The effects of estrogen and progesterone on blood glutamate levels: evidence from changes of blood glutamate levels during the menstrual cycle in women

The gonadal steroids estrogen and progesterone have been shown to have neuroprotective properties against various neurodegenerative conditions. Excessive concentrations of glutamate have been found to exert neurotoxic properties. We hypothesize that estrogen and progesterone provide neuroprotection by the autoregulation of blood and brain glutamate levels. Venous blood samples (10 ml) were taken from 31 men and 45 women to determine blood glutamate, estrogen, progesterone, glucose, glutamate-pyruvate transaminase (GPT), and glutamate-oxaloacetate transaminase (GOT) levels, collected on Days 1, 7, 12, and 21 of the female participants' menstrual cycle. Blood glutamate concentrations were higher in men than in women at the start of menstruation (P < 0.05). Blood glutamate levels in women decreased significantly on Days 7 (P < 0.01), 12 (P < 0.001), and 21 (P < 0.001) in comparison with blood glutamate levels on Day 1. There was a significant decrease in blood glutamate levels on Days 12 (P < 0.001) and 21 (P < 0.001) in comparison with blood glutamate levels on Day 7. Furthermore, there was an increase in blood glutamate levels on Day 21 compared with Day 12 (P < 0.05). In women, there were elevated levels of estrogen on Days 7 (P < 0.05), 12, and 21 (P < 0.001), and elevated levels of progesterone on Days 12 and 21 (P < 0.001). There were no differences between men and women with respect to blood glucose concentrations. Concentrations of GOT (P < 0.05) and GPT (P < 0.001) were significantly higher in men than in women during the entire cycle. The results of this study demonstrate that blood glutamate levels are inversely correlated to levels of plasma estrogen and progesterone.     

Constructions of expression vectors of polyhydroxybutyrate-co-hydroxyvalerate (PHBV) and transient expression of transgenes in immature oil palm embryos

Polyhydroxybutyrate-co-hydroxyvalerate (PHBV) is a polyhydroxyalkanoate (PHA) bioplastic group with thermoplastic properties is thus high in quality and can be degradable. PHBV can be produced by bacteria, but the process is not economically competitive with polymers produced from petrochemicals. To overcome this problem, research on transgenic plants has been carried out as one of the solutions to produce PHBV in economically sound alternative manner. Four different genes encoded with the enzymes necessary to catalyze PHBV are bktB, phaB, phaC and tdcB. All the genes came with modified CaMV 35S promoters (except for the tdcB gene, which was promoted by the native CaMV 35S promoter), nos terminator sequences and plastid sequences in order to target the genes into the plastids. Subcloning resulted in the generation of two different orientations of the tdcB, pLMIN (left) and pRMIN (right), both 17.557 and 19.967 kb in sizes. Both plasmids were transformed in immature embryos (IE) of oil palm via Agrobacterium tumefaciens. Assays of GUS were performed on one-week-old calli and 90% of the calli turned completely blue. This preliminary test showed positive results of integration. Six-months-old calli were harvested and RNA of the calli were isolated. RT-PCR was used to confirm the transient expression of PHBV transgenes in the calli. The bands were 258, 260, 315 and 200 bp in size for bktB, phaB, phaC and tdcB transgenes respectively. The data obtained showed that the bktB, phaB, phaC and tdcB genes were successfully integrated and expressed in the oil palm genome.     

Ribosome‐associated pentatricopeptide repeat proteins function as translational activators in mitochondria of trypanosomes

Mitochondrial ribosomes of Trypanosoma brucei are composed of 9S and 12S rRNAs, eubacterial‐type ribosomal proteins, polypeptides lacking discernible motifs and approximately 20 pentatricopeptide repeat (PPR) RNA binding proteins. Several PPRs also populate the polyadenylation complex; among these, KPAF1 and KPAF2 function as general mRNA 3′ adenylation/uridylation factors. The A/U‐tail enables mRNA binding to the small ribosomal subunit and is essential for translation. The presence of A/U‐tail also correlates with requirement for translation of certain mRNAs in mammalian and insect parasite stages. Here, we inquired whether additional PPRs activate translation of individual mRNAs. Proteomic analysis identified KRIPP1 and KRIPP8 as components of the small ribosomal subunit in mammalian and insect forms, but also revealed their association with the polyadenylation complex in the latter. RNAi knockdowns demonstrated essential functions of KRIPP1 and KRIPP8 in the actively respiring insect stage, but not in the mammalian stage. In the KRIPP1 knockdown, A/U‐tailed mRNA encoding cytochrome c oxidase subunit 1 declined concomitantly with the de novo synthesis of this subunit whereas polyadenylation and translation of cyb mRNA were unaffected. In contrast, the KRIPP8 knockdown inhibited A/U‐tailing and translation of both CO1 and cyb mRNAs. Our findings indicate that ribosome‐associated PPRs may selectively activate mRNAs for translation.     

Efficient target cleavage by Type V Cas12a effectors programmed with split CRISPR RNA

CRISPR RNAs (crRNAs) that direct target DNA cleavage by Type V Cas12a nucleases consist of constant repeat-derived 5′-scaffold moiety and variable 3′-spacer moieties. Here, we demonstrate that removal of most of the 20-nucleotide scaffold has only a slight effect on in vitro target DNA cleavage by a Cas12a ortholog from Acidaminococcus sp. (AsCas12a). In fact, residual cleavage was observed even in the presence of a 20-nucleotide crRNA spacer moiety only. crRNAs split into separate scaffold and spacer RNAs catalyzed highly specific and efficient cleavage of target DNA by AsCas12a in vitro and in lysates of human cells. In addition to dsDNA target cleavage, AsCas12a programmed with split crRNAs also catalyzed specific ssDNA target cleavage and non-specific ssDNA degradation (collateral activity). V-A effector nucleases from Francisella novicida (FnCas12a) and Lachnospiraceae bacterium (LbCas12a) were also functional with split crRNAs. Thus, the ability of V-A effectors to use split crRNAs appears to be a general property. Though higher concentrations of split crRNA components are needed to achieve efficient target cleavage, split crRNAs open new lines of inquiry into the mechanisms of target recognition and cleavage and may stimulate further development of single-tube multiplex and/or parallel diagnostic tests based on Cas12a nucleases.     

Benchmark of different charges for prediction of the partitioning coefficient through the hydrophilic/lipophilic index

A few different theoretical methods for assigning the partial atomic charges were benchmarked for calculation of the hydrophilic/lipophilic index (HLI). The coefficients were selected to produce the best correlation of the HLI values with the experimental octanol-water partition. Different parameters were checked in calculations of partial charges to get the best performance of the HLI values obtained. Thus, four partitioning schemes (Coulson, Mulliken, Merz-Kollman, Ford-Wang) were benchmarked for calculations of atomic charges with six semiempirical methods (AM1, PM3, RM1, PM6, PM6-D3H4, PM7). Moreover, five distinct types of partial atomic charges (Mulliken, Hirshfeld, Löwdin, CHELPG, NPA), obtained at the Hartree–Fock and DFT levels of theory with three basis sets, were tested for their ability to produce the HLI values with the best correlation to experimental logP coefficients of 50 mono-charged organic anions. In the case of the semiempirical methods, the best correlation between the HLI and logP values (the correlation coefficient r?=?0.9216) was obtained with the AM1 Ford–Wang parametric electrostatic potential charges. The Mulliken and Coulson charges calculated with the PM7 method can be used as an alternative to AM1, with the r values of 0.9107 and 0.8984, respectively. In the case of the DFT, the PBE/def2-TZVP natural population analysis charges produce the best correlation (r?=?0.9220). Nevertheless, in spite of a marginally lower performance (r?=?0.9159), the NPA charges computed at the PBE/def2-SVP level are more robust and can be regarded as the optimum choice for calculating the HLI values.

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Graphical abstract The hydrophilic/lipophilic index (HLI)

     

Synthesis and photoluminescence properties of co‐polyamides with anthrazoline‐containing units in the main chain

A series of anthrazoline‐containing monomers are synthesized, and eight co‐polyamides of different chemical structures, containing 1,9‐anthrazoline fragments in the main chain, are obtained and investigated. Photoluminescent, stress–strain, and thermal properties of these polymers are studied. It is shown that polymers with fragments of 4,4′‐(pyrido[3,2‐g]‐quinoline‐2,8‐diyl)dianiline and 4,4′‐(10‐methylpyrido[3,2‐g]quinoline‐2,8‐diyl)dianiline possess an intense luminescence in the range 550–650 nm. The performed investigations made it possible to determine the effect of substituents of various natures in the anthrazoline cycle and the position of amide group (meta‐ and para‐configurations) on optical, stress‐strain, and thermal properties of copolymers, opening up a prospect for further developments of principles of design of polymers with optimal characteristics.     

Dynamics of formation and morphological features of neutrophil extracellular traps formed under the influence of opsonized Staphylococcus aureus

In the process of performing their protective functions, neutrophils can form neutrophil extracellular traps (NETs), consisting of DNA in combination with enzymes and histones. The aim of the study was to determine the dynamics of the formation of NETs under the influence of opsonized Staphylococcus aureus and to determine the morphological features of their development in real time by atomic force microscopy. It was found that the maximum formation of NETs was observed after 3 hours of co‐incubation of neutrophils and opsonized S. aureus. For the first time, the atomic force microscopy method revealed that, at first, large blocks of parallel DNA helices are formed, which then spread in waves, and only then their bifurcation and separation can be observed. Some of the strands formed are covered by a shell, which subsequently completely disappears. Enzymes and histones become clearly visible only after 140 to 150 minutes of observation. The DNA helixes move toward the opsonized S. aureus. After NET formation, the cell remains on the substrate only in the form of traces of focal adhesion. This, and the fact that the maximum amount of NETs is formed after 3 hours of co‐incubation with opsonized S. aureus, suggests that the formation of NETs follows the classical mechanism. The study of the dynamics of formation and the microstructure of NETs makes it possible to estimate the time frame for the implementation of this protective mechanism of the human body when performing the compensatory inflammatory reaction.     

Availability of the key metabolic substrates dictates the respiratory response of cancer cells to the mitochondrial uncoupling

Active glycolysis and glutaminolysis provide bioenergetic stability of cancer cells in physiological conditions. Under hypoxia, metabolic and mitochondrial disorders, or pharmacological treatment, a deficit of key metabolic substrates may become life-threatening to cancer cells. We analysed the effects of mitochondrial uncoupling by FCCP on the respiration of cells fed by different combinations of Glc, Gal, Gln and Pyr. In cancer PC12 and HCT116 cells, a large increase in O₂ consumption rate (OCR) upon uncoupling was only seen when Gln was combined with either Glc or Pyr. Inhibition of glutaminolysis with BPTES abolished this effect. Despite the key role of Gln, addition of FCCP inhibited respiration and induced apoptosis in cells supplied with Gln alone or Gal/Gln. For all substrate combinations, amplitude of respiratory responses to FCCP did not correlate with Akt, Erk and AMPK phosphorylation, cellular ATP, and resting OCR, mitochondrial Ca^2 + or membrane potential. However, we propose that proton motive force could modulate respiratory response to FCCP by regulating mitochondrial transport of Gln and Pyr, which decreases upon mitochondrial depolarisation. As a result, an increase in respiration upon uncoupling is abolished in cells, deprived of Gln or Pyr (Glc). Unlike PC12 or HCT116 cells, mouse embryonic fibroblasts were capable of generating pronounced response to FCCP when deprived of Gln, thus exhibiting lower dependence on glutaminolysis. Overall, the differential regulation of the respiratory response to FCCP by metabolic environment suggests that mitochondrial uncoupling has a potential for substrate-specific inhibition of cell function, and can be explored for selective cancer treatment.