Population structure and linkage disequilibrium in Lupinus albus L. germplasm and its implication for association mapping

White lupin (Lupinus albus L.) has been around since 300 B.C. and is recognized for its ability to grow on poor soils and application as green manure in addition to seed harvest. The seed has very high levels of protein (33-47 %) and oil (6-13 %). It also has many secondary metabolites that are potentially of nutraceutical value to animals and humans. Despite such a great potential, lupins role in modern agriculture began only in the twentieth century. Although a large collection of Lupinus germplasm accessions is available worldwide, rarely have they been genetically characterized. Additionally, scarce genomic resources in terms of recombinant populations and genome information have been generated for L. albus. With the advancement in association mapping methods, the natural populations have the potential to replace the recombinant populations in gene mapping and marker-trait associations. Therefore, we studied the genetic similarity, population structure and marker-trait association in a USDA germplasm collection for their current and future application in this crop improvement. A total of 122 PI (Plant Inventory) lines were screened with 18 AFLP primer pairs that generated 2,277 fragments. A subset of 892 polymorphic markers with MAF >0.05 (minor allele frequency) were used for association mapping. The cluster analysis failed to group accessions on the basis of their passport information, and a weak structure and low linkage disequilibrium (LD) were observed indicating the usefulness of the collection for association mapping. Moreover, we were also able to identify two markers (a p value of 1.53 × 10(-4) and 2.3 × 10(-4)) that explained 22.69 and 20.5 % of seed weight variation determined using R (LR) (2) . The implications of lack of geographic clustering, population structure, low LD and the ability of AFLP to map seed weight trait using association mapping and the usefulness of the PI collections in breeding programs are discussed.     

Effect of mixing on reaction-diffusion kinetics for protein hydrogel-based microchips

Protein hydrogel-based microchips are being developed for high-throughput evaluation of the concentrations and activities of various proteins. To shorten the time of analysis, the reaction-diffusion kinetics on gel microchips should be accelerated. Here we present the results of the experimental and theoretical analysis of the reaction-diffusion kinetics enforced by mixing with peristaltic pump. The experiments were carried out on gel-based protein microchips with immobilized antibodies under the conditions utilized for on-chip immunoassay. The dependence of fluorescence signals at saturation and corresponding saturation times on the concentrations of immobilized antibodies and antigen in solution proved to be in good agreement with theoretical predictions. It is shown that the enhancement of transport with peristaltic pump results in more than five-fold acceleration of binding kinetics. Our results suggest useful criteria for the optimal conditions for assays on gel microchips to balance high sensitivity and rapid fluorescence saturation kinetics.     

Endurance training without weight loss lowers systemic, but not muscle, oxidative stress with no effect on inflammation in lean and obese women

Obesity is associated with oxidative stress. Endurance training (ET) in healthy individuals increases antioxidant enzyme activity and decreases oxidative stress, whereas its effects on oxidative status in obese humans have yet to be determined. We investigated the effects of obesity and ET on markers of oxidative stress, antioxidant defense, and inflammation. Obese (n=12) and lean (n=12) women underwent 12 weeks of ET with blood, 24-h urine, and muscle biopsies collected prior to and following training for determination of oxidative stress (urinary 8-hydroxy-2-deoxyguanosine and 8-isoprostanes, muscle protein carbonyls, and 4-hydroxynonenal), antioxidant enzyme protein content (muscle CuZnSOD, MnSOD, and catalase), and inflammation (C-reactive protein, leptin, adiponectin, interleukin-6). Obese women had elevated urinary 8-hydroxy-2-deoxyguanosine (P=0.03), muscle protein carbonyls (P=0.03), and 4-hydroxynonenal (P<0.001); serum C-reactive protein (P=0.01); and plasma leptin (P=0.0001) and interleukin-6 (P=0.03). ET decreased urinary 8-hydroxy-2-deoxyguanosine (P=0.006) and 8-isoprostanes (P=0.02) in all subjects and CuZnSOD protein content (P=0.04) in obese women, in the absence of changes in body weight or composition. ET without weight loss decreases systemic oxidative stress, but not markers of inflammation, in obese women.     

A high-content chemical screen identifies ellipticine as a modulator of p53 nuclear localization

p53 regulates apoptosis and the cell cycle through actions in the nucleus and cytoplasm. Altering the subcellular localization of p53 can alter its biological function. Therefore, small molecules that change the localization of p53 would be useful chemical probes to understand the influence of subcellular localization on the function of p53. To identify such molecules, a high-content screen for compounds that increased the localization of p53 to the nucleus or cytoplasm was developed, automated, and conducted. With this image-based assay, we identified ellipticine that increased the nuclear localization of GFP-mutant p53 protein but not GFP alone in Saos-2 osteosarcoma cells. In addition, ellipticine increased the nuclear localization of endogenous p53 in HCT116 colon cancer cells with a resultant increase in the transactivation of the p21 promoter. Increased nuclear p53 after ellipticine treatment was not associated with an increase in DNA double stranded breaks, indicating that ellipticine shifts p53 to the nucleus through a mechanism independent of DNA damage. Thus, a chemical biology approach has identified a molecule that shifts the localization of p53 and enhances its nuclear activity. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. G. Wei Xu and Imtiaz A. Mawji have contributed equally to this work.     

Identification of oxidative stress and Toll-like receptor 4 signaling as a key pathway of acute lung injury

Multiple lung pathogens such as chemical agents, H5N1 avian flu, or SARS cause high lethality due to acute respiratory distress syndrome. Here we report that Toll-like receptor 4 (TLR4) mutant mice display natural resistance to acid-induced acute lung injury (ALI). We show that TLR4-TRIF-TRAF6 signaling is a key disease pathway that controls the severity of ALI. The oxidized phospholipid (OxPL) OxPAPC was identified to induce lung injury and cytokine production by lung macrophages via TLR4-TRIF. We observed OxPL production in the lungs of humans and animals infected with SARS, Anthrax, or H5N1. Pulmonary challenge with an inactivated H5N1 avian influenza virus rapidly induces ALI and OxPL formation in mice. Loss of TLR4 or TRIF expression protects mice from H5N1-induced ALI. Moreover, deletion of ncf1, which controls ROS production, improves the severity of H5N1-mediated ALI. Our data identify oxidative stress and innate immunity as key lung injury pathways that control the severity of ALI.     

Investigating the biochar effects on C‐mineralization and sequestration of carbon in soil compared with conventional amendments using the stable isotope (δ13C) approach

Biomass‐derived black carbon (biochar) is considered to be an effective tool to mitigate global warming by long‐term C‐sequestration in soil and to influence C‐mineralization via priming effects. However, the underlying mechanism of biochar (BC) priming relative to conventional biowaste (BW) amendments remains uncertain. Here, we used a stable carbon isotope (δ¹³C) approach to estimate the possible biochar effects on native soil C‐mineralization compared with various BW additions and potential carbon sequestration. The results show that immediately after application, BC suppresses and then increases C‐mineralization, causing a loss of 0.14–7.17 mg‐CO₂–C g^?1‐C compared to the control (0.24–1.86 mg‐CO₂–C g^?1‐C) over 1–120 days. Negative priming was observed for BC compared to various BW amendments (?10.22 to ?23.56 mg‐CO₂–C g^?1‐soil‐C); however, it was trivially positive relative to that of the control (8.64 mg‐CO₂–C g^?1‐soil‐C). Furthermore, according to the residual carbon and δ¹³C signature of postexperimental soil carbon, BC‐C significantly increased (P < 0.05) the soil carbon stock by carbon sequestration in soil compared with various biowaste amendments. The results of cumulative CO₂–C emissions, relative priming effects, and carbon storage indicate that BC reduces C‐mineralization, resulting in greater C‐sequestration compared with other BW amendments, and the magnitude of this effect initially increases and then decreases and stabilizes over time, possibly due to the presence of recalcitrant‐C (4.92 mg‐C g^?1‐soil) in BC, the reduced microbial activity, and the sorption of labile organic carbon (OC) onto BC particles.     

A Fluorescent Dye with Low Specificity to DNA Nucleotide Sequences: Quantitative Assessment of Oligonucleotides Immobilized in Microchip Gel Pads

To assess the DNA amount in samples (e.g., in biological microchip gel pads) by means of fluorescent dyes, one should use the dyes whose fluorescence weakly depends on DNA composition and structure. With the ImD-310 dye created for this purpose, we have analyzed the staining of single- and double-stranded oligo- and polynucleotides of different nucleotide composition, length, and concentration both in solution and being immobilized in biological microchip gel pads. It turned out that ImD-310 has no pronounced specificity to the single- and double-stranded nucleotide sequences, while the intensity of fluorescence for the dye complexes with d(A)₈, d(T)₈, d(C)₈, and d(G)₈ at high temperatures (50°C) differs by less than 25%. A linear correlation has been established between the intensity of fluorescence and the amount of oligonucleotides immobilized on a biological microchip. The plots of the intensity of fluorescence against the concentration of NaCl and the temperature were obtained. By using a generic microchip containing all 4096 hexamer oligonucleotides, it has been determined that the dye has no distinct specificity to any certain motifs of the nucleotide sequence. Thus, ImD-310 may serve as an efficient fluorescent probe to quickly estimate the amount of oligonucleotides immobilized in a microchip, in an electrophoretic gel, etc.     

Hematological and serum biochemical reference intervals of rainbow trout,Oncorhynchus mykiss cultured in Himalayan aquaculture: Morphology,morphometrics and quantification of peripheral blood cells

De novo reference intervals (RIs) for a total of thirty two hematological and serum biochemical attributes were established for rainbow trout (Oncorhynchus mykiss) cultured in Himalayan aquaculture system. For this purpose, long term assessment of hemato-biochemical parameters was carried over a period of one year from March 2019 to February 2020 and a total of 444 blood samples were analysed. Blood examination results were recorded systematically and reference intervals were established, notably for erythrocyte parameters: hematocrit (Hct) 29–40%, hemoglobin 8.32–12.28 g/dL, red blood cell (RBC) count 1.01–2.04 (×10⁶/mm3); leukocyte parameters (x 10³/mm3): total leukocytes 31.32–90.60, neutrophils 4.21–18.85, total lymphocytes 20.55–63.63, small lymphocytes 14.86–46.50, large lymphocytes 6.35–22.34 and monocytes 1.22–7.56; thrombocyte count 23.00–68.00 (×10⁶/mm3). RIs were also established for red blood cell indices, vital serum constituents involved in carbohydrate, protein, lipid and nitrogen metabolism including the less known, diagnostically important, serum enzymes and electrolyte concentrations. Principal component analysis revealed that certain serum components were more efficient at distinguishing between the life stages (juvenile, adult) of fish by explaining about 92.7% of variation in the whole dataset compared to the principal hematological components which explained only about 80% of the variation. Significant (P < 0.05) differences were noted for RBC count, total leukocyte count (TLC), total protein, total cholesterol and uric acid with respect to the sex of fish. Moreover, clearly differentiable morphometric and morphological attributes were also noticed among erythrocytes, leukocytes (lymphocytes, neutrophils and monocytes) and thrombocytes. To our knowledge, the present study is the first of its kind that elucidates blood chemistry of cultured rainbow trout, O. mykiss in accordance to the guidelines framed by the American society of veterinary clinical pathologists (ASVCP). RIs reported here can help monitor the fish health status by improving the use of non-lethal diagnostics in piscine medicine.     

¹⁸F]GE-180: a novel fluorine-18 labelled PET tracer for imaging Translocator protein 18 kDa (TSPO)

A series of tricyclic compounds have been synthesised and evaluated in vitro for affinity against Translocator protein 18 kDa (TSPO) and for preferred imaging properties. The most promising of the compounds were radiolabelled and evaluated in vivo to determine biodistribution and specificity for high expressing TSPO regions. Metabolite profiling in brain and plasma was also investigated. Evaluation in an autoradiography model of neuroinflammation was also carried out for the best compound, 12a ([(18)F]GE-180).     

Microchips based on three dimensional gel cells: history and perspective

The review describes the history of creation and development of the microchip technology and its role in the human genome project in Russia. The emphasis is placed on the three-dimensional gel-based microchips developed at the Center of Biological Microchips headed by A.D. Mirzabekov since 1988. The gel-based chips of the last generation, IMAGE chips (Immobilized Micro Array of Gel Elements), have a number of advantages over the previous versions. The microchips are manufactured by photo-initiated copolymerization of gel components and immobilized molecules (DNA, proteins, and ligands). This ensures an even distribution of the immobilized probe throughout the microchip gel element with a high yield (about 50% for oligonucleotides). The use of methacrylamide as a main component of the polymerization mixture resulted in a substantial increase of gel porosity without affecting its mechanical strength and stability, which allowed one to work with the DNA fragments of up to 500 nt in length, as well as with rather large protein molecules. At present, the gel-based microchips are widely applied to address different problems. The generic microchips containing a complete set of possible hexanucleotides are used to reveal the DNA motifs binding with different proteins and to study the DNA-protein interactions. The oligonucleotide microchips are a cheap and reliable tool of diagnostics designed for mass application. Biochips have been developed for identification of the tuberculosis pathogen and its antibiotic-resistant forms; for diagnostics of orthopoxviruses, including the smallpox virus; for diagnostics of the anthrax pathogen; and for identification of chromosomal rearrangements in leukemia patients. The protein microchips can be adapted for further use in proteomics. Bacterial and yeast cells were also immobilized in the gel, maintaining their viability, which open a wide potential for creation biosensors on the basis of microchips.