Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Simulation of the Energy Performance of a Building with Green Roofs and Green Walls in a Tropical Climate

Global temperatures have continued to rise for decades, partly due to human-caused greenhouse gas emissions and subsequent urban heat island (UHI) effects. This current research examines the benefits of urban greenery by studying the impact of green roofs and walls of a building on thermal behavior and heat transfer in a warm and humid climate. This simulation study discusses the importance of greening systems in improving thermal comfort and minimizing the causes of UHI by assessing an integrated green building design. Using the simulation software DesignBuilder, the significance of greening systems, green roofs, and walls in enhancing thermal comfort and reducing the factors that contribute to UHI is investigated. The simulation results are based on the building’s energy usage in hot and humid regions while featuring green roofs and walls. The simulation results indicate a considerable positive impact of greening systems in improving the urban environment in hot and humid tropical climates. Air temperature, radiant temperature, humidity, and solar gain are decreased by urban greening. The total energy consumption and district cooling demand of buildings with green roofs and walls are reduced by 10.5% and 13%, respectively. The greening systems substantially improve air quality and building’s energy efficiency. Thus, the present study‘s findings can benefit urban designers and dwellers in devising strategies for establishing green spaces in congested urban environments by integrating green technologies and systems into built environments

Daylight performance analysis of a residential building in a tropical climate

The present study investigates the effects of visible light transmittance in glazing and window-to-wall ratios on the ground floor daylighting performance in a two-storey residential building in a warm-humid climate. The metrics used to optimize daylighting performance with minimal glare are useful daylight illuminance, annual sunlight exposure, and spatial daylight autonomy. The daylighting performance of a residential building is assessed by empirical method and Design-Builder simulation, focusing on overcast sky situations. The useful daylight illuminance is the primary metric for analysing the amount of daylight throughout the year. Annual sunlight exposure and spatial daylight autonomy complement useful daylight illuminance in evaluating the daylighting performance. A window-to-wall ratio of 16%, a visible light transmittance of 0.62, and a glare of 0.52 can meet the daylighting requirements and standards. A design change in the window position helps to obtain annual sunlight exposure within 10% while maintaining high daylighting performance. When installed in the upper position of the wall with a higher sill and lintel height, glazing with a window-to-wall ratio of 16% and a visible light transmittance of 0.62 functions well without creating glare. The significant findings benefit all stakeholders in improving daylighting strategies in tropical climates and satisfying building standards

Real-Time, Wearable, Biomechanical Movement Capture of Both Humans and Robots with Metal-Free Electrodes

We demonstrate an all-carbon-based, flexible, conformal movement-capturing device capable of precisely monitoring biomechanical movements of both humans and robots. Mechanically robust, metal-free electrodes form a unique component of the device responsible for qualitatively and quantitatively transducing biomechanical movements without any signal artifacts. Importantly, the device withstands and operates in a wide dynamic range for both stretching (25% strain) and bending (140°) actions with minimal cycling hysteresis (2.0), high repeatability (>100 cycles), low creep, and humidity-independent rapid response (∼200 ms). Furthermore, the device qualitatively distinguishes movements such as bending of finger, knuckle, and wrist and also provides quantitative information on the extent of such movements. We establish that single-wall carbon nanotubes (CNTs) embedded in ultralow concentration (0.016 wt %) within an elastomeric matrix undergo three-dimensional conformational changes during biomechanical movements that are subsequently transduced as signals. In addition, such CNT–elastomer strips exhibit enhanced stretchability (>100%) and elasticity (∼77%) in comparison to those of pure elastomers, leading to a wider dynamic working range of the device. Furthermore, seamless integration of a versatile gesture tracker on ubiquitous platforms, such as human skin, kinesiologic tapes, gloves, and robotic arms, is achieved, thereby catering to applications ranging from healthcare monitoring and physiotherapy to robotics and wearable technologies

Growth of gold nanoparticles in human cells

Gold nanoparticles of 20-100 nm diameter were synthesized within HEK-293 (human embryonic kidney), HeLa (human cervical cancer), SiHa (human cervical cancer), and SKNSH (human neuroblastoma) cells. Incubation of 1 mM tetrachloroaurate solution, prepared in phosphate buffered saline (PBS), pH 7.4, with human cells grown to ~80% confluency yielded systematic growth of nanoparticles over a period of 96 h. The cells, stained due to nanoparticle growth, were adherent to the bottom of the wells of the tissue culture plates, with their morphology preserved, indicating that the cell membrane was intact. Transmission electron microscopy of ultrathin sections showed the presence of nanoparticles within the cytoplasm and in the nucleus, the latter being much smaller in dimension. Scanning near field microscopic images confirmed the growth of large particles within the cytoplasm. Normal cells gave UV-visible signatures of higher intensity than the cancer cells. Differences in the cellular metabolism of cancer and noncancer cells were manifested, presumably in their ability to carry out the reduction process

Layer-by-layer coating of alginate matrices with chitosan–alginate for the improved survival and targeted delivery of probiotic bacteria after oral administration

The oral administration of probiotic bacteria has shown potential in clinical trials for the alleviation of specific disorders of the gastrointestinal tract. However, cells must be alive in order to exert these benefits. The low pH of the stomach can greatly reduce the number of viable microorganisms that reach the intestine, thereby reducing the efficacy of the administration. Herein, a model probiotic, Bifidobacterium breve, has been encapsulated into an alginate matrix before coating in multilayers of alternating alginate and chitosan. The intention of this formulation was to improve the survival of B. breve during exposure to low pH and to target the delivery of the cells to the intestine. The material properties were first characterized before in vitro testing. Biacore™ experiments allowed for the polymer interactions to be confirmed; additionally, the stability of these multilayers to buffers simulating the pH of the gastrointestinal tract was demonstrated. Texture analysis was used to monitor changes in the gel strength during preparation, showing a weakening of the matrices during coating as a result of calcium ion sequestration. The build-up of multilayers was confirmed by confocal laser-scanning microscopy, which also showed the increase in the thickness of coat over time. During exposure to in vitro gastric conditions, an increase in viability from <3 log(CFU) per mL, seen in free cells, up to a maximum of 8.84 ± 0.17 log(CFU) per mL was noted in a 3-layer coated matrix. Multilayer-coated alginate matrices also showed a targeting of delivery to the intestine, with a gradual release of their loads over 240 min

The <em>C. elegans</em> Rab Family: Identification, Classification and Toolkit Construction

<div><p>Rab monomeric GTPases regulate specific aspects of vesicle transport in eukaryotes including coat recruitment, uncoating, fission, motility, target selection and fusion. Moreover, individual Rab proteins function at specific sites within the cell, for example the ER, golgi and early endosome. Importantly, the localization and function of individual Rab subfamily members are often conserved underscoring the significant contributions that model organisms such as <em>Caenorhabditis elegans</em> can make towards a better understanding of human disease caused by Rab and vesicle trafficking malfunction. With this in mind, a bioinformatics approach was first taken to identify and classify the complete <em>C. elegans</em> Rab family placing individual Rabs into specific subfamilies based on molecular phylogenetics. For genes that were difficult to classify by sequence similarity alone, we did a comparative analysis of intron position among specific subfamilies from yeast to humans. This two-pronged approach allowed the classification of 30 out of 31 <em>C. elegans</em> Rab proteins identified here including <em>Rab31/Rab50</em>, a likely member of the last eukaryotic common ancestor (LECA). Second, a molecular toolset was created to facilitate research on biological processes that involve Rab proteins. Specifically, we used Gateway-compatible <em>C. elegans</em> ORFeome clones as starting material to create 44 full-length, sequence-verified, dominant-negative (DN) and constitutive active (CA) <em>rab</em> open reading frames (ORFs). Development of this toolset provided independent research projects for students enrolled in a research-based molecular techniques course at California State University, East Bay (CSUEB).</p> </div

Comparative analysis of intron position among diverse Rab subfamily members.

<p>A) Cladogram indicating evolutionary relationships of 18 species examined here <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049387#pone.0049387-Dunn1" target="_blank">[128]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049387#pone.0049387-Csuros1" target="_blank">[130]</a>. Ecdy. = Ecdysozoa, Chromal. = Chromalveolata. For species abbreviations see Methods. B) An ML tree of Opisthokonts created from the MSA used to map intron positions. Bootstrap support (100 replicates) is indicated for each subfamily cluster. C) For each subfamily, the number of times a <u>S</u>ubfamily <u>S</u>pecific <u>C</u>onserved <u>I</u>ntron <u>P</u>osition (SSCIP) involving the indicated number of species was observed (gray bars), compared to what is expected by chance (black diamonds). The difference between observed and expected is statistically significant where indicated. *P(Monte Carlo) <0.05. ***P(Monte Carlo)≤0.00001. The Rab<i>31, 6, 5, 22, 34, 21</i> and <i>23</i> subfamilies include 17, 18, 17, 9, 9, 10, 14 and 12 species, respectively. D) and E) Heat map indicating number of introns within <i>Rab31</i> (D) or <i>Rab6</i> (E) that match SSCIPs from <i>Rab31, 5, 22, 21, 6, 34</i> and <i>23</i>. The circled number indicates the number of introns present in the MSA for each gene. % equals the percentage of introns that are shared with the true SSCIP. <i>C56E6.2</i> (D) and <i>Y71H2AM.12</i> (E) are highlighted red. Genbank Descriptions (if any) and RABDB! classifications are included. Classification abbreviations include: HypoRabX1 (H.RabX1), HypoRabX2 (H.RabX2), HypoRabX3 (H.RabX3) and MetazoaRabX3 (M.RabX3). F) A pairwise comparison of intron position conservation between specific genes (Rab31 at left, Rab6 at right) and their corresponding set of SSCIPs. Black diamonds plot the probability that a specific number of intron matches would be expected by chance for each set of conditions. Chart 1 plots a comparison of 5 introns with 7 SSCIPs (5×7). Chart 2∶4×7. Chart 3∶2×7. Chart 4∶4×6. Observed values for a subset of genes are indicated with P values estimated from the Monte Carlo simulation data (See text and methods). Species abbreviations are as in A. C) and F) 72 protosplice sites assumed.</p

A list and description of Rab isolates created for the <i>C. elegans</i> ORFeome-based toolkit.

<p>WT, DN and CA clones included in the Rab Toolkit are given isolate names otherwise an explanation for its absence is provided. Subfamily classifications are based on Diekmann et al. 2011 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049387#pone.0049387-Diekmann1" target="_blank">[33]</a> and/or data presented here. The majority of <i>C. elegans rab</i> genes are predicted to have only one splice variant with the following exceptions: WormBase describes two splice variants for <i>rab-3</i> that code for proteins 233 and 219 amino acids (aa) in length. ORFeome project primers were designed to amplify the shorter isoform only. WormBase describes two splice variants for <i>4R79.2 (Rab44)</i> that code for proteins 311 aa and 395 aa in length. ORFeome project primers were designed to amplify the longer isoform only. Names listed under “other” are from WormBase or Pereira-Leal and Seabra (2001). Finally, while <i>rab-37</i> shows 100% identity with the Refseq protein NP_001041293 it contains an additional 5 amino acids at its N-terminus. See text for details.</p

A chladogram of Rab family members from <i>C. elegans</i> and <i>H. sapien</i>s.

<p>The evolutionary history was inferred using the Neighbor-Joining phylogenetic reconstruction method. The tree is rooted with the natural outlying clade, Rab28. The optimal tree is shown with the percentage of replicate trees (>40) in which the associated genes cluster together in the bootstrap test (500 replicates) provided next to each branch. The tree is drawn to emphasize topology. The evolutionary distances were computed using the JTT amino acid substitution method and are in the units of the number of amino acid differences per site. Evolutionary analyses were conducted using MEGA5. Clades marked with red, orange or yellow circles indicate their degree of stability under a variety of phylogenetic reconstruction parameters (see text and methods for details). Red = 14/14, orange = 13/14 and yellow = 12/14 trees. Genes highlighted with black circles represent putative orphan <i>C. elegan</i> Rabs (lacking a human ortholog). For simplicity, closely related splice variants and well-supported human-specific clades were deleted (see methods for details).</p