed of the seven micro-scale assays and benthic invertebrate bioassays, are proposed for cost-effective appraisal of freshwater sediment toxicity.

Comparisons between microscale and whole-sediment assays for freshwater sediment toxicity assessment

The use of several microscale assays for evaluating freshwater sediment toxicity was investigated to develop a representative and cost-effective test battery. The bioassays evaluated (18 assays total) included microscale assays performed on solid-phase, pore water, and organic extracts as well as conventional standardized whole-sediment assays with Chironomus riparius and Hyalella azteca on 10 sediment samples collected in the St. Lawrence/Great Lakes system. Selected sediment physical and chemical characteristics were also conducted to aid in the interpretation of results. Benthic invertebrate assays such as C. riparius and H. azteca endpoints were found to correlate well to contaminant levels. Microscale assays such as ATP (adenosine triphosphate) measurement of sediment microbial biomass gave promising results to predict whole-sediment toxicity to benthic invertebrates. Most assays on pore water did not adequately represent whole-sediment toxicity, whereas organic extracts appeared to be more representative, alleviating some potential confounding factors such as interference due to toxicity caused by ammonia.

Analysis of the carbohydrate-binding specificity of lectin-conjugated lipid vesicles which interact with polysaccharide fragments

In order to obtain some insight into understanding of the multivalent interactions between polysaccharide fragments as ligands and lectins conjugated on the membrane surface of lipid vesicles as receptors, two kinds of lectin-conjugated lipid vesicles were prepared, and studies on agglutination and binding were performed with two kinds of microscale assays by applying these to two model systems consisting of different ligands and receptors. The agglutination studies based on the interaction between plant galactomannan fragments and Ricinus communis agglutinin-conjugated lipid vesicles indicated three possible cases of interaction; strong agglutination was caused by large-size fragments with high side chain density, moderate agglutination by medium-size fragments with low side chain density and no agglutination by small-size fragments. The binding studies based on the interaction between yeast mannan fragments and concanavalin A lectin-conjugated lipid vesicles, together with glycosyl-linkage composition analyses, suggested that binding efficiency should be correlated with the structural characteristics of ligands such as size of fragments, rate of branching, and number of high-affinity residues.