Graphical Abstract Highlights d Tandem paired nicking promotes targeted knockin with substantial efficiency d Tandem paired nicking scarcely creates indels at the edited genomic loci d DNA recombination elicited by tandem paired nicking is analogous to Holliday's model d Targeted knockin by tandem paired nicking shows no reciprocal interference with p53 SUMMARYTargeted knockin mediated by double-stranded DNA cleavage is accompanied by unwanted insertions and deletions (indels) at on-target and off-target sites. A nick-mediated approach scarcely generates indels but exhibits reduced efficiency of targeted knockin. Here, we demonstrate that tandem paired nicking, a method for targeted knockin involving two Cas9 nickases that create nicks at the homologous regions of the donor DNA and the genome in the same strand, scarcely creates indels at the edited genomic loci, while permitting the efficiency of targeted knockin largely equivalent to that of the Cas9-nuclease-based approach. Tandem paired nicking seems to accomplish targeted knockin by DNA recombination analogous to Holliday's model and creates intended genomic changes without introducing additional nucleotide changes, such as silent mutations. Targeted knockin through tandem paired nicking neither triggers significant p53 activation nor occurs preferentially in p53-suppressed cells. These properties of tandem paired nicking demonstrate its utility in precision genome engineering.

Malaria, the exterminator of ~1.5 to 2.7 million human lives yearly, is a notorious disease known throughout the world. The eradication of this disease is difficult and a challenge to scientists. Vector elimination and effective chemotherapy for the patients are key tactics to be used in the fight against malaria. However, drug resistance and environmental and social concerns are the main hurdles in this fight against malaria. Overcoming these limitations is the major challenge for the 21st-century malarial researchers. Adapting the principles of nano-biotechnology to both vector control and patient therapy is the only solution to the problem. Several compounds such as lipids, proteins, nucleic acid and metallic nanoparticles (NPs) have been successfully used for the control of this lethal malaria disease. Other useful natural reagents such as microbes and their products, carbohydrates, vitamins, plant extracts and biodegradable polymers, are also used to control this disease. Among these particles, the plant-based particles such as leaf, root, stem, latex, and seed give the best antagonistic response against malaria. In the present review, we describe certain efforts related to the control, prevention and treatment of malaria. We hope that this review will open new doors for malarial research.

Ameloblastoma is a rare odontogenic benign tumor accounting for less than 1% of head and neck tumors. Advanced next generation sequencing (NGS) analyses identified high frequency of BRAF V600E and SMO L412F mutations in ameloblastoma.Despite the existence of whole genomic sequence information from patients with ameloblastoma, entire molecular signature of and the characteristics of ameloblastoma cells are still obscure. In this study, we sought to uncover the molecular basis of ameloblastoma and to determine the cellular phenotype of ameloblastoma cells with BRAF mutations. Our comparative cDNA microarray analysis and gene set enrichment analysis (GSEA) showed that ameloblastoma exhibited a distinct gene expression pattern from the normal tissues: KRAS-responsive gene set is significantly activated in ameloblastoma. Importantly, insulin like growth factor 2 (IGF2), a member of KRAS-responsive genes, enhances the proliferation of an ameloblastoma cell line AMU-AM1 with BRAF mutation. In addition, Toll-like receptor 2 (TLR2) knockdown readily inactivated KRAS-responsive gene sets as well as increases caspase activities, suggesting that TLR2 signaling may mediate cell survival signaling in ameloblastoma cells. Collectively, the findings may help to further clarify the pathophysiology of ameloblastoma and lead to the development of precision medicine for patients with ameloblastoma.

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Overexpression of epithelial cell adhesion molecule (EpCAM) has been associated with chemotherapeutic resistance, leads to aggressive tumor behavior, and results in an adverse clinical outcome. The molecular mechanism by which EpCAM enrichment is linked to therapeutic resistance via Nrf2, a key regulator of antioxidant genes is unknown. We have investigated the link between EpCAM and the Nrf2 pathway in light of therapeutic resistance using head and neck squamous cell carcinoma (HNSCC) patient tumor samples and cell lines. We report that EpCAM was highly expressed in Nrf2-positive and HPV-negative HNSCC cells. In addition, cisplatin-resistant tumor cells consisted of a higher proportion of EpCAM high cells compared to the cisplatin sensitive counterpart. EpCAM high populations exhibited resistance to cisplatin, a higher efficiency in colony formation, sphere growth and invasion capacity, and demonstrated reduced reactive oxygen species (ROS) activity. Furthermore, Nrf2 expression was significantly higher in EpCAM high populations. Mechanistically, expression of Nrf2 and its target genes were most prominently observed in EpCAM high populations. Silencing of EpCAM expression resulted in the attenuation of expressions of Nrf2 and SOD1 concomitant with a reduction of Sox2 expression. On the other hand, silencing of Nrf2 expression rendered EpCAM high populations sensitive to cisplatin treatment accompanied by the inhibition of colony formation, sphere formation, and invasion efficiency and increased ROS activity. The molecular mechanistic link between EpCAM expression and activation of Nrf2 was found to be a concerted interaction of interleukin-6 (IL-6) and p62. Silencing of p62 expression in EpCAM high populations resulted in the attenuation of Nrf2 pathway activation suggesting that Nrf2 pathway activation promoted resistance to cisplatin in EpCAM high populations. We propose that therapeutic targeting the Nrf2-EpCAM axis might be an excellent approach to modulate stress resistance and thereby survival of HNSCC patients enriched in EpCAM high populations.