Optimization and calibration of 384-well kinetic Ca mobilization assays for the human transient receptor potential cation channels TRPM8, TRPV1, and TRPA1.

Development, optimization, and calibration of human transient receptor potential (TRP) channel Ca mobilization assays for TRPM8, TRPV1, and TRPA1 are described. Heterologous expression of hTRPM8 in HEK293T cells was required for anti-TRPM8 antibody staining and TRPM8 agonist induced Ca mobilization signals which were both used to optimize transfection efficiency. FLIPR Calcium 6 dye concentration, loading time, and TRPM8 transfected cell seeding density were optimized and a DMSO tolerance of ≤0.

A review of the emerging technologies and systems to mitigate food fraud in supply chains.

Food fraud has serious consequences including reputational damage to businesses, health and safety risks and lack of consumer confidence. New technologies targeted at ensuring food authenticity has emerged and however, the penetration and diffusion of sophisticated analytical technologies are faced with challenges in the industry. This review is focused on investigating the emerging technologies and strategies for mitigating food fraud and exploring the key barriers to their application.

Subcutaneous administration of a novel TRPM8 antagonist reverses cold hypersensitivity while attenuating the drop in core body temperature.

Background And Purpose: We extend the characterization of the TRPM8 antagonist VBJ103 with tests of selectivity, specificity and distribution, therapeutic efficacy of systemic administration against oxaliplatin-induced cold hyperalgesia and the impact of systemic administration on core body temperature (CBT).

Experimental Approach: Selectivity at human TRPA1 and TRPV1 as well as in vitro safety profiling was determined. Effects of systemic administration of VBJ103 were evaluated in a model of oxaliplatin-induced cold hyperalgesia.

Replacement of the hydroxamic acid group in the selective HDAC8 inhibitor PCI-34051.

PCI-34051 is a valuable tool to interrogate the therapeutic effects of selective inhibition of HDAC8. However, it has not advanced to clinical trials, perhaps due to poor PK or off-target effects. We hypothesized that the presence of a hydroxamic acid (HA) group in PCI-34051 contributed to its lack of advancement.

WITHDRAWN: Detection and impact of hypoxic regions in multicellular tumor spheroid cultures formed by head and neck squamous cell carcinoma cells lines.

The Publisher regrets that this article is an accidental duplication of an article previously published at http://dx.doi.org/10.

Investigating how nitrogen nutrition and pruning impacts on CBD and THC concentration and plant biomass of Cannabis sativa.

Precise crop fertilization requires an in-depth understanding of plant uptake and utilisation to optimise sustainable production. This study investigated the influence of nitrogen (N) nutrition and pruning on the cannabinoid concentrations and biomass of a commercial cannabis cultivar; the rationale for this study is how N supply and pruning affect cannabinoid yields and concentration in a commercial setting. Clones of a Cannabis sativa L.

Characterization of allosteric modulators that disrupt androgen receptor co-activator protein-protein interactions to alter transactivation-Drug leads for metastatic castration resistant prostate cancer.

Three series of compounds were prioritized from a high content screening campaign that identified molecules that blocked dihydrotestosterone (DHT) induced formation of Androgen Receptor (AR) protein-protein interactions (PPIs) with the Transcriptional Intermediary Factor 2 (TIF2) coactivator and also disrupted preformed AR-TIF2 PPI complexes; the hydrobenzo-oxazepins (S1), thiadiazol-5-piperidine-carboxamides (S2), and phenyl-methyl-indoles (S3). Compounds from these series inhibited AR PPIs with TIF2 and SRC-1, another p160 coactivator, in mammalian 2-hybrid assays and blocked transcriptional activation in reporter assays driven by full length AR or AR-V7 splice variants. Compounds inhibited the growth of five prostate cancer cell lines, with many exhibiting differential cytotoxicity towards AR positive cell lines.

Direct selection of functional fluorescent-protein antibody fusions by yeast display.

Antibodies are important reagents for research, diagnostics, and therapeutics. Many examples of chimeric proteins combining the specific target recognition of antibodies with complementing functionalities such as fluorescence, toxicity or enzymatic activity have been described. However, antibodies selected solely on the basis of their binding specificities are not necessarily ideal candidates for the construction of chimeras.

The Effect of the Height of Coppicing and Harvest Season on the Yield and Quality of the Essential Oil of .

is a small shrub belonging to the family and the leaves are steam-distilled to produce a therapeutically active essential oil. With production moving from wild-harvested to orchardised stands, there is a need for harvest management of kunzea oil. This study compared the regrowth, essential oil content and composition of plants after harvesting vegetative material to a depth of 0.

Sap flow of sweet cherry reveals distinct effects of humidity and wind under rain covered and netted protected cropping systems.

Protected cropping systems (PCS) alter the plant growing environment, though understanding of this in ventilated systems and how the new climate affects tree water uptake is limited. Sap flow sensors and weather stations were deployed in 16-year-old 'Lapins' on 'Colt' rootstock cherry trees under a ventilated Voen PCS and in an adjacent bird netted PCS. Average and maximum temperatures were consistently higher (14.

Orchard microclimate, tree water uptake and sweet cherry fruit quality under protected cropping.

Protected cropping systems (PCS) de-risk adverse climatic effects in intensive horticultural production but alter the growing environment. The objectives of this study were to investigate the effects of modern, commercial-scale PCS on sweet cherry orchard microclimate, tree water uptake and fruit quality. Sap flow sensors and weather stations were positioned at four locations under a 21 ha PCS at varying elevations (125, 114, 111, 102 m above sea level) and distances from the block boundary (105, 75, 60 or 50 m, referred to hereafter as Locations 1 to 4, respectively).

Algorithmic improvements for discovery of germline copy number variants in next-generation sequencing data.

Background: Copy number variants (CNVs) play a significant role in human heredity and disease. However, sensitive and specific characterization of germline CNVs from NGS data has remained challenging, particularly for hybridization-capture data in which read counts are the primary source of copy number information.

Results: We describe two algorithmic adaptations that improve CNV detection accuracy in a Hidden Markov Model (HMM) context.

Validation of HDAC8 Inhibitors as Drug Discovery Starting Points to Treat Acute Kidney Injury.

Acute kidney injury (AKI), a sudden loss of kidney function, is a common and serious condition for which there are no approved specific therapies. While there are multiple approaches to treat the underlying causes of AKI, no targets have been clinically validated. Here, we assessed a series of potent, selective competitive inhibitors of histone deacetylase 8 (HDAC8), a promising therapeutic target in an AKI setting.

Protoplast fusion in Bacillus species produces frequent, unbiased, genome-wide homologous recombination.

In eukaryotes, fine-scale maps of meiotic recombination events have greatly advanced our understanding of the factors that affect genomic variation patterns and evolution of traits. However, in bacteria that lack natural systems for sexual reproduction, unbiased characterization of recombination landscapes has remained challenging due to variable rates of genetic exchange and influence of natural selection. Here, to overcome these limitations and to gain a genome-wide view on recombination, we crossed Bacillus strains with different genetic distances using protoplast fusion.

Detection and impact of hypoxic regions in multicellular tumor spheroid cultures formed by head and neck squamous cell carcinoma cells lines.

In solid tumors like head and neck cancer (HNC), chronic and acute hypoxia have serious adverse clinical consequences including poorer overall patient prognosis, enhanced metastasis, increased genomic instability, and resistance to radiation-, chemo-, or immuno-therapies. However, cells in the two-dimensional monolayer cultures typically used for cancer drug discovery experience 20%-21% O levels (normoxic) which are 4-fold higher than O levels in normal tissues and ≥10-fold higher than in the hypoxic regions of solid tumors. The oxygen electrodes, exogenous bio-reductive markers, and increased expression of endogenous hypoxia-regulated proteins like HIF-1α generally used to mark hypoxic regions in solid tumors are impractical in large sample numbers and longitudinal studies.

Improvements in Smartphone and Night Vision Imaging Technologies Enable Low Cost, On-Site Assays of Bioluminescent Cells.

Technologies enabling on-site environmental detection or medical diagnostics in resource-limited settings have a strong disruptive potential compared to current analytical approaches that require trained personnel in laboratories with immobile, resource intensive instrumentation. Handheld devices, such as smartphones, are now routinely produced with CPUs, RAM, wireless data transfer capabilities, and high-resolution complementary metal oxide semiconductor (CMOS) cameras capable of supporting the capture and processing of bioluminescent signals. In theory, combining the capabilities of these devices with continuously bioluminescent human cell-based bioreporters would allow them to replicate the functionality of more expensive, more complex, and less flexible platforms while supporting human-relevant conclusions.

Screening for androgen agonists using autonomously bioluminescent HEK293 reporter cells.

Due to the public health concerns of endocrine-disrupting chemicals, there is an increasing demand to develop improved high-throughput detection assays for enhanced exposure control and risk assessment. A substrate-free, autobioluminescent HEK293 assay was developed to screen compounds for their ability to induce androgen receptor (AR)-mediated transcriptional activation. The assay was validated against a group of 40 recommended chemicals and achieved an overall 87.

Focus on the role of seed tannins and pectolytic enzymes in the color development of Pinot noir wine.

Maceration techniques which promote the extraction of color pigments and tannin from grapes are often sought in Pinot noir winemaking to optimise color stability; alternatively, exogenous grape tannins may be included during fermentation. To examine the effect of seed-derived tannins and the use of pectolytic enzymes on color development in wines, conventional must preparations of L. cv Pinot noir grapes were compared with wines made using a supplementary addition of either a commercial seed tannin product or previously fermented seeds, while in a complementary experiment, seeds were sequentially removed during fermentation.

Nitrogen Use Efficiency, Allocation, and Remobilization in Apple Trees: Uptake Is Optimized With Pre-harvest N Supply.

Optimizing the utilization of applied nitrogen (N) in fruit trees requires N supply that is temporally matched to tree demand. We investigated how the timing of N application affected uptake, allocation, and remobilization within 14-year-old "Gala"/M26 apple trees ( Borkh) over two seasons. In the 2017-2018 season, 30 g N tree of 5.

Cross-oncopanel study reveals high sensitivity and accuracy with overall analytical performance depending on genomic regions.

Background: Targeted sequencing using oncopanels requires comprehensive assessments of accuracy and detection sensitivity to ensure analytical validity. By employing reference materials characterized by the U.S.

Evaluating the analytical validity of circulating tumor DNA sequencing assays for precision oncology.

Circulating tumor DNA (ctDNA) sequencing is being rapidly adopted in precision oncology, but the accuracy, sensitivity and reproducibility of ctDNA assays is poorly understood. Here we report the findings of a multi-site, cross-platform evaluation of the analytical performance of five industry-leading ctDNA assays. We evaluated each stage of the ctDNA sequencing workflow with simulations, synthetic DNA spike-in experiments and proficiency testing on standardized, cell-line-derived reference samples.

Construction, characterization and crystal structure of a fluorescent single-chain Fv chimera.

In vitro display technologies based on phage and yeast have a successful history of selecting single-chain variable fragment (scFv) antibodies against various targets. However, single-chain antibodies are often unstable and poorly expressed in Escherichia coli. Here, we explore the feasibility of converting scFv antibodies to an intrinsically fluorescent format by inserting the monomeric, stable fluorescent protein named thermal green, between the light- and heavy-chain variable regions.