Ploidia levels in pyrenic and apyrenic “pitangueira” accessions

Pitangueira (E. uniflora) is considered a diploid species with n = 11 and 2n = 22 chromosomes, in genotypes with the presence of seeds in the fruits. With the pitangueira production of apyrenic fruits existence, such behavior may be related to the ploidy level. The objective of this study was to determine the ploidy level of the pitangueira accessions producing fruit with and without seed, as well as to observe the meiotic behavior and possible chromosomal abnormalities. To check the ploidy level of the pitangueira, freshly expanded mature leaves were collected from the pyrenic pitangueira and from two pyrenic accessions, with suspensions of intact nuclei being prepared. Samples were analyzed in a flow cytometer equipped with multiple parameters data acquisition and UV laser. All analyses were performed using peak-height detection (>6000 fluorescent events, for example, nuclei, were analyzed per sample) and logarithmic amplification. The data were presented as histograms of the number of nuclei along the y-axis and the relative fluorescence intensity on the x-axis. The pyrenean pitangueira and other two pyrenic accessions were characterized as diploid.

Functional Analysis of Haplotypes and Promoter Activity at the 5’ Region of the ADH7 Gene

Background: The function of the 5’ regulatory region and the role of the different SNP loci have not been well characterized. This study investigated the effect of several ADH7 haplotypes on the regulation of gene expression in vitro and the functional sequences in the 5’ regulatory region of ADH7. Three SNPs (rs17537595, rs2851028, and rs2654847) and four different haplotypes (T-C-T, T-T-T, C-T-T, T-C-A) were identified by cloning and sequencing. Methods: Effects of 4 different haplotypes and 8 truncated fragments of 5’ regulatory region on ADH7 gene expression were detected using a dual-luciferase reporter assay system. All recombinant plasmids were transfected into HEK-293, U87, and SH-SY5Y cells, respectively, and their relative fluorescence intensity was measured.Results: In HEK-293, U87, and SH-SY5Y cell lines, the relative fluorescence intensity of haplotype T-C-T was significantly higher than that of haplotype T-T-T, C-T-T, and T-C-A. Additionally, we found that regions from -83 to -310bp (ATG, +1), -560 to -768bp , and -987bp to -1203bp up-regulated gene expression. In contrast, the region from -768 to -987bp down-regulated gene expression. The gene expression of regions from -1203 to -1369bp and -1369 to -1626bp was down-regulated in U87 and SH-SY5Y cell lines, but the trend was opposite in HEK-293 cell line. The region from -310 to -560bp up-regulated gene expression in SH-SY5Y cell line, but down-regulated gene expression in HEK-293 and U87 cell lines.Conclusions: This study has shown that the polymorphisms of ADH7 5’ regulatory region play an important role in the regulation of gene expression.

Transcription Factor CEBPB Inhibits the Expression of the Human HTR1A by Binding to 5’ Regulatory Region in Vitro

This study identified a transcription factor that might bind to the 5’ regulatory region of the HTR1A and explored the potential effect on 5-HT1A receptor expression. Based on JASPAR predictions, the binding of the transcription factor was demonstrated using the electrophoretic mobility shift assay (EMSA). Vectors over-expressing the transcription factor were co-transfected into HEK-293 and SK-N-SH cells with the recombinant pGL3 vector, and relative fluorescence intensity was measured to determine regulatory activity. Additionally, the qRT-PCR and Western blot were also used to identify whether the transcription factor modulated the endogenous expression of 5-HT1A receptor. The results suggest that the transcription factor CCAA/T enhancer binding protein beta (CEBPB) likely binds to the -1219 to -1209 bp (ATG+1) region of the HTR1A. Two sequences located in the -722 to -372 bp and -119 to +99 bp were also identified. Although the effect of CEBPB on endogenous 5-HT1A receptor expression was not significant, it exhibited the strong inhibition on the relative fluorescence intensity and the mRNA level of HTR1A. CEBPB inhibited the human HTR1A expression by binding to the sequence -1219 - -1209 bp. This is useful and informative for ascertaining the regulation of 5-HT1A receptor and mental diseases.

Neutral Lipid Content in Lipid Droplets: Potential Biomarker of Cordycepin Accumulation in Cordycepin-Producing Fungi

To clarify the relationship between neutral lipid content and cordycepin accumulation in Cordyceps militaris, mutants were generated from mixed spores of two C. militaris strains with varying cordycepin-producing capacities. Fifteen stable mutants producing from 0.001 to 2.363 mg/mL cordycepin were finally selected. The relative fluorescence intensities of the 15 mutants, two C. militaris strains and an Aspergillus nidulans strain at different concentrations of lyophilized mycelium powder were then investigated using the Nile red method. The mutant CM1-1-1 with the highest relative fluorescence intensity among the eighteen strains was selected for optimizing the Nile red method. Relative fluorescence intensity was linearly correlated with cordycepin concentration in liquid broth (R2 = 0.9514) and in lyophilized mycelium powder (R2 = 0.9378) for the 18 cordycepin-producing strains under identical culture conditions and with cordycepin concentration in liquid broth (R2 = 0.9727) and in lyophilized mycelium powder (R2 = 0.9613) for CM1-1-1 under eight different sets of conditions. In addition, the cordycepin content in lyophilized mycelium powder measured by the Nile red method was linearly correlated with that determined by an HPLC method (R2 = 0.9627). In conclusion, neutral lipids in lipid droplets are required during cordycepin accumulation; these neutral lipids are potential biomarkers of cordycepin biosynthesis.

Novel terpolymers containing carbazole, coumarin and Alq3 complexes

In this paper, the synthesis of terpolymers bearing carbazole, coumarin and 8-hydroxyquinoline moiety as pendant groups with content of coumarin and 8-hydroxyquinoline between 0.5 and 4 mol% was elaborated. Then the terpolymers underwent complexation reactions with aluminum bis(8-hydroxyquinoline)isopropoxylate (Alq2ispr) to form polymeric hybrid materials with aluminum tris(8-hydroxyquinoline) (Alq3) as side groups. The presence of carbazole, coumarin (MK) and 8-hydroxyquinoline (QM) as well as Alq3 in the terpolymers was confirmed by NMR, UV, photoluminescence, and size exclusion chromatography. The results indicates that these hybrid polymers have moderate molecular weights and solubility in common organic solvents. For photoluminescence spectra, an evidence for energy transfer from carbazole to coumarin and Alq3 groups was observed and highest relative fluorescence intensity was exhibited by solutions of the terpolymers containing the lowest content of MK, QM and Alq3.

Flow Cytometry and Sperm Sexing in Animals

Flow cytometry is a useful technology in the sexed sperm, which measures and analyzes simultaneously, multiple physical characteristics of the cell, as they flow in a stream flow, through a light beam. The measured properties are the size of a particle, relative internal granularity, relative complexity and relative fluorescence intensity. Currently, hundreds of calves have been gestated through artificial insemination with sexed sperm in animal production. Since 1992, flow cytometry has been used, a technique that allows spermatozoa X and Y differentiation by DNA content. There is no other practical technique for sperm sexing to keep sperm functionality. The objectives of this review are to explain: (1) why the sperm containing the X or Y chromosome are phenotypically similar, but differ among themselves, (2) the principles and procedures used for sexing sperm by flow cytometry and sorting ( 3) accuracy, speed and efficiency of current procedure sperm sexing, (4) sperm damage occurred during sperm sexing and consequently the effects on fertility.

Semi-quantitative analysis on the content of berberine hydrochloride in compound berberine tablets with the fluorescence spectral imaging method

The content of berberine hydrochloride (BH) in compound berberine tablets (CBTs) is subject to strict requirements. Its content is usually measured based on chemical analysis. In this paper, the fluorescence spectral imaging method was used to study the relative content of BH from a physics perspective. By comparing the relative fluorescence intensity of self-made CBTs with different mass percentages of BH, a linear positive relationship was observed between the BH content and the relative fluorescence intensity, and accordingly the quality of CBTs of different brands was evaluated. The results indicate that the fluorescence spectral imaging method can be a simple, fast and nondestructive semi-quantitative analysis method to determine the content of BH in CBTs, and this method has great potential in the quality control of CBTs.