A high-throughput method for unbiased quantitation and categorisation of nuclear morphology

AbstractThe physical arrangement of chromatin in the nucleus is cell type and species specific. This is particularly evident in sperm, in which most of the cytoplasm has been lost; the shape of the nucleus reflects the shape of the cell. Mice have distinctive falciform (‘hook shaped’) sperm heads and nuclei. Quantification of the differences in shape variation between mouse species and lines often relies on manual measurement and classification that leads to subjective results, making comparisons within and between samples difficult.We have developed an analysis program for assessing the morphology of asymmetric nuclei, and characterised the sperm of mice from a range of inbred, outbred and wild-derived mouse lines. We find that laboratory lines have elevated sperm shape variability both within and between samples in comparison to wild-derived inbred lines, and that sperm shape in the F1 offspring of CBA and C57Bl6J lines is subtly affected by the direction of the cross.Hierarchical clustering can distinguish distinct sperm shapes with greater efficiency and reproducibility than even experienced manual assessors. We quantified the range of morphological defects in the inbred BALB/c line, demonstrating we can identify different morphological subgroups. This approach has applications for studies of sperm development, infertility and toxicology.

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A high-throughput method for unbiased quantitation and categorization of nuclear morphology

Biology of Reproduction ◽

10.1093/biolre/ioz013 ◽

2019 ◽

Vol 100 (5) ◽

pp. 1250-1260 ◽

Cited By ~ 11

Author(s):

Benjamin Matthew Skinner ◽

Claudia Cattoni Rathje ◽

Joanne Bacon ◽

Emma Elizabeth Philippa Johnson ◽

Erica Lee Larson ◽

...

Keyword(s):

Morphometric Analysis ◽

Hybrid Sterility ◽

Inbred Strains ◽

Mouse Strains ◽

Nuclear Morphology ◽

Analysis Program ◽

Cell Type ◽

High Throughput Method ◽

Sperm Development ◽

Species Specific

Abstract The physical arrangement of chromatin in the nucleus is cell type and species-specific, a fact particularly evident in sperm, in which most of the cytoplasm has been lost. Analysis of the characteristic falciform (“hook shaped”) sperm in mice is important in studies of sperm development, hybrid sterility, infertility, and toxicology. However, quantification of sperm shape differences typically relies on subjective manual assessment, rendering comparisons within and between samples difficult. We have developed an analysis program for morphometric analysis of asymmetric nuclei and characterized the sperm of mice from a range of inbred, outbred, and wild-derived mouse strains. We find that laboratory strains have elevated sperm shape variability both within and between samples in comparison to wild-derived inbred strains, and that sperm shape in F1 offspring from a cross between CBA and C57Bl6J strains is subtly affected by the direction of the cross. We further show that hierarchical clustering can discriminate distinct sperm shapes with greater efficiency and reproducibility than even experienced manual assessors, and is useful both to distinguish between samples and also to identify different morphological classes within a single sample. Our approach allows for the analysis of nuclear shape with unprecedented precision and scale and will be widely applicable to different species and different areas of biology.

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Evolutionary origin of the cell nucleus and its functional architecture

Essays in Biochemistry ◽

10.1042/bse0480001 ◽

2010 ◽

Vol 48 ◽

pp. 1-24 ◽

Cited By ~ 15

Author(s):

Jan Postberg ◽

Hans J. Lipps ◽

Thomas Cremer

Keyword(s):

Cell Nucleus ◽

Interdisciplinary Research ◽

Nuclear Organization ◽

Nuclear Architecture ◽

Evolutionary Origin ◽

Cell Type ◽

Research Strategies ◽

Evolutionarily Conserved ◽

Species Specific ◽

The Impact

Understanding the evolutionary origin of the nucleus and its compartmentalized architecture provides a huge but, as expected, greatly rewarding challenge in the post-genomic era. We start this chapter with a survey of current hypotheses on the evolutionary origin of the cell nucleus. Thereafter, we provide an overview of evolutionarily conserved features of chromatin organization and arrangements, as well as topographical aspects of DNA replication and transcription, followed by a brief introduction of current models of nuclear architecture. In addition to features which may possibly apply to all eukaryotes, the evolutionary plasticity of higher-order nuclear organization is reflected by cell-type- and species-specific features, by the ability of nuclear architecture to adapt to specific environmental demands, as well as by the impact of aberrant nuclear organization on senescence and human disease. We conclude this chapter with a reflection on the necessity of interdisciplinary research strategies to map epigenomes in space and time.

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Uptake of fluorescent-labeled BSA into root cells: endocytosis?

Acta Societatis Botanicorum Poloniae ◽

10.5586/asbp.1997.037 ◽

2014 ◽

Vol 66 (3-4) ◽

pp. 319-324 ◽

Cited By ~ 1

Author(s):

A. Kaźmierczak ◽

J. Maszewski

Keyword(s):

Zea Mays ◽

Bovine Serum Albumin ◽

Serum Albumin ◽

Bovine Serum ◽

Cytochalasin B ◽

Fluorescein Isothiocyanate ◽

Cell Type ◽

Root Cells ◽

Root Zones ◽

Species Specific

Incorporation of rhodamine- and fluorescein-isothiocyanate labeled bovine serum albumin (BSA-TRITC, BSA-FITC) was examined in different root zones of the 3-day-old seedlings in Melandrium noctiflorum, Allium cepa and Zea mays. The uptake of fluorescent-labeled BSA was found: (1) species-specific, (2) cell-type dependent, and (3) cytochalasin B-sensitive. The characteristic punctute distribution of vesicles within the cytoplasm suggests the internalization of labeled proteins by endocytosis.

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Obatoclax, Saliphenylhalamide, and Gemcitabine Inhibit Influenza A Virus Infection

Journal of Biological Chemistry ◽

10.1074/jbc.m112.392142 ◽

2012 ◽

Vol 287 (42) ◽

pp. 35324-35332 ◽

Cited By ~ 59

Author(s):

Oxana V. Denisova ◽

Laura Kakkola ◽

Lin Feng ◽

Jakob Stenman ◽

Ashwini Nagaraj ◽

...

Keyword(s):

Influenza A ◽

Treatment Options ◽

Antiviral Agents ◽

Cell Type ◽

Influenza A Viruses ◽

Novel Host ◽

Chemical Screen ◽

Species Specific ◽

Host Target ◽

Transcription And Replication

Influenza A viruses (IAVs) infect humans and cause significant morbidity and mortality. Different treatment options have been developed; however, these were insufficient during recent IAV outbreaks. Here, we conducted a targeted chemical screen in human nonmalignant cells to validate known and search for novel host-directed antivirals. The screen validated saliphenylhalamide (SaliPhe) and identified two novel anti-IAV agents, obatoclax and gemcitabine. Further experiments demonstrated that Mcl-1 (target of obatoclax) provides a novel host target for IAV treatment. Moreover, we showed that obatoclax and SaliPhe inhibited IAV uptake and gemcitabine suppressed viral RNA transcription and replication. These compounds possess broad spectrum antiviral activity, although their antiviral efficacies were virus-, cell type-, and species-specific. Altogether, our results suggest that phase II obatoclax, investigational SaliPhe, and FDA/EMEA-approved gemcitabine represent potent antiviral agents.

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Species specificity and organ, cellular and subcellular localization of the 100 kDa Ras GTPase activating protein

Journal of Cell Science ◽

10.1242/jcs.107.3.427 ◽

1994 ◽

Vol 107 (3) ◽

pp. 427-435 ◽

Cited By ~ 2

Author(s):

P. Mollat ◽

A. Fournier ◽

C.Z. Yang ◽

E. Alsat ◽

Y. Zhang ◽

...

Keyword(s):

Amino Terminus ◽

Specific Function ◽

Precise Localization ◽

Cell Type ◽

Gtpase Activating Protein ◽

Ras Gtpase ◽

Organ Specific ◽

Hydrophobic Amino Acids ◽

Species Specific ◽

Fraction Bound

A p100-GAP isoform, generated by an alternative splicing mechanism that eliminates the 180 hydrophobic amino acids at the amino terminus of p120-GAP, has been described in human placenta, in addition to the known p120GAP and neurofibromin. This p100-GAP possesses full Ras-GTPase stimulating activity. p120-GAP is ubiquitously localized in the cytosol while the localization of p100-GAP is unknown. Here we have explored the precise localization of p100-GAP and show that p100-GAP is present only in extracts of primate placenta. It is abundant in both human and Maccaca Rhesus placentae, where it is present in far larger amounts than p120-GAP. The p100-GAP is species-specific since it was not detected in the placenta of pig, sheep, mouse or rat. p100-GAP was also found to be organ-specific, since it was not detectable in organs other than the placenta. In this connection, we substantiated our previous finding that p100-GAP is mainly localized in the trophoblasts. Both subcellular trophoblast fractionation and immunofluorescence analyses showed that this protein was distributed between the cytosol, plasma membrane and a fraction bound to the nucleus, but not inside it. This highly restrictive specificity of p100-GAP localization in relation to species, organ and cell type, confirms the extreme singularity of this protein, and strongly suggests a particular specific function in the trophoblast.

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Breeding synthetic varieties in annual caraway: observations on the outcrossing rate in a polycross using a high-throughput genotyping system

Euphytica ◽

10.1007/s10681-020-02732-5 ◽

2020 ◽

Vol 217 (1) ◽

Author(s):

Daniel von Maydell ◽

Julia Brandes ◽

Heike Lehnert ◽

Wolfram Junghanns ◽

Frank Marthe

Keyword(s):

Medicinal Plant ◽

Dna Extraction ◽

High Throughput ◽

Inbred Lines ◽

High Variability ◽

Outcrossing Rate ◽

Breeding Method ◽

High Throughput Method ◽

Synthetic Varieties ◽

Seed Yields

AbstractCaraway (Carum carvi) is an economically important spice and medicinal plant of the Apiaceae family (syn. Umbelliferrae). Farmers often favor annual cultivation of caraway. However, the annual varieties, which are currently available, do not provide satisfying seed yields. Employing heterosis can be a promising approach to increase yield. Breeding of synthetic varieties utilizes heterosis and may be the method of choice for future caraway breeding. Knowledge of the outcrossing rate is important to evaluate the effectiveness of this breeding method. However, the outcrossing rate of caraway is unknown so far. We estimated the outcrossing rate of seven inbred lines under field conditions in a neighbor-balanced polycross design. For this purpose, we implemented a high-throughput genotyping system (PACE), accompanied by a high-throughput method for DNA extraction adapted to caraway. In total, more than 1300 individual plants were genotyped. We found a high variability of lines regarding outcrossing rate and other traits associated with flowering. The outcrossing rate was on average 66.5% and ranged from 51.6 to 82%. We discussed implications of our findings on the targeted breeding method.

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Cellular site for prothrombin synthesis

American Journal of Physiology-Legacy Content ◽

10.1152/ajplegacy.1960.199.2.360 ◽

1960 ◽

Vol 199 (2) ◽

pp. 360-366 ◽

Cited By ~ 41

Author(s):

Marion I. Barnhart

Keyword(s):

Plasma Proteins ◽

Fluorescent Antibody ◽

Parenchymal Cell ◽

Bovine Liver ◽

Fluorescent Antibody Technique ◽

Cell Type ◽

Antibody Technique ◽

Natural Fluorescence ◽

Species Specific ◽

Parenchymal Cells

The cell type responsible for synthesis of any of the trace plasma proteins concerned in blood coagulation has not been identified before. In this study the fluorescent antibody technique was used to find out the cellular site for prothrombin synthesis. Fluorescent antiprothrombin precipitated out on bovine liver parenchymal cells containing adequate concentrations of prothrombin. The specificity of the reaction was established using various absorptive and blocking techniques. The antiprothrombins produced were species specific. Not all parenchymal cells reacted uniformly with fluorescent antiprothrombin so that groups of brilliantly fluorescing cells were seen among cells displaying dim natural fluorescence. This may mean that only a certain type of parenchymal cell produces prothrombin or that there is cyclic production of prothrombin.

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Nucleofection Efficiency of Sheep Primary Fibroblasts Established from Refrigerated Skin

International Journal of Biology ◽

10.5539/ijb.v10n4p12 ◽

2018 ◽

Vol 10 (4) ◽

pp. 12

Author(s):

Mahipal Singh ◽

Xiaoling Ma

Keyword(s):

Fluorescent Protein ◽

Transfection Efficiency ◽

Genetically Engineered ◽

Dermal Fibroblasts ◽

Biologically Active ◽

Cell Type ◽

Gfp Expression ◽

Primary Fibroblasts ◽

Species Specific ◽

Green Fluorescent

Dermal fibroblasts are useful for production of genetically engineered biologically active factors for development of cellular therapies and tissue engineering products for regenerative medicine. However, their transfection efficiencies using traditional non-viral methods are low and vary based on cell-type and species-specific differences. Using nucleofection technology, here we show that the transfection efficiency of primary fibroblasts established after 0-, 35-, and 65-days of postmortem storage of sheep skin tissues in a refrigerator was 59.49 % ± 9.66 %, 59.33 % ± 11.59 %, and 43.48 % ± 8.09 % respectively, as determined by analysis of green fluorescent protein (GFP) expression.

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