Ploidy Number Research Articles

Precise capture of circulating endometrial cells in endometriosis.

Endometriosis (EM) is a complex benign gynecological disease, but it has malignant biological behavior and can invade any part of the body. Clinical manifestations include pelvic pain, dysmenorrhea, infertility, pelvic nodules, and masses. Our previous study successfully detected circulating endometrial cells (CECs) in the peripheral blood of patients with EM. The purpose of this study is to overcome the limitation of cell size in the previous microfluidic chip method, to further accurately capture CECs, understand the characteristics of these cells, and explore the relationship between CECs and the clinical course characteristics of patients with EM. Human peripheral venous blood used to detect CECs and circulating vascular endothelial cells (CVECs) was taken from EM patients ( n = 34) hospitalized in the Peking University People's Hospital. We used the subtraction enrichment and immunostaining fluorescence in situ hybridization (SE-iFISH) method to exclude the interference of red blood cells, white blood cells, and CVECs, so as to accurately capture the CECs in the peripheral blood of patients with EM. Then we clarified the size and ploidy number of chromosome 8 of CECs, and a second grouping of patients was performed based on clinical characteristics to determine the relationship between CECs and clinical course characteristics. The peripheral blood of 34 EM patients and 12 non-EM patients was evaluated by SE-iFISH. Overall, 34 eligible EM patients were enrolled. The results showed that the detection rates of CECs were 58.8% in EM patients and 16.7% in the control group. However, after classification according to clinical characteristics, more CECs could be detected in the peripheral blood of patients with rapidly progressive EM, with a detection rate of 94.4% (17/18). In total, 63.5% (40/63) of these cells were small cells with diameters below 5 μm, and 44.4% (28/63) were aneuploid cells. No significant association was found between the number of CECs and EM stage. The number and characteristics of CECs are related to the clinical course characteristics of patients with EM, such as pain and changes in lesion size, and may be used as biomarkers for personalized treatment and management of EM in the future.

Abstract LB390: Enhancing prostate cancer diagnosis: A deep learning approach for DNA ploidy prediction from whole slide images

Abstract Prostate cancer is distinguished by unique histological alterations in glandular architecture, observable in Whole Slide Images (WSIs). The disease is further characterized by pronounced genomic instability, exemplified by biomarkers such as DNA ploidy. While the Gleason Score effectively assesses the histological changes, the manual evaluation of DNA ploidy is subjective and time-consuming. Addressing the need for an objective diagnostic tool, our study developed a deep learning model aimed at accurately predicting DNA ploidy using publicly available WSI from the Cancer Genome Atlas (TCGA). Utilizing a ResNet-18 convolutional neural network, we trained the model on WSIs from 200 TCGA patients to extract features and predict total mRNA expression. The model was further refined using data from 19 TCGA patients, focusing on ploidy number prediction. Comparative analysis against a random reference model revealed significant improvements of ploidy prediction in Mean Absolute Error (MAE) by 43.52%, Mean Absolute Percentage Error (MAPE) by 43.59%, and Mean Squared Error (MSE) by 67.86%. These enhancements were statistically validated (p-values: MAE - 7.07E-06, MAPE - 7.69E-06, MSE - 3.14E-05) and substantiated by large Cohen's d values (MAE - 2.88, MAPE - 2.86, MSE - 2.42), confirming the model's advanced predictive accuracy and practical utility. This approach, validated further on a separate TCGA hold-out test set, signifies a major leap in prostate cancer diagnostics. By integrating mRNA prediction checkpoints, our model not only refines ploidy assessment but also sets the stage for correlating these predictions with patient clinical outcomes. These developments promise to enhance existing diagnostic methods, offering a more objective and efficient tool for DNA ploidy assessment in prostate cancer prognosis and treatment planning. Citation Format: Josselyn Sofia Vergara Cobos, Francisco Carrillo-Perez, Marija Pizurica, Noemi Andor, Olivier Gevaert. Enhancing prostate cancer diagnosis: A deep learning approach for DNA ploidy prediction from whole slide images [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB390.

Cytogenetic Characterization and Metabolomic Differences of Full-Sib Progenies of Saccharum spp.

Sugarcane smut is a worldwide fungal disease. Disease resistance breeding is the most economical and effective measure to prevent and control sugarcane smut. The cytogenetic characteristics and metabolomic differences of sugarcane F1s are closely related to disease resistance. Zhongzhe 1 and G160 sugarcane from the same parents (ROC25 and Yunzhe89-7) were used; the plants were grown in accordance with the barrel method. When the seedlings had 4-5 leaves, genomic in situ hybridization (GISH) was performed; digoxigenin (DIG)-labeled female parental (ROC25)DNA and biotin-labeled male parental (Yunzhe89-7) DNA were used as probes, and the karyotypes of two hybrids were analyzed. The new sugarcane smut-resistant variety (Zhongzhe 1) and the susceptible variety (G160) derived from the same parent were analyzed via gas chromatography-mass spectrometry technology (GC-MS) to compare the metabolomic differences between them. GISH analysis revealed that the chromosome ploidy number of Zhongzhe 1 sugarcane and G160 sugarcane were 114 and 110, respectively. However, the two contain different numbers of chromosomes from the female (ROC25) and male (Yunzhe89-7) parents. Moreover, 258 significantly changed metabolites were identified in smut-resistant Zhongzhe 1, as compared with the smut-susceptible G160 sugarcane: 56 flavonoids, 52 phenolic acids, 30 lipids, 26 organic acids, 26 amino acids and derivatives, 19 nucleotides and derivatives, 5 alkaloids, 9 terpenoids, and 35 others. Multivariate statistical analysis revealed a distinct difference in metabolic pathways between Zhongzhe 1 sugarcane and G160, and both of these varieties had unique functional metabolites. Differences in chromosome composition may constitute the genetic basis for the difference in resistance to smut disease between Zhongzhe 1 sugarcane and G160 sugarcane, and a high accumulation of flavonoids, lipids, terpenoids and tannins may constitute the basis of resistance to smut disease for the Zhongzhe 1 variety.

Replication Origin Deletion Enhances Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Synthesis in Haloarchaea.

Although the use of multiple replication origins for chromosome replication has been widely characterized in haloarchaea, whether it is possible to manipulate the chromosome copy number by their genetic engineering is not known, and how it would affect the cell functioning is poorly understood. Here, we demonstrate that deletion of the three active chromosomal origins in Haloferax mediterranei remarkably reduces its DNA amounts and ploidy numbers. Consequently, the mutant strain H. mediterranei Δ123 is more sensitive to UV and mitomycin C. Surprisingly, the cell size increases by 21.2%, and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production in shake flask culture enhances from 7.23 to 8.11 g/L in ΔEPSΔ123, although there is also a decrease in cell growth. In this mutant, the chromosomal copy number decreases, whereas the pha-encoding pHM300 megaplasmid copy number increases. Moreover, our transcriptome analysis reveals that the genes involved in primary metabolisms are significantly downregulated in ΔEPSΔ123, whereas those responsible for starch utilization and precursor supplying for PHBV monomers are upregulated. This indicates that more energy and carbon flux is redirected from primary metabolism to PHBV synthesis, thereby enhancing its PHBV accumulation. These findings may therefore provide a rational design to enhance PHBV synthesis by simply tuning the replication origins to modulate the chromosome/megaplasmid copy number ratio and subsequently influence cellular metabolism and physiological functions. IMPORTANCE The haloarchaeon Haloferax mediterranei is a potential producer of PHBV (100% biodegradable plastic) from inexpensive carbon sources. We previously reported that H. mediterranei possessed three active chromosomal origins and, when these origins were deleted, a dormant origin was activated to initiate the replication of chromosome. In this context, in the present study, we first found a close connection between replication initiation and PHBV accumulation. We describe the potential industrial advantages of the strain H. mediterranei ΔEPSΔ123, which includes the enlargement of cell volume by 21.2% and enhancement of PHBV production by 11.2%. We further reveal the possible mechanism that contributes to the greater PHBV production in the ΔEPSΔ123 strain. Overall, we provide here a conceptual advance in the field of synthetic biology by modulating chromosome replication to improve the production of bio-based chemicals.

The hybrid number of a ploidy profile

Polyploidization, whereby an organism inherits multiple copies of the genome of their parents, is an important evolutionary event that has been observed in plants and animals. One way to study such events is in terms of the ploidy number of the species that make up a dataset of interest. It is therefore natural to ask: How much information about the evolutionary past of the set of species that form a dataset can be gleaned from the ploidy numbers of the species? To help answer this question, we introduce and study the novel concept of a ploidy profile which allows us to formalize it in terms of a multiplicity vector indexed by the species the dataset is comprised of. Using the framework of a phylogenetic network, we present a closed formula for computing the hybrid number (i.e. the minimal number of polyploidization events required to explain a ploidy profile) of a large class of ploidy profiles. This formula relies on the construction of a certain phylogenetic network from the simplification sequence of a ploidy profile and the hybrid number of the ploidy profile with which this construction is initialized. Both of them can be computed easily in case the ploidy numbers that make up the ploidy profile are not too large. To help illustrate the applicability of our approach, we apply it to a simplified version of a publicly available Viola dataset.

O-077 The chromosomal constitution of embryos developing from 0PN zygotes

Abstract Study question To compare the ploidy status of embryos originated from zygotes with absence of pronuclei (0PN) with those developing from normal two pronuclei (2PN) fertilization. Summary answer We did not find significant differences in euploidy rates between blastocysts from 0PN and 2PN zygotes. What is known already Information regarding the ploidy number of 0PN zygotes, even when the pronuclei number is assessed by time-lapse microscope coupled incubators, is still insufficient. Because of this, embryos originating from 0PN zygotes are usually discarded or individualized in order not to include them in first transfers. Even more, abnormal fertilization is considered as a proxy of poor embryo quality. Study design, size, duration A retrospective observational study from March 2016 and December 2021 including 80 PGT-A cycles in which at least one of the blastocysts studied developed from oocytes in which pronuclei were not seen at the time of fertilization check. Participants/materials, setting, methods 307 blastocysts were studied, of which 115 originated from 0PN and 192 from 2PN zygotes. Pronuclear number was assessed 16-20 h after insemination. Zygotes with 0PN were cultured separately from 2PN ones of the same cohort. After extended culture, a biopsy was performed and the material obtained was genetically studied using Next Generation Sequencing. Statistical comparisons were made with Fisher's exact and Chi-square tests. Significance level was set at P<0.05. Main results and the role of chance No differences were observed in euploidy rates between the 2PN and 0PN groups [55.2% (106/193) VS 43.5% (50/115)]. In aneuploid embryos originating from 2PN zygotes, a similar amount of trisomies and monosomies was observed (56 and 56 respectively). In contrast, aneuploid embryos from 0PN oocytes showed a greater number of trisomies than monosomies (55 and 35 respectively, P < 0.05). None of the blastocysts resulting from 0PN was haploid, that is, we did not find parthenogenic embryos. Blastocysts derived from 0PN zygotes showed similar quality scoring than those from 2PN ones. To date, 20 euploid blastocysts from the 0PN group have been transferred, producing 12 pregnancies: 6 still ongoing and 4 healthy live births. Limitations, reasons for caution Non-visualization of pronuclei in oocytes that later generate normal diploid blastocysts may be due to a faster or slower cell cycle progression. Modifications in the daily fertilization check schedules of busy laboratories should also be considered. Wider implications of the findings Contrary to expectations, aneuploid embryos from 0PN oocytes showed a greater number of trisomies than monosomies. A significant fraction of the embryos generated from 0PN zygotes were actually diploid, with similar quality and outcome results than 2PN ones. This makes them perfectly usable in IVF routines. Trial registration number Not applicable

Circulating KRAS variant-specific shedding and association with survival in patients with metastatic pancreatic ductal adenocarcinoma (mPDAC) receiving chemoimmunotherapy.

2548 Background: Circulating tumor DNA (ctDNA) is increasingly used as a prognostic marker with high ctDNA shedding associated with poor survival. Gene-, but not variant-specific, differences in ctDNA shedding have been reported. Tumor burden, mitotic rate, and cell death rate have been proposed as contributors to ctDNA shedding. Here we investigate associations of ctDNA shedding for the two most common mPDAC KRAS variants, G12D and G12V, with tumor burden, mitotic score, and overall survival (OS). Methods: Pretreatment (baseline) ctDNA was analyzed by droplet digital PCR for 86 (including 44 G12D, 30 G12V) patients with mPDAC receiving front-line chemoimmunotherapy in a randomized open-label Phase II study (NCT03214250). Baseline tumor burden in total, within the pancreas, and distally, was assessed by sum of RECIST target lesion diameters. Tumor tissue variant allele fraction (tVAF) and mitotic score (geometric mean expression of 65 mitosis-associated genes) were calculated from DNA and RNA sequencing. Results: ctKRAS shedding (dichotomized at median mutant copies/mL) was associated with OS for G12D bearing tumors (p = 0.03) but not G12V (p = 0.17, log-rank test). To identify variant-specific features of shedding, we examined the Spearman correlation for total tumor burden and ctKRAS shedding; G12D but not G12V shedding was correlated with tumor burden (p = 0.01 and p = 0.22 respectively). However, the higher tVAF in G12V compared to G12D tumors (p = 0.048, Mann-Whitney test) could result from differences in purity, ploidy, and KRAS copy number. Thus, we used tVAF as a scalar to calculate an adjusted tumor burden which was significantly correlated with both G12D and G12V ctDNA shedding (p = 0.004 and 0.02, respectively). When a patient’s distal vs. pancreatic lesions were analyzed separately, pancreatic tumor burden was not correlated with G12D or G12V shedding (p = 0.10 and 0.33, respectively) but distal burden was correlated with both (p = 0.001 and 0.02, respectively). While there was no difference by KRAS variant for the correlation between adjusted tumor burden and shedding, these results do suggest that, in patients with mPDAC, distal rather than primary tumor burden may drive ctDNA shedding. Finally, tumor mitotic rate was combined with adjusted total tumor burden in a linear regression model; both were significant for predicting G12D shedding (p = 0.007 and p < 0.0001, respectively) but not for G12V (p = 0.045 and p = 0.16, respectively). Conclusions: These data suggest that ctDNA shedding and survival associations may be KRAS variant-specific in mPDAC. Tumor mitotic score and location of tumors may explain some variant-specific differences in shedding. As clinical ctDNA tests become more widely used, further investigation of variant-specific shedding in KRAS and other genes may be key for proper interpretation of ctDNA tests.

In silico analysis enabling informed design for genome editing in medicinal cannabis; gene families and variant characterisation.

BackgroundCannabis has been used worldwide for centuries for industrial, recreational and medicinal use, however, to date no successful attempts at editing genes involved in cannabinoid biosynthesis have been reported. This study proposes and develops an in silico best practices approach for the design and implementation of genome editing technologies in cannabis to target all genes involved in cannabinoid biosynthesis.ResultsA large dataset of reference genomes was accessed and mined to determine copy number variation and associated SNP variants for optimum target edit sites for genotype independent editing. Copy number variance and highly polymorphic gene sequences exist in the genome making genome editing using CRISPR, Zinc Fingers and TALENs technically difficult. Evaluation of allele or additional gene copies was determined through nucleotide and amino acid alignments with comparative sequence analysis performed. From determined gene copy number and presence of SNPs, multiple online CRISPR design tools were used to design sgRNA targeting every gene, accompanying allele and homologs throughout all involved pathways to create knockouts for further investigation. Universal sgRNA were designed for highly homologous sequences using MultiTargeter and visualised using Sequencher, creating unique sgRNA avoiding SNP and shared nucleotide locations targeting optimal edit sites.ConclusionsUsing this framework, the approach has wider applications to all plant species regardless of ploidy number or highly homologous gene sequences.Significance statementUsing this framework, a best-practice approach to genome editing is possible in all plant species, including cannabis, delivering a comprehensive in silico evaluation of the cannabinoid pathway diversity from a large set of whole genome sequences. Identification of SNP variants across all genes could improve genome editing potentially leading to novel applications across multiple disciplines, including agriculture and medicine.

Abstract 2190: Integrative genomic and transcriptomic characterization of metastatic urothelial carcinoma

Abstract Metastatic urothelial carcinoma (mUC) is a lethal cancer with limited therapeutic options available. To identify novel targets for therapy, large-scale sequencing efforts are needed. The Cancer Genome Atlas (TCGA) initiative substantially improved our knowledge on the genomic and transcriptomic characteristics of primary UC (Robertson et al. Cell 2017), however, the molecular landscape of mUC still remains largely unexplored. We performed whole genome sequencing (WGS) on 116 biopsies of UC metastases and matched mRNA sequencing (RNAseq) for 90 samples. We applied genomic alteration analysis on the WGS data and consensus clustering on the RNAseq data. We compared the molecular alterations of mUC with the data of the TCGA cohort and found that most affected driver genes were similar between primary UC and mUC. However, we identified CNTNAP5, RARG and MGP as exclusively mutated driver genes in the metastatic setting, and TP53 alterations were more prevalent in mUC than in primary UC. APOBEC induced mutation signatures in coding and non-coding regions were identified in 91% of the mUC samples and correlated with ploidy, mutational burden and copy number alterations. Based on the etiology of observed mutational signatures, five genomic subtypes were identified in mUC and validated in the TCGA cohort. APOBEC mutagenesis was the main mutational process in genomic subtype 1, which was the most common subtype (67%, N = 78). Moreover, APOBEC deamination mutations were found to be increased over time in paired biopsies from eight patients, indicating that APOBEC enzyme activity is ongoing in the metastatic setting. Unsupervised clustering and comparing phenotypic markers between the groups revealed five clinically-relevant transcriptomic subtypes: luminal-a (20%), luminal-b (20%), stroma-rich (24%), basal/squamous (23%) and a non-specified phenotype (12%). The basal/squamous and stroma-rich subtypes showed highly similar molecular characteristics as the TCGA subtypes, but with enrichment of the stroma-rich and reduction of the basal/squamous subtype. Based on genomic alterations, potential therapeutic targets were identified in 111/116 mUC patients, of which FGFR3 alterations (18%) and fusions (6%), and CDKN2A (44%), ERBB2 (20%), and TSC1 (14%) alterations were most common. By integrating the genomic and transcriptomic data, we propose potential novel therapeutic options per transcriptomic subtype, like addition of a TGF-β inhibitor to immune checkpoint inhibition for the stroma-rich and basal/squamous subtypes that showed the highest TGF-β pathway activity. Using WGS and RNAseq analyses, we provide a comprehensive overview of the molecular landscape of mUC that may serve as a reference for more subgroup-oriented and patient-specific research on the etiology of mUC and future drug development. Citation Format: Maud Rijnders, J Alberto Nakauma-González, Job van Riet, Michiel S. van der Heijden, Jens Voortman, Edwin P. J. G Cuppen, Niven Mehra, Sandra van Wilpe, Sjoukje F. Oosting, L Lucia Rijstenberg, Ellen C. Zwarthoff, Ronald de Wit, Astrid A. van der Veldt, Harmen J. van de Werken, Martijn P. Lolkema, Joost L. Boormans. Integrative genomic and transcriptomic characterization of metastatic urothelial carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2190.

Cytological diversity in colchiploid of Pontianak tangerine citrus

Pontianak tangerine citrus (Siam Pontianak) is the most popular tangerine citrus in Indonesia. Induced mutation with colchicine treatment on Pontianak tangerine was intended to produce polyploid plants. The study aimed to analyze the cytological diversity in colchiploid of Pontianak tangerine citrus. The study was conducted at ICISFRI from January – May 2018. The cytological analysis was performed on a five-year-old of 36 colchiploids of Pontianak tangerine and a mother plant as control. The results showed that the colchiploid plants had higher chromosome numbers, stomatal sizes, and chloroplast numbers than the control. However, the stomatal density in several genotypes did not differ from that of the control. Colchicine treatment resulted in an aneuploid plant with chromosome number 18+x. Based on the clustering analysis results, all colchiploid plants differed from the control with a variability coefficient of 0.65. The colchiploid plants were not grouped according to colchicine concentration and duration of treatment. The increase of colchicine concentration and treatment duration did not correlate with the increasing ploidy number of colchiploid plants. The implication of the research result was the stomatal density, stomatal size, and chloroplast number were useful tools for rapid pre-screening of plant polyploidy.

The effect of ploidy number on vigor, productivity, and potential adaptation to climate change in annual Medicago species

AbstractTetraploid plants, including those induced from diploid barrel medic (Medicago truncatula Gaertn.) and the snail medic [Medicago scutellata (L.) Mill.], have the potential to increase vigor and leaf size to improve adaptation to environments with low and variable rainfall in Mediterranean climates. The growth of tetraploid lines induced from barrel medic cultivar Sultan‐SU was evaluated against the diploid parent line and a natural tetraploid snail medic cultivar Sava in field conditions in Adelaide, South Australia. In field conditions, tetraploid lines had up to 106% greater autumn biomass than Sultan‐SU, 58% larger seed weight, and a 127% increase in leaf area. In a controlled environment experiment, snail medic Sava was the only entry to show improved early season (assessed at 6 wk) shoot biomass and canopy cover under short days and low temperatures that were designed to simulate a late start to the growing season. An examination of fresh flower buds showed that the fertility and stability of induced tetraploid lines varied between lines and generations. Hardseed breakdown patterns differed among tetraploid mutant lines, with two lines having much harder seed than Sultan‐SU. Some tetraploid Medicago spp. produced greater early biomass and larger leaves than diploids. Increasing ploidy level provides plant breeders with a promising tool in the development of new cultivars better suited to future climate scenarios.

A Recently Formed Triploid Cardamine insueta Inherits Leaf Vivipary and Submergence Tolerance Traits of Parents.

Contemporary speciation provides a unique opportunity to directly observe the traits and environmental responses of a new species. Cardamine insueta is an allotriploid species that appeared within the past 150 years in a Swiss village, Urnerboden. In contrast to its two progenitor species, Cardamine amara and Cardamine rivularis that live in wet and open habitats, respectively, C. insueta is found in-between their habitats with temporal water level fluctuation. This triploid species propagates clonally and serves as a triploid bridge to form higher ploidy species. Although niche separation is observed in field studies, the mechanisms underlying the environmental robustness of C. insueta are not clear. To characterize responses to a fluctuating environment, we performed a time-course analysis of homeolog gene expression in C. insueta in response to submergence treatment. For this purpose, the two parental (C. amara and C. rivularis) genome sequences were assembled with a reference-guided approach, and homeolog-specific gene expression was quantified using HomeoRoq software. We found that C. insueta and C. rivularis initiated vegetative propagation by forming ectopic meristems on leaves, while C. amara did not. We examined homeolog-specific gene expression of three species at nine time points during the treatment. The genome-wide expression ratio of homeolog pairs was 2:1 over the time-course, consistent with the ploidy number. By searching the genes with high coefficient of variation of expression over time-course transcriptome data, we found many known key transcriptional factors related to meristem development and formation upregulated in both C. rivularis and rivularis-homeolog of C. insueta, but not in C. amara. Moreover, some amara-homeologs of these genes were also upregulated in the triploid, suggesting trans-regulation. In turn, Gene Ontology analysis suggested that the expression pattern of submergence tolerant genes in the triploid was inherited from C. amara. These results suggest that the triploid C. insueta combined advantageous patterns of parental transcriptomes to contribute to its establishment in a new niche along a water-usage gradient.

Application of next generation sequencing (NGS) for descriptive analysis of 30 genomes of Leishmania infantum isolates in Middle-North Brazil

Visceral leishmaniasis (VL) is a life-threatening disease caused by the protozoa Leishmania donovani and L. infantum. Likely, L. infantum was introduced in the New World by the Iberic colonizers. Due to recent introduction, the genetic diversity is low. Access to genomic information through the sequencing of Leishmania isolates allows the characterization of populations through the identification and analysis of variations. Population structure information may reveal important data on disease dynamics. Aiming to describe the genetic diversity of L. infantum from the Middle-North, Brazil, next generation sequencing of 30 Leishmania isolates obtained in the city of Teresina, from where the disease dispersed, was performed. The variations were categorized accordingly to the genome region and impact and provided the basis for chromosomal ploidy and population structure analysis. The results showed low diversity between the isolates and the Iberic reference genome JPCM5. Most variations were seen in non-coding regions, with modifying impact. The ploidy number analysis showed aneuploid profile. The population structure analysis revealed the presence of two L. infantum populations identified in Teresina. Further population genetics studies with a larger number of isolates should be performed in order to identify the genetic background associated with virulence and parasite ecology.

Pronuclear Number does Not Fully Reflect Ploidy Number in Human Embryos

Pronuclear Number does Not Fully Reflect Ploidy Number in Human Embryos

A flow cytometry-based analysis to establish a cell cycle synchronization protocol for Saccharum spp.

Modern sugarcane is an unusually complex heteroploid crop, and its genome comprises two or three subgenomes. To reduce the complexity of sugarcane genome research, the ploidy level and number of chromosomes can be reduced using flow chromosome sorting. However, a cell cycle synchronization (CCS) protocol for Saccharum spp. is needed that maximizes the accumulation of metaphase chromosomes. For flow cytometry analysis in this study, we optimized the lysis buffer, hydroxyurea(HU) concentration, HU treatment time and recovery time for sugarcane. We determined the mitotic index by microscopic observation and calculation. We found that WPB buffer was superior to other buffers for preparation of sugarcane nuclei suspensions. The optimal HU treatment was 2 mM for 18 h at 25 °C, 28 °C and 30 °C. Higher recovery treatment temperatures were associated with shorter recovery times (3.5 h, 2.5 h and 1.5 h at 25 °C, 28 °C and 30 °C, respectively). The optimal conditions for treatment with the inhibitor of microtubule polymerization, amiprophos-methyl (APM), were 2.5 μM for 3 h at 25 °C, 28 °C and 30 °C. Meanwhile, preliminary screening of CCS protocols for Badila were used for some main species of genus Saccharum at 25 °C, 28 °C and 30 °C, which showed that the average mitotic index decreased from 25 °C to 30 °C. The optimal sugarcane CCS protocol that yielded a mitotic index of >50% in sugarcane root tips was: 2 mM HU for 18 h, 0.1 X Hoagland’s Solution without HU for 3.5 h, and 2.5 μM APM for 3.0 h at 25 °C. The CCS protocol defined in this study should accelerate the development of genomic research and cytobiology research in sugarcane.

Embryo fragmentation and its relationship with aneuploidy

Objective: Selecting embryos is an important step in the in vitro fertilization process before transferring them to the uterus. There are some invasive methods for choosing a good quality embryo, such as embryo grading. This method evaluates the equality and fragmentation of an embryo. However, this method does not adequately evaluate the chromosomal status of the embryos, which is often necessary for high-risk embryos. Here, we evaluated embryo fragmentation and chromosomal numbers using next-generation sequencing. Materials and Methods: Each embryo was biopsied on the 3rd or 5th culture day to obtain a single blastomere cell. DNA was then extracted from each blastomere and whole-genome amplification was carried out. Amplification products were then sequenced to obtain a ploidy number. Results: Among the 30 embryos that were evaluated, 19 embryos had no fragments, 10 embryos had small fragments, and 1 embryo had moderate fragments. However, 12 of 19 embryos, 57.9% with no fragments were detected to have chromosomal abnormality. Aneuploidy was increased in 7 of 10 embryos (70%) with mild fragments. One moderately fragmented embryo included was surprisingly found to have normal ploidy (100%). Gamma correlation test showed that there was no correlation between fragmentation and the incidence rate of aneuploidy (P > 0.05). Although there was no correlation, the study's result exemplifies that aneuploidy rate increased along with higher fragmentation. Conclusion: This research concluded that embryo fragmentation was not correlated with aneuploidy.

Chromosome Characterization of Festival Strawberry (Fragaria x ananassa D. var. Festival) Result of Polyploidization

Strawberry is fruit commodities which originating from the member of Rosaceae and has been widely cultivated in several countries of the world including Indonesia. One of the strawberry cultivars which consumed and cultivate in Indonesia is the strawberry Festival (Fragaria x ananassa D. var. Festival), which was developed in the area of Banyuroto, Magelang. In order to improve the quality and quantity of strawberry plants, polyploidization using colchicine has been employed in previous studies. The objectives of this research were study the chromosome character which include number, size, shape, and karyotype of normal strawberries and treatment strawberries with colchicine (0.05% 36 hours of root and leaf induction, 0.05% 24 hours of root and leaf induction, 0.05% 36 hours of leaf induction, 0.01% 36 hours of root and leaf induction). The results of this research shown that chromosome number difference and degree of ploidy between Fragaria x ananassa var Chandler in America with Fragaria x ananassa var. Festival in Indonesia due to natural mutation. While Festival strawberry control and treatment colchicine have same number chromosome is 2n=4x=28M and there are a difference long sleeve and short sleeve chromosome that may affect the content of chromosome expression and phenotype character are produced. The analysis of flow cytometry showed that treatment strawberry with colchicine has two peaks with intensity fluorescence equal to strawberry control so that it can be interpreted that the ploidy number of strawberries treatment with colchicine is tetraploid.

The development and prevalidation of an in vitro mutagenicity assay based on MutaMouse primary hepatocytes, Part I: Isolation, structural, genetic, and biochemical characterization.

To develop an improved in vitro mammalian cell gene mutation assay, it is imperative to address the known deficiencies associated with existing assays. Primary hepatocytes isolated from the MutaMouse are ideal for an in vitro gene mutation assay due to their metabolic competence, their “normal” karyotype (i.e., neither transformed nor immortalized), and the presence of the MutaMouse transgene for rapid and reliable mutation scoring. The cells were extensively characterized to confirm their utility. Freshly isolated cells were found to have a hepatocyte‐like morphology, predominantly consisting of binucleated cells. These cells maintain hepatocyte‐specific markers for up to 3 days in culture. Analyses revealed a normal murine hepatocyte karyotype with a modal ploidy number of 4n. Fluorescence in situ hybridization analysis confirmed the presence of the lambda shuttle vector on chromosome 3. The doubling time was determined to be 22.5 ± 3.3 h. Gene expression and enzymatic activity of key Phase I and Phase II metabolic enzymes were maintained for at least 8 and 24 h in culture, respectively. Exposure to β‐naphthoflavone led to approximately 900‐ and 9‐fold increases in Cyp1a1 and Cyp1a2 gene expression, respectively, and approximately twofold induction in cytochrome P450 (CYP) 1A1/1A2 activity. Exposure to phenobarbital resulted in an approximately twofold increase in CYP 2B6 enzyme activity. Following this characterization, it is evident that MutaMouse primary hepatocytes have considerable promise for in vitro mutagenicity assessment. The performance of these cells in an in vitro gene mutation assay is assessed in Part II. Environ. Mol. Mutagen. 60:331–347, 2019. © 2018 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.

Retroelements and DNA Methylation Could Contribute to Diversity of 5S rDNA in Agave L.

Agave L. is a genus of economic importance, and many of the 166 species in the American plant genus Agave L. inhabit high-stress environments, which makes the genus promising for facing global climate change. However, sustainable use of economically important species without interfering with their ecology and evolution requires generating knowledge about the factors responsible for their genetic variation and diversity and, on this basis, their adaptation and speciation. Few genetic studies exploring the evolutionary relationships, speciation processes, genetic variability and diversity within species of Agave are currently available. Analyses of rDNA loci have been performed with the purpose of determining the genetic variability and diversity of the genus Agave, and these loci have been used as genetic markers of ploidy. However, the factors involved in the diversity of 5S rDNA regions in Agave have not yet been studied in depth. Our study explored the possible mechanisms of genetic (retroelements) and epigenetic (DNA methylation) diversity in 5S rDNA regions in Agave. We characterized the 5S rDNA gene tandem in species of the genus with different ploidy numbers and determined the levels of methylation in 13 haplotypes of 5S rDNA and in four non-transcribed spacers (NTS). Our results showed highly dynamic methylation with a high percentage in haplotypes and NTS of 5S rDNA regions in Agave. The characterization of the 5S rDNA tandem array in Agave revealed vestigial remains of the Cassandra terminal-repeat retrotransposon in miniature (TRIM). Our analysis supported previous results suggesting that in species of Agave L., regulation and diversity of 5S rDNA regions are controlled by coordinated genetic and epigenetic events, which will vary according to the species and the level of ploidy. The artificial pressure to which some agave crops are subjected may affect the mechanisms of evolution of gene 5S rDNA.

The ploidy of Vibrio cholerae is variable and is influenced by growth phase and nutrient levels

The ploidy of Vibrio cholerae was quantified under different growth conditions. The V. cholerae was found to be (mero-) oligoploid or polyploid. The ploidy levels per cell were found to be growth phase regulated. The ploidy is highest during the early stationary phase (56-72 per cell) and lowest in the long-term starved state. In addition to growth phase, an external parameter such as nutrient level influences the ploidy, i.e. ploidy reduces rapidly at the onset of the starvation. The reduction is significant with P-value < 0.05 within 2 h of starvation. Even after prolonged starvation of 10 days, the ploidy number remained above 2 per cell. Failure to obtain a monoploid V. cholerae indicates that during starvation the genome is not distributed equally to daughter cells. The activity of DNase enzyme increased during starvation that decreased the ploidy. The ploidy was restored to the pre-starvation levels with nutrient supplementation.