Analysis of Hypericum accessions by DNA fingerprinting and flow cytometry

Hypericum perforatum, H. umbellatum, H. maculatum, and H. hircinum accessions originating from botanical gardens across Europe were examined by flow cytometry and molecular markers. 2C DNA content of 17 Hypericum perforatum accessions (Hp) and the H. perforatum cultivar Topaz amounted to between 1.56 pg and 1.62 pg. In four Hp accessions some individual plants were found with a DNA content corresponding to 6Cx (2.34 - 2.39 pg). All plants of accession Hp8 showed a DNA content of 6Cx (2.41 pg). In root tips of Hp plants with an average DNA amount of 1.58 pg, 32 chromosomes were detected, corresponding to 2n = 4x. This is the first ploidy and/or DNA content report for H. umbellatum, H. maculatum and H. hircinum. H. umbellatum and H. maculatum, each contained 0.76 pg DNA and 16 chromosomes were counted. The 2C DNA content of H. hircinum was 1.00 pg with the best metaphase plate revealing 32 chromosomes. Additionally, a combined marker analysis, based on inter-simple sequence repeats (ISSR) and sequence related amplified polymorphism (SRAP), was conducted to gain a better understanding of diversity especially within the accessions of H. perforatum. A total of 27 (11 ISSR and 16 SRAP) primer combinations were screened, showing 699 bands, of which 661 were polymorphic. UPGMA clustering revealed that accessions from the same geographic area tended to be more closely related, while H. maculatum was grouped separately from all H. perforatum accessions. Both methods have shown similar sensitivities in detecting the genetic diversity of the analyzed genotypes. Our results may be useful for Hypericum breeding programs and the development of effective conservation strategies.

Control of nuclear size by osmotic forces in Schizosaccharomyces pombe

The size of the nucleus scales robustly with cell size so that the nuclear-to-cell volume ratio (N/C ratio) is maintained during cell growth in many cell types. The mechanism responsible for this scaling remains mysterious. Previous studies have established that the N/C ratio is not determined by DNA amount, but is instead influenced by factors such as nuclear envelope mechanics and nuclear transport. Here, we developed a quantitative model for nuclear size control based upon colloid osmotic pressure and tested key predictions in the fission yeast Schizosaccharomyces pombe. This model posits that the N/C ratio is determined by the numbers of macromolecules in the nucleoplasm and cytoplasm. Osmotic shift experiments showed that the fission yeast nucleus behaves as an ideal osmometer whose volume is primarily dictated by osmotic forces. Inhibition of nuclear export caused accumulation of macromolecules and an increase in crowding in the nucleoplasm, leading to nuclear swelling. We further demonstrated that the N/C ratio is maintained by a homeostasis mechanism based upon synthesis of macromolecules during growth. These studies demonstrate the functions of colloid osmotic pressure in intracellular organization and size control.

Contamination of grain in West Siberia by Alternaria fungi and their mycotoxins

The ubiquitous occurrence of Alternaria fungi belonging to sections Alternaria and Infectoriae was confirmed using real-time PCR in wheat, barley and oat grain grown in West Siberia in 2018‒2019. The DNA amount of Alternaria section Alternaria fungi varied from 53×10-4 to 21731×10-4 pg/ng and on average exceeded the DNA amount of Alternaria section Infectoriae fungi by 4.5‒14.6 times, depending on the crop and harvest year.The average DNA amount of Alternaria fungi belonging to both sections in the oat grain was lower than in wheat and barley grain. The grain samples from Altay region were the most infected with Alternaria fungi. The alternariol (AOH), alternariol monomethyl ether (AME), tentoxin (TEN), and tenuazonic acid (TeA) mycotoxins produced by Alternaria fungi were detected by HPLC-MS/MS in 23 %, 6 %, 85 %, and 83 % of analyzed grain samples, respectively. The majority (61 %) of the samples contained two Alternaria mycotoxins in the grain (mainly TEN and TeA), 19 % of the samples three mycotoxins, and only one sample all four together. In the most of samples the content of Alternaria mycotoxins did not exceed 100 μg/kg, and only TeA content was higher (from 113 to 14963 μg/kg) than others. The significant differences in grain crops by the Alternaria mycotoxins content were revealed: more amounts of AOH, AME, and less amount of TEN were found in oat grain then in barley grain. A high positive significant correlation between the DNA amount of Alternaria section Alternaria fungi and TeA was established that indicates the role of these fungi as the main producers of TeA in the grain.

Elevated 2HG does not cause features of tumorigenesis

Aims Gliomas are the most frequent brain tumours, representing 75% of all primary malignant brain tumours in adults. IDH1 (and IDH2) driver mutations occur in >80% of low grade gliomas and secondary GBMs, in <10% of primary GBMs and other cancers. How IDH1/2 mutations contribute to tumorigenesis is mostly unknown. IDH1/2 convert isocitrate to α-ketoglutarate, but when mutated possess a novel enzymatic function that reduces α-ketoglutarate to D2-hydroxyglutarate (2HG). Indeed 2HG accumulates in IDH1/2-mutant tumours, and this discovery suggested that 2HG may have a role in IDH1/2-mutant tumours onset and progression, possibly by causing dysregulations of various enzymes in the cells. Studies are undergoing to clarify the causative role of 2HG in IDH1/2-mutant tumours, but it is still not clear whether 2HG is the driver/oncometabolite. Our aim is to understand the role of 2HG in developing and adult mouse tissues and whether its accumulation might cause features of gliomagenesis. Method A constitutive D2hgdh Knock-out mouse (D2hgdh KO) was generated and the relative molecular and cellular analysis were performed. Results Brains dissected from D2hgdh KO mice appeared to be histologically normal. No differences were found in the proliferation and labelling retaining capacity of neural stem and progenitors cells (NSC/NPC) of the D2hgdh KO mice compared to controls. A comprehensive metabolites analysis showed that D2hgdh KO mouse accumulated 2HG in various organs and tissues, included total brains and in the NSC/NPC microdissected from the subventricular zone, the site of origin of many human gliomas. The DNA amount of 5mC and 5hmC extracted from brains of D2hgdh KO mice was similar to controls. A normal number of haematopoietic progenitors was also found. Conclusion Although D2hgdh KO mice accumulated 2HG in all tissues analysed, they did not develop any abnormalities and remained completely asymptomatic. This suggests that a mere increment of 2HG in developing and adult tissues may be not sufficient to cause tumorigenesis (and gliomagenesis), leading some doubts on the oncogenic roles of the 2HG in IDH1/2-mutant tumours.

Linker histone H1.8 inhibits chromatin-binding of condensins and DNA topoisomerase II to tune chromosome length and individualization

DNA loop extrusion by condensins and decatenation by DNA topoisomerase II (topo II) are thought to drive mitotic chromosome compaction and individualization. Here, we reveal that the linker histone H1.8 antagonizes condensins and topo II to shape mitotic chromosome organization. In vitro chromatin reconstitution experiments demonstrate that H1.8 inhibits binding of condensins and topo II to nucleosome arrays. Accordingly, H1.8 depletion in Xenopus egg extracts increased condensins and topo II levels on mitotic chromatin. Chromosome morphology and Hi-C analyses suggest that H1.8 depletion makes chromosomes thinner and longer through shortening the average loop size and reducing the DNA amount in each layer of mitotic loops. Furthermore, excess loading of condensins and topo II to chromosomes by H1.8 depletion causes hyper-chromosome individualization and dispersion. We propose that condensins and topo II are essential for chromosome individualization, but their functions are tuned by the linker histone to keep chromosomes together until anaphase.

Impact of SNP microarray analysis of compromised DNA on kinship classification success in the context of investigative genetic genealogy

Single nucleotide polymorphism (SNP) data generated with microarray technologies have been used to solve murder cases via investigative leads obtained from identifying relatives of the unknown perpetrator included in accessible genomic databases, referred to as investigative genetic genealogy (IGG). However, SNP microarrays were developed for relatively high input DNA quantity and quality, while SNP microarray data from compromised DNA typically obtainable from crime scene stains are largely missing. By applying the Illumina Global Screening Array (GSA) to 264 DNA samples with systematically altered quantity and quality, we empirically tested the impact of SNP microarray analysis of deprecated DNA on kinship classification success, as relevant in IGG. Reference data from manufacturer-recommended input DNA quality and quantity were used to estimate genotype accuracy in the compromised DNA samples and for simulating data of different degree relatives. Although stepwise decrease of input DNA amount from 200 nanogram to 6.25 picogram led to decreased SNP call rates and increased genotyping errors, kinship classification success did not decrease down to 250 picogram for siblings and 1st cousins, 1 nanogram for 2nd cousins, while at 25 picogram and below kinship classification success was zero. Stepwise decrease of input DNA quality via increased DNA fragmentation resulted in the decrease of genotyping accuracy as well as kinship classification success, which went down to zero at the average DNA fragment size of 150 base pairs. Combining decreased DNA quantity and quality in mock casework and skeletal samples further highlighted possibilities and limitations. Overall, GSA analysis achieved maximal kinship classification success from 800-200 times lower input DNA quantities than manufacturer-recommended, although DNA quality plays a key role too, while compromised DNA produced false negative kinship classifications rather than false positive ones.

Circulating Tumor DNA as a Marker for Treatment Response in Metastatic Melanoma Patients Using Next-Generation Sequencing—A Prospective Feasibility Study

We prospectively performed a longitudinal analysis of circulating tumor DNA (ctDNA) from 149 plasma samples and CT scans in Stage III and IV metastatic melanoma patients (n = 20) treated with targeted agents or immunotherapy using two custom next-generation sequencing (NGS) Ion AmpliSeq™ HD panels including 60 and 81 amplicons in 18 genes, respectively. Concordance of matching cancer-associated mutations in tissue and plasma was 73.3%. Mutant allele frequency (MAF) levels showed a range from 0.04% to 28.7%, well detectable with NGS technologies utilizing single molecule tagging like the AmpliSeq™ HD workflow. Median followup time of the tissue and/or plasma positive cohort (n = 15) was 24.6 months and median progression-free survival (PFS) was 7.8 months. Higher MAF ≥ 1% at baseline was not significantly associated with a risk of progression (Odds Ratio = 0.15; p = 0.155). Although a trend could be seen, MAF levels did not differ significantly over time between patients with and without a PFS event (p = 0.745). Depending on the cell-free DNA amount, NGS achieved a sensitivity down to 0.1% MAF and allowed for parallel analysis of multiple mutations and previously unknown mutations. Our study indicates that NGS gene panels could be useful for monitoring disease burden during therapy with ctDNA in melanoma patients.

Evaluation of the effects of library preparation procedure and sample characteristics on the accuracy of metagenomic profiles

Shotgun metagenomic sequencing has transformed our understanding of microbial community ecology. However, preparing metagenomic libraries for high-throughput DNA sequencing remains a costly, labor-intensive, and time-consuming procedure, which in turn limits the utility of metagenomes. Several library preparation procedures have recently been developed to offset these costs, but it is unclear how these newer procedures compare to current standards in the field. In particular, it is not clear if all such procedures perform equally well across different types of microbial communities, or if features of the biological samples being processed (e.g., DNA amount) impact the accuracy of the approach. To address these questions, we assessed how five different shotgun DNA sequence library preparation methods, including the commonly used Nextera® Flex kit, perform when applied to metagenomic DNA. We measured each method's ability to produce metagenomic data that accurately represents the underlying taxonomic and genetic diversity of the community. We performed these analyses across a range of microbial community types (e.g., soil, coral-associated, mouse-gut-associated) and input DNA amounts. We find that the type of community and amount of input DNA influence each method’s performance, indicating that careful consideration may be needed when selecting between methods, especially for low complexity communities. However, cost-effective preparation methods we assessed are generally comparable to the current gold standard Nextera® DNA Flex kit for high-complexity communities. Overall, the results from this analysis will help expand and even facilitate access to metagenomic approaches in future studies.

Dominant ACO2 mutations are a frequent cause of isolated optic atrophy

Biallelic mutations in ACO2, encoding the mitochondrial aconitase 2, have been identified in individuals with neurodegenerative syndromes, including infantile cerebellar retinal degeneration and recessive optic neuropathies (locus OPA9). By screening European cohorts of individuals with genetically unsolved inherited optic neuropathies, we identified 61 cases harboring variants in ACO2, among whom 50 carried dominant mutations, emphasizing for the first time the important contribution of ACO2 monoallelic pathogenic variants to dominant optic atrophy. Analysis of the ophthalmological and clinical data revealed that recessive cases are affected more severely than dominant cases, while not significantly earlier. In addition, 27% of the recessive cases and 11% of the dominant cases manifested with extraocular features in addition to optic atrophy. In silico analyses of ACO2 variants predicted their deleterious impacts on ACO2 biophysical properties. Skin derived fibroblasts from patients harboring dominant and recessive ACO2 mutations revealed a reduction of ACO2 abundance and enzymatic activity, and the impairment of the mitochondrial respiration using citrate and pyruvate as substrates, while the addition of other Krebs cycle intermediates restored a normal respiration, suggesting a possible short-cut adaptation of the tricarboxylic citric acid cycle. Analysis of the mitochondrial genome abundance disclosed a significant reduction of the mitochondrial DNA amount in all ACO2 fibroblasts. Overall, our data position ACO2 as the third most frequently mutated gene in autosomal inherited optic neuropathies, after OPA1 and WFS1, and emphasize the crucial involvement of the first steps of the Krebs cycle in the maintenance and survival of retinal ganglion cells. By screening European cohorts of individuals with genetically unsolved inherited optic neuropathies, Charif et al. report 61 new cases harboring variants in ACO2, among whom 50 with dominant mutations, emphasizing for the first time the important contribution of ACO2 monoallelic pathogenic variants to dominant optic atrophy.