Multiplex gene removal by two-step polymerase chain reactions

Precise DNA manipulation is critical for molecular biotechnology. Restriction enzyme-based approaches are limited by their requirement of specific enzyme sites. Restriction-free cloning has greatly improved the flexibility and speed of precise DNA assembly. Most of these approaches focus on DNA assembly rather than gene removal. Here we present a polymerase chain reaction (PCR)-based cloning method that allows removal of multiple gene segments from plasmids without using restriction enzymes and thermostable ligase. We demonstrate simultaneous removal of three gene segments from a plasmid. This approach could be beneficial to DNA library construction, genetic and protein engineering, and synthetic biology.     

CO2 Saturation and Trophic Shift Induced by Microbial Metabolic Processes in a River-Dominated Ocean Margin (Tropical Shallow Lagoon,Chilika, India)

An effort has been made for the first time in Asia's largest brackish water lagoon, Chilika, to investigate the spatio-temporal variability in primary productivity (PP), bacterial productivity (BP), bacterial abundance (BA), bacterial respiration (BR) and bacterial growth efficiency (BGE) in relation to partial pressure of CO₂ (pCO₂) and CO₂ air–water flux and the resultant trophic switchover. Annually, PP ranged between 24 and 376 µg C L^?1 d^?1 with significantly low values throughout the monsoon (MN), caused by light limitation due to inputs of riverine suspended matter. On the contrary, BP and BR ranged from 11.5 to 186.3 µg C L^?1 d^?1 and from 14.1 to 389.4 µg C L^?1 d^?1, respectively, with exceptionally higher values during MN. A wide spatial and temporal variation in the lagoon trophic status was apparent from BP/PP (0.05–6.4) and PP/BR (0.10–18.2) ratios. The seasonal shift in net pelagic production from autotrophy to heterotrophy due to terrestrial organic matter inputs via rivers, enhanced the bacterial metabolism during the MN, as evident from the high pCO₂ (10,134 µatm) and CO₂ air–water flux (714 mm m^?2 d^?1). Large variability in BGE and BP/PP ratios especially during MN led to high bacteria-mediated carbon fluxes which was evident from significantly high bacterial carbon demand (BCD >100% of PP) during this season. This suggested that the net amount of organic carbon (either dissolved or particulate form) synthesized by primary producers in the lagoon was not sufficient to satisfy the bacterial carbon requirements. Lagoon sustained low to moderate autotrophic–heterotrophic coupling with annual mean BCD of 231% relative to the primary production, which depicted that bacterioplankton are the mainstay of the lagoon biogeochemical cycles and principal players that bring changes in trophic status. Study disclosed that the high CO₂ supersaturation and oxygen undersaturation during MN was attributed to the increased heterotrophic respiration (in excess of PP) fuelled by allochthonous organic matter. On a spatial scale, lagoon sectors such as south sector, central sector and outer channel recorded “net autotrophic,” while the northern sector showed “net heterotrophic” throughout the study period.     

Nuclear import and export signals in control of Nrf2

Nrf2 binds to the antioxidant response element and regulates expression and antioxidant induction of a battery of chemopreventive genes. In this study, we have identified nuclear import and export signals of Nrf2 and show that the nuclear import and export of Nrf2 is regulated by antioxidants. We demonstrate that Nrf2 contains a bipartite nuclear localization signal (NLS) and a leucine-rich nuclear export signal, which regulate Nrf2 shuttling in and out of the nucleus. Immunofluorescence and immunoblot analysis revealed that Nrf2 accumulates in the nucleus within 15 min of antioxidant treatment and is exported out of nucleus by 8 h after treatment. Nrf2 mutant lacking the NLS failed to enter the nucleus and displayed diminished expression and induction of the downstream NAD(P)H:quinone oxidoreductase 1 gene. The Nrf2 NLS sequence, when fused to green fluorescence protein, resulted in the nuclear accumulation of green fluorescence protein, indicating that this signal sequence was sufficient to direct nuclear localization of Nrf2. A nuclear export signal (NES) was characterized in the C terminus of Nrf2, the deletion of which caused Nrf2 to accumulate predominantly in the nucleus. The Nrf2 NES was sensitive to leptomycin B and could function as an independent export signal when fused to a heterologous protein. Further studies demonstrate that NES-mediated nuclear export of Nrf2 is required for degradation of Nrf2 in the cytosol. These results led to the conclusion that Nrf2 localization between cytosol and nucleus is controlled by both nuclear import and export of Nrf2, and the overall distribution of Nrf2 is probably the result from a balance between these two processes. Antioxidants change this balance in favor of nuclear accumulation of Nrf2, leading to activation of chemopreventive proteins. Once this is achieved, Nrf2 exits the nucleus for binding to INrf2 and degradation.     

Nucleic acid-based vaccine for ovarian cancer cells; bench to bedside

Ovarian cancer continues to be a difficult medical issue that affects millions of individuals worldwide. Important platforms for cancer immunotherapy include checkpoint inhibitors, chimeric antigen receptor T cells, bispecific antibodies, cancer vaccines, and other cell-based treatments. To avoid numerous infectious illnesses, conventional vaccinations based on synthetic peptides, recombinant subunit vaccines, and live attenuated and inactivated pathogens are frequently utilized. Vaccine manufacturing processes, however, are not entirely safe and carry a significant danger of contaminating living microorganisms. As a result, the creation of substitute vaccinations is required for both viral and noninfectious illnesses, including cancer. Recently, there has been testing of nucleic acid vaccines, or NAVs, as a cancer therapeutic. Tumor antigens (TAs) are genetically encoded by DNA and mRNA vaccines, which the host uses to trigger immune responses against ovarian cancer cells that exhibit the TAs. Despite being straightforward, safe, and easy to produce, NAVs are not currently thought to be an ideal replacement for peptide vaccines. Some obstacles to this strategy include selecting the appropriate therapeutic agents (TAs), inadequate immunogenicity, and the immunosuppressive characteristic of ovarian cancer. We focus on strategies that have been employed to increase NAVs' effectiveness in the fight against ovarian cancer in this review.