Is lower intensity aquaculture a valuable means of producing food? An evaluation of its effects on near‐shore and inland waters

The effects of aquaculture on the environment have been the subject of much examination, but most of the focus has been on shrimp and salmon. These are not the most common species grown in aquaculture, nor the most common systems used. About 60% of production today uses lower intensity culture to produce organisms in natural systems such as ponds. This paper is an overview of the positive and negative environmental impacts of lower intensity aquaculture. The ranked positive impacts of lower intensity aquaculture include: conservation aquaculture that supplements reproduction in natural populations; improving the quality of natural waters through filtering or consuming wastes by cultured organisms; reducing pressure on wild stocks by providing alternative sources in the market; and replacing damaging employment with more sustainable aquaculture jobs. Negative impacts include: escapement of alien species that become invasive; eutrophication of receiving waters from pond effluents; release of parasites and diseases into natural communities; escapement of unique genotypes resulting in genetic alteration of native stocks; land degradation due to pond construction; release of antibiotics or other drugs into receiving waters; depletion of natural resources such as water; loss of benthic biodiversity from settling of sediments; and reductions in natural populations by collection of larval or juvenile fish. Some impacts, especially the use of fishmeal and the transmission of disease, are much less common in lower intensity aquaculture systems. Aquaculture has an important role in current and future food production, and in many cases lower intensity aquaculture provides a sustainable solution to increased aquaculture production.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/94475/1/j.1753-5131.2012.01079.x.pd

Drug-resilient cancer cell phenotype is acquired via polyploidization associated with early stress response coupled to HIF-2α transcriptional regulation

Therapeutic resistance and recurrence remain core challenges in cancer therapy. How therapy resistance arises is currently not fully understood with tumors surviving via multiple alternative routes. Here, we demonstrate that a subset of cancer cells survives therapeutic stress by entering a transient state characterized by whole genome doubling. At the onset of the polyploidization program, we identified an upregulation of key transcriptional regulators, including the early stress-response protein AP-1 and normoxic stabilization of HIF-2α. We found altered chromatin accessibility, ablated expression of RB1, and enrichment of AP-1 motif accessibility. We demonstrate that AP-1 and HIF-2α regulate a therapy resilient and survivor phenotype in cancer cells. Consistent with this, genetic or pharmacologic targeting of AP-1 and HIF-2α reduced the number of surviving cells following chemotherapy treatment. The role of AP-1 and HIF-2α in stress-response by polyploidy suggest a novel avenue for tackling chemotherapy-induced resistance in cancer

CLUE: a bioinformatic and wet-lab pipeline for multiplexed cloning of custom sgRNA libraries.

Becker M, Noll-Puchta H, Amend D, et al. CLUE: a bioinformatic and wet-lab pipeline for multiplexed cloning of custom sgRNA libraries. Nucleic acids research. 2020;48(13).The systematic perturbation of genomes using CRISPR/Cas9 deciphers gene function at an unprecedented rate, depth and ease. Commercially available sgRNA libraries typically contain tens of thousands of pre-defined constructs, resulting in a complexity challenging to handle. In contrast, custom sgRNA libraries comprise gene sets of self-defined content and size, facilitating experiments under complex conditions such as in vivo systems. To streamline and upscale cloning of custom libraries, we present CLUE, a bioinformatic and wet-lab pipeline for the multiplexed generation of pooled sgRNA libraries. CLUE starts from lists of genes or pasted sequences provided by the user and designs a single synthetic oligonucleotide pool containing various libraries. At the core of the approach, a barcoding strategy for unique primer binding sites allows amplifying different user-defined libraries from one single oligonucleotide pool. We prove the approach to be straightforward, versatile and specific, yielding uniform sgRNA distributions in all resulting libraries, virtually devoid of cross-contaminations. For in silico library multiplexing and design, we established an easy-to-use online platform at www.crispr-clue.de. All in all, CLUE represents a resource-saving approach to produce numerous high quality custom sgRNA libraries in parallel, which will foster their broad use across molecular biosciences. © The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research

Fluorescent labeling of CRISPR/Cas9 RNP for gene knockout in HSPCs and iPSCs reveals an essential role for GADD45b in stress response

CRISPR/Cas9-mediated gene editing of stem cells and primary cell types has several limitations for clinical applications. The direct delivery of ribonucleoprotein (RNP) complexes consisting of Cas9 nuclease and guide RNA (gRNA) has improved DNA- and virus-free gene modifications, but it does not enable the essential enrichment of the gene-edited cells. Here, we established a protocol for the fluorescent labeling and delivery of CRISPR/Cas9-gRNA RNP in primary human hematopoietic stem and progenitor cells (HSPCs) and induced pluripotent stem cells (iPSCs). As a proof of principle for genes with low-abundance transcripts and context-dependent inducible expression, we successfully deleted growth arrest and DNA-damage-inducible β (GADD45B). We found that GADD45B is indispensable for DNA damage protection and survival in stem cells. Thus, we describe an easy and efficient protocol of DNA-free gene editing of hard-to-target transcripts and enrichment of gene-modified cells that are generally difficult to transfect

Drug-resilient cancer cell phenotype is acquired via polyploidization associated with early stress response coupled to HIF-2α transcriptional regulation

Therapeutic resistance and recurrence remain core challenges in cancer therapy. How therapy resistance arises is currently not fully understood with tumors surviving via multiple alternative routes. Here, we demonstrate that a subset of cancer cells survives therapeutic stress by entering a transient state characterized by whole genome doubling. At the onset of the polyploidization program, we identified an upregulation of key transcriptional regulators, including the early stress-response protein AP-1 and normoxic stabilization of HIF-2α. We found altered chromatin accessibility, ablated expression of RB1, and enrichment of AP-1 motif accessibility. We demonstrate that AP-1 and HIF-2α regulate a therapy resilient and survivor phenotype in cancer cells. Consistent with this, genetic or pharmacologic targeting of AP-1 and HIF-2α reduced the number of surviving cells following chemotherapy treatment. The role of AP-1 and HIF-2α in stress-response by polyploidy suggest a novel avenue for tackling chemotherapy-induced resistance in cancer.</p

Combined proteomics and CRISPR‒Cas9 screens in PDX identify ADAM10 as essential for leukemia in vivo

Abstract Background Acute leukemias represent deadly malignancies that require better treatment. As a challenge, treatment is counteracted by a microenvironment protecting dormant leukemia stem cells. Methods To identify responsible surface proteins, we performed deep proteome profiling on minute numbers of dormant patient-derived xenograft (PDX) leukemia stem cells isolated from mice. Candidates were functionally screened by establishing a comprehensive CRISPR‒Cas9 pipeline in PDX models in vivo. Results A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) was identified as an essential vulnerability required for the survival and growth of different types of acute leukemias in vivo, and reconstitution assays in PDX models confirmed the relevance of its sheddase activity. Of translational importance, molecular or pharmacological targeting of ADAM10 reduced PDX leukemia burden, cell homing to the murine bone marrow and stem cell frequency, and increased leukemia response to conventional chemotherapy in vivo. Conclusions These findings identify ADAM10 as an attractive therapeutic target for the future treatment of acute leukemias