Interpreting Sequence-Levenshtein distance for determining error type and frequency between two embedded sequences of equal length

Levenshtein distance is a commonly used edit distance metric, typically applied in language processing, and to a lesser extent, in molecular biology analysis. Biological nucleic acid sequences are often embedded in longer sequences and are subject to insertion and deletion errors that introduce frameshift during sequencing. These frameshift errors are due to string context and should not be counted as true biological errors. Sequence-Levenshtein distance is a modification to Levenshtein distance that is permissive of frameshift error without additional penalty. However, in a biological context Levenshtein distance needs to accommodate both frameshift and weighted errors, which Sequence-Levenshtein distance cannot do. Errors are weighted when they are associated with a numerical cost that corresponds to their frequency of appearance. Here, we describe a modification that allows the use of Levenshtein distance and Sequence-Levenshtein distance to appropriately accommodate penalty-free frameshift between embedded sequences and correctly weight specific error types.Comment: 10 pages, 8 figure

Workflow optimization for identification of female germline or oogonial stem cells in human ovarian cortex using single-cell RNA sequence analysis

In 2004, the identification of female germline or oogonial stem cells (OSCs) that can support post–natal oogenesis in ovaries of adult mice sparked a major paradigm shift in reproductive biology. Although these findings have been independently verified, and further extended to include identification of OSCs in adult ovaries of many species ranging from pigs and cows to non–human primates and humans, a recent study rooted in single–cell RNA sequence analysis (scRNA-seq) of adult human ovarian cortical tissue claimed that OSCs do not exist, and that other groups working with OSCs following isolation by magnetic-assisted or fluorescence-activated cell sorting have mistaken perivascular cells (PVCs) for germ cells. Here we report that rare germ lineage cells with a gene expression profile matched to OSCs but distinct from that of other cells, including oocytes and PVCs, can be identified in adult human ovarian cortical tissue by scRNA-seq after optimization of analytical workflow parameters. Deeper cell-by-cell expression profiling also uncovered evidence of germ cells undergoing meiosis-I in adult human ovaries. Lastly, we show that, if not properly controlled for, PVCs can be inadvertently isolated during flow cytometry protocols designed to sort OSCs because of inherently high cellular autofluorescence. However, human PVCs and human germ cells segregate into distinct clusters following scRNA-seq due to non–overlapping gene expression profiles, which would preclude the mistaken identification and use of PVCs as OSCs during functional characterization studies

Activity of Bdellovibrio Hit Locus Proteins, Bd0108 and Bd0109, Links Type IVa Pilus Extrusion/Retraction Status to Prey-Independent Growth Signalling

Bdellovibrio bacteriovorus are facultatively predatory bacteria that grow within gram-negative prey, using pili to invade their periplasmic niche. They also grow prey-independently on organic nutrients after undergoing a reversible switch. The nature of the growth switching mechanism has been elusive, but several independent reports suggested mutations in the hit (host-interaction) locus on the Bdellovibrio genome were associated with the transition to preyindependent growth. Pili are essential for prey entry by Bdellovibrio and sequence analysis of the hit locus predicted that it was part of a cluster of Type IVb pilus-associated genes, containing bd0108 and bd0109. In this study we have deleted the whole bd0108 gene, which is unique to Bdellovibrio, and compared its phenotype to strains containing spontaneous mutations in bd0108 and the common natural 42 bp deletion variant of bd0108. We find that deletion of the whole bd0108 gene greatly reduced the extrusion of pili, whereas the 42 bp deletion caused greater pilus extrusion than wild-type. The pili isolated from these strains were comprised of the Type IVa pilin protein; PilA. Attempts to similarly delete gene bd0109, which like bd0108 encodes a periplasmic/secreted protein, were not successful, suggesting that it is likely to be essential for Bdellovibrio viability in any growth mode. Bd0109 has a sugar binding YD- repeat motif and an N-terminus with a putative pilin-like fold and was found to interact directly with Bd0108. These results lead us to propose that the Bd0109/Bd0108 interaction regulates pilus production in Bdellovibrio (possibly by interaction with the pilus fibre at the cell wall), and that the presence (and possibly retraction state) of the pilus feeds back to alter the growth state of the Bdellovibrio cell. We further identify a novel small RNA encoded by the hit locus, the transcription of which is altered in different bd0108 mutation background

Female Fertility Preservation through Stem Cell-based Ovarian Tissue Reconstitution In Vitro and Ovarian Regeneration In Vivo

Historically, approaches designed to offer women diagnosed with cancer the prospects of having a genetically matched child after completion of their cytotoxic treatments focused on the existing oocyte population as the sole resource available for clinical management of infertility. In this regard, elective oocyte and embryo cryopreservation, as well as autologous ovarian cortical tissue grafting posttreatment, have gained widespread support as options for young girls and reproductive-age women who are faced with cancer to consider. In addition, the use of ovarian protective therapies, including gonadotropin-releasing hormone agonists and sphingosine-1-phosphate analogs, has been put forth as an alternative way to preserve fertility by shielding existing oocytes in the ovaries in vivo from the side-effect damage caused by radiotherapy and many chemotherapeutic regimens. This viewpoint changed with the publication of now numerous reports that adult ovaries of many mammalian species, including humans, contain a rare population of oocyte-producing germ cells—referred to as female germline or oogonial stem cells (OSCs). This new line of study has fueled research into the prospects of generating new oocytes, rather than working with existing oocytes, as a novel approach to sustain or restore fertility in female cancer survivors. Here, we overview the history of work from laboratories around the world focused on improving our understanding of the biology of OSCs and how these cells may be used to reconstitute “artificial” ovarian tissue in vitro or to regenerate damaged ovarian tissue in vivo as future fertility-preservation options

Influence of Maternal Aging on Mitochondrial Heterogeneity, Inheritance, and Function in Oocytes and Preimplantation Embryos

Contrasting the equal contribution of nuclear genetic material from maternal and paternal sources to offspring, passage of mitochondria, and thus mitochondrial DNA (mtDNA), is uniparental through the egg. Since mitochondria in eggs are ancestral to all somatic mitochondria of the next generation and to all cells of future generations, oocytes must prepare for the high energetic demands of maturation, fertilization and embryogenesis while simultaneously ensuring that their mitochondrial genomes are inherited in an undamaged state. Although significant effort has been made to understand how the mtDNA bottleneck and purifying selection act coordinately to prevent silent and unchecked spreading of invisible mtDNA mutations through the female germ line across successive generations, it is unknown if and how somatic cells of the immediate next generation are spared from inheritance of detrimental mtDNA molecules. Here, we review unique aspects of mitochondrial activity and segregation in eggs and early embryos, and how these events play into embryonic developmental competency in the face of advancing maternal age

Implications and Current Limitations of Oogenesis from Female Germline or Oogonial Stem Cells in Adult Mammalian Ovaries

A now large body of evidence supports the existence of mitotically active germ cells in postnatal ovaries of diverse mammalian species, including humans. This opens the possibility that adult stem cells naturally committed to a germline fate could be leveraged for the production of female gametes outside of the body. The functional properties of these cells, referred to as female germline or oogonial stem cells (OSCs), in ovaries of women have recently been tested in various ways, including a very recent investigation of the differentiation capacity of human OSCs at a single cell level. The exciting insights gained from these experiments, coupled with other data derived from intraovarian transplantation and genetic tracing analyses in animal models that have established the capacity of OSCs to generate healthy eggs, embryos and offspring, should drive constructive discussions in this relatively new field to further exploring the value of these cells to the study, and potential management, of human female fertility. Here, we provide a brief history of the discovery and characterization of OSCs in mammals, as well as of the in-vivo significance of postnatal oogenesis to adult ovarian function. We then highlight several key observations made recently on the biology of OSCs, and integrate this information into a broader discussion of the potential value and limitations of these adult stem cells to achieving a greater understanding of human female gametogenesis in vivo and in vitro

Mitochondrial membrane depolarization enhances TRAIL-induced cell death in adult human granulosa tumor cells, KGN, through inhibition of BIRC5

Abstract Background Cellular metabolic changes that accompany malignant transformation have been heralded as hallmark features of cancer. However, metabolic signatures between neoplasms can be unique, allowing for distinctions in malignancy, invasion and chemoresistance between cancer types and subtypes. Mitochondria are central metabolic mediators, as cellular bioenergetics veers from oxidative phosphorylation to glycolysis. Herein, we evaluate the role of mitochondria in maintenance of cellular metabolism, proliferation, and survival in the adult granulosa tumor cell line, KGN, as well as three epithelial ovarian cancer cell lines to determine distinctions in specific features. Results Notably, KGN cells were susceptible to TRAIL- and cisplatin-induced death following pretreatment with the metabolic inhibitor FCCP, but not oligomycin A. Collapse of mitochondrial membrane potential was found concomitant with cell death via apoptosis, independent from extrinsic canonical apoptotic routes. Rather, treatment with FCCP resulted in elevated cytochrome c release from mitochondria and decreased responsiveness to BIRC5. Following knockdown of BIRC5, mitochondrial membrane depolarization further sensitized KGN cells to induction of apoptosis via TRAIL. Conclusions These results indicate an essential role, distinct from metabolism, for mitochondrial membrane potential in KGN cells to sense and respond to external mediators of apoptotic induction

A method for freeze-fracture and scanning electron microscopy of isolated mitochondria

Electron microscopy as a methodology for the study of mitochondria based on morphological features is a standard technique that has experienced little evolution over the course of several decades. This technology has identified heterogeneity of mitochondria populations across both whole tissues, as well between individual cells, using primarily ultrathin sections for transmission electron microscopy (TEM). However, this technique constrains the evaluation of a sample to a single two-dimensional plane. To overcome this limitation, scanning electron microscopy (SEM) has been successfully utilized to observe three-dimensional mitochondria structures within the complex microenvironment containing total cellular components. In response to these dual technical caveats of existing electron microscopy protocols, we developed a methodology to evaluate the three-dimensional ultrastructure of isolated mitochondria, utilizing a freeze-fracture step and rigorous preservation of sample morphology. This protocol allows for a more high-throughput analysis of mitochondria populations from a specimen of interest, as the sample has been previously purified, as well as a finer resolution of complex intra-mitochondrial structures, using the depth of field created by SEM. • Protocol designed for SEM of isolated mitochondria samples. • SEM visualizes mitochondria ultrastructure in 3-D. • Freeze-fracture creates cross-sectional plane for view of interior organelle structures. Method name: Freeze-fracture scanning electron microscopy of isolated mitochondria, Keywords: Organelle, Ultrastructure, Protocol, Mitochondria, Scanning electron microscop

Postnatal oogenesis through ongoing oogonial stem cell (OSC) mitosis explains increasing oocyte depth with age.

<p>(a) Following primordial germ cell (PGC) expansion starting at embryonic day 7.5 (e7.5) in the mouse, proliferation of female germ cells (oogonia; <i>pink</i>) ceases at e13.5 concomitant with a 5-day period of germ cell meiotic commitment that drives formation of oocytes (<i>blue</i>); since all oocytes produced during this time are of equivalent “depth”, the production-line hypothesis of postnatal oocyte maturation cannot logically explain increasing oocyte depth as females age. (b) If continued proliferation of OSCs (<i>red</i>) and their subsequent differentiation into oocytes (<i>blue</i>) during postnatal life is superimposed on the production-line hypothesis, the emerging picture is consistent with a progressive increase in oocyte depth in females as they age.</p