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Forensic Science International. Genetics May 2024In forensics investigations, it is common to encounter biological mixtures consisting of homogeneous or heterogeneous components from multiple individuals and with...
In forensics investigations, it is common to encounter biological mixtures consisting of homogeneous or heterogeneous components from multiple individuals and with different genetic contributions. One promising mixture deconvolution strategy is the DEPArray™ technology, which enables the separation of cell populations before genetic analysis. While technological advances are fundamental, their reliable validation is crucial for successful implementation and use for casework. Thus, this study aimed to 1) systematically validate the DEPArray™ system concerning specificity, sensitivity, repeatability, and contamination occurrences for blood, epithelial, and sperm cells, and 2) evaluate its potential for single-cell analysis in the field of forensic science. Our findings confirmed the effective identification of different cell types and the correct assignment of successfully genotyped single cells to their respective donor(s). Using the NGM Detect™ Amplification Kit, the average profile completeness for diploid cells was approximately 80%, with ∼ 290 RFUs. In contrast, haploid sperm analysis yielded an average completeness of 51% referring to the haploid reference profile, accompanied by mean peak heights of ∼ 176 RFUs. Although certain alleles of heterozygous loci in diploid cells showed strong imbalances, the overall peak balances yielded acceptable values above ≥ 60% with a mean value of 72% ± 0.21, a median of 77%, but with a maximum imbalance of 9% between heterozygous peaks. Locus dropouts were considered stochastic events, exhibiting variations among donors and cell types, with a notable failure incidence observed for TH01. Within the wet-lab experimentation with >500 single cells for the validation, profiling was performed using the consensus approach, where profiles were selected randomly from all data to better mirror real casework results. Nevertheless, complete profiles could be achieved with as few as three diploid cells, while the average success rate increased to 100% when using profiles of 6-10 cells. For sperms, however, a consensus profile with completeness >90% of the autosomal diploid genotype could be attained using ≥15 cells. In addition, the robustness of the consensus approach was evaluated in the absence of the respective reference profile without severe deterioration. Here, increased stutter peaks (≥ 15%) were found as the main artifact in single-cell profiles, while contamination and drop-ins were ascertained as rare events. Lastly, the technique's potential and limitations are discussed, and practical guidance is provided, particularly valuable for cold cases, multiple perpetrator rapes, and analyses of homogeneous mixed evidence.
Topics: Humans; Male; DNA Fingerprinting; Semen; Microsatellite Repeats; Polymerase Chain Reaction; Spermatozoa
PubMed: 38412740
DOI: 10.1016/j.fsigen.2024.103026 -
Plant Science : An International... Sep 2023Novel approaches to optimize the production of plant specialized metabolites are crucial to reach maximum productivity of plant biofactories. Plant polyploidization...
Novel approaches to optimize the production of plant specialized metabolites are crucial to reach maximum productivity of plant biofactories. Plant polyploidization frequently enhances protein synthesis and thereby increases the biosynthesis of specialized metabolites. Paclitaxel is a valuable anticancer agent scarcely produced in nature. Therefore, plant biofactories represent a sustainable alternative source of this compound and related taxanes. With the aim of improving the productivity of Taxus spp. cell cultures, we induced polyploidy in vitro by treating immature embryos of Taxus baccata with colchicine. To obtain the polyploid cell lines, calli were induced from T. baccata plantlets previously treated with colchicine and ploidy levels were accurately identified using flow cytometry. In terms of cell morphology, tetraploid cells were about 3-fold bigger than the diploid cells. The expression of taxane pathway genes was higher in the tetraploid cell line compared to the diploid cells. Moreover, taxane production was 6.2-fold higher and the production peak was achieved 8 days earlier than in the diploid cell line, indicating a higher productivity. The obtained tetraploid cell line proved to be highly productive, constituting a step forward towards the development of a bio-sustainable production system for this chemotherapeutic drug.
Topics: Taxus; Tetraploidy; Taxoids; Cell Culture Techniques; Cell Line; Colchicine
PubMed: 37343603
DOI: 10.1016/j.plantsci.2023.111776 -
Autophagy Nov 2023Polyploidy is an extended phenomenon in biology. However, its physiological significance and whether it defines specific cell behaviors is not well understood. Here we...
Polyploidy is an extended phenomenon in biology. However, its physiological significance and whether it defines specific cell behaviors is not well understood. Here we study its connection to macroautophagy/autophagy, using the larval respiratory system of as a model. This system comprises cells with the same function yet with notably different ploidy status, namely diploid progenitors and their polyploid larval counterparts, the latter destined to die during metamorphosis. We identified an association between polyploidy and autophagy and found that higher endoreplication status correlates with elevated autophagy. Finally, we report that tissue histolysis in the trachea during metamorphosis is mediated by autophagy, which triggers the apoptosis of polyploid cells. APF: after pupa formation; : autophagy related; : breathless; : Cyclin E; DT: dorsal trunk; : fizzy-related; L3: larval stage 3; PBS: phosphate-buffered saline; RI: RNAi; Tr: tracheal metamere; : yorkie.
Topics: Animals; Drosophila; Trachea; Drosophila melanogaster; Drosophila Proteins; Larva; Autophagy; Polyploidy
PubMed: 37424089
DOI: 10.1080/15548627.2023.2231828 -
JCI Insight Feb 2024Genetic modifications leading to pain insensitivity phenotypes, while rare, provide invaluable insights into the molecular biology of pain and reveal targets for...
Genetic modifications leading to pain insensitivity phenotypes, while rare, provide invaluable insights into the molecular biology of pain and reveal targets for analgesic drugs. Pain insensitivity typically results from Mendelian loss-of-function mutations in genes expressed in nociceptive (pain-sensing) dorsal root ganglion (DRG) neurons that connect the body to the spinal cord. We document a pain insensitivity mechanism arising from gene overexpression in individuals with the rare 7q11.23 duplication syndrome (Dup7), who have 3 copies of the approximately 1.5-megabase Williams syndrome (WS) critical region. Based on parental accounts and pain ratings, people with Dup7, mainly children in this study, are pain insensitive following serious injury to skin, bones, teeth, or viscera. In contrast, diploid siblings (2 copies of the WS critical region) and individuals with WS (1 copy) show standard reactions to painful events. A converging series of human assessments and cross-species cell biological and transcriptomic studies identified 1 likely candidate in the WS critical region, STX1A, as underlying the pain insensitivity phenotype. STX1A codes for the synaptic vesicle fusion protein syntaxin1A. Excess syntaxin1A was demonstrated to compromise neuropeptide exocytosis from nociceptive DRG neurons. Taken together, these data indicate a mechanism for producing "genetic analgesia" in Dup7 and offer previously untargeted routes to pain control.
Topics: Child; Humans; Ganglia, Spinal; Neurons; Pain; Synaptic Transmission; Williams Syndrome
PubMed: 38261410
DOI: 10.1172/jci.insight.176147 -
Integrative and Comparative Biology Dec 2023A review of the fossil record coupled with insights gained from molecular and developmental biology reveal a series of body plan transformations that gave rise to the... (Review)
Review
A review of the fossil record coupled with insights gained from molecular and developmental biology reveal a series of body plan transformations that gave rise to the first land plants. Across diverse algal clades, including the green algae and their descendants, the plant body plan underwent a unicellular $\to $ colonial $\to $ simple multicellular → complex multicellular transformation series. The colonization of land involved increasing body size and associated cell specialization, including cells capable of hydraulic transport. The evolution of the life-cycle that characterizes all known land plant species involved a divergence in body plan phenotypes between the haploid and diploid generations, one adapted to facilitate sexual reproduction (a free-water dependent gametophyte) and another adapted to the dissemination of spores (a more water-independent sporophyte). The amplification of this phenotypic divergence, combined with indeterminate growth in body size, resulted in a desiccation-adapted branched sporophyte with a cuticularized epidermis, stomates, and vascular tissues. Throughout the evolution of the land plants, the body plans of the sporophyte generation involved "axiation," i.e., the acquisition of a cylindrical geometry and subsequent organographic specializations.
Topics: Animals; Fossils; Plants; Embryophyta; Molecular Biology; Water; Biological Evolution; Phylogeny
PubMed: 36316013
DOI: 10.1093/icb/icac150 -
Telomerase Activity in Somatic Tissues and Ovaries of Diploid and Triploid Rainbow Trout () Females.Cells Jul 2023Telomerase activity has been found in the somatic tissues of rainbow trout. The enzyme is essential for maintaining telomere length but also assures homeostasis of the...
Telomerase activity has been found in the somatic tissues of rainbow trout. The enzyme is essential for maintaining telomere length but also assures homeostasis of the fish organs, playing an important role during tissue regeneration. The unique morphological and physiological characteristics of triploid rainbow trout, when compared to diploid specimens, make them a promising model for studies concerning telomerase activity. Thus, in this study, we examined the expression of the gene in various organs of subadult and adult diploid and triploid rainbow trout females. Upregulated mRNA transcription was observed in all the examined somatic tissues sampled from the triploid fish when compared to diploid individuals. Contrastingly, expression in the ovaries was significantly decreased in the triploid specimens. Within the diploids, the highest expression of was observed in the liver and in the ovaries of the subadult individuals. In the triploids, expression was increased in the somatic tissues, while the ovaries exhibited lower activity of telomerase compared to other organs and decreased compared to the ovaries in the diploids. The ovaries of triploid individuals were underdeveloped, consisting of only a few oocytes. The lack of germ cells, which are usually characterized by high expression, might be responsible for the decrease in telomerase activity in the triploid ovaries. The increase in expression in triploid somatic tissues suggests that they require higher telomerase activity to cope with environmental stress and maintain internal homeostasis.
Topics: Animals; Female; Triploidy; Ovary; Diploidy; Oncorhynchus mykiss; Telomerase
PubMed: 37443805
DOI: 10.3390/cells12131772 -
Translational Oncology Nov 2023Epithelial-mesenchymal plasticity (EMP) is a hallmark of cancer. By enabling cells to shift between different morphological and functional states, EMP promotes invasion,...
Epithelial-mesenchymal plasticity (EMP) is a hallmark of cancer. By enabling cells to shift between different morphological and functional states, EMP promotes invasion, metastasis and therapy resistance. We report that near-diploid non-cancerous human epithelial lung cells spontaneously shift along the EMP spectrum without genetic changes. Strikingly, more than half of single cell-derived clones adopt a mesenchymal morphology. We independently characterise epithelial-like and mesenchymal-like clones. Mesenchymal clones lose epithelial markers, display larger cell aspect ratios and lower motility, with mostly unaltered proliferation rates. Stemness marker expression and metabolic rewiring diverge independently of phenotypes. In 3D culture, more epithelial clones become mesenchymal-like. Thus, non-cancerous epithelial cells may acquire cancer metastasis-associated features prior to genetic alterations and cancerous transformation.
PubMed: 37611490
DOI: 10.1016/j.tranon.2023.101760 -
The Journal of Biological Chemistry Sep 2023Voltage-gated sodium (Na) channels drive the upstroke of the action potential and are comprised of a pore-forming α-subunit and regulatory β-subunits. The β-subunits...
Voltage-gated sodium (Na) channels drive the upstroke of the action potential and are comprised of a pore-forming α-subunit and regulatory β-subunits. The β-subunits modulate the gating, trafficking, and pharmacology of the α-subunit. These functions are routinely assessed by ectopic expression in heterologous cells. However, currently available expression systems may not capture the full range of these effects since they contain endogenous β-subunits. To better reveal β-subunit functions, we engineered a human cell line devoid of endogenous Na β-subunits and their immediate phylogenetic relatives. This new cell line, β-subunit-eliminated eHAP expression (BeHAPe) cells, were derived from haploid eHAP cells by engineering inactivating mutations in the β-subunits SCN1B, SCN2B, SCN3B, and SCN4B, and other subfamily members MPZ (myelin protein zero(P0)), MPZL1, MPZL2, MPZL3, and JAML. In diploid BeHAPe cells, the cardiac Na α-subunit, Na1.5, was highly sensitive to β-subunit modulation and revealed that each β-subunit and even MPZ imparted unique gating properties. Furthermore, combining β1 and β2 with Na1.5 generated a sodium channel with hybrid properties, distinct from the effects of the individual subunits. Thus, this approach revealed an expanded ability of β-subunits to regulate Na1.5 activity and can be used to improve the characterization of other α/β Na complexes.
Topics: Humans; Action Potentials; Cell Line; Intracellular Signaling Peptides and Proteins; NAV1.5 Voltage-Gated Sodium Channel; Phosphoproteins; Protein Subunits; Voltage-Gated Sodium Channel beta Subunits; Mutation
PubMed: 37544648
DOI: 10.1016/j.jbc.2023.105132 -
World Journal of Stem Cells Jul 2023Haploid embryonic stem cells (haESCs) have been established in many species. Differentiated haploid cell line types in mammals are lacking due to spontaneous...
BACKGROUND
Haploid embryonic stem cells (haESCs) have been established in many species. Differentiated haploid cell line types in mammals are lacking due to spontaneous diploidization during differentiation that compromises lineage-specific screens.
AIM
To derive human haploid neural stem cells (haNSCs) to carry out lineage-specific screens.
METHODS
Human haNSCs were differentiated from human extended haESCs with the help of Y27632 (ROCK signaling pathway inhibitor) and a series of cytokines to reduce diploidization. Neuronal differentiation of haNSCs was performed to examine their neural differentiation potency. Global gene expression analysis was con-ducted to compare haNSCs with diploid NSCs and haESCs. Fluorescence activated cell sorting was performed to assess the diploidization rate of extended haESCs and haNSCs. Genetic manipulation and screening were utilized to evaluate the significance of human haNSCs as genetic screening tools.
RESULTS
Human haESCs in extended pluripotent culture medium showed more compact and smaller colonies, a higher efficiency in neural differentiation, a higher cell survival ratio and higher stability in haploidy maintenance. These characteristics effectively facilitated the derivation of human haNSCs. These human haNSCs can be generated by differentiation and maintain haploidy and multipotency to neurons and glia in the long term . After PiggyBac transfection, there were multiple insertion sites in the human haNSCs' genome, and the insertion sites were evenly spread across all chromosomes. In addition, after the cells were treated with manganese, we were able to generate a list of manganese-induced toxicity genes, demonstrating their utility as genetic screening tools.
CONCLUSION
This is the first report of a generated human haploid somatic cell line with a complete genome, proliferative ability and neural differentiation potential that provides cell resources for recessive inheritance and drug targeted screening.
PubMed: 37545755
DOI: 10.4252/wjsc.v15.i7.734 -
Molecular Biology and Evolution Aug 2023The masking theory states that genes expressed in a haploid stage will be under more efficient selection. In contrast, selection will be less efficient in genes...
The masking theory states that genes expressed in a haploid stage will be under more efficient selection. In contrast, selection will be less efficient in genes expressed in a diploid stage, where the fitness effects of recessive deleterious or beneficial mutations can be hidden from selection in heterozygous form. This difference can influence several evolutionary processes such as the maintenance of genetic variation, adaptation rate, and genetic load. Masking theory expectations have been confirmed in single-cell haploid and diploid organisms. However, in multicellular organisms, such as plants, the effects of haploid selection are not clear-cut. In plants, the great majority of studies indicating haploid selection have been carried out using male haploid tissues in angiosperms. Hence, evidence in these systems is confounded with the effects of sexual selection and intraspecific competition. Evidence from other plant groups is scarce, and results show no support for the masking theory. Here, we have used a gymnosperm Scots pine megagametophyte, a maternally derived seed haploid tissue, and four diploid tissues to test the strength of purifying selection on a set of genes with tissue-specific expression. By using targeted resequencing data of those genes, we obtained estimates of genetic diversity, the site frequency spectrum of 0-fold and 4-fold sites, and inferred the distribution of fitness effects of new mutations in haploid and diploid tissue-specific genes. Our results show that purifying selection is stronger for tissue-specific genes expressed in the haploid megagametophyte tissue and that this signal of strong selection is not an artifact driven by high expression levels.
Topics: Haploidy; Selection, Genetic; Mutation; Biological Evolution; Diploidy; Plants
PubMed: 37565532
DOI: 10.1093/molbev/msad183