Induction of <i>DrePR</i> recombinase activity by RU486.

<p>(<b>A</b>) Schematic of <i>ubb-DrePR</i> driver line and <i>Rox-Nuc-mCherry-Rox</i> reporter. Additional <i>cryaa:eCFP</i> cassette facilitates identification of transgene-expressing embryos. Open triangles indicate Tol2 arms. (<b>B</b>) Tight control of <i>DrePR</i> recombinase activity by RU486. <i>ubb-DrePR</i>; <i>Rox-Nuc-mCherry-Rox-eGFP</i> embryos were treated with and without 4 µM RU486 between 24 and 48 hpf, and imaged at 96 hpf. No expression of eGFP is observed in untreated (-RU486) or tamoxifen (4-OHT)-treated embryos, while treatment with RU486 results in potent induction of eGFP expression indicating successful recombination of <i>Rox-Nuc-mCherry-Rox-eGFP</i> allele. (<b>C</b>) To quantify the efficiency of <i>Dre</i> recombination in <i>Dre</i>-expressing embryos and <i>DrePR</i> recombination in <i>DrePR</i>-expressing embryos, the intestine of larval zebrafish (4 dpf) were dissected following treatment with RU486 at the indicated concentration between 24–48 hpf. DAPI-labeled cells also labeled by either nucleus mCherry or cytoplasmic eGFP were counted. Maximal recombination frequency is achieved an an RU486 concentration of 4 µM, at a level comparable with Dre lacking the PR fusion. Scale bar: 25 µm.</p

Combinatorial activation of <i>DrePR</i> and <i>CreER^T2</i>.

<p>(<b>A</b>) Schematic of <i>ubb:lox-stop-lox-rox-Nuc-mCherry-stop-rox-eGFP</i> dual reporter for assessment of both <i>Cre</i>- and <i>Dre</i>-mediated recombination, along with <i>ubb-CreER^T2</i> and <i>ubb-DrePR</i> driver lines. Ocular and cardiac fluorescence conveyed by additional <i>cryaa:mCherry</i>, <i>cmlc2:eGFP and cryaa:eCFP</i> cassettes facilitates identification of transgene-expressing embryos. Open triangles indicate Tol2 arms. (<b>B</b>) Triple transgenic fish were treated for 24 hrs with or without 4-OHT and RU486 as indicated, and imaged at 96 hpf. While untreated embryos showed no transgene-specific fluorescence besides that provided by the ocular mCherry, ocular eCFP and cardiac eGFP markers (Fig. 3B a1, a2, a3, and a4), embryos treated with only 4-OHT displayed widespread activation of nuc-mCherry, but no activation of eGFP (Fig. 3B b1, b2, b3, b4). In contrast, embryos simultaneously treated with both 4-OHT and RU486 displayed expression of both nuc-mCherry and eGFP (Fig. 3B c1, c2, c3, and c4). Scale bar: 200 µm. (<b>C</b>) Confocal imaging of dissected intestine, liver, and pancreas, confirming patterns of mCherry and eGFP expression observed in whole embryos. Following combined treatment with 4-OHT and RU486, a majority of cells in each tissue express either nuclear mCherry or cytoplasmic eGFP, with a smaller fraction of cells expressing both. Scale bar: 25 µm.</p

Heterospecific recombination of <i>rox</i> and <i>lox</i> sites by <i>Dre</i> and <i>Cre</i> in zebrafish embryos.

<p>(<b>A, B</b>) Schematic of <i>ubb-Dre</i> (A) and <i>ubb-Cre</i> (B) driver lines and corresponding <i>Rox-Nuc-mCherrys-Rox</i> and <i>Lox-Nuc-mCherry-Lox</i> reporters. Additional <i>cryaa:Venus</i> cassette facilitates identification of transgene-expressing embryos. Open triangles indicate Tol2 arms. (<b>C</b>) Images from double transgenic embryos produced by indicated crosses of <i>ubb-Dre</i> and <i>ubb-Cre</i> driver lines with either <i>Rox-Nuc-mCherry-Rox-eGFP</i> or <i>Lox-Nuc-mCherry-Lox-eGFP</i> reporter lines. Activation of eGFP confirms <i>Dre</i>-specific recombination of <i>Rox-Nuc-mCherry-Rox</i> reporter and <i>Cre</i>-specific recombination of <i>Lox-Nuc-mCherry-Lox</i> reporter. Scale bar: 200 µm.</p

Sequential transgene activation and inactivation using <i>lox</i> and <i>rox</i> (TAILOR).

<p>(<b>A</b>) <i>ubb:lox-stop-lox-rox-Nuc-mCherry-stop-rox-eGFP; ubb-CreER^T2; ubb-Dre</i> triple transgenic fish were subjected to treatment with 4-OHT beginning at 24 hpf, followed by removal and replacement with RU486 at 48 hpf. The untreated triple transgenic embryos (24 hpf) showed no transgene-specific fluorescence besides that provided by the ocular mCherry, ocular eCFP and cardiac eGFP markers (Fig. 4A a1, a2, a3, and a4). The effective induction of nuclear mCherry expression was observed following 24 hrs of 4-OHT treatment (Fig. 4A b1 and b2). Following staged treatment with RU486 initiated at 48 hpf and left in place for 24 hrs, effective activation of eGFP expression was observed (Fig. 4A c1 and c4). Scale bar: 200 µm. (<b>B</b>) Quantification of relative numbers of intestinal, liver and pancreatic cells expressing mCherry (red), eGFP (green) or neither (blue) at 7 days following sequential 4OHT and RU486 exposure as above. Note high fraction of cells undergoing sequential <i>CreER^T2</i>-mediated activation and <i>DrePR</i>-mediated inactivation of mCherry expression, as indicated by eGFP expression. Scale bar: 25 µm.</p

Method To Purify and Analyze Heterogeneous Senescent Cell Populations Using a Microfluidic Filter with Uniform Fluidic Profile

To precisely purify and study aged (senescent) cells, we have designed, fabricated, and demonstrated a novel diamond-structure (DS) microfluidic filter. Nonuniform flow velocities within the microfilter channel can compromise microfluidic filter performance, but with this new diamond structure, further optimized via simulation, we achieve a uniform microfilter flow field, improving the throughput of size-based separation of senescent cells, as obtained by 39-passaged human dermal fibroblasts. After separating these aged cells into two groups, consisting of large- and small-sized cells, we assessed senescence by measuring lipofuscin accumulation and β-galactosidase activity. Our results reveal that even though these senescent cells had been equivalently passaged in culture, a high degree of size distribution and senescent phenotype heterogeneity was observed. In particular, the smaller-sized cells tended to express a younger phenotype while the larger aged cells demonstrated an older phenotype. We suggest that size-based separation of senescent cells, subtyped into small- and large-sized cohorts, offers an alternative method to purify such aged cells, thereby enabling more precise study of the mechanisms of aging, autophagy impairment, and rejuvenation

Harnessing the power of tidal flat diatoms to combat climate change

In approximately one decade, global temperatures will likely exceed a warming level that a United Nations Intergovernmental Panel on Climate Change report considers a “red alert for humanity”. We propose exploring tidal flat diatoms to address climate change challenges. Tidal flats are extensive coastal ecosystems crucial to the provisioning and regulation of aquatic environments. Diatoms contribute to tidal flat biomass production and account for 20% of global primary productivity and 40% of annual marine biomass production, making them crucial for nutrient cycling and sediment stabilization. Potential CO2 removal from Korean tidal flats by diatoms is estimated to be 598,457–683,171 t CO2 equivalents (CO2e) annually, with the economic value of blue carbon (BC) resulting from diatom activity being approximately US$ 17.95–20.50 million. Dissemination of this potential could incentivize coastal wetland protection and climate change mitigation measures. The global estimated CO2e removal potential of tidal flat diatoms is 40,957,346–46,754,961 t CO2e, representing 0.11–0.13% of the annual global greenhouse gas emissions, even though tidal flats cover 0.0025% of the Earth’s surface and diatoms represent less than 0.5% (by weight) of all photosynthetic plants. Researchers should combine ecology and economics to develop standardized approaches for carbon input monitoring and quantification. Further, spatiotemporal analyses of environmental threats to tidal flat diatoms are necessary for conserving their biodiversity and function as a critical BC source. Land-based cultivation for large-scale biomass production and biorefinery processes can contribute to a greener, more prosperous future for humanity and the marine ecosystems upon which we rely.</p