Lossless Altered Histone Modification Analysis System (LAHMAS).

Cell concentration via centrifugation is a ubiquitous step in many cell culture procedures. At the macroscale, centrifugation suffers from a number of limitations, particularly when dealing with small numbers of cells (e.g., less than 50,000). On the other hand, typical microscale methods for cell concentration can affect cell physiology and bias readouts of cell behavior and function. In this paper, we present a microfluidic concentrator device that utilizes the effects of gravity to allow cells to gently settle out of a suspension into a collection region without the use of specific adhesion ligands. Dimensional analysis was performed to compare different device designs and was verified with flow modeling to optimize operational parameters. We are able to concentrate low-density cell suspensions in a microfluidic chamber, achieving a cell loss of only 1.1 ± 0.6% (SD, n = 7) with no observed loss during a subsequent cell staining protocol which incorporates ∼36 complete device volume replacements. This method provides a much needed interface between rare cell samples and microfluidic culture assays.

Introduction: The Oncomine® Cancer Panel (OCP) is designed to simultaneously detect and report hotspot mutations, Copy Number Variants (CNVs) and gene fusions in 143 genes with minimal DNA/RNA sample input. The OCP utilizes the AmpliSeq™ library preparation chemistry for the Ion Torrent™ Next Generation Sequencing (NGS) platform, in combination with data annotations by the Oncomine® Knowledgebase. Here we report analytical validation of OCP. Methods: DNA and RNA, extracted from the FFPE processed GM12878, was used as the negative control to evaluate the specificity of OCP. A RNA sample containing multiple oncogenic gene fusions, and a DNA sample containing multiple hotspot SNV and indels were used as the positive control. Fresh DNA from cancer cell lines (HCC1143 or NCI-H2122) along with DNA from matched normal cell lines, HorizonDx NGS standards TruQ-1, TruQ-2, several engineered FFPE samples with gene fusions or copy number changes, and 26 clinical FFPE samples of a variety of solid tumor types (lung, breast, colon, ovary, stomach, uterus and larynx) were used to evaluate the OCP performance. The analysis of the sequencing data was primarily performed with the OCP pipeline integrated with Oncomine® Knowledgebase from Life Technologies, supplemented by the MolecularMD proprietary pipeline. Only the genetic alterations with clinical utility were selected as the final output from the data pipeline. Of these detected by OCP, the SBS and indels were confirmed by the Illumina TruSeq, Ion Torrent AmpliSeq commercial cancer panels, or by Sanger sequencing. Any CNVs detected were confirmed by FISH, if possible, and detected fusions were confirmed by RT-PCR or FISH. Results: No clinically relevant genetic alterations were detected from the negative control FFPE-GM12878, indicating the high specificity of the OCP. High specificity was achieved, in part, using stringent filters, which removed error prone regions from analysis. The LOD for SBS and short indel detection was 5%, as assessed by TruQ-1 and TruQ-2, each containing 15 variants. While the exact LOD for gene fusion detection is currently under evaluation, the 100%, 50% and 20% fusion were detected in the RNA samples from serially diluted EML4-ALK or SL34A2-ROS1 fusion into the FFPE-GM12878 RNA. Four known gene amplifications (MYC, CCND1, MDM2, AKT1) with CNV from 5.4X to 14.5X in HCC1143, and a 15.7X MYC amplification in NCI-H2122 were detected. In addition, an EML4-ALK.E13A20 fusion was detected in a lung cancer FFPE sample, and confirmed by FISH. The analytical sensitivity and the specificity of the OCP are currently assessed with 26 clinical samples and three engineered FFPE samples. Conclusions: These results demonstrate the high sensitivity and specificity of OCP. With as little as 10 ng RNA and 20 ng DNA, OCP provides comprehensive screening of patient FFPE tumor specimens for the detection of a broad spectrum of clinically relevant genetic alterations. Citation Format: Peng Fang, Zhenyu Yan, Weihua Liu, Jennifer Biroschak, Paul Labrousse, Jennifer Wright, Cindy Spittle, Chad Galderisi, Li Jin. Oncomine® Cancer Panel: simultaneous detection of clinically relevant hotspot mutations, CNVs, and gene fusions in solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4261. doi:10.1158/1538-7445.AM2015-4261

Metabolism, thermoregulation and evaporative water loss in the Chaotung Vole (Eothenomys olitor) in Yunnan-Kweichow Plateau in summer

in an engineering capacity upon his arrival in the US, and he and his wife are now US citizens. He is currently Manager of the Engineering Development Division of the Linde Co., employing about 600 people including 200 engineers. His responsibilities include the design and construction of gas separation equipment and devices, improvement of their processes and economic analyses of the results. It was his group which developed the new improved distillation columns which have permitted extraordinary increases in through-put and efficiency in air separation plants, and which he would use in any purification system that he would design for Hanford. The important point to be emphasized in relation to the previous proposal for gas purification is that this break-through in distillation technology will now permit the almost complete removal of the last 500 parts per million of Argon to a new low level of less than 20 parts per million.

The measurement of accurate temperature profiles is critical for transport analysis and equilibrium reconstruction in the DIII-D tokamak. Recent refinements in the Michelson interferometer diagnostic have produced more precise electron temperature measurements from electron cyclotron emission and made them available for a wider range of discharge conditions. Replacement of a lens-relay with a low-loss corrugated waveguide transmission system resulted in an increase in throughput of 6 dB and a reduction of calibration error from 15% to 5%. The waveguide exhibits a small polarization scrambling fraction of 0.05 at the quarter-wavelength frequency and very stable transmission characteristics over time. Further reduction in error was realized through special signal processing of the calibration and plasma interferograms.

Simultaneous determination of the primary prostanoids prostaglandin E 2, prostaglandin F 2α and 6-oxoprostaglandin F 1α by immunoaffinity chromatography in combination with negative ion chemical ionization gas chromatography—tandem mass spectrometry

Single‐source energy harvesters that convert solar, thermal, or kinetic energy into electricity for small‐scale smart electronic devices and wireless sensor networks have been under development for decades. When an individual energy source is insufficient for the required electricity generation, multi‐source energy harvesting is indicated. Current technology usually combines different individual harvesters to achieve the capability of harvesting multiple energy sources simultaneously. However, this increases the overall size of the multi‐source harvester, but in microelectronics miniaturization is a critical consideration. Herein, an advanced approach is demonstrated to solve this issue. A single‐material energy harvesting/sensing device is fabricated using a (K0.5Na0.5)NbO3‐Ba(Ni0.5Nb0.5)O3–Δ (KNBNNO) ceramic as the sole energy‐conversion component. This single‐material component is able simultaneously to harvest or sense solar (visible light), thermal (temperature fluctuation), and kinetic (vibration) energy sources by incorporating its photovoltaic, pyroelectric, and piezoelectric effects, respectively. The interactions between different energy conversion effects, e.g., the influence of dynamic behavior on the photovoltaic effect and alternating current–direct current (AC–DC) signal trade‐offs, are assessed and discussed. This research is expected to stimulate energy‐efficient design of electronic devices by integrating both harvesting and sensing functions in the same material/component.

Genomic resources for Platyhelminthes of the class Monogenea are scarce, despite the diversity of these parasites, some species of which are highly pathogenic to their fish hosts. This work aimed to generate de novo-assembled transcriptomes of two monogenean species, Scutogyrus longicornis (Dactylogyridae) and Rhabdosynochus viridisi (Diplectanidae), providing a protocol for cDNA library preparation with low input samples used in single cell transcriptomics. This allowed us to work with sub-microgram amounts of total RNA with success. These transcriptomes consist of 25,696 and 47,187 putative proteins, respectively, which were further annotated according to the Swiss-Prot, Pfam, GO, KEGG, and COG databases. The completeness values of these transcriptomes evaluated with BUSCO against Metazoa databases were 54.1% and 73%, respectively, which is in the range of other monogenean species. Among the annotations, a large number of terms related to G-protein-coupled receptors (GPCRs) were found. We identified 109 GPCR-like sequences in R. viridisi, and 102 in S. longicornis, including family members specific for Platyhelminthes. Rhodopsin was the largest family according to GRAFS classification. Two putative melatonin receptors found in S. longicornis represent the first record of this group of proteins in parasitic Platyhelminthes. Forty GPCRs of R. viridisi and 32 of S. longicornis that were absent in Vertebrata might be potential drug targets. The present study provides the first publicly available transcriptomes for monogeneans of the subclass Monopisthocotylea, which can serve as useful genomic datasets for functional genomic research of this important group of parasites.

Tandem mass tags (TMT) and tribrid mass spectrometers are a powerful combination for high-throughput proteomics with high quantitative accuracy. Increasingly, this technology is being used to map the effects of drugs on the proteome. However, the depth of proteomic profiling is still limited by sensitivity and speed. The new Orbitrap Ascend mass spectrometer was designed to address these limitations with a combination of hardware and software improvements. We evaluated the performance of the Ascend in multiple contexts including deep proteomic profiling. We found that the Ascend exhibited increased sensitivity, yielding higher signal-to-noise ratios than the Orbitrap Eclipse with shorter injection times. As a result, higher numbers of peptides and proteins were identified and quantified, especially with low sample input. TMT measurements had significantly improved signal-to-noise ratios, improving quantitative precision. In a fractionated 16plex sample that profiled proteomic differences across four human cell lines, the Ascend was able to quantify hundreds more proteins than the Eclipse, many of them low-abundant proteins, and the Ascend was able to quantify >8000 proteins in 30% less instrument time. We used the Ascend to analyze 8881 proteins in HCT116 cancer cells treated with covalent sulfolane/sulfolene inhibitors of peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1), a phosphorylation-specific peptidyl-prolyl cis-trans isomerase implicated in several cancers. We characterized these PIN1 inhibitors' effects on the proteome and identified discrepancies among the different compounds, which will facilitate a better understanding of the structure-activity relationship of this class of compounds. The Ascend was able to quantify statistically significant, potentially therapeutically relevant changes in proteins that the Eclipse could not detect.

Zwitter-ionic monolith-based spintip column coupled with Evosep One liquid chromatography for high-throughput proteomic analysis

Short-beaked echidnas, Tachyglossus aculeatus, are heterothermic, prototherian mammals whose distribution extends across Australia and parts of New Guinea. They are reputed to be poor physiological thermoregulators in the heat as they lack the evaporative cooling mechanisms associated with other mammals and can tolerate ambient temperatures in excess of 35 oC to 37 oC for only short periods in laboratory experiments. However, in the field in western Queensland, they routinely tolerate ambient temperatures in some of their daytime retreats that exceed 35 oC for several hours and peak at 42 oC. In other, and colder, situations, except when incubating, body temperatures of echidnas vary on a daily basis, increasing with activity and decreasing with inactivity. They appear to be fully coordinated even with body temperatures as low as 20 oC. This suggests that the classical model of mammalian thermoregulation, assuming as it does,the maintenance of high and stable body temperatures, is of little relevance to non-incubating echidnas.The primary aim of this project was to investigate physiological adjustments that allow echidnas to tolerate ambient temperatures in the field that would be lethally high in laboratories. The secondary aim of this project was to develop simple heat budget modelling that could be used to analyse physiological adjustments and responses in animals with significant thermal inertia, endothermy and heterothermy in complex, dynamically fluctuating (field) thermal environments. The third aim of the project was to investigate the relevance of traditional concepts about mammalian thermoregulation to echidnas within the context of their heterothermy.The first aim was addressed by experiments conducted under field conditions where metabolic rate, evaporative heat loss, thermal conductance and body temperature were determined in response to ambient temperature fluctuations similar to those experienced by echidnas in the wild. It was found that echidnas in these conditions utilised the low ambient temperatures of the early morning to lose body heat passively to their environment which then gave them scope to absorb heat as ambient temperatures increased during the day. In this way, resting echidnas tolerated ambient temperatures that peaked in the range 30 oC to 49 oC as long as their body temperatures remained below 33 oC: slightly higher than the modal body temperatures of foraging echidnas. Because body temperatures rose only slowly in these relatively large animals, echidnas were frequently unperturbed by experimental conditions until after ambient temperatures had reached their daytime peak.The second aim of the project was addressed by developing integrated parameter, a priori heat budget modelling to predict body temperature responses of animals in general to combined variations of metabolic heat production, thermal conductance and other physiological factors in dynamically fluctuating (field) thermal environments. This modelling was found to predict well the body temperature fluctuations in a wide range of ectotherms and endotherms of various sizes in various environments. When applied todata collected from echidnas under field experimental conditions, this modelling highlighted the importance of maintaining low thermal conductance and low metabolic heat production in the ability of echidnas to tolerate high ambient temperatures. It also showed that there was no evidence of aestivation or metabolic suppression in echidnas exposed to a high heat load while sheltering inside artificial retreats.The third aim of the project was addressed by experiments conducted under laboratory conditions where thermoregulatory responses of echidnas to constant ambient temperatures in the range 13 oC to 31 oC were determined. In these, similarities and differences between echidna and other mammalian thermoregulation were found. In common with many mammals, echidnas varied their thermal conductance from low levels in low ambient temperatures to higher levels in warmer conditions. Thermoneutrality was difficult to identify precisely but metabolic rate was minimal at ambient temperatures of between 25 oC and 28 oC and increased at higher temperatures. Below ambient temperatures of 25 oC, echidnas sometimes demonstrated shivering thermogenesis and sometimes torpor, both common responses in other mammals. However, unusually for an endotherm, echidnas also demonstrated varying metabolic rates intermediate to these two extremes. This resulted in variable or labile body temperatures with this variability increasing at lower ambient temperatures. Also unusual for an endotherm, echidnas continue to show apparently normal activity and behaviour with body temperatures as low as 20 oC without becoming torpid.These results suggest that echidnas have alternative thermoregulatory strategies to many mammals. Thermogenesis and evaporative cooling are usually associated with mammals as physiological mechanisms resulting in the maintenance of high and stable body temperatures. However echidnas are not only capable of tolerating fluctuations in body temperature, they use them to advantage. On the one hand, they use cyclic storage of heat to tolerate short term exposure to high ambient temperatures in some day time retreats without the need for cooling mechanisms which require energy. On the other hand, in cooler conditions, they frequently dispense with energetically expensive shivering thermogenesis, allowing their body temperature to fall as a result, apparently without compromise to their foraging activities. This energy economy likely gives to echidnas advantages unavailable to many mammals and may allow them to persist in relatively resource poor environments.

In this paper, the design of distributed arithmetic (DA) finite impulse response (FIR) filter using block least mean square algorithm (BLMS) based on systolic array architecture with parallel data processing units is presented. A high level of parallelism is observed in the proposed work, improving the efficiency of variable coefficient FIR structure. The parallel look-up tables (LUT) in combination with the shift accumulator emulate multiply and accumulate operations. In order to reduce the number of clock cycles, B parallel LUTs are used, where B is the coefficient size. The block processing in BLMS Adaptive FIR Filter of block length L gives L times high throughput. This structure accepts block of input samples and generates block of output in each clock cycle. It requires less number of registers for computing filter output response and weight increment vector as memory reuse concept is used for implementing registers. This structure doesn’t require multiplexer. The designed DA based FIR filter is implemented on FPGA. The implementation results shows that the manuscript presents a high speed and low power architecture. The proposed structure provides 17.3 times less power and 9.5 times increase in throughput when compared to existing designs.

The interest on outdoor photocatalytic materials is growing in the last years. Nevertheless, most of the experimental devices designed for the assessment of their performance operate at controlled laboratory conditions, i.e., pollutant concentration, temperature, UV irradiation, and water vapor contents, far from those of real outdoor environments. The aim of the present study was the design and development of an experimental device for the continuous test of photocatalytic outdoor materials under sun irradiation using real outdoor air as feed, with the concomitant fluctuation of pollutant concentration, temperature, and water vapor content. A three-port measurement system based on two UV-transparent chambers was designed and built. A test chamber contained the photoactive element and a reference chamber to place the substrate without the photoactive element were employed. The third sampling point, placed outdoors, allowed the characterization of the surrounding air, which feeds the test chambers. Temperature, relative humidity (RH), and UV-A irradiance were monitored at each sampling point with specific sensors. NO x concentration was measured by a chemiluminescence NO x analyzer. Three automatic valves allowed the consecutive analysis of the concentration at the three points at fixed time intervals. The reliability of the analytical system was demonstrated by comparing the NO x concentration data with those obtained at the nearest weather station to the experimental device location. The use of a chamber-based reaction system leads to an attenuation of NO x and atmospheric parameter profiles, but maintaining the general trends. The air characterization results showed the wide operating window under which the photoactive materials should work outdoors, depending on the traffic intensity and the season, which are reproduced inside the test chambers. The designed system allows the measurement of the photoactivity of outdoor materials or the comparison of several samples at the same time. The suitability of the system for the evaluation of the DeNO x properties of construction elements at realistic outdoor conditions was demonstrated. The designed experimental device can be used 24/7 for testing materials under real fluctuations of NO x concentration, temperature, UV irradiation, and relative humidity and the presence of other outdoor air pollutants such as VOCs, SO x , or NH3. The chamber-based design allows comparing a photocatalytic material with respect to a reference substrate without the photoactive phase, or even the comparison of several outdoor elements at the same time.

Chapter 13 - Strategies to Enhance Enzyme Activity for Industrial Processes in Managing Agro-Industrial Waste

SYNOPSIS. Terrestrial and semi-terrestrial crustaceans are exposed to fluctuations in ambient temperature and conditions that favor evaporative water loss. These environmental stresses alter performance limits in the laboratory and behavior in the field. The maximal rate of oxygen consumption, maximum aerobic speed, and endurance capacity are greater at a body temperature (Tb) of 24°C than at 15°C or 30°C in the ghost crab, Ocypode quadrata . The total metabolic cost to move at the same relative speed is greater at a Tb of 24°C than at 15°C. Slower aerobic kinetics at 15°C result in a smaller relative contribution of oxidative metabolism to total metabolic cost. However, the relative contributions from accelerated glycolysis are similar at both temperatures. When locomotion is intermittent, the total distance traveled before fatigue can be similar at Tbs of 15 and 24°C but result from different movement and pause durations at these temperatures. Performance limits of the ghost crab are negatively affected by dehydration and are sensitive to rates of water loss. In the laboratory, endurance capacity of the fiddler crab, Uca pugilator, is greater at a Tb of 30°C than at 25°C. In the field, freely moving fiddler crabs with a Tb of 30°C travel at faster mean preferred speeds, as determined by motion analysis, than crabs at 25°C. Data for land crabs support and advance general ectothermic models for the effects of temperature and dehydration on locomotor performance.