Next-generation sequencing technologies have enabled many advances across biology, with microbial ecology benefiting primarily through expanded sample sizes. Although the cost of running sequencing ...instruments has decreased substantially over time, the price of library preparation methods has largely remained unchanged. In this study, we developed a low-cost miniaturized (5-µl volume) high-throughput (384-sample) amplicon library preparation method with the Echo 550 acoustic liquid handler. Our method reduces costs of library preparation to $1.42 per sample, a 58% reduction compared to existing automated methods and a 21-fold reduction from commercial kits, without compromising sequencing success or distorting the microbial community composition analysis. We further validated the optimized method by sampling five body sites from 46 Pacific chub mackerel fish caught across 16 sampling events over seven months from the Scripps Institution of Oceanography pier in La Jolla, CA. Fish microbiome samples were processed with the miniaturized 5-µl reaction volume with 0.2 µl of genomic DNA (gDNA) and the standard 25-µl reaction volume with 1 µl of gDNA. Between the two methods, alpha diversity was highly correlated (
> 0.95), while distances of technical replicates were much lower than within-body-site variation (
< 0.0001), further validating the method. The cost savings of implementing the miniaturized library preparation (going from triplicate 25-µl reactions to triplicate 5-µl reactions) are large enough to cover a MiSeq sequencing run for 768 samples while preserving accurate microbiome measurements.
Reduced costs of sequencing have tremendously impacted the field of microbial ecology, allowing scientists to design more studies with larger sample sizes that often exceed 10,000 samples. Library preparation costs have not kept pace with sequencing prices, although automated liquid handling robots provide a unique opportunity to bridge this gap while also decreasing human error. Here, we take advantage of an acoustic liquid handling robot to develop a high-throughput miniaturized library preparation method of a highly cited and broadly used 16S rRNA gene amplicon reaction. We evaluate the potential negative effects of reducing the PCR volume along with varying the amount of gDNA going into the reaction. Our optimized method reduces sample-processing costs while continuing to generate a high-quality microbiome readout that is indistinguishable from the original method.
Corrosion of reinforcing steel presents a major durability issue worldwide and is the focus of much research activity. The long time periods involved in replicating reinforcement corrosion within ...laboratories has resulted in a number of accelerated test methods being developed. The basis of the research presented in this paper was to examine the impressed current technique often used to induce reinforcement corrosion. The suitability of the technique to model chloride-induced corrosion was investigated by examining the electrochemical nature of the test method. Corrosion was induced in prisms of differing characteristic strengths and cover thicknesses by applying a current between 3 days and 17 days. The acoustic emission (AE) technique detected the onset of corrosion. The gravimetrical and theoretical mass losses are compared and a modified expression based on Faraday's law relating the electrical current to the mass loss is also proposed, which accounts for the localized nature of chloride-induced corrosion. This improved method is dependent upon a reliable method of detecting the onset of corrosion, which can be achieved by AE. On balance, the impressed current technique appears suitable to simulate reinforcement corrosion.
Purpose Subureteral injection of dextranomer/hyaluronic acid copolymer is a minimally invasive method to treat vesicoureteral reflux. We report short and long-term success in treating secondary ...vesicoureteral reflux in patients with neurogenic bladder dysfunction or severe voiding dysfunction. Materials and Methods We performed a retrospective chart review of all subureteral injection procedures done to identify patients with neurogenic bladder or severe voiding dysfunction. Short (less than 12 months) and long-term vesicoureteral reflux results for patients and ureters were recorded. Preoperative urodynamics and radiographic findings were reviewed. Preoperative factors were evaluated to identify patients with greater chances of success. Results A total of 12 patients (17 ureters) were identified (10 with neurogenic bladder and 2 with Hinman syndrome). Short-term success (no vesicoureteral reflux) was achieved in 50% of patients and 58% of ureters. At a median followup of 4.5 years (range 1 to 9) success decreased to 35% of ureters. Overall, long-term success was found in 25% of patients who were free of vesicoureteral reflux and required no additional surgery. Of the patients 41% required additional urological surgery for vesicoureteral reflux or related conditions. Conclusions With long-term followup many patients who had initial improvement in vesicoureteral reflux ultimately experienced treatment failure and recurrence of reflux. At a median of 4.5 years 25% of patients with neurogenic bladder and vesicoureteral reflux were successfully treated with endoscopic injection of dextranomer/hyaluronic acid copolymer.
Increases in neuronal activity induce local increases in cerebral perfusion. However, our understanding of the processes underlying this neurovascular coupling remains incomplete and, particularly, ...how these vary across the brain. Recent work supports an important role for astrocytes in neurovascular coupling, in large part via activation of their metabotropic glutamate receptors (mGluR). Here, using a combination of functional magnetic resonance imaging (fMRI) and electrophysiology we demonstrate regional heterogeneity in the mechanisms underlying neurovascular coupling. Direct electrical stimulation of the rat hindpaw sensorimotor cortex induces blood oxygenation level dependent (BOLD) and cerebral blood volume (CBV) fMRI responses in several anatomically distinct cortical and subcortical structures. Following intraperitoneal administration of the type 5 mGluR antagonist, MPEP, both BOLD and CBV responses to cortical stimulation were significantly reduced, whilst the local field potential (LFP) responses remained largely constant. Spatially, the degree of reduction in fMRI responses varied between cortical and subcortical regions (primary cortex ~
18%
vs. striatum ~
66%), and also between primary and secondary cortical areas (~
18%
vs. ~
55%). Similarly, greater decreases in response amplitude were seen in the contralateral secondary cortex (~
91%) and ipsilateral striatum (~
70%), compared to the primary cortex (~
44%). Following MPEP, a negative component of the BOLD and CBV responses became more apparent, suggesting that different mechanisms mediate vasodilatory and vasoconstrictory responses. Interestingly, under baseline conditions the quantitative relationship between fMRI and LFP responses in cortical and subcortical regions was markedly different. Our data indicate that coupling between neuronal and fMRI responses is neither empirically nor mechanistically consistent across the brain.
►Neurovascular uncoupling by mGluR5 blockade is regionally heterogenous. ►BOLD–LFP relationship varies across different brain regions. ►Different mechanisms mediate vasodilatory and vasoconstrictory responses.
Summary
Two prominent characteristics of marine coccolithophores are their secretion of coccoliths and their susceptibility to infection by coccolithoviruses (EhVs), both of which display variation ...among cells in culture and in natural populations. We examined the impact of calcification on infection by challenging a variety of Emiliania huxleyi strains at different calcification states with EhVs of different virulence. Reduced cellular calcification was associated with increased infection and EhV production, even though calcified cells and associated coccoliths had significantly higher adsorption coefficients than non‐calcified (naked) cells. Sialic acid glycosphingolipids, molecules thought to mediate EhV infection, were generally more abundant in calcified cells and enriched in purified, sorted coccoliths, suggesting a biochemical link between calcification and adsorption rates. In turn, viable EhVs impacted cellular calcification absent of lysis by inducing dramatic shifts in optical side scatter signals and a massive release of detached coccoliths in a subpopulation of cells, which could be triggered by resuspension of healthy, calcified host cells in an EhV‐free, ‘induced media’. Our findings show that calcification is a key component of the E. huxleyi‐EhV arms race and an aspect that is critical both to the modelling of these host–virus interactions in the ocean and interpreting their impact on the global carbon cycle.
Each year, underwater remotely operated vehicles (ROVs) collect thousands of hours of video of unexplored ocean habitats revealing a plethora of information regarding biodiversity on Earth. However, ...fully utilizing this information remains a challenge as proper annotations and analysis require trained scientists’ time, which is both limited and costly. To this end, we present a Dataset for Underwater Substrate and Invertebrate Analysis (DUSIA), a benchmark suite and growing large-scale dataset to train, validate, and test methods for temporally localizing four underwater substrates as well as temporally and spatially localizing 59 underwater invertebrate species. DUSIA currently includes over ten hours of footage across 25 videos captured in 1080p at 30 fps by an ROV following pre-planned transects across the ocean floor near the Channel Islands of California. Each video includes annotations indicating the start and end times of substrates across the video in addition to counts of species of interest. Some frames are annotated with precise bounding box locations for invertebrate species of interest, as seen in Fig. 1. To our knowledge, DUSIA is the first dataset of its kind for deep sea exploration, with video from a moving camera, that includes substrate annotations and invertebrate species that are present at significant depths where sunlight does not penetrate. Additionally, we present the novel context-driven object detector (CDD) where we use explicit substrate classification to influence an object detection network to simultaneously predict a substrate and species class influenced by that substrate. We also present a method for improving training on partially annotated bounding box frames. Finally, we offer a baseline method for automating the counting of invertebrate species of interest.
Although a promising technique, phytoextraction has yet to see significant commercialization. Major limitations include metal uptake rates and subsequent processing costs. However, it has been shown ...that liquid-culture-grown Arabidopsis can take up and store palladium as nanoparticles. The processed plant biomass has catalytic activity comparable to that of commercially available catalysts, creating a product of higher value than extracted bulk metal. We demonstrate that the minimum level of palladium in Arabidopsis dried tissues for catalytic activity comparable to commercially available 3% palladium-on-carbon catalysts was achieved from dried plant biomass containing between 12 and 18 g·kg–1 Pd. To advance this technology, species suitable for in-the-field application: mustard, miscanthus, and 16 willow species and cultivars, were tested. These species were able to grow, and take up, palladium from both synthetic and mine-sourced tailings. Although levels of palladium accumulation in field-suitable species are below that required for commercially available 3% palladium-on-carbon catalysts, this study both sets the target, and is a step toward, the development of field-suitable species that concentrate catalytically active levels of palladium. Life cycle assessment on the phytomining approaches described here indicates that the use of plants to accumulate palladium for industrial applications has the potential to decrease the overall environmental impacts associated with extracting palladium using present-day mining processes.
This paper reports the influence of sulfate concentration on chloride-induced reinforcement corrosion in Portland cement concretes (with C
3A varying from 3.6% to 9.65%). The concrete specimens were ...exposed to mixed chloride and sulfate solutions for a period of 1200 days. The chloride was fixed at 5% NaCl for all solutions, while the sulfate concentration was varied to represent that noted in the sulfate-bearing soil and ground water. The study included an assessment of the effect of cation type associated with sulfate ions, namely Na
+ and Mg
2+, on chloride-induced reinforcement corrosion, an important factor that has received little attention. Reinforcement corrosion was evaluated by measuring corrosion potentials and corrosion current density at regular intervals. The results indicate that the presence of sulfate ions in the chloride solution did not influence the time to initiation of chloride-induced reinforcement corrosion, but the rate of corrosion increased with increasing sulfate concentration. Further, the rate of chloride-induced reinforcement corrosion in the concrete specimens exposed to sodium chloride plus magnesium sulfate solutions was more than that in the concrete specimens exposed to sodium chloride plus sodium sulfate solutions.
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