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Lake microbiome and trophy fluctuations of the ancient hemp rettery
Get PDFLake sediments not only store the long-term ecological information including pollen and microfossils but are also a source of sedimentary DNA (sedDNA). Here, by the combination of traditional multi-proxy paleolimnological methods with the whole-metagenome shotgun-sequencing of sedDNA we were able to paint a comprehensive picture of the fluctuations in trophy and bacterial diversity and metabolism of a small temperate lake in response to hemp retting, across the past 2000 years. Hemp retting (HR), a key step in hemp fibre production, was historically carried out in freshwater reservoirs and had a negative impact on the lake ecosystems. In Lake Slone, we identified two HR events, during the late stage of the Roman and Early Medieval periods and correlated these to the increased trophy and imbalanced lake microbiome. The metagenomic analyses showed a higher abundance of Chloroflexi, Planctomycetes and Bacteroidetes and a functional shift towards anaerobic metabolism, including degradation of complex biopolymers such as pectin and cellulose, during HR episodes. The lake eutrophication during HR was linked to the allochthonous, rather than autochthonous carbon supply—hemp straws. We also showed that the identification of HR based on the palynological analysis of hemp pollen may be inconclusive and we suggest the employment of the fibre count analysis as an additional and independent proxy
Potentiating Vγ9Vδ2 T cell proliferation and assessing their cytotoxicity towards adherent cancer cells at the single cell level
Get PDFVγ9Vδ2 T cells is the dominant γδ T cell subset in human blood. They are cytotoxic and activated by phosphoantigens whose concentrations are increased in cancer cells, making the cancer cells targets for Vγ9Vδ2 T cell immunotherapy. For successful immunotherapy, it is important both to characterise Vγ9Vδ2 T cell proliferation and optimise the assessment of their cytotoxic potential, which is the aim of this study. We found that supplementation with freshly thawed human serum potentiated Vγ9Vδ2 T cell proliferation from peripheral mononuclear cells (PBMCs) stimulated with (E)-4-Hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) and
consistently enabled Vγ9Vδ2 T cell proliferation from cryopreserved PBMCs. In cryopreserved PBMCs the proliferation was higher than in freshly prepared PBMCs. In a panel of short-chain prenyl alcohols, monophosphates and diphosphates, most diphosphates and also dimethylallyl monophosphate stimulated Vγ9Vδ2 T cell proliferation. We
developed a method where the cytotoxicity of Vγ9Vδ2 T cells towards adherent cells is assessed at the single cell level using flow cytometry, which gives more clear-cut results than the traditional bulk release assays. Moreover, we found that HMBPP enhances the Vγ9Vδ2 T cell cytotoxicity towards colon cancer cells. In summary, we have developed an easily interpretable method to assess the cytotoxicity of Vγ9Vδ2 T cells towards adherent cells, found that Vγ9Vδ2 T cell
proliferation can be potentiated by media-supplementation and how misclassification of non-responders may be avoided. Our findings will be useful in the further development of Vγ9Vδ2 T cell immunotherapy
An Adenosine Triphosphate- Dependent 5′-3′ DNA Helicase From sk1-Like Lactococcus lactis F13 Phage
Get PDFHere, we describe functional characterization of an early gene (gp46) product of a virulent Lactococcus lactis sk1-like phage, vB_Llc_bIBBF13 (abbr. F13). The GP46F13 protein carries a catalytically active RecA-like domain belonging to the P-loop NTPase superfamily. It also retains features characteristic for ATPases forming oligomers. In order to elucidate its detailed molecular function, we cloned and overexpressed the gp46 gene in Escherichia coli. Purified GP46F13 protein binds to DNA and exhibits DNA unwinding activity on branched substrates in the presence of adenosine triphosphate (ATP). Size exclusion chromatography with multi-angle light scattering (SEC-MALS) experiments demonstrate that GP46F13 forms oligomers, and further pull-down assays show that GP46F13 interacts with host proteins involved in replication (i.e., DnaK, DnaJ, topoisomerase I, and single-strand binding protein). Taking together the localization of the gene and the obtained results, GP46F13 is the first protein encoded in the early-expressed gene region with helicase activity that has been identified among lytic L. lactis phages up to date
Ni2+-Assisted Hydrolysis May Affect the Human Proteome; Filaggrin Degradation Ex Vivo as an Example of Possible Consequences
No full textDeficiency in a principal epidermal barrier protein, filaggrin (FLG), is associated with multiple allergic manifestations, including atopic dermatitis and contact allergy to nickel. Toxicity caused by dermal and respiratory exposures of the general population to nickel-containing objects and particles is a deleterious side effect of modern technologies. Its molecular mechanism may include the peptide bond hydrolysis in X1-S/T-c/p-H-c-X2 motifs by released Ni2+ ions. The goal of the study was to analyse the distribution of such cleavable motifs in the human proteome and examine FLG vulnerability of nickel hydrolysis. We performed a general bioinformatic study followed by biochemical and biological analysis of a single case, the FLG protein. FLG model peptides, the recombinant monomer domain human keratinocytes in vitro and human epidermis ex vivo were used. We also investigated if the products of filaggrin Ni2+-hydrolysis affect the activation profile of Langerhans cells. We found X1-S/T-c/p-H-c-X2 motifs in 40% of human proteins, with the highest abundance in those involved in the epidermal barrier function, including FLG. We confirmed the hydrolytic vulnerability and pH-dependent Ni2+-assisted cleavage of FLG-derived peptides and FLG monomer, using in vitro cell culture and ex-vivo epidermal sheets; the hydrolysis contributed to the pronounced reduction in FLG in all of the models studied. We also postulated that Ni-hydrolysis might dysregulate important immune responses. Ni2+-assisted cleavage of barrier proteins, including FLG, may contribute to clinical disease associated with nickel exposure
cis‐prenyltransferase 3 and α/β‐hydrolase are new determinants of dolichol accumulation in Arabidopsis
Get PDFDolichols (Dols), ubiquitous components of living organisms, are indispensable for cell survival. In plants, as well as other eukaryotes, Dols are crucial for posttranslational protein glycosylation, aberration of which leads to fatal metabolic disorders in humans and male sterility in plants. Until now, the mechanisms underlying Dol accumulation remain elusive. In this study, we have analysed the natural variation of the accumulation of Dols and six other isoprenoids among more than 120 Arabidopsis thaliana accessions. Subsequently, by combining QTL and GWAS approaches, we have identified several candidate genes involved in the accumulation of Dols, polyprenols, plastoquinone and phytosterols. The role of two genes implicated in the accumulation of major Dols in Arabidopsis—the AT2G17570 gene encoding a long searched for cis‐prenyltransferase (CPT3) and the AT1G52460 gene encoding an α/β‐hydrolase—is experimentally confirmed. These data will help to generate Dol‐enriched plants which might serve as a remedy for Dol‐deficiency in humans
Editorial: Protein posttranslational modifications in plant responses to abiotic stress - Women in plant science series
Get PDFSelected ALKBH dioxygenases are overexpressed in salivary gland tumours.
Get PDFSalivary gland tumours (SGTs) are a heterogeneous group of benign tumours of various origins and pathologies, showing a number of DNA modifications. Previously, in malignant head and neck cancer (HNSCC), we found overexpression of ALKBH proteins, the homologs of Escherichia coli AlkB 2-oxoglutarate and Fe(II) dependent dioxygenase. Moreover, we proved the connection of some of these dioxygenases with cancer development. Here, we studied the expression of five of these ALKBH dioxygenases: 1, 3, 4, 5, and FTO in benign SGTs. Using Western blot analysis, we found overexpression of three proteins: ALKBH1, 4, and FTO in SGT as compared to the surrounding, unaffected tissue. ALKBH4 was overexpressed in 76% of patient samples, whereas ALKBH1 and FTO in 65% of the samples. These results differ from those obtained in HNSCC, where FTO overexpression has been observed in 90% of patient samples. We also investigated the relationships between ALKBHs’ expression levels in normal and SGT tissues and identified two correlated pairs: ALKBH1-ALKBH3 and ALKBH1-ALKBH5. Additionally, in tumour tissue ALKBHs: ALKBH1, ALKBH3, ALKBH4, and ALKBH5 levels were correlated with each other. Together, these findings show that the ALKBH proteins exhibit pro cancerogenic action in SGT, even though the levels ALKBHs are generally lower in benign SGT than in malignant HNSCC. We suggest that the overexpression of the ALKBHs, especially FTO, may be used as a cancer marker and for its grading
The importance of prenol lipids in mitigating salt stress in the leaves of Tilia × euchlora trees
Get PDFDe-icing salt has been used on streets and pavements in most northern countries since the 1960s. Salt stress limits all vital functions of trees. Tilia × euchlora is planted in many cities given its unique decorative qualities. The aim of this study was to determine the tree strategy to mitigate salt stress due to the synthesis of polyprenols in leaves. Many years of observations have demonstrated that trees of the same species growing in the same street conditions may have extremely different health statuses. The study consisted of two experiments: a field experiment with urban street trees growing in saline soils and a controlled pot experiment with young trees exposed to increasing doses of salt. The differences between the young trees from the pot experiment and older trees from the field experiment were expressed in their ability to synthesize polyprenols. In urban conditions, the tree leaves with less damage contained signifcantly more polyprenols than did those with more damage. The salt stress mitigation strategy may be related to the ability to synthesize polyprenols. This ability
can be acquired through adaptation by older trees. The mechanism involves limiting the transport of Cl− and Na+ to leaves. In the pot experiment, the young trees did not exhibit this ability
The use of drone-based aerial photogrammetry in population monitoring of Southern Giant Petrels in ASMA 1, King George Island, maritime Antarctica
Get PDFSouthern Giant Petrels (SGPs) are surface nesting birds with a circumpolar Southern Hemisphere breeding distribution. The species tends to have no natural enemies on land, but is sensitive to human disturbance. The search for new methods is crucial and may minimize or exclude stress and risk of nest disturbance, related to ground-based research activities. The aim of this study was to conduct a population census of the SGP of the Antarctic Specially Managed Area no. 1 (ASMA no. 1), Admiralty Bay, King George Island, using an unoccupied aerial system (UAS) based on aerial photogrammetry and to determine the optimal parameters of the aerial mission for the identification of SGP adults and chicks on orthophotos while simultaneously not causing behavioural changes. To this end, in a preliminary survey in the 2019/20 season, the locations of all breeding areas for SGPs in ASMA no. 1 were determined, and the presence of 3 colonies, Llano Point/Rescuers Hills (LP/RH), Vaureal (V) and Petrel Hill (PH), was confirmed. Terrain models for two of the colonies (LP/RH and V) were established, and the flight parameters for the next season were determined. In 2020/21, a total of 23 (DJI Inspire 2 with a Zenmuse X5S camera) drone missions were performed at various stages of the breeding period over the LP/RH and V colonies. This assessment yielded estimation of the number of active nests and chicks over the entire ASMA no. 1 area, and included 508 active nests and 380 chicks for the 2020/21 season. To determine the minimum flight altitude at which no SGP behavioural response was observed, an experiment was performed that showed the vertical distance between the potential nest of SGP and the drone should be greater than 21 m given that lowering the altitude yielded statistically significant differences in bird behaviour. Image analyses showed the possibility of identifying adults and chicks at a ground sampling distance of 2.15 cm, which corresponded to an altitude of 130 m based on the equipment used and the terrain characteristics. The proposed method requires several missions during the incubation phase to determine a reliable number of active nests without using correction factors. To obtain a nesting success factor, it is recommended to perform at least one raid in the post-brooding phase of chick rearing (when the chick is not covered by an adult and is visible in orthophotos). The proposed method is not able to replace traditional methods in the context of many ongoing surveys, but we believe that it may provide a less bird-invasive, human-intensive and time-consuming option to replace ground-based census surveys
The GDAP1 p.Glu222Lys Variant-Weak Pathogenic Effect, Cumulative Effect of Weak Sequence Variants, or Synergy of Both Factors?
Get PDFCharcot–Marie–Tooth disorders (CMT) represent a highly heterogeneous group of diseases
of the peripheral nervous system in which more than 100 genes are involved. In some CMT patients,
a few weak sequence variants toward other CMT genes are detected instead of one leading CMT
mutation. Thus, the presence of a few variants in different CMT-associated genes raises the question
concerning the pathogenic status of one of them. In this study, we aimed to analyze the pathogenic
effect of c.664G>A, p.Glu222Lys variant in the GDAP1 gene, whose mutations are known to be
causative for CMT type 4A (CMT4A). Due to low penetrance and a rare occurrence limited to five
patients from two Polish families affected by the CMT phenotype, there is doubt as to whether
we are dealing with real pathogenic mutation. Thus, we aimed to study the pathogenic effect of
the c.664G>A, p.Glu222Lys variant in its natural environment, i.e., the neuronal SH-SY5Y cell line.
Additionally, we have checked the pathogenic status of p.Glu222Lys in the broader context of the
whole exome. We also have analyzed the impact of GDAP1 gene mutations on the morphology of the
transfected cells. Despite the use of several tests to determine the pathogenicity of the p.Glu222Lys
variant, we cannot point to one that would definitively solve the problem of pathogenici