There are many proteomic applications that require large collections of purified protein, but parallel production of large numbers of different proteins remains a very challenging task. To help meet the needs of the scientific community, we have developed a human protein production pipeline. Using high-throughput (HT) methods, we transferred the genes of 31 full-length proteins into three expression vectors, and expressed the collection as N-terminal HaloTag fusion proteins in Escherichia coli and two commercial cell-free (CF) systems, wheat germ extract (WGE) and HeLa cell extract (HCE). Expression was assessed by labeling the fusion proteins specifically and covalently with a fluorescent HaloTag ligand and detecting its fluorescence on a LabChip[superscript ®] GX microfluidic capillary gel electrophoresis instrument. This automated, HT assay provided both qualitative and quantitative assessment of recombinant protein. E. coli was only capable of expressing 20% of the test collection in the supernatant fraction with ≥20 μg yields, whereas CF systems had ≥83% success rates. We purified expressed proteins using an automated HaloTag purification method. We purified 20, 33, and 42% of the test collection from E. coli, WGE, and HCE, respectively, with yields ≥1 μg and ≥90% purity. Based on these observations, we have developed a triage strategy for producing full-length human proteins in these three expression systems.

Pathogen reduction is a viable approach to ensure the continued safety of the blood supply against emerging pathogens. However, the currently licensed pathogen reduction techniques are ineffective against non-enveloped viruses such as hepatitis A virus, and they introduce chemicals with concerns of side effects which prevent their widespread use. In this report, we demonstrate the inactivation of both enveloped and non-enveloped viruses in human plasma using a novel chemical-free method, a visible ultrashort pulsed laser. We found that laser treatment resulted in 2-log, 1-log, and 3-log reductions in human immunodeficiency virus, hepatitis A virus, and murine cytomegalovirus in human plasma, respectively. Laser-treated plasma showed ≥70% retention for most coagulation factors tested. Furthermore, laser treatment did not alter the structure of a model coagulation factor, fibrinogen. Ultrashort pulsed lasers are a promising new method for chemical-free, broad-spectrum pathogen reduction in human plasma.

DNA can be programmed to self-assemble into high molecular weight 3D assemblies with precise nanometer-scale structural features. Although numerous sequence design strategies exist to realize these assemblies in solution, there is currently no computational framework to predict their 3D structures on the basis of programmed underlying multi-way junction topologies constrained by DNA duplexes. Here, we introduce such an approach and apply it to assemblies designed using the canonical immobile four-way junction. The procedure is used to predict the 3D structure of high molecular weight planar and spherical ring-like origami objects, a tile-based sheet-like ribbon, and a 3D crystalline tensegrity motif, in quantitative agreement with experiments. Our framework provides a new approach to predict programmed nucleic acid 3D structure on the basis of prescribed secondary structure motifs, with possible application to the design of such assemblies for use in biomolecular and materials science.

Using supported ionic-liquid membrane (SILM)-inspired methodologies, we have synthesized, characterized, and developed a humidity sensor by coating a liquid composite material onto a hygroscopic, porous substrate. Similar to pH paper, the sensor responds to the environment’s relative humidity and changes color accordingly. The humidity indicator is prepared by casting a few microliters of low-toxicity reagents on a nontoxic substrate. The sensing material is a newly synthesized liquid composite that comprises a hygroscopic medium for environmental humidity capture and a color indicator that translates the humidity level into a distinct color change. Sodium borohydride was used to form a liquid composite medium, and DenimBlu30 dye was used as a redox indicator. The liquid composite medium provides a hygroscopic response to the relative humidity, and DenimBlu30 translates the chemical changes into a visual change from yellow to blue. The borate–redox dye-based humidity sensor was prepared, and then Fourier transform infrared spectroscopy, differential scanning calorimetry, and image analysis methods were used to characterize the chemical composition, optimize synthesis, and gain insight into the sensor reactivity. Test results indicated that this new sensing material can detect relative humidity in the range of 5–100% in an irreversible manner with good reproducibility and high accuracy. The sensor is a low-cost, highly sensitive, easy-to-use humidity indicator. More importantly, it can be easily packaged with products to monitor humidity levels in pharmaceutical and food packaging.

Nanoscale zero-valent iron (nZVI) is a strong nonspecific reducing agent that is used for in situ degradation of chlorinated solvents and other oxidized pollutants. However, there are significant concerns regarding the risks posed by the deliberate release of engineered nanomaterials into the environment, which have triggered moratoria, for example, in the United Kingdom. This critical review focuses on the effect of nZVI injection on subsurface microbial communities, which are of interest due to their important role in contaminant attenuation processes. Corrosion of ZVI stimulates dehalorespiring bacteria, due to the production of H₂ that can serve as an electron donor for reduction of chlorinated contaminants. Conversely, laboratory studies show that nZVI can be inhibitory to pure bacterial cultures, although toxicity is reduced when nZVI is coated with polyelectrolytes or natural organic matter. The emerging toolkit of molecular biological analyses should enable a more sophisticated assessment of combined nZVI/biostimulation or bioaugmentation approaches. While further research on the consequences of its application for subsurface microbial communities is needed, nZVI continues to hold promise as an innovative technology for in situ remediation of pollutants It is particularly attractive. for the remediation of subsurface environments containing chlorinated ethenes because of its ability to potentially elicit and sustain both physical–chemical and biological removal despite its documented antimicrobial properties.

Background: To be effective, orally administered live Salmonella vaccines must first survive their encounter with the low pH environment of the stomach. To enhance survival, an antacid is often given to neutralize the acidic environment of the stomach just prior to or concomitant with administration of the vaccine. One drawback of this approach, from the perspective of the clinical trial volunteer, is that the taste of a bicarbonate-based acid neutralization system can be unpleasant. Thus, we explored an alternative method that would be at least as effective as bicarbonate and with a potentially more acceptable taste. Because ingestion of protein can rapidly buffer stomach pH, we examined the possibility that the protein-rich Ensure® Nutrition shakes would be effective alternatives to bicarbonate.

Results: We tested one Salmonella enterica serovar Typhimurium and three Salmonella Typhi vaccine strains and found that all strains survived equally well when incubated in either Ensure® or bicarbonate. In a low gastric pH mouse model, Ensure® worked as well or better than bicarbonate to enhance survival through the intestinal tract, although neither agent enhanced the survival of the S. Typhi test strain possessing a rpoS mutation.

Conclusions: Our data show that a protein-rich drink such as Ensure® Nutrition shakes can serve as an alternative to bicarbonate for reducing gastric pH prior to administration of a live Salmonella vaccine.

We report a device to fill an array of small chemical reaction chambers (microreactors) with reagent and then seal them using pressurized viscous liquid acting through a flexible membrane. The device enables multiple, independent chemical reactions involving free floating intermediate molecules without interference from neighboring reactions or external environments. The device is validated by protein expressed in situ directly from DNA in a microarray of ~10,000 spots with no diffusion during three hours incubation. Using the device to probe for an autoantibody cancer biomarker in blood serum sample gave five times higher signal to background ratio compared to standard protein microarray expressed on a flat microscope slide. Physical design principles to effectively fill the array of microreactors with reagent and experimental results of alternate methods for sealing the microreactors are presented.

Sera from patients with ovarian cancer contain autoantibodies (AAb) to tumor-derived proteins that are potential biomarkers for early detection. To detect AAb, we probed high-density programmable protein microarrays (NAPPA) expressing 5177 candidate tumor antigens with sera from patients with serous ovarian cancer (n = 34 cases/30 controls) and measured bound IgG. Of these, 741 antigens were selected and probed with an independent set of ovarian cancer sera (n = 60 cases/60 controls). Twelve potential autoantigens were identified with sensitivities ranging from 13 to 22% at >93% specificity. These were retested using a Luminex bead array using 60 cases and 60 controls, with sensitivities ranging from 0 to 31.7% at 95% specificity. Three AAb (p53, PTPRA, and PTGFR) had area under the curve (AUC) levels >60% (p < 0.01), with the partial AUC (SPAUC) over 5 times greater than for a nondiscriminating test (p < 0.01). Using a panel of the top three AAb (p53, PTPRA, and PTGFR), if at least two AAb were positive, then the sensitivity was 23.3% at 98.3% specificity. AAb to at least one of these top three antigens were also detected in 7/20 sera (35%) of patients with low CA 125 levels and 0/15 controls. AAb to p53, PTPRA, and PTGFR are potential biomarkers for the early detection of ovarian cancer.

Quantifying the interactions of bacteria with external ligands is fundamental to the understanding of pathogenesis, antibiotic resistance, immune evasion, and mechanism of antimicrobial action. Due to inherent cell-to-cell heterogeneity in a microbial population, each bacterium interacts differently with its environment. This large variability is washed out in bulk assays, and there is a need of techniques that can quantify interactions of bacteria with ligands at the single bacterium level. In this work, we present a label-free and real-time plasmonic imaging technique to measure the binding kinetics of ligand interactions with single bacteria, and perform statistical analysis of the heterogeneity. Using the technique, we have studied interactions of antibodies with single Escherichia coli O157:H7 cells and demonstrated a capability of determining the binding kinetic constants of single live bacteria with ligands, and quantify heterogeneity in a microbial population.

Aquaculture production has nearly tripled in the last two decades, bringing with it a significant increase in the use of antibiotics. Using liquid chromatography/tandem mass spectrometry (LC–MS/MS), the presence of 47 antibiotics was investigated in U.S. purchased shrimp, salmon, catfish, trout, tilapia, and swai originating from 11 different countries. All samples (n = 27) complied with U.S. FDA regulations and five antibiotics were detected above the limits of detection: oxytetracycline (in wild shrimp, 7.7 ng/g of fresh weight; farmed tilapia, 2.7; farmed salmon, 8.6; farmed trout with spinal deformities, 3.9), 4-epioxytetracycline (farmed salmon, 4.1), sulfadimethoxine (farmed shrimp, 0.3), ormetoprim (farmed salmon, 0.5), and virginiamycin (farmed salmon marketed as antibiotic-free, 5.2). A literature review showed that sub-regulatory levels of antibiotics, as found here, can promote resistance development; publications linking aquaculture to this have increased more than 8-fold from 1991 to 2013. Although this study was limited in size and employed sample pooling, it represents the largest reconnaissance of antibiotics in U.S. seafood to date, providing data on previously unmonitored antibiotics and on farmed trout with spinal deformities. Results indicate low levels of antibiotic residues and general compliance with U.S. regulations. The potential for development of microbial drug resistance was identified as a key concern and research priority.