Appendage-resident epithelial cells expedite wound healing response in adult zebrafish.

Adult zebrafish are able to heal large-sized cutaneous wounds in hours with little to no scarring. This rapid re-epithelialization is crucial for preventing infection and jumpstarting the subsequent regeneration of damaged tissues. Despite significant progress in understanding this process, it remains unclear how vast numbers of epithelial cells are orchestrated on an organismic scale to ensure the timely closure of millimeter-sized wounds. Here, we report an unexpected role of adult zebrafish appendages (fins) in accelerating the re-epithelialization process. Through whole-body monitoring of single-cell dynamics in live animals, we found that fin-resident epithelial cells (FECs) are highly mobile and migrate to cover wounds in nearby body regions. Upon injury, FECs readily undergo organ-level mobilization, allowing for coverage of body surfaces of up to 4.78 mm2 in less than 8 h. Intriguingly, long-term fate-tracking experiments revealed that the migratory FECs are not short-lived at the wound site; instead, the cells can persist on the body surface for more than a year. Our experiments on "fin-less" and "fin-gaining" individuals demonstrated that the fin structures are not only capable of promoting rapid re-epithelialization but are also necessary for the process. We further found that fin-enriched extracellular matrix laminins promote the active migration of FECs by facilitating lamellipodia formation. These findings lead us to conclude that appendage structures in regenerative vertebrates, such as fins, may possess a previously unrecognized function beyond serving as locomotor organs. The appendages may also act as a massive reservoir of healing cells, which speed up wound closure and tissue repair.

A Smartphone-Based Whole-Cell Array Sensor for Detection of Antibiotics in Milk.

We present an integral smartphone-based whole-cell biosensor, LumiCellSense (LCS), which incorporates a 16-well biochip with an oxygen permeable coating, harboring bioluminescent Escherichia coli bioreporter cells, a macro lens, a lens barrel, a metal heater tray, and a temperature controller, enclosed in a light-impermeable case. The luminescence emitted by the bioreporter cells in response to the presence of the target chemicals is imaged by the phone's camera, and a dedicated phone-embedded application, LCS_Logger, is employed to calculate photon emission intensity and plot it in real time on the device's screen. An alert is automatically given when light intensity increases above the baseline, indicating the presence of the target. We demonstrate the efficacy of this system by the detection of residues of an antibiotic, ciprofloxacin (CIP), in whole milk, with a detection threshold of 7.2 ng/mL. This value is below the allowed maximum as defined by European Union regulations.

Microbial biosensing of ciprofloxacin residues in food by a portable lens-free CCD-based analyzer.

We present a rapid and simple approach for sensitive detection of antibiotic residues in food samples based on luminescence induction by live bacterial sensor strains integrated into a CCD-based lens-free optical analyzer (LumiSense). Using ciprofloxacin as a model antibiotic, we demonstrate response times of between 20 and 80 min, and detection thresholds of 8 ng/mL for milk, egg white, and chicken essence, and 64 ng/mL for egg yolk. These values are below the minimal allowed values as defined by European Union regulations. Although not intended to replace traditional analytical equipment and regulation-approved methods, LumiSense and similar systems, sample preparation for which involves only simple mixing, dilution, and homogenization, may nevertheless provide a simple means for high-throughput food sample screening. Graphical abstract Detection of bioluminescence from genetically modified bacteria offers a simple and effective way for monitoring an antibiotic, ciprofloxacin, in milk without prior sample preparation.

Bio-butanol production from glycerol with Clostridium pasteurianum CH4: the effects of butyrate addition and in situ butanol removal via membrane distillation.

BACKGROUND: Clostridium pasteurianum CH4 was used to produce butanol from glycerol. The performance of butanol fermentation was improved by adding butyrate as the precursor to trigger the metabolic pathway toward butanol production, and by combining this with in situ butanol removal via vacuum membrane distillation (VMD) to avoid the product inhibition arising from a high butanol concentration. RESULTS: Adding 6 g L(-1) butyrate as precursor led to an increase in the butanol yield from 0.24 to 0.34 mol butanol (mol glycerol)(-1). Combining VMD and butyrate addition strategies could further enhance the maximum effective butanol concentration to 29.8 g L(-1), while the yield was also improved to 0.39 mol butanol (mol glycerol)(-1). The butanol concentration in the permeate of VMD was nearly five times higher than that in the feeding solution. CONCLUSIONS: The proposed butyrate addition and VMD in situ butanol removal strategies are very effective in enhancing both butanol titer and butanol yield. This would significantly enhance the economic feasibility of fermentative production of butanol. The VMD-based technology not only alleviates the inhibitory effect of butanol, but also markedly increases butanol concentration in the permeate after condensation, thereby making downstream processing easier and more cost-effective.

Rapid single cell detection of Staphylococcus aureus by aptamer-conjugated gold nanoparticles.

Staphylococcus aureus is one of the most important human pathogens, causing more than 500,000 infections in the United States each year. Traditional methods for bacterial culture and identification take several days, wasting precious time for patients who are suffering severe bacterial infections. Numerous nucleic acid-based detection methods have been introduced to address this deficiency; however, the costs and requirement for expensive equipment may limit the widespread use of such technologies. Thus, there is an unmet demand of new platform technology to improve the bacterial detection and identification in clinical practice. In this study, we developed a rapid, ultra-sensitive, low cost, and non-polymerase chain reaction (PCR)-based method for bacterial identification. Using this method, which measures the resonance light-scattering signal of aptamer-conjugated gold nanoparticles, we successfully detected single S. aureus cell within 1.5 hours. This new platform technology may have potential to develop a rapid and sensitive bacterial testing at point-of-care.

Enhancing butanol production with Clostridium pasteurianum CH4 using sequential glucose-glycerol addition and simultaneous dual-substrate cultivation strategies.

Adding butyrate significantly enhanced butanol production from glycerol with Clostridium pasteurianum CH4, which predominantly produces butyrate (instead of butanol) when grown on glucose. Hence, the butyrate produced from assimilating glucose can be used to stimulate butanol production from glycerol under dual-substrate cultivation with glucose and glycerol. This proposed butanol production process was conducted by employing sequential or simultaneous addition of the two substrates. The latter approach exhibited better carbon source utilization and butanol production efficiencies. Under the optimal glucose to glycerol ratio (20 g L(-1) to 60 g L(-1)), the simultaneous dual-substrate strategy obtained maximum butanol titer, productivity and yield of 13.3 g L(-1), 0.28 g L(-1) h(-1), and 0.38 mol butanol/mol glycerol, respectively. Moreover, bagasse and crude glycerol as dual-substrates were also converted into butanol efficiently with a maximum butanol concentration, productivity and yield of 11.8 g L(-1), 0.14 g L(-1) h(-1), and 0.33 mol butanol/mol glycerol, respectively.

Identification and characterization of oligonucleotides that inhibit Toll-like receptor 2-associated immune responses.

Toll-like receptors (TLRs) play important roles in the immune responses against invading microorganisms. Development of TLR antagonists is recognized as a promising direction in suppressing the associated inflammatory reactions of the TLRs. Aptamers are single-stranded RNA or DNA molecules isolated through an in vitro selection process. Using a novel molecular evolution strategy that combines immunoprecipitation (IP) with systematic evolution of ligands by exponential enrichment (SELEX), we developed an IP-SELEX selection method to facilitate the screening of high-affinity aptamers for the Toll-like receptor 2 (TLR2). Also, human TLR2 functional aptamers were identified and characterized using NF-kappaB reporter assays. Among the functional aptamers, the most effective, AP177, with a dissociation constant of 73 pM, was characterized with TLR2-expressing cells challenged with bacterial cells and purified ligands. The aptamer could effectively antagonize TLR2, significantly inhibit NF-kappaB activity, and suppress the secretion of the cytokines by >80%. In addition, the precise region within the functional aptamer that specifically bound TLR2 was resolved using aptamer microarray analysis. The results of functional assays showed that AP177 acted as a TLR2 antagonist and may hold therapeutic potential in the treatment of diseases related to dysregulated TLR2 immune responses.

Cadmium biosorption by polyvinyl alcohol immobilized recombinant Escherichia coli.

Recombinant Escherichia coli expressing human metallothionein protein was immobilized with polyvinyl alcohol (PVA) for the removal of cadmium from solution. The adsorption ability was strongly affected by pH with optimal performance at pH 5.0, while it was less sensitive to temperature over the range of 20-42 degrees C. The adsorption kinetics and equilibrium of PVA-immobilized cells was best described by pseudo-second order model and Langmuir isotherm, respectively. Over the Cd concentrations range of 10-150 mg/l, PVA-cells had the highest Cd removal percentage (82.7%) at 10mg Cd/l and a biomass loading of 15.4 wt.%. Better adsorption ability was obtained when biomass loading was increased, as the highest adsorption capacity of 4.29 mg/g was achieved at 33.0 wt.% of biomass (initial Cd concentration=100mg/l). An aqueous solution of 0.01 M Na(3)NTA displayed the best desorption efficiency (57-89%) for four A/D cycles, while 51-61% of the original adsorption capacity was retained after regeneration.

Biosorption of nickel, chromium and zinc by MerP-expressing recombinant Escherichia coli.

Escherichia coli hosts able to over-express metal-binding proteins (MerP) originating from Gram-positive (Bacillus cereus RC607) and Gram-negative (Pseudomonas sp. K-62) bacterial strains were used to adsorb Ni(2+), Zn(2+) and Cr(3+) in aqueous solutions. The initial adsorption rate and adsorption capacity were determined to evaluate the performance of the biosorbents. With the expression of MerP protein, the metal adsorption capacity of the recombinant strains for Ni(2+), Zn(2+) and Cr(3+) significantly improved. The cells carrying Gram-positive merP gene (GB) adsorbed Zn(2+) and Cr(3+) at a capacity of 22.3 and 0.98 mmol/g biomass, which is 121% and 72% higher, respectively, over that of the MerP-free host cells. Adsorption capacity of the cells carrying Gram-negative merP gene (GP) also increased 144% and 126% for Zn(2+) and Cr(3+), respectively. Both recombinant strains also exhibited 24% and 5% enhancement in adsorption of Ni(2+) for GB and GP, respectively. The initial adsorption rate of the recombinant biosorbents was also higher than that of the MerP-free host, suggesting an increased metal-binding affinity with MerP expression. Severe cell damage on GB biosorbent was observed after Cr(3+) adsorption, probably due to the metal toxicity effect on the cells.

Exploring multi-metal biosorption by indigenous metal-hyperresistant Enterobacter sp. J1 using experimental design methodologies.

A novel experimental design, combining mixture design and response surface methodology (RSM), was developed to investigate the competitive adsorption behavior of lead, copper and cadmium by an indigenous isolate Enterobacter sp. J1 able to tolerate high concentrations of a variety of heavy metals. Using the proposed combinative experimental design, two different experiment designs in a ternary metal biosorption system can be integrated to a succinct experiment and the number of experimental trials was markedly reduced from 38 to 26 by reusing the mutual experimental data. Triangular contour diagrams and triangular three-dimensional surface plots were generated to describe the ternary metal biosorption equilibrium data in mixture design systems. The results show that the preference of metal sorption of Enterobacter sp. J1 decreased in the order of Pb(2+)>Cu(2+)>Cd(2+). The presence of other metals resulted in a competitive effect. The influence of the other two metals in ternary metal biosorption system can be easily determined by comparing the stray distance from the single metal biosorption. The behavior of competitive biosorption was successfully described and predicted using a combined Langmuir-Freundlich model along with new three-dimensional contour-surface plots.

Localization effect on the metal biosorption capability of recombinant mammalian and fish metallothioneins in Escherichia coli.

In this study, we examined the expression of mammalian and fish metallothioneins (MTs) in Escherichia coli as a strategy to enhance metal biosorption efficiency of bacterial biosorbents for lead (Pb), copper (Cu), cadmium (Cd), and zinc (Zn). In addition, MT proteins were expressed in either the cytoplasmic or periplasmic compartment of host cells to explore the localization effect on metal biosorption. The results showed that MT expression led to a significant increase (5-210%) in overall biosorption efficiency (eta(ads)), especially for biosorption of Cd. The MT-driven improvement in metal biosorption relied more on the increase in the biosorption rates (r(2), a kinetic property) than on the equilibrium biosorption capacities (q(max), a thermodynamic property), despite a 10-45% and 30-80% increase in q(max) of Cd and Zn, respectively. Periplasmic expression of MTs appeared to be more effective in facilitating the metal-binding ability than the cytoplasmlic MT expression. Notably, disparity of the impacts on biosorption ability was observed for the origin of MT proteins, as human MT (MT1A) was the most effective biosorption stimulator compared to MTs originating from mouse (MT1) and fish (OmMT). Moreover, the overall biosorption efficiency (eta(ads)) of the MT-expressing recombinant biosorbents was found to be adsorbate-dependent: the eta(ads) values decreased in the order of Cd > Cu > Zn > Pb.

Cartridge-based high-throughput purification of oligonucleotides for reliable oligonucleotide arrays.

A novel, cartridge-based procedure for the efficient and irreversible detritylation of oligonucleotides is reported. This method, combined with a process for the elimination of depurinated fragments produces, in a highly parallel fashion, oligonucleotides with better purity than those traditionally obtained using reversed-phase high-performance liquid chromotography purification. Our combined detritylation and purification methodology compares favorably with commercial cartridge-based purification systems. The benefits of working with pure oligonucleotides, with regard to higher signal and better signal linearity, are shown in array-based hybridization experiments.

A novel algal toxicity testing technique for assessing the toxicity of both metallic and organic toxicants.

This study presents a closed-system algal toxicity test technique that is capable of detecting the effects of both organic and metallic toxicants. Toxicity testing was conducted by transferring adequate amounts of algal suspension, dilution water (with culture growth medium), and toxicants into 300-mL BOD bottles. The BOD bottles were completely filled up with no head-space left. The initial cell density and the exposure time were 15,000 cells/mL and 48 h, respectively. The performance of the above test method was evaluated using three heavy metals and six organic toxicants based on three different test endpoints, i.e., dissolved oxygen production, algal growth rate, and cell density. The proposed test revealed excellent test sensitivity and reproducibility. Currently, none of the existing algal toxicity test protocols is adequate for assessing the toxicity of organic chemicals. The closed-system algal toxicity tests developed by previous researchers also may not be ideal because the enlarged headspace and/or enriched bicarbonate buffer may result in either underestimations of the exposure concentrations or insensitive responses to both heavy metals and organic toxicants. Compared to the aforementioned algal toxicity test methods, the proposed technique in the present study has a more general applicability under conditions such as effluent samples containing both metals and organic toxicants or samples with unknown compositions.

High throughput parallel synthesis of oligonucleotides with 1536 channel synthesizer.

A 1536 channel oligonucleotide synthesizer, the MultiSyn, was developed with the capability to simultaneously synthesize 1536 oligonucleotides of 20mer length in 10 h. The instrument was designed to synthesize different sequences of various lengths in micro-wells and has synthesized oligonucleotides as long as 119 nt with reasonably good yields using CPG beads of 1000 A pore size. The instrument consists of four 384 channel synthesis modules. Phosphoramidite chemistry was employed and step yields as high as 99.3% were achieved. The enhancement of oligonucleotide synthesis throughput is accomplished by increasing the spatial density of reaction wells. We have identified several parameters that are critical in achieving a good synthesis yield and negligible failure rate in small reaction wells. The coefficient of variation (CV) of product yields in 1536 reaction wells was 20%. The quality of the product was examined by capillary electrophoresis and mass spectrometry. The instrument has robustly synthesized oligonucleotides of various lengths for use as primers and probes for PCR amplifications, oligonucleotide microarrays and genotyping applications. This high throughput oligonucleotide synthesizer is a useful instrument for genomic applications, which require tens of thousands of probes or primers in a short time.