A mechanistic study on the tolerance of PAM distal end mismatch by SpCas9.

The therapeutic application of CRISPR-Cas9 is limited due to its off-target activity. To have a better understanding of this off-target effect, we focused on its mismatch-prone PAM distal end. The off-target activity of SpCas9 depends directly on the nature of mismatches, which in turn results in deviation of the active site of SpCas9 due to structural instability in the RNA-DNA duplex strand. In order to test the hypothesis, we designed an array of mismatched target sites at the PAM distal end and performed in vitro and cell line-based experiments, which showed a strong correlation for Cas9 activity. We found that target sites having multiple mismatches in the 18th to 15th position upstream of the PAM showed no to little activity. For further mechanistic validation, Molecular Dynamics simulations were performed, which revealed that certain mismatches showed elevated root mean square deviation values that can be attributed to conformational instability within the RNA-DNA duplex. Therefore, for successful prediction of the off-target effect of SpCas9, along with complementation-derived energy, the RNA-DNA duplex stability should be taken into account.

Differential carbonic anhydrase activities control EBV-induced B-cell transformation and lytic cycle reactivation.

Epstein-Barr virus (EBV) contributes to ~1% of all human cancers including several B-cell neoplasms. A characteristic feature of EBV life cycle is its ability to transform metabolically quiescent B-lymphocytes into hyperproliferating B-cell blasts with the establishment of viral latency, while intermittent lytic cycle induction is necessary for the production of progeny virus. Our RNA-Seq analyses of both latently infected naïve B-lymphocytes and transformed B-lymphocytes upon lytic cycle replication indicate a contrasting expression pattern of a membrane-associated carbonic anhydrase isoform CA9, an essential component for maintaining cell acid-base homeostasis. We show that while CA9 expression is transcriptionally activated during latent infection model, lytic cycle replication restrains its expression. Pharmacological inhibition of CA-activity using specific inhibitors retards EBV induced B-cell transformation, inhibits B-cells outgrowth and colony formation ability of transformed B-lymphocytes through lowering the intracellular pH, induction of cell apoptosis and facilitating degradation of CA9 transcripts. Reanalyses of ChIP-Seq data along with utilization of EBNA2 knockout virus, ectopic expression of EBNA2 and sh-RNA mediated knockdown of CA9 expression we further demonstrate that EBNA2 mediated CA9 transcriptional activation is essential for EBV latently infected B-cell survival. In contrast, during lytic cycle reactivation CA9 expression is transcriptionally suppressed by the key EBV lytic cycle transactivator, BZLF1 through its transactivation domain. Overall, our study highlights the dynamic alterations of CA9 expression and its activity in regulating pH homeostasis act as one of the major drivers for EBV induced B-cell transformation and subsequent B-cell lymphomagenesis.

Exploration of Variable Solvent Directed Self-Healable Supramolecular M(II)-Metallogels (M = Co, Ni, Zn) of Azelaic Acid: Investigating Temperature-Dependent Ion Conductivity and Antibacterial Efficiency.

Molecular self-assembly assisted self-healing supramolecular metallogels of azelaic acid with cobalt(II)-, nickel(II)-, and zinc(II)-based metal acetate salts were successfully fabricated. Individually, N,N'-dimethylformamide and dimethyl sulfoxide were immobilized within these distinctly synthesized soft-scaffolds of metallogels to attain their semisolid viscoelastic nature. Rheological experiments such as amplitude sweep, frequency sweep, and thixotropic measurements were executed for these metallogels to ratify their gel features. The different extents of supramolecular interactions operating within these solvent-directed metallogels were clearly reflected in terms of their distinct morphological patterns as investigated through field emission scanning electron microscopy. Comparative infrared (IR) spectral properties of metallogels along with individual metal salts and azelaic acid were analyzed. These experimental data clearly depict the significant shifting of Fourier transform (FT)-IR peaks of xerogel samples of different metallogels from the gel-forming precursors. The networks present within the soft-scaffold are evidently illustrated by the electrospray ionization-mass experimental data. The temperature-dependent ionic conductivity studies with these solvent-directed versatile metallogel systems were investigated through impedance spectroscopy. The temperature-dependent impedance spectroscopic studies clearly demonstrate that the ion-transportation within the gel matrix depends not only on the types of cations but also on the dielectric properties of the immobilized solvents. The antipathogenic effect of these metallogel systems has also been explored by testing their effectiveness against human pathogenic Gram-negative bacteria Klebsiella pneumoniae (MTCC 109) and Vibrio parahemolyticus, and Gram-positive bacteria like Bacillus cereus (MTCC 1272). These gel soft-scaffolds show no significant cytotoxicity against both the human neuroblastoma cell line-SH-SY5Y and the human embryonic kidney cell line-HEK 293.

A Review on the Current Status of Homeopathy in the Clinical Manage-ment of Cancer.

Homeopathy is a widely practiced alternate system of medicine around the world that employs small doses of various medicines to promote auto-regulation and self-healing. It is among the most commonly used alternative approaches in cancer and other diseases and alternative therapeutic systems. It is widely used as palliative and as supportive therapy in cancer patients. Few cases have been reported on patients using homeopathy after surgery, radiotherapy, and chemotherapy, generally for overcoming side effects. The dose of Homoeopathic medicines and their mechanism of action in cancer has also been documented, while clinical trials on the effects of Homoeopathy in cancer treatment are rare. It is found that the anticancer potential of homeopathic medicines is reported for different cancer types, which show their efficacy through apoptosis and immune system modulation. Homeopathic treatment is an add-on to conventional therapy, with almost no interaction with the conventional drugs due to the small dose, and is largely attributed to improving lives by providing symptomatic relief, increasing survival time and boosting patient immunity. This review explores the accountability of the homeopathic system of medicine by highlighting some of the most commonly used homeopathic drugs for different types of cancers.

Etiologic Role of Kinases in the Progression of Human Cancers and Its Targeting Strategies.

Cancer is one of the dominant causes of death worldwide while lifelong prognosis is still inauspicious. The maturation of the cancer is seen as a process of transformation of a healthy cell into a tumor-sensitive cell, which is held entirely at the cellular, molecular, and genetic levels of the organism. Tyrosine kinases can play a major, etiologic role in the inception of malignancy and devote to the uncontrolled proliferation of cancerous cells and the progression of a tumor as well as the development of metastatic disease. Angiogenesis and oncogene activation are the major event in cell proliferation. The growth of a tumor and metastasis are fully depending on angiogenesis and lymphangiogenesis triggered by chemical signals from tumor cells in a phase of rapid growth. Tyrosine kinase inhibitors are compounds that inhibit tyrosine kinases and effective in targeting angiogenesis and blocking the signaling pathways of oncogenes. Small molecule tyrosine kinase inhibitors like afatinib, erlotinib, crizotinib, gefitinib, and cetuximab are shown to a selective cut off tactic toward the constitutive activation of an oncogene in tumor cells, and thus contemplated as promising therapeutic approaches for the diagnosis of cancer and malignancies.