The effect of ancymidol on hyperhydricity, regeneration, starch and antioxidant enzymatic activities in liquid-cultured Narcissus

 Addition of the growth retardant ancymidol to Narcissus shoots and lower inner leaf sections isolated from shoots cultured in liquid medium induced hyperhydric malformations associated with morphogenetic changes. Meristematic centers initiated on the basal proximal ends appeared over the entire surface of the hyperhydric leaf sections after 6 weeks in culture. The meristematic centers which formed clusters on the leaf sections developed later into buds. In leaf sections grown in the liquid medium lacking ancymidol, hyperhydricity was not induced, and regeneration was not observed. Starch and protein levels and ascorbate peroxidase and catalase activities were examined in shoots and isolated leaf sections that were either hyperhydric or non-hyperhydric. In ancymidol-treated, hyperhydric leaf sections, ascorbate peroxidase and catalase activities were lower than in control, untreated leaf sections. The changes in starch and protein levels and in antioxidant enzymatic activities appeared to be related to the onset of meristematic-center initiation and further bud development on Narcissus hyperhydric leaf sections. Received: 6 May 2000 / Revision received: 21 August 2000 / Accepted: 22 August 2000     

Improving large-scale proteomics by clustering of mass spectrometry data

Tandem mass spectrometry (MS/MS), coupled with liquid chromatography (LC), is a powerful tool for the analysis and comparison of complex protein and peptide mixtures. However, the extremely large amounts of data that result from the process are very complex and difficult to analyze. We show how the clustering of similar spectra from multiple LC-MS/MS runs can help in data management and improve the analysis of complex peptide mixtures. The major effect of spectrum clustering is the reduction of the huge amounts of data to a manageable size. As a result, analysis time is shorter and more data can be stored for further analysis. Furthermore, spectrum quality improvement allows the identification of more peptides with greater confidence, the comparison of complex peptide mixtures is facilitated, and the entire proteomics project is presented in concise form. Pep-Miner is an advanced software tool that implements these clustering-based applications. It proved useful in several comparative proteomics projects involving lung cancer cells and various other cell types. In one of these projects, Pep-Miner reduced 517 000 spectra to 20 900 clusters and identified 2518 peptides derived from 830 proteins. Clustering and identification lasted less than two hours on an IBM Thinkpad T23 computer (laptop). Pep-Miner's unique properties make it a very useful tool for large-scale shotgun proteomics projects.     

Systematic bias and the phylogeny of Coleoptera—A response to Cai et al. (2022) following the responses to Cai et al. (2020)

Systematic bias is one of the major phylogenetic issues arising over the last two decades. Using methods designed to reduce compositional and rate heterogeneity, hence systematic bias, Cai and co-workers (2022) (= CEA22) reanalyzed the DNA sequence dataset for Coleoptera of Zhang et al. (2018) (= ZEA). CEA22 suggest that their phylogenetic results and major evolutionary hypotheses about the Coleoptera should be favoured over other recently published studies. Here, we discuss the methodology of CEA22 with particular attention to how their perfunctory reanalysis of ZEA obfuscates rather than illuminates beetle phylogeny. Similar to published rebuttals of an earlier study of theirs, we specifically find that many of their claims are misleading, unsupported, or false. Critically, CEA22 fail to establish the stated premise for their reanalysis. They fail to demonstrate how composition or rate heterogeneity supposedly impacted the phylogeny estimate of ZEA, let alone the results of other recent studies. Moreover, despite their claim of comprehensive sampling of Coleoptera, their dataset is neither the most diverse with respect to species and higher taxa included, nor anywhere near the largest in terms of sequence data and sampled loci. Although CEA22 does contribute additional fossils for calibration, those seeking the best available estimate for Coleoptera phylogeny and evolution based on molecular data are advised to look elsewhere.     

Inhibition of de novo pyrimidine synthesis augments Gemcitabine induced growth inhibition in an immunocompetent model of pancreatic cancer

Leflunomide (Lef) is an agent used in autoimmune disorders that interferes with DNA synthesis. De Novo pyrimidine synthesis is a mechanism of Gemcitabine (Gem) resistance in pancreatic cancer. This study aims to assess the efficacy and changes in the tumor microenvironment of Lef monotherapy and in combination with Gem, in a syngeneic mouse model of pancreatic cancer.Methods: MTS proliferation assays were conducted to assess growth inhibition by Gem (0-20 nM), Lef (0-40 uM) and Gem+Lef in KPC (KrasLSL.G12D/+;p53R172H/+; PdxCretg/+) cells in vitro. An in vivo heterotopic KPC model was used and cohorts were treated with: PBS (control), Gem (75 mg/kg/q3d), Lef (40 mg/kg/d), or Gem+Lef. At d28 post-treatment, tumor burden, proliferation index (Ki67), and vascularity (CD31) were measured. Changes in the frequency of peripheral and intratumoral immune cell subsets were evaluated via FACS. Liquid chromatography-mass spectrometry was used for metabolomics profiling.Results: Lef inhibits KPC cell growth and synergizes with Gem in vitro (P<0.05; Combination Index 0.44 (<1 indicates synergy). In vivo, Lef alone and in combination with Gem delays KPC tumor progression (P<0.001). CTLA-4+T cells are also significantly decreased in tumors treated with Lef, Gem or in combination (Gem+Lef) compared to controls (P<0.05). Combination therapy also decreased the Ki67 and vascularity (P<0.01). Leflunomide inhibits de novo pyrimidine synthesis both in vitro (p<0.0001) and in vivo (p<0.05).Conclusions: In this study, we demonstrated that Gem+Lef inhibits pancreatic cancer growth, decrease T cell exhaustion, vascularity and as proof of principle inhibits de novo pyrimidine synthesis. Further characterization of changes in adaptive immunity are necessary to characterize the mechanism of tumor growth inhibition and facilitate translation to a clinical trial.     

Targeting double-strand breaks to replicating DNA identifies a subpathway of DSB repair that is defective in ataxia-telangiectasia cells.

The critical cellular defect(s) and basis for cell killing by ionizing radiation in ataxia-telangiectasia (A-T) are unknown. We use the topoisomerase I inhibitor camptothecin (CPT), which kills mainly S-phase cells and induces DSBs predominantly in replication forks, to show that A-T cells are defective in the repair of this particular subclass of DSBs. CPT-treated A-T cells reaching G2 have abnormally high levels of chromatid exchanges (viewed as prematurely condensed G2 chromosomes); aberrations in normal cells are mostly chromatid breaks. Transfectants of A-T cells with the wild-type ATM cDNA are corrected for CPT sensitivity, chromatid aberrations, and the DSB repair defect. These data suggest that in normal cells ATM, the A-T protein, probably recognizes DSBs in active replicons and targets the repair machinery to the breaks; in addition, the ATM protein is involved in the suppression of low-fidelity, adventitious rejoining between replication-associated DSBs. The loss of ATM functions therefore leads to genome destabilization, sensitivity to DSB-inducing agents and to the cancer-promoting illegitimate exchange events that follow.     

Successful Treatment of Human Visceral Leishmaniasis Restores Antigen-Specific IFN-γ, but not IL-10 Production

One of the key immunological characteristics of active visceral leishmaniasis (VL) is a profound immunosuppression and impaired production of Interferon-γ (IFN-γ). However, recent studies from Bihar in India showed using a whole blood assay, that whole blood cells have maintained the capacity to produce IFN-γ. Here we tested the hypothesis that a population of low-density granulocytes (LDG) might contribute to T cell responses hyporesponsiveness via the release of arginase. Our results show that this population is affected by the anticoagulant used to collect blood: the frequency of LDGs is significantly lower when the blood is collected with heparin as compared to EDTA; however, the anticoagulant does not impact on the levels of arginase released. Next, we assessed the capacity of whole blood cells from patients with active VL to produce IFN-γ and IL-10 in response to antigen-specific and polyclonal activation. Our results show that whole blood cells produce low or levels below detection limit of IFN-γ and IL-10, however, after successful treatment of VL patients, these cells gradually regain their capacity to produce IFN-γ, but not IL-10, in response to activation. These results suggest that in contrast to VL patients from Bihar, India, whole blood cells from VL patients from Gondar, Ethiopia, have lost their ability to produce IFN-γ during active VL and that active disease is not associated with sustained levels of IL-10 production following stimulation.     

<Emphasis Type="Italic">N</Emphasis>-glycan moieties of the crustacean egg yolk protein and their glycosylation sites

Vitellogenin (Vg) is the precursor of the egg yolk glycoprotein of crustaceans. In the prawn Macrobrachium rosenbergii, Vg is synthesized in the hepatopancreas, secreted to the hemolymph, and taken up by means of receptor-mediated endocytosis into the oocytes. The importance of glycosylation of Vg lies in its putative role in the folding, processing and transport of this protein to the egg yolk and in the fact that the N-glycan moieties could provide a source of carbohydrate during embryogenesis. The present study describes, for the first time, the structure of the glycan moieties and their sites of attachment to the Vg of M. rosenbergii. Bioinformatics analysis revealed seven putative N-glycosylation sites in M. rosenbergii Vg; two of these glycosylation sites are conserved throughout the Vgs of decapod crustaceans from the Pleocyemata suborder (N ¹⁵⁹ and N ⁶⁶⁰). The glycosylation of six putative sites of M. rosenbergii Vg (N ¹⁵¹, N ¹⁵⁹, N _,¹⁶⁸ N _,⁶¹⁴ N ⁶⁶⁰ and N ²³⁰⁰) was confirmed; three of the confirmed glycosylation sites are localized around the N-terminally conserved N-glycosylation site N ¹⁵⁹. From a theoretical three-dimensional structure, these three N-glycosylated sites N ¹⁵¹, N ¹⁵⁹, and N ¹⁶⁸ were localized on the surface of the Vg consensus sequence. In addition, an uncommon high mannose N-linked oligosaccharide structure with a glucose cap (Glc₁Man₉GlcNAc₂) was characterized in the secreted Vg. These findings thus make a significant contribution to the structural elucidating of the crustacean Vg glycan moieties, which may shed light on their role in protein folding and transport and in recognition between Vg and its target organ, the oocyte.     

Evaluating soil seed banks of phosphate mining restoration in the hyper-arid Negev desert

Ecological systems are severely damaged by the anthropogenic disturbance of mining. Phosphate open-pit mining fields cover over 200 km² of the Negev desert, Israel. However, the effects of the ongoing mine site restoration efforts on the plant community have not been studied. Plants and their seed banks have a major role in ecosystem processes and restoration. In this study, we focused on three mining sites, restored in different years, along Zin River valley. We compared the plant community of restored mining plots within these areas to adjacent natural plots. We asked whether plant community germination potential from the soil seed bank differs between the restored plots and the adjacent natural plots within a mining site. We hypothesized that: (1) there is a lack of seed bank in the restored plots; (2) the altered soil composition at the restored plots inhibits germination. We used soil samples collected from the different mining sites and set up greenhouse experiments. One experiment compared natural and restored areas with different soil treatments. In another experiment, we added native seeds to test their germination potential on restored soil. Our results indicate that lack of seed bank is the major limiting factor hindering germination and not the composition of the soil after restoration. Our findings shed light on the constraints of seed bank establishment in post-mining areas of hyper-arid regions. We suggest considering active restoration practices to facilitate natural dispersal and improve seed bank establishment.