Total Sample Volume Research Articles

Array Tomography Workflow for the Targeted Acquisition of Volume Information using Scanning Electron Microscopy.

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission Electron Microscopy (TEM) provided insight into cell ultrastructure, but in the recent decade, the development of modern Scanning Electron Microscopes (SEM) has changed the way of looking inside the cells. Even though the resolution of TEM is superior when protein-level structural details are needed, SEM-resolution is sufficient for the majority of the organelle-level cell biology-related questions. The advancement in technology enabled automatic volume acquisition solutions such as Serial block-face imaging (SBF-SEM) and Focused ion beam SEM (FIB-SEM). Nevertheless, to this day, these methods remain inefficient when the identification and navigation to areas of interest are crucial. Without the means for precise localization of target areas before imaging, operators need to acquire much more data than they need (in SBF-SEM), or, even worse, prepare many grids and image them all (in TEM). We propose the strategy of "lateral screening" using Array Tomography in SEM, which facilitates the localization of areas of interest, followed by automated imaging of the relevant fraction of the total sample volume. Array tomography samples are conserved during imaging, and they can be arranged into section libraries ready for repeated imaging. Several examples are shown in which lateral screening enables us to analyze structural details that are incredibly challenging to access with any other method.

MATLAB modeling to evaluate the ratio of packed cell volume

<p>There are many direct and indirect methods of measuring the volumetric ratioof red blood cells relative to sample size and total blood, but the manual carcinogenic method remains the most stable and least expensive method despite the possibility of human error.The ratio percent of the packed red blood cell volume to the total volume of the blood sample is known as the packed cell volume (PCV). Our efforts were directed at finding a precise and economic method for PCV determination. There by, we designed a simple device consisting of a digital camera that is connected to a computer which uses a specific MATLAB program to process the blood images. As a result,the image can be read and the PCV can be calculated using a special code system. This new measuring system provides the capabilities and advantagesto frequently comment on measured PCV with normal or abnormal, in comparison with normal values of PCV, which are stored in the computer. Our measurement method provides accurate results compared to the ruler results to alleviate the effort of clearing the hepatitis C virus from the blood (PCV). This system enables a swift and simple sample analysis and PCV identification.</p>

Effects of A low-carbon emission additive on mechanical properties of fine-grained soil under freeze-thaw cycles

The microstructural and resistance properties of fine-grained soils can remarkably alter under freeze-thaw cycles in seasonally frozen regions. Although treated soils have better mechanical properties than natural ones to withstand severe environmental damage, CO2 emitted from additives is not usually regarded. In this research, by analyzing the amount of carbon dioxide releases from some widely used materials, glass fiber was selected as an eco-friendly and low-carbon reinforcement additive. The strength properties of natural and reinforced samples were studied under freeze-thaw conditions via a series of unconfined compression and direct shear tests. The microstructural changes and interfacial interaction of fiber-clay particles was also evaluated by Brunauer-Emmett-Teller (BET) and scanning electron microscopy (SEM) analyses. The results revealed that increasing freeze-thaw cycles reduces the resistance properties of the soil. Despite the favorable impact of glass fiber on compressive strength and elastic modulus, its effect on preventing the resistance reduction due to freeze-thaw cycles was not significant and the best performance of fiber was achieved for shear strength. After 12 cycles, natural clay and soil treated with 2.5% fiber showed 42 and 19% reduction in shear strength, respectively. The results of BET test showed a 30 and 42% increase in total pore volume and mean pore diameter of clay sample due to the freeze-thaw cycle. Adding fiber had no effect on reducing the number and size of micro-voids. However, the mechanical effect of entanglement and high strength of fiber against tensile stresses improved tensile strength of the soil. The stresses were caused by external loading or internal pressure due to water volume changes which were triggered by ice lenses. SEM images showed that glass fiber prevented the formation and expansion of micro-voids and crack lines during freeze-thaw conditions. The presence of fiber preserves integrity, improves bonding and solidarity, and widens the contact surface between the soil particles, finally leading to enhanced frictional resistance between soil particles. Furthermore, glass fiber produces less strength than popular additives such as lime and cement, but it was found to emit very low carbon dioxide to the environment compared to other additive materials.

Plasmonic and label-free real-time quantitative PCR for point-of-care diagnostics.

In response to the world's medical community's need for accurate and immediate infectious pathogen detection, many researchers have focused on adapting the standard molecular diagnostic method of polymerase chain reaction (PCR) for point-of-care (POC) applications. PCR technology is not without its shortcomings; current platforms can be bulky, slow, and power-intensive. Although there have been some advances in microfluidic PCR devices, a simple-to-operate and fabricate PCR device is still lacking. In the first part of this paper, we introduce a compact plasmonic PCR thermocycler in which fast DNA amplification is derived from efficient photothermal heating of a colloidal reaction mixture containing gold nanorods (AuNRs) using a small-scale vertical-cavity surface-emitting laser (VCSEL). Using this method, we demonstrate 30 cycle-assay time of sub-ten minutes for successful Chlamydia trachomatis DNA amplification in 20 μL total PCR sample volume. In the second part, we report an ultrasensitive real-time amplicon detection strategy which is based on cycle-by-cycle monitoring of 260 nm absorption of the PCR sample. This was accomplished by irradiating the PCR sample using a UV LED and collecting the transmitted optical power with a photodetector. The UV absorption dependency on the nucleotides' structural degree of freedom gives rise to distinctive features in the shape of UV amplification curves for the determination of PCR results, thus circumventing the need for the complicated design of target-specific probes or intercalating fluorophores. This amplicon quantification method has a high detection sensitivity of one DNA copy. This is the first demonstration of a compact plasmonic thermocycler combined with a real-time fluorophore-free quantitative amplicon detection system. The small footprint of our PCR device stems from hardware miniaturization, while abundant sample volume facilitates highly sensitive detection and fluid handling required for in-field sample analysis, thereby making it an excellent candidate for POC molecular diagnostics.

Chest computed tomography for outcome prediction in laboratory-confirmed COVID-19: A retrospective analysis of 38,051 cases

BACKGROUND:In the current epidemiological situation, computed tomography (CT) of chest organs plays an important role in disease diagnosis. Clinical and CT data allow physicians to quickly establish the probability of the presence and prognosis of patients with coronavirus disease 2019 (COVID-19).
 AIMS:This study aimed to predict outcomes in patients with laboratory-confirmed COVID-19 based on chest CT and a semi-quantitative visual pulmonary lesion grading system (CT 04).
 MATERIALS AND METHODS:A retrospective analysis of the Unified Medical Information and Analytical Service and Unified Radiological Information Service records from March 01, 2020 to July 30, 2020 was performed. The inclusion criteria were as follows: patients diagnosed with U07.1 (laboratory-verified coronavirus infection) from March 01, 2020 to July 30, 2020 and referred for a chest CT by a physician with suspected community-acquired pneumonia caused by COVID-19; the maximum period between laboratory verification and CT was not more than five days. The observation period for each patient was at least till 30 days from the date of CT. CT was performed in 48 medical organizations providing primary medical care to adults in Moscow. The exclusion criterion was a negative reverse transcription-polymerase chain reaction results by July30, 2020. The CT 04 scale is recommended for use in the Russian Federation to estimate the volume of lung parenchyma lesions when COVID-19 is suspected.
 RESULTS:The total sample volume was 38,051 patients. In this study, the risk of death was three times higher for CT-4 than for CT-0. In the KaplanMeier survival curve, the survival rate of patients in the CT-3 category was almost three times lower (hazard ratio = 2.94) than in the CT 02 categories; in addition, the higher the initial category of CT, the lower the risk of deterioration. The time for hospitalization decreased with the increase in the CT grade.
 CONCLUSION:The visual CT 04 scale can be used to predict outcomes, such as hospitalizations and deaths, in patients suspected of COVID-19 who underwent chest CT in primary health care.

Entrainment of fish eggs and larvae at an operating nuclear generating station using improved methodology

Patrick PH, Di Giuseppe M, Manolopoulos H, Tai M-K, Poulton S, Wright J. 2020. Entrainment of fish eggs and larvae at an operating nuclear generating station using improved methodology. Lake Reserv Manage. 37:186–198. Entrainment data collected from 7 December 2015 to 22 November 2016 at the Darlington Nuclear Generating Station (DNGS) using automated sampling methodology are discussed in this article, including numbers of fish eggs and larvae entrained. We used a more robust sampling design than previously used in 2004 and 2006, which involved more frequent sampling over a longer, 12 month period. The design allowed collection of daily samples with a longer sampling duration and higher total sample volumes that reduced variability compared to previous studies with less robust sampling throughout a day. Our study resulted in the capture of deepwater sculpin and burbot that were not observed in previous entrainment studies. The maximum entrainment density for eggs was about 17 times higher than that for larvae. The entrainment of fish eggs was highest in the summer months (June and July) and did not vary diurnally. The highest entrainment rates for larvae occurred during the months of August and September, with higher densities entrained at night. We recommend the use of both increased sampling frequency and sampling volumes to characterize fish entrainment for water users in the Great Lakes watershed.

Structure of Alloys for (Sm,Zr)(Co,Cu,Fe)z Permanent Magnets: II. Composition, Magnetization Reversal, and Magnetic Hardening of Main Structural Components.

Experimental series of alloys for (Sm,Zr)(Co,Cu,Fe)Z permanent magnets are presented in the concentration ranges that provide wide variations of (4f)/(4d)/(3d) ratios of comprising elements. Optical metallographic analysis, observation of the surface domain structure upon magnetization reversal (Kerr effect), electron microprobe analysis, and measuring the major hysteresis loops of samples at different stages of heat treatment are used to study processes related to the development of the highly coercive state of these samples. It was found that the volume fractions of two main structural components A and B, which comprise 90% of the total sample volume, rigorously control the coercivity at all stages of thermal aging. At the same time, structural components A and B themselves in samples being in the high-coercivity state differ qualitatively and quantitatively in the chemical composition, domain structure and its development in external magnetic fields and, therefore, are characterized by different morphologies of the phases comprising the structural components. Two stages of phase transformations in the sample structure are observed. During isothermal annealing, the cellular structure develops within the B component, whereas, during stepwise (slow) cooling or quenching from the isothermal aging temperature to 400 °C, a phase structure evolves within both the cell boundaries in B and the structural component A. The degree of completion of the phase transformations within micro- and nano-volumes of the components determines the ultimate hysteretic characteristics of the material.

Mechanical Properties and Microstructure of Lime-Treated Red Clay

This research conducted a triaxial test on lime-treated clay with different contents to analyze the effect of mechanical properties. Scanning electron microscope (SEM) and low-field nuclear magnetic resonance (NMR) analysis were used to analyze the effect. X-ray diffraction (XRD) was used to analyze the chemical changes of calcium oxide on red clay. The results show that with the increase of calcium oxide content, the strength increases, and it can be increased by 282% at 200 kPa confining pressure. The calcium oxide in the red clay reacted with the water in the soil to form calcium hydroxide. The alkaline environment will erode the edge of the soil particles and cause pores to become larger. SEM observed that the addition of calcium oxide caused the agglomeration of soil particles and changed the pore structure of red clay. Low-field NMR showed that calcium oxide had a significant effect on the pore structure of red clay. Calcium oxide increased the total pore volume of the soil sample. Calcium oxide had a substantial influence on the three pore distribution ranges, I (1.1–11 µm), II (15–137 µm), and III (137–512 µm). The porosity fitted with shear strength, it negatively correlated with I and III, and positively correlated with II.

Detection of urinary podocytes by flow cytometry in idiopathic membranous nephropathy

Idiopathic membranous nephropathy (iMN) is considered an immune-mediated disease where circulating autoantibodies against podocyte targets (mainly the PLA2R) cause the deposition of in-situ subepithelial immune-complexes. The consequent podocyte damage may cause cell detachment in urine (Podocyturia-PdoU). PdoU has been assessed in different kidney diseases, but limited data are available in iMN. In this study all patients with a diagnosis of iMN between 15/12/1999–16/07/2014 were tested for PLA2R antibodies (Ab anti-PLA2R, ELISA kit) and PdoU by flow cytometry with anti-podocalyxin antibody. A semi-quantitative PdoU score was defined according to the percentage of podocalyxin positive cells normalized to the total volume of sample and set relative to the urine creatinine measured in the supernatant. PdoU was positive in 17/27 patients (63%; 1+ score in 6/27—22.2%, 2+ in 4/27—14.8%, 3+ in 2/27—7.4%, 4+ in 5/27—18.5%). Only 2/7 patients with complete remission showed a positive PdoU (1+) while all six patients without remission have significant PdoU. PdoU+ was statistically correlated with the absence of remission and Ab anti-PLA2R + (p < 0.05) but PdoU, analysed as a continuous variable, showed a non-linear correlation with proteinuria or PLA2R antibody levels also in the cohort of patients with two available PdoU tests. In conclusion, PdoU could be detected in iMN and seems to be associated with commonly considered markers of disease activity (proteinuria and Ab anti-PLA2R) with a non-linear correlation. Despite data should be confirmed in large and prospective cohorts, according to the podocyte depletion hypothesis PdoU may represent an early marker of immunological activation with potential prognostic utility.

Multiple Approaches to Quantifying the Effective Porosity of Lacustrine Shale Oil Reservoirs in Bohai Bay Basin, East China

An effective porosity is defined as the ratio of volume of interconnected pore space to total volume of a porous sample. It controls the magnitude of fluid flow and is a key parameter in the assessment of recoverable resources. However, its accurate measurement in tight formations is challenging, due to their complex pore structure and lithofacies heterogeneity. In this study, porosities of sixteen lacustrine shale samples from the second Member of the Kongdian Formation (Ek2) in the Cangdong Sag, Bohai Bay Basin were measured and compared using multiple methods and sample sizes to compare and contrast the effective porosity results. The methods included helium pycnometry (HP; cubes of 1 cm3 and grains at 500-841 μm), water immersion porosimetry (WIP; cubes), mercury intrusion porosimetry (MIP; cubes), and nuclear magnetic resonance (NMR; cubes). Finally, samples were completely sealed using paraffin for bulk density measurements to evaluate the extent of potential clay swelling in shale samples involving probing fluids. Results from the HP, WIP, and MIP methods for skeletal density, bulk density, and effective porosity with cubic samples were compared. While very similar skeletal densities were found for all three methods, a lower bulk density, and therefore lower porosity, from the MIP approach can be attributed to the experimental conditions (e.g., vacuum efficiency, applied pressure, wettability of water/helium vs. mercury) and the probable presence of pores with diameters larger than 50 μm not measurable by MIP. Furthermore, the HP porosity of granular samples with 500-841 μm grain sizes can be regarded as approaching the total porosity. The complicated relationship between WIP and NMR porosities may result from the heat-induced volatilization of moisture in pores during NMR tests, and countercurrent imbibition of water replacing the residual hydrocarbons during the saturation process for sample preparation in both tests. The swelling behavior of the lacustrine Ek2 shale with water is not significant because of the low content of expansive clay minerals. In summary, the WIP and HP methods are recommended for effective porosity measurement, whereas the NMR and MIP methods are invaluable for the measurement of pore-size distribution, with additional information on the effective porosity.

Yield Stress Enhancement of a Ternary Colloidal Suspension via the Addition of Minute Amounts of Sodium Alginate to the Interparticle Capillary Bridges.

Capillary suspensions are ternary solid-liquid-liquid systems produced via the addition of a small amount of secondary fluid to the bulk fluid that contained the dispersed solid particles. The secondary fluid could exert strong capillary forces between the particles and dramatically change the rheological properties of the suspension. So far, research has focused on capillary suspensions that consist of additive-free fluids, whereas capillary suspensions with additives, particularly those of large molecular weight that are highly relevant for industrial purposes, have been relatively less studied. In this study, we performed a systematic analysis of the properties of capillary suspensions that consist of paraffin oil (bulk phase), water (secondary phase), and α-Al2O3 microparticles (particle phase), in which the aqueous secondary phase contained an important eco-friendly polymeric binder, sodium alginate (SA). It was determined that the yield stress of the suspension increased significantly with the increase in the SA content in the aqueous secondary phase, which was attributed to the synergistic effect of the capillary force and hydrogen bonding force that may be related to the increase in the number of capillary bridges. The amounts of SA used to induce a significant change in the yield stress in this study were very small (<0.02% of the total sample volume). The addition of Ca2+ ions to the SA-containing secondary phase further increased the yield stress with possible gelation of the SA chains-in the presence of excess Ca2+ ions, however, the yield stress decreased because of the microscopic phase separation that occurred in the aqueous secondary phase. The microstructures of the sintered porous materials that were produced by using capillary suspensions as precursors were qualitatively well correlated to the rheological behavior of the precursor suspensions, suggesting a new method for the subtle control of the microstructures of porous materials using the addition of minute amounts of polymeric additives.

Determination of citalopram in fish brain tissue: benefits of coupling laser diode thermal desorption with low- and high-resolution mass spectrometry.

Recent state-of-the-art methods developed for the analysis of polar xenobiotics from different types of biological matrices usually employ liquid chromatography with mass spectrometry. However, there are limitations when a small amount of sample mass is available. For example, individual benthic invertebrates or fish tissue samples often weigh less than 100mg (e.g., brain, liver) but are necessary to understand environmental fate and bioaccumulation dynamics. We developed ultra-fast methods based on a direct sample introduction technique. This included coupling laser diode thermal desorption with atmospheric pressure chemical ionization mass spectrometry (LDTD-APCI-MS). We then quantitated a common selective serotonin reuptake inhibitor (citalopram) in brain tissues of individual juvenile fish after in vivo exposure to environmentally relevant concentration. Two mass spectrometric methods based on low (LDTD-APCI-triple quadrupole (QqQ)-MS/MS) and high (LDTD-APCI-high-resolution product scan (HRPS)) resolutions were developed and evaluated. Individual instrument conditions were optimized to achieve an accurate and robust analytical method with minimum sample preparation requirements. We achieved very good recovery (97-108%) across the range of 1-100ngg-1 for LDTD-APCI-HRPS. LDTD-APCI-QqQ-MS/MS showed poorer performance due to interferences from the matrix at the lowest concentration level. LDTD-APCI ionization was successfully validated for analysis of non-filtered sample extracts. Evaluation of final methods was performed for a set of real fish brain samples, including comparison of LDTD-APCI-HRPS with a previously validated LC-heated electrospray ionization-HRPS method. This new LDTD-APCI-HRPS method avoids the chromatographic step and provides important benefits such as analysis of limited sample masses, lower total sample volume (typically μL), and reduction in analysis time per sample run to a few seconds. Graphical abstract.

On an algorithm for generating inhomogeneous porous media

In the article, the results of the development of an algorithm for creating inhomogeneous three-dimensional porous media based on stochastic fractals are presented. The issues of estimating the parameters of porosity of the media are considered. It has been shown that the fractal dimension of porous media is preserved for any slice scales and the size of the fractal grid used to build porous media, as well as the fractal dimension of matrices and porous media depends on the binary filtration parameter that forms the given porosity by changing the ratio of matrices, open and closed pores, to the total sample volume.

The analysis of dissolved inorganic carbon in liquid using a microfluidic conductivity sensor with membrane separation of CO2

Autonomous continuous analysis of oceanic dissolved inorganic carbon (DIC) concentration with depth is of great significance with regard to ocean acidification and climate change. However, miniaturisation of in situ analysis systems is hampered by the size, cost and power requirements of traditional optical instrumentation. Here, we report a low-cost microfluidic alternative based on CO2 separation and conductance measurements that could lead to integrated lab-on-chip systems for ocean float deployment, or for moored or autonomous surface vehicle applications. Conductimetric determination of concentration, in the seawater range of 1000–3000 µmol kg−1, has been achieved using a microfluidic thin-film electrode conductivity cell and a membrane-based gas exchange cell. Sample acidification released CO2 through the membrane, reacting in a NaOH carrier, later drawn through a sub-µL conductivity cell, for impedance versus time measurements. Precision values (relative standard deviations) were ~ 0.2% for peak height measurements at 2000 µmol kg−1. Comparable precision values of ~ 0.25% were obtained using a C4D electrophoresis headstage with similar measurement volume. The required total sample and reagent volumes were ~ 500 µL for the low volume planar membrane gas exchange cell. In contrast, previous conductivity-based DIC analysis systems required total volumes between 5000 and 10,000 µL. Long membrane tubes and macroscopic wire electrodes were avoided by incorporating a planar membrane (PDMS) in the gas exchange cell, and by sputter deposition of Ti/Au electrodes directly onto a thermoplastic (PMMA) manifold. Future performance improvements will address membrane chemical and mechanical stability, further volume reduction, and component integration into a single manifold.

A conceptual model for unbiased calculations of invertebrate abundances from freeze core samples

Freeze coring is a commonly used method for the investigation of the bed sediment fauna of rivers. It is considered to produce quantitative numbers of invertebrate abundance in different depth layers. Calculations of abundance use total volume of the freeze core sample as spatial reference. This definition of sample volume is incorrect. In the present, study freeze core samples are shown to consist of two parts: (1) a cylindrical inner core in which the pore water has turned into ice and (2) all parts of sediment, which protrude from this inner core. Invertebrates are fixed only within the inner core since the protruding parts contain no pores and therefore no invertebrate habitat. In samples from gravel rivers, the protruding parts form a considerable bias, which depends on the size of the core and the coarseness of the sediment. In the present study, total volume of individual core segments varied between 97 and 248% of actual sample volume. The inner core can be measured directly to avoid the bias. The procedure is proposed for future studies to produce unbiased, comparable values of invertebrate abundance and consequently reliable data on vertical distribution.

Efficient enrichment of total flavonoids from Pteris ensiformis Burm. extracts by macroporous adsorption resins and in vitro evaluation of antioxidant and antiproliferative activities

The aim of this work is to develop an efficient and economical method for the enrichment of total flavonoids from Pteris ensiformis Burm. extracts. Resin screening, adsorption kinetics, adsorption isotherms and thermodynamics were successively researched prior to the dynamic adsorption and desorption tests. NKA-II resin was chosen as the best adsorbent, and the adsorption data were best described by the pseudo-second-order kinetics model and Langmuir isotherm model. The optimum enrichment conditions were as follows: for adsorption the total flavonoids concentration, flow rate and volume of sample were 1.84 mg/mL, 2 BV/h and 5 BV, respectively, and for desorption the flavonoids-loaded NKA-II resin column was desorbed by 7 BV of 50% ethanol at a rate of 2 BV/h. The product had a 6.63-fold higher total flavonoids content than crude extracts, and the recovery yield of total flavonoids was 80.65%. Furthermore, flavonoids-enriched extracts exhibited higher in vitro scavenging activity against superoxide anion radical and hydroxyl radical than crude extracts. In addition, higher antiproliferative activity of flavonoids-enriched extracts against MCF-7 and HepG-2 cell lines was also found as compared to the crude extracts. The developed method is appropriate for large-scale enrichment of total flavonoids from Pteris ensiformis Burm. extracts in the food and pharmaceutical industries.

Energy dependent structure of Xe ion tracks in YBCO and the effect on the superconductive properties in magnetic fields

The morphology and structure of damaged regions (tracks) produced by swift heavy 167, 77, and 46 MeV 132Xe23+ ions in YBCO-based second generation industrial high temperature superconductors wires (2G HTS) were studied using transmission electron microscopy. It was shown that ions produce tracks aligned along the ion trajectory but of morphology depending on their energy: continuous, nearly continuous, or discontinuous tracks like prolate ellipsoids of 10–15 nm in length and spheroids of 5 nm in diameter. The damaged regions of about 5 nm in diameter contain an amorphous material with a lower density compared to the pristine YBCO. The material density drops from 6300 kg m−3 for YBCO matrix to 3600 kg m−3 inside the tracks. Barium enrichment was found in the vicinity of the track at a distance of about 10 nm from the center of the defect. Ion damage tracks with different morphologies showed different effectivenesses as pinning centers. Tracks composed of discontinuous pinning centers—spheroids of about 5 nm in diameter—bring the best enhancement of the critical current density. It occurs for the lowest ion energy (46 MeV) in the range of energy loss of 8.9 keV/nm–4.7 keV/nm for Xe ions. The samples showed highest critical current densities of 56 MA/cm2 (4.2 K) and 3 MA/cm2 (77 K) in self-field, while in magnetic fields of 8 T, the values of 17 MA/cm2 (4.2 K) and 1.6 MA/cm2 (77 K) were achieved. The reduction of the superconducting volume corresponding to the amorphous radiation defects did not exceed 4% from the total sample volume.

The Development and Characterization of IFT20 knockout Mice

A primary cilium is a singular cilium on a cell that functions in cell signaling, development and homeostasis. Intraflagellar transport proteins are important for formation of primary cilia and trafficking of proteins to and from the distal tip of the cilium. In mice, tamoxifen‐inducible Cre recombinase‐mediated knockout (KO) of one of these intraflagellar transport proteins, IFT20, results in decreased formation of primary cilia and associated developmental defects. Phenotypic differences, including polydactyly, craniofacial defects, fluid accumulation, and lymphatic defects, were observed between the wildtype and the IFT20 KO, but the exact penetrance of tamoxifen on the inducible Cre‐mechanism that disrupts IFT20 was not known as the induction protocol was relatively conservative. A single dose of tamoxifen was delivered by gavage to pregnant dams at E7 and embryos were harvested and analyzed at E16.5. To infer penetrance of the IFT20 KO, we studied the penetrance of tamoxifen on dual‐reporter mTmG mouse embryos, which followed a similar mechanism as the IFT20 KO model. In this reporter system, cells constitutively expressed membrane‐bound tomato (mT) fluorescent protein; activation of the Cre enzyme in cells exposed to tamoxifen induced expression of green fluorescent protein (mG). Differential expression of mT and mG was assessed by two photon microscopy of whole mount E16.5 embryonic skin. This difference in fluorescence allowed the determination of the penetrance of recombination based on the volume of mG+ cells to the total volume of the tissue sample. The penetrance was quantified manually through ImageJ software. Quantification was difficult due to high background and bright red blood cell autofluorescence in whole mount skin samples. A series of MATLAB‐based clustering and other image analysis methods were used to improve detection of mG+ cells in these tissue samples. In related work, we have identified primary cilia on lymphatic endothelial cells for the first time both in vitro and in vivo and have developed a lymphatic‐specific inducible model of IFT20 disruption.Support or Funding InformationThe funding for this project was provided by the South Dakota State University Department of Chemistry and Biochemistry and BioSNTR of South Dakota.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Vortex- and Centrifugation-Free Extraction of HIV-1 RNA.

HIV viral load measurements play a critical role in monitoring disease progression in those who are on antiretroviral treatment. In order to obtain an accurate measurement, rapid sample preparation techniques are required. There is an unmet need for HIV extraction instruments in resource-limited settings, where HIV prevalence is high. Therefore, the objective of our study was to develop a three-dimensional (3D) microfluidic system to extract HIV-1 RNA with minimal electricity and without complex laboratory instruments. A 3D microfluidic system was designed in which magnetic beads bound with nucleic acids move through immiscible oil-water interfaces to separate HIV-1 RNA from the sample. Polymerase chain reaction (PCR) amplification was used to quantify the total amount of HIV-1 RNA extracted as we optimized the system through chip design, bead type, carry-over volume, carrier RNA concentration, and elution buffer temperature. Additionally, the extraction efficiency of the 3D microfluidic system was evaluated by comparing with a Qiagen EZ1 Advanced XL instrument using 20 HIV-1-positive plasma samples. Our method has near-perfect (100%) extraction efficiency in spiked serum samples with as little as 50copies/mL starting sample. Furthermore, we report carry-over volumes of 0.31%±0.006% of total sample volume. Using the EZ1 Advanced XL as a gold standard, the average percentage HIV-1 RNA extracted using the microchip was observed to be 65.4% ± 24.6%. From a clinical perspective, the success of our method opens up its possible use in diagnostic tests for HIV in the remote areas where access to vortexes and centrifuges is not available. Here we present a proof-of-concept device which, with further development, could be used for sample preparation at the point of care.

Representative elementary volume in limestone sample

The study of fluid flow through porous media with complex characteristics is of great relevance in applications related to exploration of oil and gas reserves. With growth of computer simulation combined with the use of reconstructed images it became increasingly common to use a representative volume, popularly known as REV. The use of a REV is necessary since the total volume of reconstructed image is too heavy to be loaded into simulation software, increasing data processing time and in some cases making total volume simulation impracticable. However, when investigating different rocks types, some main issues are addressed related to REV, such as how reliable is representative volume compared to the total sample volume. Quantitative and qualitative results such as porosity and permeability vary with the region chosen for the REV as well as configuration of pore connected vary depending on region, showing the importance in choosing a region so that REV represents total results of the sample.