Demonstration of targeted crossovers in hybrid maize using CRISPR technology

Naturally occurring chromosomal crossovers (CO) during meiosis are a key driver of genetic diversity. The ability to target CO at specific allelic loci in hybrid plants would provide an advantage to the plant breeding process by facilitating trait introgression, and potentially increasing the rate of genetic gain. We present the first demonstration of targeted CO in hybrid maize utilizing the CRISPR Cas12a system. Our experiments showed that stable and heritable targeted CO can be produced in F1 somatic cells using Cas12a at a significantly higher rate than the natural CO in the same interval. Molecular characterization of the recombinant plants demonstrated that the targeted CO were driven by the non-homologous end joining (NHEJ) or HDR repair pathways, presumably during the mitotic cell cycle. These results are a step towards the use of RNA-guided nuclease technology to simplify the creation of targeted genome combinations in progeny and accelerate breeding.

Naturally Sourced CDK Inhibitors and Current Trends in Structure-Based Synthetic Anticancer Drug Design by Crystallography

: Cyclin-dependent kinases (CDKs) are the chief regulators in cell proliferation; the kinase activities are largely regulated by their interactions with CDK inhibitors (CKIs) and Cyclins. The association of different CDKs with CDKIs and Cyclins at the cell-cycle checkpoints of different stages of mitotic cell cycle function act more likely as the molecular switches that regulate different transcriptional events required for progression through the cell cycle. A fine balance in response to extracellular and intracellular signals is highly maintained in the orchestrated function of CDKs along with Cyclins and CDKIs for normal cell proliferation. This fine-tuning in mitotic cell cycle progression sometimes gets lost due to dysregulation of CDKs. The aberrant functioning of the CDKIs is therefore studied for its contributions as a vital hallmark of cancers. It has attracted our focus to maneuver cancer therapy. Hence, several synthetic CDKIs and their crystallography-based drug design have been explained to understand their mode of action with CDKs. Since most of the synthetic drugs function by inhibiting the CDK4/6 kinases by competitively binding to their ATP binding cleft, these synthetic drugs are reported to attack the normal, healthy growing cells adjacent to the cancer cells leading to the decrease in the life span of the cancer patients. The quest for traditional natural medicines may have a great impact on the treatment of cancer. Therefore, in the present studies, a search for naturally sourced CDK inhibitors has been briefly focused. Additionally, some synthetic crystallography-based drug design has been explained to elucidate different avenues to develop better anticancer chemotherapeutics, converting natural scaffolds into inhibitors of the CDK mediated abnormal signal transduction with lesser side effects.

SCL28 promotes cell expansion and endoreplication in Arabidopsis by activating SIAMESE-RELATED cyclin-dependent kinase inhibitors.

The processes that contribute to plant organ morphogenesis are spatial-temporally organized. Within the meristem the mitotic cell cycle produces new cells that subsequently engage in specific cell expansion and differentiation programs once they exit the division competent zone. The latter is frequently accompanied by endoreplication, being an alternative cell cycle that replicates the DNA without nuclear division, causing a stepwise increase in somatic ploidy. We have previously shown that the Arabidopsis SCL28 transcription factor promotes progression through G2/M and modulates division plane orientation. Here, we demonstrate that SCL28 co-express and regulates genes specific to cell elongation and differentiation, including genes related to cell wall and cytoskeleton assembly. Consistently, this correlates with defects in post-mitotic cell expansion in a scl28 mutant. Strikingly, SCL28 controls expression of 6 members of the SIAMESE/SIAMESE-RELATED (SIM/SMR) family, encoding cyclin-dependent kinase inhibitors with a role in promoting mitotic cell cycle exit and endoreplication onset, both in response to developmental and environmental cues. Consistent with this role, scl28 mutants displayed reduced endoreplication, both in roots and leaves. Altogether, these results suggest that SCL28 controls cell expansion and differentiation by promoting endoreplication onset and by modulating aspects of the biogenesis, assembly and remodeling of the cytoskeleton and cell wall.

Natural sourced inhibitors of EGFR, PDGFR, FGFR and VEGFR-mediated signaling pathways as potential anticancer agents

: The molecular mechanisms of mitotic cell cycle progression involve very tightly restricted types of machinery which are highly regulated by a fine balance between the positive and negative accelerators (or regulators). These regulators include several checkpoints that have proteins acting as enzymes and their activating partners. These checkpoints incessantly monitor the external as well as internal environments such as growth signals, favorable conditions for growth, cell size, DNA integrity of the cell and hence function to maintain the highly ordered cell cycle progression by sustaining cell homeostasis and promotes error-free DNA replication and cell cycle, division. To progress through the mitotic cell cycle, the cell has to successfully drive past the cell cycle checkpoints. Due to the abnormal behavior of some cell cycle proteins, the cells tend to divide continuously overcoming the tight regulation of cell cycle checkpoints. Such anomalies may lead to unwanted cell division and this deregulation of cell cycle events is considered as one of the main reasons behind tumor development and thus cancer progression. So the understanding of the molecular mechanisms in cancer progression might be insightful for designing several cancer treatment strategies. The deregulation in the checkpoints is caused due to the changes brought in the tyrosine residues of TPKs via PDGFR, EGFR, FGFR, and VEGFR-mediated signalling pathways. Therefore, the inhibitors of PDGFR, EGFR, FGFR, and VEGFR-mediated signalling pathways would be potential anticancer agents. The resistance and toxicity in the existing synthetic anticancer chemotherapeutics may decrease the life span of a patient. For a long, natural products have always played an essential alternative source of therapeutic agents due to having the least or no side effect and toxicity. The present study is an attempt to promote the natural anticancer drug development focusing on the updated structural information of PDGFR, EGFR, FGFR, and VEGFR inhibitors isolated from the plant sources. The data used in this review has been collected from internet resources viz. GOOGLE Web, GOOGLE SCHOLAR, and PubMed Central. The citation of each report was first checked after which the articles were selected as an authentic reference for the present study. Around 200 journal articles were selected of which around 142 were selected finally for presenting the study on the natural sourced inhibitors of EGFR, PDGFR, FGFR, and VEGFR-mediated signaling pathways which would help in the potential cancer treatment.

Comprehensive Analysis of Aerobic Exercise-Related Genes Identifies CDCA4 That Promotes the Progression of Osteosarcoma

BackgroundExercise has a positive impact on patients with osteosarcoma, improving function, reducing disability, maintaining independence and quality of life. Exercise may also directly affect the effectiveness of cancer treatment. Cell division cycle-associated protein 4 (CDCA4) is reported to function importantly during numerous human cancers development. Nevertheless, the details toward CDCA4 function are still to be investigated.MethodsThis study comprehensively analyzed the GSE74194 database and obtained aerobic exercise-related genes. Protein-protein interaction network (PPI) and Gene Ontology (GO) analysis were performed on the differentially expressed genes (DEGs). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and tumor genome atlas (TCGA) data mining were applied to measure aerobic exercise-related gene CDCA4 level in osteosarcoma tissue. We conducted lots of functional experiments to uncover CDCA4 function and its corresponding mechanism in osteosarcoma.ResultsWe screened a total of 547 DEGs related to aerobic exercise, of which 373 were up-regulated and 174 were down-regulated. PPI analysis revealed 90 genes that might play key roles. GO analysis showed that aerobic exercise-related DEGs were significantly enriched during the mitotic cell cycle, cell division, mitotic nuclear division and sister chromatid segregation, nuclear division, microtubule cytoskeleton organization involved protein, microtubule-based process, spindle organization, G2/M transition of mitotic cell cycle. Our results indicated that CDCA4 was increased in osteosarcoma tissues and cell lines, and its level had association with high mortality of osteosarcoma patients. Further studies revealed that absence of CDCA4 largely hindered osteosarcoma cancer cell proliferation, invasion, and migration.ConclusionComprehensive bioinformatics analysis improves our understanding of the underlying molecular mechanisms of aerobic exercise on osteosarcoma. This provides evidence for the effect of aerobic exercise on CDCA4 expression. Our data suggested that CDCA4 could facilitate osteosarcoma development, and gave a hint that CDCA4 was a candidate target in the treatment of osteosarcoma, aerobic exercise might help the treatment and prognosis of patients with osteosarcoma.

OsMre11 Is Required for Mitosis during Rice Growth and Development

Meiotic recombination 11 (Mre11) is a relatively conserved nuclease in various species. Mre11 plays important roles in meiosis and DNA damage repair in yeast, humans and Arabidopsis, but little research has been done on mitotic DNA replication and repair in rice. Here, it was found that Mre11 was an extensively expressed gene among the various tissues and organs of rice, and loss-of-function of Mre11 resulted in severe defects of vegetative and reproductive growth, including dwarf plants, abnormally developed male and female gametes, and completely abortive seeds. The decreased number of cells in the apical meristem and the appearance of chromosomal fragments and bridges during the mitotic cell cycle in rice mre11 mutant roots revealed an essential role of OsMre11. Further research showed that DNA replication was suppressed, and a large number of DNA strand breaks occurred during the mitotic cell cycle of rice mre11 mutants. The expression of OsMre11 was up-regulated with the treatment of hydroxyurea and methyl methanesulfonate. Moreover, OsMre11 could form a complex with OsRad50 and OsNbs1, and they might function together in non-homologous end joining and homologous recombination repair pathways. These results indicated that OsMre11 plays vital roles in DNA replication and damage repair of the mitotic cell cycle, which ensure the development and fertility of rice by maintaining genome stability.

Significant transcriptomic changes are associated with differentiation of bone marrow-derived mesenchymal stem cells into neural progenitor-like cells in the presence of bFGF and EGF

Introduction Mesenchymal stem cells (MSCs) isolated from bone marrow have different developmental origins, including neural crest. MSCs can differentiate into neural progenitor-like cells (NPCs) under the influence of bFGF and EGF. NPCs can terminally differentiate into neurons that express beta-III-tubulin and elicit action potential. The main aim of the study was to identify key genetic markers involved in differentiation of MSCs into NPCs through transcriptomic analysis. Method Total RNA was isolated from MSCs and MSCs-derived NPCs followed by cDNA library construction for transcriptomic analysis. Sample libraries that passed the quality and quantity assessments were subjected to high throughput mRNA sequencing using NextSeq®500. Differential gene expression analysis was performed using the DESeq2 R package with MSC samples being a reference group. The expression of eight differentially regulated genes was counter validated using real-time PCR. Results In total, of the 3,252 differentially regulated genes between MSCs and NPCs with two or more folds, 1,771 were upregulated genes, whereas 1,481 were downregulated in NPCs. Amongst these differential genes, 104 transcription factors were upregulated, and 45 were downregulated in NPCs. Neurogenesis related genes were upregulated in NPCs and the main non-redundant gene ontology (GO) terms enriched in NPCs were the autonomic nervous system, cell surface receptor signalling pathways), extracellular structure organisation, and programmed cell death. The main non-redundant GO terms enriched in MSCs included cytoskeleton organisation cytoskeleton structural constituent, mitotic cell cycle), and the mitotic cell cycle process Gene set enrichment analysis also confirmed cell cycle regulated pathways as well as Biocarta integrin pathway were upregulated in MSCs. Transcription factors enrichment analysis by ChEA3 revealed Foxs1 and HEYL, amongst the top five transcription factors, inhibits and enhances, respectively, the NPCs differentiation of MSCs. Conclusions The vast differences in the transcriptomic profiles between NPCs and MSCs revealed a set of markers that can identify the differentiation stage of NPCs as well as provide new targets to enhance MSCs differentiation into NPCs.

Mosaic changes to the global transcriptome in response to inhibiting ribosome formation versus inhibition of ribosome function

Cell fate is susceptible to several internal and external stresses. Stress resulting from mutations in genes for ribosomal proteins and assembly factors leads to many congenital diseases, collectively called ribosomopathies. Even though such mutations all depress the cell’s protein synthesis capacity, they are manifested in many different phenotypes. This prompted us to use Saccharomyces cerevisiae to explore whether reducing the protein synthesis capacity by different mechanisms result in the same or different changes to the global transcriptome. We have compared the transcriptome after abolishing the assembly of new ribosomes and inhibiting the translocation of ribosomes on the mRNA. Our results show that these alternate obstructions generate different mosaics of expression for several classes of genes, including genes for ribosomal proteins, mitotic cell cycle, cell wall synthesis, and protein transport.

APOBEC3B and SPAG5, the Target Genes of miR-539, Involved into Imatinib-Resistant of Gastrointestinal Stromal Tumors

Background: Although imatinib can effectively treat gastrointestinal stromal tumor (GIST), some patients are still resistant to it or cannot tolerate the adverse reactions of the drug. This study aimed to investigate potential miRNAs, mRNA and tumor-infiltrating lymphocytes (TILs) associated with the prognosis of imatinib-resistant GIST.Methods: mRNA, miRNA sequencing data and patient clinic traits of primary (imatinib-naive) and imatinib-resistant GIST were obtained from GEO (Gene Expression Omnibus) database. A systems biology approach combining Weighted co-expression network analysis (WGCNA) and differential expression analysis were utilized to detect the imatinib-resistant-related miRNA and gene modules and construct a miRNA-gene network. Tumor-infiltrating immune cells were analyzed by Estimating the Proportion of Immune and Cancer cells (EPIC) and Tumor-Immune System Interactions (TISIDB). miR-539 was measured by qRT-PCR. SPAG5 and APOBEC3B was measured by qRT-PCR and WB. Transforming growth factor (TGF)-β and interleukin (IL)-10 were assessed with enzyme-linked immunosorbent assay (ELISA). The proportions of CD4+ T cells, CD8+ T cells , NK cells and B cells in tumor-infiltrating lymphocyteswere analyzed via flow cytometry (FCM).Results: Two gene modules (brown and yellow) and one miRNA module were associated with the imatinib-resistant. Two hub genes (APOBEC3B and SPAG5) were associated with the imatinib-resistant. Three hub miRNAs were identified to be related to imatinib-resistant (miR-539, miR-376b and miR-18b). G1/S transition of mitotic cell cycle, G2/M transition of mitotic cell cycle, and cell proliferation were common pathways of the gene modules and miRNA module. apolipoprotein B mRNA editing catalytic polypeptide-like 3B (APOBEC3B) and Sperm-associated antigen 5 (SPAG5), which were both target genes of miR-539 was located at the core of miRNA–gene network. APOBEC3B (rho=0.509, p < 2.2e-16) and SPAG5 (rho=0.468, p < 2.2e-16) was positive correlated with the infiltration levels of activated CD4+ T cells. he proportions of CD4+ T cells, and the mRNA and protein relative expression of APOBEC3B and SPAG5 in imatinib-resistant tumor samples significantly increased as compared with tumor samples in imatinib-naive group. Imatinib-resistant tumor samples exhibited significantly downregulation of miR-539 and TGF-β1. Over-expression of miR-539 sensitized imatinib resistant GIST48 cells and increased the secretion of TGF-β1 by inhibiting APOBEC3B and SPAG5.Conclusion: APOBEC3B and SPAG5, the target genes of miR-539 may play as key factor for imatinib-resistant GIST by increasing the proportion of tumor-infiltrated activated CD4+ T cells via TGF-β1. These findings help to advance the understanding of imatinib-resistant GIST and provide potential therapeutic targets.