High genome plasticity and frequent genetic exchange in Leishmania tropica isolates from Afghanistan, Iran and Syria

Background The kinetoplastid protozoan Leishmania tropica mainly causes cutaneous leishmaniasis in humans in the Middle East, and relapse or treatment failure after treatment are common in this area. L. tropica’s digenic life cycle includes distinct stages in the vector sandfly and the mammalian host. Sexual reproduction and genetic exchange appear to occur more frequently than in other Leishmania species. Understanding these processes is complicated by chromosome instability during cell division that yields aneuploidy, recombination and heterozygosity. This combination of rare recombination and aneuploid permits may reveal signs of hypothetical parasexual mating, where diploid cells fuse to form a transient tetraploid that undergoes chromosomal recombination and gradual chromosomal loss. Methodology/principal findings The genome-wide SNP diversity from 22 L. tropica isolates showed chromosome-specific runs of patchy heterozygosity and extensive chromosome copy number variation. All these isolates were collected during 2007–2017 in Sweden from patients infected in the Middle East and included isolates from a patient possessing two genetically distinct leishmaniasis infections three years apart with no evidence of re-infection. We found differing ancestries on the same chromosome (chr36) across multiple samples: matching the reference genome with few derived alleles, followed by blocks of heterozygous SNPs, and then by clusters of homozygous SNPs with specific recombination breakpoints at an inferred origin of replication. Other chromosomes had similar marked changes in heterozygosity at strand-switch regions separating polycistronic transcriptional units. Conclusion/significance These large-scale intra- and inter-chromosomal changes in diversity driven by recombination and aneuploidy suggest multiple mechanisms of cell reproduction and diversification in L. tropica, including mitotic, meiotic and parasexual processes. It underpins the need for more genomic surveillance of Leishmania, to detect emerging hybrids that could spread more widely and to better understand the association between genetic variation and treatment outcome. Furthering our understanding of Leishmania genome evolution and ancestry will aid better diagnostics and treatment for cutaneous leishmaniasis caused by L.tropica in the Middle East.

Tingkat Pengetahuan Mahasiswa Fakultas Kedokteran Universitas Yarsi Mengenai Penggunaan Human Diploid Cell Dalam Proses Produksi Vaksin Polio

Latar Belakang : Vaksin merupakan suspensi mikroorganisme yang dilemahkan atau dimatikan, atau protein antikgenik dari berbagai organisme tadi yang diberikan untuk mencegah, meringankan, atau mengobati penyakit-penyakit menular. Vaksin pertama kali tercatat pada tahun 1769, yang dipublikasikan oleh Edward Jenner, yaitu specimen yang berasal dari lesi lengan seseorang yang terinfeksi Cowpox. Human Diploid Cells (HDC) merupakan salah satu sel yang digunakan untuk mengkultur virus yang akan dijadikan vaksin. HDC yang berasal dari aborsi manusia ini banyak digunakan untuk mengkultur virus Polio IPV dan OPV, Rabies, Rubella, Measles, Varicella-Zooster, dan Hepatitis A. Tujuan : Vaksin polio merupakan vaksin yang diwajibkan pada anak yang dijadwalkan dari Ikatan Dokter Anak Indonesia (IDAI) yang dibagi menjadi dua jenis, IPV (Inactivated Polio Vaccine) dan OPV (Oral Polio Vaccine). Metode : Jenis Penelitian yang digunakan adalah deskriptif dengan pendekatan cross sectional menggunakan kuesioner. Populasi yang digunakan adalah mahasisa Fakultas Kedokteran Universitas YARSI tahun pertama dan tahun ketiga yang memenuhi syarat. Cara pemilihan sampel dengan simple random sampling. Hasil : Penelitian yang dilaksanakan selama 3 hari dengan menggunakan kuesioner, dari 100 responden didapatkan persentase jumlah kuesioner Pengetahuan mengenai Human Diploid Cell berdasarkan Tingkat Pendidikan didapatkan pengetahuan baik sebanyak 5% pada tahun ketiga dan 7% pada tahun pertama. Pengetahuan cukup sebanyak 23% pada tingkat ketiga dan 28% pada tahun pertama. Pengetahuan kurang sebanyak 9% pada tingkat ketiga dan 28% pada tahun pertama. Persentase jumlah kuesioner Pengetahuan mengenai Polio berdasarkan Tingkat Pendidikan didapatkan pengetahuan baik sebanyak 15% pada tahun ketiga dan 19% pada tahun pertama. Pengetahuan cukup sebanyak 18% pada tingkat ketiga dan 31% pada tahun pertama. Pengetahuan kurang sebanyak 4% pada tingkat ketiga dan 13% pada tahun pertama. Kesimpulan : Tidak terdapat hubungan antara tingkat pendidikan dengan pengetahuan mengenai Human Diploid Cell dalam vaksin Polio. Dalam pandangan Islam, penggunaan vaksin Polio hukumnya mubah karena prinsip Dharuriyat bertujuan untuk mempertahankan nyawa atau Hifdz an-nafs anak dari ancaman penyakit.

DExCon, DExogron, LUXon: on-demand expression control of endogenous genes reveals differential dynamics of Rab11 family members

CRISPR technology has made generation of gene knockouts widely achievable in cells. However, once inactivated, their reactivation remains difficult, especially in diploid cells. Here, we present DExCon (Doxycycline-mediated endogenous gene Expression Control), DExogron (DExCon combined with auxin-mediated targeted protein degradation) and LUXon (light responsive DExCon), approaches which combine one-step CRISPR-Cas9 mediated targeted knock-in of fluorescent proteins with an advanced Tet-inducible TRE3GS promoter. These approaches combine blockade of active gene transcription with the ability to reactivate transcription on demand, including activation of silenced genes. Systematic control can be exerted using doxycycline or spatiotemporally by light, and we demonstrate functional knockout/rescue in the closely related Rab11 family of vesicle trafficking regulators. Fluorescent protein knock-in results in bright signals compatible with low-light live microscopy from monoallelic modification, the potential to simultaneously image different alleles of the same gene and bypasses the need to work with clones. Protein levels are easily tunable to correspond with endogenous expression through cell sorting (DExCon), timing of light illumination (LUXon) or by exposing cells to different levels of auxin (DExogron). Furthermore, our approach allowed us to quantify previously unforeseen differences in vesicle dynamics, expression kinetics and protein stability among highly similar endogenous Rab11 family members and their colocalization in triple knock-in cells.GRAPHICAL ABSTRACTIN BRIEFWe describe development of DExCon, LUXon and DExogron approaches, where a single CRIPR/Cas9-mediated gene editing event can block endogenous gene expression, with the ability to reactivate expression encoded such that even silent genes can be expressed. Expression can be controlled systematically using doxycycline, or spatiotemporally by light, allowing fluorescent tagging of endogenous proteins and quantification of expression kinetics, protein dynamics and stability for highly similar genes such as members of the Rab11 family.

High clonal diversity and spatial genetic admixture in early prostate cancer and surrounding normal tissue

Copy number alterations (CNAs) are pervasive in advanced human cancers, but their prevalence in early-stage, localized tumors and their surrounding normal tissues is poorly characterized. To investigate this phenomenon, here we developed a method for spatially resolved single-cell CNA profiling and applied it to characterize the CNA landscape in 10,007 nuclei extracted from 70 tumor and normal tissue regions (~125 mm3 tissue cubes) from prostatectomies performed in six patients with localized prostate cancer. We identified two distinct groups of cells with abnormal karyotype, one mainly consisting of sparse alterations (‘pseudo-diploid’ cells) and the other characterized by genome-wide karyotypic changes (‘monster’ cells). Pseudo-diploid cells displayed high clonal diversity and formed numerous small sized clones ranging from highly spatially localized to broadly spread clones, whereas monster cells were singular events detected throughout the prostate. We observed a remarkable correlation between the fraction of the genome affected by CNAs and the number of tissue regions in which pseudo-diploid cells were found. Highly localized pseudo-diploid clones were enriched in tumor regions and carried deletions of known or putative tumor suppressors, including APC, CDKN1B, FOXO1, FOXP1, and RB1. Spatially resolved targeted deep sequencing of 523 cancer genes detected non-synonymous mutations in both normal and tumor regions, including mutations in FOXA1, FOXP1, and SPOP genes previously implicated in prostate cancer. Strikingly, in two regions in which targeted deep sequencing detected a point mutation affecting the DNA-binding activity of the FOXA1 transcription factor, we also found a co-deletion of FOXO1 and FOXO3 genes in cells from two different pseudo-diploid clones, implicating combinatorial perturbations of Forkhead transcription factors as an early driver of prostate carcinogenesis. Our study reveals that CNAs and mutations are widespread across normal and tumor regions in the prostate glands of patients with localized prostate cancer and suggests that a subset of alterations—most likely small deletions causing the loss of key tumor suppressors—confer a fitness advantage and channel cells towards tumorigenesis.

Trehalose pathway regulates filamentation response in Saccharomyces cerevisiae

In Saccharomyces cerevisae , the diploid cells undergo either pseudohyphal differentiation or sporulation in response to carbon and nitrogen source depletion. Distinct pathways are known to regulate the processes of filamentation and sporulation in response to nutritional stress. Here, we report the novel finding that the trehalose pathway which is essential for sporulation, is involved in pseudohyphae formation both via GPR1 as well as RAS2 mediated signaling. Our observations indicate that GPR1 is epistatic over TPS1 in signaling for filamentation. Further, we have demonstrated that the pseudohyphal defect of the ras2 mutant is overcome upon disruption of TPS2 . Thus, our results indicate that TPS1 and TPS2 may be involved in cell fate decision between meiosis and filamentation response under nutrient depleting conditions. Further, monitoring pseudohyphae formation under limiting glucose condition unravelled the possibility that TPS1 and TPS2 exert opposing effects to trigger filamentation response.

CBIO-11. HISTONE H3.3 G34R/V MUTATIONS STIMULATE PEDIATRIC HIGH-GRADE GLIOMA FORMATION THROUGH THE INDUCTION OF CHROMOSOMAL INSTABILITY

H3.3 G34R/V mutations are drivers of high-grade pediatric glioma (pHGG). H3.3 G34R/V mutations are linked to altered H3.3 K36 trimethylation (H3K36me3); implicating epigenetic gene regulation as a possible contributor to pHGG formation. Here we show that H3.3 G34R/V also induces chromosomal instability (CIN); a hallmark of pHGG. If CIN promotes pHGG formation is unknown. We observed that H3.3 G34 mutant pHGG cells have reduced mitotic H3.3 S31 phosphorylation compare to WT H3.3 cell lines. And, H3.3 G34R reduced Chk1 phosphorylation at S31 by >90% in an in vitro kinase assay. Chk1 regulates chromosome segregation through phosphorylation of pericentromeric H3.3 S31 during early mitosis. Overexpression of H3.3 G34R or non-phosphorylatable S31A in H3.3 WT, diploid cells caused a significant increase in CIN. Likewise, H3.3 G34 mutant pHGG cells have significantly elevated rates of CIN as compare to H3.3 WT pHGG cells. During normal cell division, phospho-S31 is lost in anaphase. However, following chromosome missegregation, phospho-S31 spreads and stimulates p53-induced cell cycle arrest. Here we show that WT p53 cells expressing mutant G34 fail to arrest following chromosome mis-segregation. These studies demonstrate that H3.3 G34R/V mutations are sufficient to transform normal, diploid cells into proliferating CIN cells. To determine if this process contributes to tumorigenesis, we used RCAS Nestin-TVA mice to overexpress H3.3 WT, G34R, or S31A – P2A-linked to PDGFB expression in glial precursor cells of newborn mice. Over 100 days, S31A and G34R mice had drastically reduced survival (averaging 77, 81, and 100 days for S31A, G34R, and WT). Furthermore, most G34R and S31A mice developed HGG, while H3.3 WT mice remained tumor-free. Our work implicates CIN as a driver of H3.3 G34 mutant pHGG formation. Our ongoing studies utilize K36M and double mutants to further define the contributions of S31 phosphorylation (CIN) and H3K36me3 (epigenetic gene regulation) to tumorigenesis.

The Formation of Neochromosomes during Experimental Evolution in the Yeast Saccharomyces cerevisiae

Novel, large-scale structural mutations were previously discovered during the cultivation of engineered Saccharomyces cerevisiae strains in which essential tRNA synthetase genes were replaced by their orthologs from the distantly related yeast Yarrowia lipolytica. Among those were internal segmental amplifications forming giant chromosomes as well as complex segmental rearrangements associated with massive amplifications at an unselected short locus. The formation of such novel structures, whose stability is high enough to propagate over multiple generations, involved short repeated sequences dispersed in the genome (as expected), but also novel junctions between unrelated sequences likely triggered by accidental template switching within replication forks. Using the same evolutionary protocol, we now describe yet another type of major structural mutation in the yeast genome, the formation of neochromosomes, with functional centromeres and telomeres, made of extra copies of very long chromosomal segments ligated together in novel arrangements. The novel junctions occurred between short repeated sequences dispersed in the genome. They first resulted in the formation of an instable neochromosome present in a single copy in the diploid cells, followed by its replacement by a shorter, partially palindromic neochromosome present in two copies, whose stability eventually increased the chromosome number of the diploid strains harboring it.

Role of CD36 in Palmitic Acid Lipotoxicity in Neuro-2a Neuroblastoma Cells

Elevated level of palmitic acid (PA), a long-chain saturated fatty acid (SFA), is lipotoxic to many different types of cells including Neuro-2a (N2a) neuroblastoma cells. CD36 is a multifunctional membrane glycoprotein that acts as a fatty acid translocase (FAT) facilitating the transport of long-chain free fatty acids (FFAs) into cells, serves a fatty acid (FA) sensing function in areas including taste buds and the proximal gut, and acts as a scavenger receptor that binds to many ligands, including FAs, collagen, oxidized low-density lipoproteins, and anionic phospholipids. However, the involvement of CD36 in FA uptake and PA lipotoxicity in N2a cells remains unclear. In this study, we examined FA uptake in BSA- and PA-treated N2a cells and investigated the involvement of CD36 in FA uptake and PA lipotoxicity in N2a cells. Our data showed that PA treatment promoted FA uptake in N2a cells, and that treatment with sulfo-N-succinimidyl oleate (SSO), a CD36 inhibitor, significantly decreased FA uptake in BSA- and PA-treated N2a cells, and ameliorated PA-induced decrease of cell viability, decrease of diploid cells, and increase of tetraploid cells. We also found that CD36 knockdown significantly decreased FA uptake in both BSA- and PA-treated cells as compared to their corresponding wild-type controls, and dramatically attenuated PA-induced cell cycle defects in N2a cells. Our data suggest that CD36 may play a critical role in FA uptake and PA lipotoxicity in N2a cells. CD36 may therefore represent a regulatory target against pathologies caused by excess FAs.

Chromosomal mutations in high-yield Holstein cows

The results of the research of somatic chromosomal instability: aneuploidy and polyploidy in the blood cells of high-yield Holstein cows with the productivity of more than 9000 kg have been presented in the article. The research was conducted in JSC Vaganovo, Promyshlennovsky District, Kemerovo Region on the population of Holstein cows. Sample preparation was carried out accord- ing to P. Moorhead et al. Samples were stained using the Romanowsky-Giemsa method. The authors studied 6068 metaphase plates in a group of 30 healthy animals. Aneuploidy was counted according to the practice of N.P. Bochkov et al. The authors also analysed the ecological situation in the area of keeping and breeding cattle. Studies of soil, fodder, organs and tissues in farm animals of different species confirm that heavy metal and chemical pollutants are not contaminated in Western Siberia, and their levels are within sanitary norms. Therefore, the ecological situation in the study area should be considered safe. The study shows that the frequency of polyploidy was 0.59% in the studied sample. Tetraploidy, triploidy, and hexaploidy rates were 0.33; 0.18 and 0.06, respectively, the frequency of true hypoploidy was 0.73, and the number of diploid cells was 84.7%. The data obtained can be tentatively applied as average values and can also be used to evaluate the interior of the animals.

Imputation-free reconstructions of three-dimensional chromosome architectures in human diploid single-cells using allele-specified contacts

The sparseness of chromosomal contact information and the presence of homologous chromosomes with very similar nucleotide sequences make Hi-C analysis difficult. We propose a new algorithm using allele-specific single-nucleotide variations (SNVs) to reconstruct the three-dimensional (3D) chromosomal architectures from the Hi-C dataset of single diploid cells. Our algorithm has a function to discriminate SNVs specifically found between homologous chromosomes to our “recurrence plot”-based algorithm to estimate the 3D chromosome structure, which does not require imputation for ambiguous segment information. The new algorithm can efficiently reconstruct 3D chromosomal structures in single human diploid cells by employing only Hi-C segment pairs containing allele-specific SNVs. The datasets of the remaining pairs of segments without allele-specific SNVs are used to validate the estimated chromosome structure. This approach was used to reconstruct the 3D structures of human chromosomes in single diploid cells at a 1-Mb resolution. Introducing a subsequent mathematical measure further improved the resolution to 40-kb or 100-kb. The reconstruction data reveals that human chromosomes form chromosomal territories and take fractal structures where the mean dimension is a non-integer value. We also validate our approach by estimating 3D protein/polymer structures.