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Decreasing cytosolic translation is beneficial to yeast and human Tafazzin-deficient cells
Get PDFCardiolipin (CL) optimizes diverse mitochondrial processes, including oxidative phosphorylation (OXPHOS). To function properly, CL needs to be unsaturated, which requires the acyltransferase Tafazzin (TAZ). Loss-of-function mutations in the TAZ gene are responsible for the Barth syndrome (BTHS), a rare X-linked cardiomyopathy, presumably because of a diminished OXPHOS capacity. Herein we show that a partial inhibition of cytosolic protein synthesis, either chemically with the use of cycloheximide or by specific genetic mutations, fully restores biogenesis and the activity of the oxidative phosphorylation system in a yeast BTHS model (taz1Δ). Interestingly, the defaults in CL were not suppressed, indicating that they are not primarily responsible for the OXPHOS deficiency in taz1Δ yeast. Low concentrations of cycloheximide in the picomolar range were beneficial to TAZ-deficient HeLa cells, as evidenced by the recovery of a good proliferative capacity. These findings reveal that a diminished capacity of CL remodeling deficient cells to preserve protein homeostasis is likely an important factor contributing to the pathogenesis of BTHS. This in turn, identifies cytosolic translation as a potential therapeutic target for the treatment of this disease
Study of fauna population changes on Penguin Island and Turret Point Oasis (King George Island, Antarctica) using an unmanned aerial vehicle
Get PDFAn unmanned aerial vehicle (UAV) as an alternative to manned aircrafts is an excellent, less invasive, safe tool, especially
in sensitive polar regions. Here we used a fixed-wing UAV to collect data on seabird and pinniped populations in hardly
accessible Antarctic areas. The implementation of an auto-piloted UAV equipped with a digital camera (Canon EOS 700D,
35 mm f/2.0 lens) allowed us to collect high-quality material applicable to a quantitative analysis of the fauna populations.
A successful photogrammetric mission, at an altitude of 550 m above sea level, was accomplished during one Beyond Visual
Line of Sight flight above hard-to-access Penguin Island and Turret Point Oasis (King George Island). Obtained selected
RGB images were processed to generate a panoramic image stitch with resolution of 0.07 m ground sampling distance. A
total of 4290 (SD = 33.08) breeding individuals of two penguin species, Adélie (Pygoscelis adeliae) and chinstrap (Pygoscelis
antarcticus), 426 (SD = 7.78) individuals of the southern elephant seal (Mirounga leonina) and 6 individuals of the Weddell
seal (Leptonychotes weddellii) were identified in both study areas. Additionally, 222 (SD = 2.0) individuals of the southern
giant petrel (Macronectes giganteus) and 76 (SD = 1.0) of the Antarctic shag (Phalacrocorax atriceps bransfieldensis) in the
Turret Point area were recognized. The presented observations on the natural history of the investigated fauna together with
the available literature may be useful in future research on population trends. A comparison with available historical data
for both investigated areas suggests a decrease of 68.29% in both penguin species in the 1980–2016 period. The presented
results confirmed that UAVs are useful for remote census work for Antarctic seabirds
Inhibition of hydrogen-yielding dark fermentation by ascomycetous yeasts
Get PDFHydrogen-yielding fermentation conducted in bioreactors is an alternative method of hydrogen production. However, unfavourable processes can seriously inhibit bio-hydrogen
generation during the acidogenic step of anaerobic digestion. Here, ascomycetous yeasts were identified as a major factor inhibiting the production of bio-hydrogen by fermentation.Changes in the performance of hydrogen-producing bioreactors including metabolic shift,quantitative changes in the fermentation products, decreased pH, instability of the microbialcommunity and consequently a dramatic drop in bio-hydrogen yield were observed following
yeast infection. Ascomycetous yeasts from the genera Candida, Kazachstania and Geotrichum were isolated from hydrogen-producing bioreactors. Yeast metabolites secreted into the growth medium showed antibacterial activity. Our studies indicate that yeast infection of hydrogen-producing microbial communities is one of the serious obstacles to use dark fermentation as an alternative method of bio-hydrogen production. It also explains why studies on hydrogen fermentation are still limited to the laboratory or pilot-scale systems
Molecular characterization of central cytoplasmic loop in Aspergillus nidulans AstA transporter
Get PDFAstA (alternative sulfate transporter) belongs to a large, but poorly characterized, Dal5 family of allantoate permeases of the Major Facilitator Superfamily. The astA gene has been cloned from an IAM 2006 Japanese strain of Aspergillus nidulans by complementation of a sulfate permease-deficient mutant. In this study we show that conserved lysine residues in Central Cytoplasmic Loop (CCL) of the AstA protein may participate in anion selectivity, and control kinetic properties of the AstA transporter. A three-dimensional model containing four clustered lysine residues was created, showing a novel substrate-interacting structure in Major Facilitator Superfamily transporters. The assimilation constant (Kτ) of wild type AstA protein is 85 μM, while Vmax/mg of DW of AstA is twice that of the main sulfate transporter SB per mg of dry weight (DW) of mycelium (1.53 vs. 0.85 nmol/min, respectively). Amino acid substitutions in CCL did not abolish sulfate uptake, but affected its kinetic parameters. Mutants affected in the lysine residues forming the postulated sulfate-interacting pocket in AstA were able to grow and uptake sulfate, indicating that CCL is not crucial for sulfate transportation. However, these mutants exhibited altered values of Kτ and Vmax, suggesting that CCL is involved in control of the transporter activity
Inhibition of protein disulfide isomerase induces differentiation of acute myeloid leukemia cells
Get PDFAcute myeloid leukemia is a malignant disease of immature myeloid cells. Despite significant therapeutic effects of differentiation-inducing agents in some acute myeloid leukemia subtypes, the disease remains incurable in a large fraction of patients. Here we show that SK053, a thioredoxin inhibitor, induces differentiation and cell death of acute myeloid leukemia cells. Considering that thioredoxin knock-down with short hairpin RNA failed to exert antiproliferative effects in one of the acute myeloid leukemia cell lines, we used a biotin affinity probe-labeling approach to identify potential molecular targets for the effects of SK053. Mass spectrometry of proteins precipitated from acute myeloid leukemia cells incubated with biotinylated SK053 used as a bait revealed protein disulfide isomerase as a potential binding partner for the compound. Biochemical, enzymatic and functional assays using fluorescence lifetime imaging confirmed that SK053 binds to and inhibits the activity of protein disulfide isomerase. Protein disulfide isomerase knockdown with short hairpin RNA was associated with inhibition of cell growth, increased CCAAT enhancer-binding protein α levels, and induction of differentiation of HL-60 cells. Molecular dynamics simulation followed by the covalent docking indicated that SK053 binds to the fourth thioredoxin-like domain of protein disulfide isomerase. Differentiation of myeloid precursor cells requires the activity of CCAAT enhancer-binding protein α, the function of which is impaired in acute myeloid leukemia cells through various mechanisms, including translational block by protein disulfide isomerase. SK053 increased the levels of CCAAT enhancer-binding protein α and upregulated mRNA levels for differentiation-associated genes. Finally, SK053 decreased the survival of blasts and increased the percentage of cells expressing the maturation-associated CD11b marker in primary cells isolated from bone marrow or peripheral blood of patients with acute myeloid leukemia. Collectively, these results provide a proof-of-concept that protein disulfide isomerase inhibition has potential as a therapeutic strategy for the treatment of acute myeloid leukemia and for the development of small-molecule inhibitors of protein disulfide isomerase
Detailed characterization of Synechocystis PCC 6803 ferredoxin:NADP+ oxidoreductase interaction with model membranes
Get PDFDirect interaction of ferredoxin:NADP+ oxidoreductase (FNR) with thylakoid membranes was postulated as a part of the cyclic electron flow mechanism. In vitro binding of FNR to digalactosyldiacylglycerol and monogalactosyldiacylglycerol membranes was also shown. In this paper we deal with the latter interaction in more detail describing the effect for two FNR forms of Synechocystis PCC 6803. The so-called short FNR (sFNR) is homologous to FNR from higher plant chloroplasts. The long FNR (lFNR) form contains an additional domain, responsible for the interaction with phycobilisomes. We compare the binding of both sFNR and lFNR forms to native and non-native lipids. We also include factors which could modulate this process: pH change, temperature change, presence of ferredoxin, NADP+ and NADPH and heavy metals. For the lFNR, we also include phycobilisomes as a modulating factor. The membrane binding is generally faster at lower pH. The sFNR was binding faster than lFNR. Ferredoxin isoforms with higher midpoint potential, as well as NADPH and NADP+, weakened the binding. Charged lipids and high phosphate promoted the binding. Heavy metal ions decreased the rate of membrane binding only when FNR was preincubated with them before injection beneath the monolayer. FNR binding was limited to surface lipid groups and did not influence hydrophobic chain packing. Taken together, FNR interaction with lipids appears to be non-specific, with an electrostatic component. This suggests that the direct FNR interaction with lipids is most likely not a factor in directing electron transfer, but should be taken into account during in vitro studies
PlasFlow: predicting plasmid sequences in metagenomic data using genome signatures
Get PDFPlasmids are mobile genetics elements that play an important role in the environmental adaptation of microorganisms. Although plasmids are usually analyzed in cultured microorganisms, there is a need for methods that allow for the analysis of pools of plasmids (plasmidomes) in environmental samples. To that end, several molecular biology and bioinformatics methods have been developed; however, they are limited to environments with low diversity and cannot recover large plasmids. Here, we present PlasFlow, a novel tool based on genomic signatures that employs a neural network approach for identification of bacterial plasmid sequences in environmental samples. PlasFlow can recover plasmid sequences from assembled metagenomes without any prior knowledge of the taxonomical or functional composition of samples with an accuracy up to 96%. It can also recover sequences of both circular and linear plasmids and can perform initial taxonomical classification of sequences. Compared to other currently available tools, PlasFlow demonstrated significantly better performance on test datasets. Analysis of two samples from heavy metal-contaminated microbial mats revealed that plasmids may constitute an important fraction of their metagenomes and carry genes involved in heavy-metal homeostasis, proving the pivotal role of plasmids in microorganism adaptation to environmental conditions
Low Transfer of Tacrolimus and Its Metabolites into Colostrum of Graft Recipient Mothers
Get PDFCurrently, the majority of neonates born to organ recipient mothers on chronic
immunosuppressive therapy are formula fed. However, over the past few years, evidence has
grown, suggesting that breastfeeding might be possible and beneficial. We designed a study assessing
the transfer of tacrolimus into the colostrum of posttransplant mothers. We assessed the amount
of tacrolimus and its metabolites, M-1 and M-3, that would be ingested by the breastfed neonates.
Concentrations of tacrolimus and its metabolites were measured in colostrum from 14 posttransplant
mothers as well as in venous cord blood and venous blood of the neonates. Test material analysis
was performed by liquid chromatography coupled with mass spectrometry (LC/MS). The amount
of ingested formula was registered, which allowed for estimation of the amount of tacrolimus and
its metabolites that would be ingested by breastfed infants. The mean amount of tacrolimus that
would be ingested by the neonates in maternal milk was 151.4 ng/kg/24 h (standard deviation
SD � 74.39); metabolite M-1: 23.80 ng/kg/24 h (SD � 14.53); and metabolite M-3: 13.25 ng/kg/24 h
(SD � 9.05). The peak level of tacrolimus and metabolite M-1 in colostrum was noted 8 h after an
oral dose (3.219 ng/mL SD � 2.22 and 0.56 ng/mL SD � 0.60, respectively) and metabolite M-3 after
6 h (0.29 ng/mL SD � 0.22). Low concentrations of tacrolimus and its metabolites, M-1 and M-3,
in colostrum show that neonates will ingest trace amounts of the drug. Further studies are required
to fully assess the safety of breastfeeding by posttransplant mothers
Fidelity of DNA replication - a matter of proofreading
Get PDFDNA that is transmitted to daughter cells must be accurately duplicated to maintain genetic integrity and to promote genetic continuity. A major function of replicative DNA polymerases is to replicate DNA with the very high accuracy. The fidelity of DNA replication relies on nucleotide selectivity of replicative DNA polymerase, exonucleolytic proofreading and postreplicative DNA mismatch repair (MMR). Proofreading activity that assists most of the replicative polymerases is responsible for removal of incorrectly incorporated nucleotides from the primer terminus before further primer extension. It is estimated that proofreading improves the fidelity by a 2-3 orders of magnitude. The primer with the incorrect terminal nucleotide has to be moved to exonuclease active site, and after removal of the wrong nucleotide must be transferred back to polymerase active site. The mechanism that allows the transfer of the primer between pol and exo site is not well understood.
While defects in MMR are well known to be linked with increased cancer incidence only recently, the replicative polymerases that have alterations in the exonuclease domain have been associated with some sporadic and hereditary human cancers. In this review, we would like to emphasize the importance of proofreading (3’-5’ exonuclease activity) in the fidelity of DNA replication and highlight what is known about switching from polymerase to exonuclease active site
Cell Size Influences the Reproductive Potential and Total Lifespan of the Saccharomyces cerevisiae Yeast as Revealed by the Analysis of Polyploid Strains
Get PDFThe total lifespan of the yeast Saccharomyces cerevisiae may be divided into two phases: the reproductive phase, during which the cell undergoes mitosis cycles to produce successive buds, and the postreproductive phase, which extends from the last division to cell death. These phases may be regulated by a common mechanism or by distinct ones. In this paper, we proposed a more comprehensive approach to reveal the mechanisms that regulate both reproductive potential and total lifespan in cell size context. Our study was based on yeast cells, whose size was determined by increased genome copy number, ranging from haploid to tetraploid. Such experiments enabled us to test the hypertrophy hypothesis, which postulates that excessive size achieved by the cell—the hypertrophy state—is the reason preventing the cell from further proliferation. This hypothesis defines the reproductive potential value as the difference between the maximal size that a cell can reach and the threshold value, which allows a cell to undergo its first cell cycle and the rate of the cell size to increase per generation. Here, we showed that cell size has an important impact on not only the reproductive potential but also the total lifespan of this cell. Moreover, the maximal cell size value, which limits its reproduction capacity, can be regulated by different factors and differs depending on the strain ploidy. The achievement of excessive size by the cell (hypertrophic state) may lead to two distinct phenomena: the cessation of reproduction without “mother” cell death and the cessation of reproduction with cell death by bursting, which has not been shown before