Rapid and precise genome engineering in a naturally short-lived vertebrate.

The African turquoise killifish is a powerful vertebrate system to study complex phenotypes at scale, including aging and age-related disease. Here, we develop a rapid and precise CRISPR/Cas9-mediated knock-in approach in the killifish. We show its efficient application to precisely insert fluorescent reporters of different sizes at various genomic loci in order to drive cell-type- and tissue-specific expression. This knock-in method should allow the establishment of humanized disease models and the development of cell-type-specific molecular probes for studying complex vertebrate biology.

Husbandry of the African Turquoise Killifish Nothobranchius furzeri.

The African turquoise killifish (Nothobranchius furzeri) is an extremely short-lived vertebrate that has emerged as a powerful model organism for several research areas, including aging and embryonic diapause, which is the temporary suspension of embryonic development. The killifish research community is expanding and developing new solutions to improve the tractability of the killifish as a model system. Starting a killifish colony from scratch can present numerous challenges. In this protocol, we aim to highlight critical elements in building and maintaining a killifish colony. This protocol should help laboratories start a killifish colony and standardize aspects of killifish husbandry.

A cryptic oxidoreductase safeguards oxidative protein folding in Corynebacterium diphtheriae.

In many gram-positive Actinobacteria, including Actinomyces oris and Corynebacterium matruchotii, the conserved thiol-disulfide oxidoreductase MdbA that catalyzes oxidative folding of exported proteins is essential for bacterial viability by an unidentified mechanism. Intriguingly, in Corynebacterium diphtheriae, the deletion of mdbA blocks cell growth only at 37 °C but not at 30 °C, suggesting the presence of alternative oxidoreductase enzyme(s). By isolating spontaneous thermotolerant revertants of the mdbA mutant at 37 °C, we obtained genetic suppressors, all mapped to a single T-to-G mutation within the promoter region of tsdA, causing its elevated expression. Strikingly, increased expression of tsdA-via suppressor mutations or a constitutive promoter-rescues the pilus assembly and toxin production defects of this mutant, hence compensating for the loss of mdbA. Structural, genetic, and biochemical analyses demonstrated TsdA is a membrane-tethered thiol-disulfide oxidoreductase with a conserved CxxC motif that can substitute for MdbA in mediating oxidative folding of pilin and toxin substrates. Together with our observation that tsdA expression is upregulated at nonpermissive temperature (40 °C) in wild-type cells, we posit that TsdA has evolved as a compensatory thiol-disulfide oxidoreductase that safeguards oxidative protein folding in C. diphtheriae against thermal stress.

Cervical Cancer Screening Postpandemic: Self-Sampling Opportunities to Accelerate the Elimination of Cervical Cancer.

The persisting burden of cervical cancer in underserved populations and low-resource regions worldwide, worsened by the onset of the COVID-19 pandemic, requires proactive strategies and expanded screening options to maintain and improve screening coverage and its effects on incidence and mortality from cervical cancer. Self-sampling as a screening strategy has unique advantages from both a public health and individual patient perspective. Some of the barriers to screening can be mitigated by self-sampling, and resources can be better allocated to patients at the highest risk of developing cervical cancer. This review summarizes the implementation options for self-sampling and associated challenges, evidence in support of self-sampling, the available devices, and opportunities for expansion beyond human papillomavirus testing.

OncomiR-10b hijacks the small molecule inhibitor linifanib in human cancers.

The pervasive role of microRNAs (miRNAs) in cancer pathobiology drives the introduction of new drug development approaches such as miRNA inhibition. In order to advance miRNA-therapeutics, meticulous screening strategies addressing specific tumor targets are needed. Small molecule inhibitors represent an attractive goal for these strategies. In this study, we devised a strategy to screen for small molecule inhibitors that specifically inhibit, directly or indirectly, miR-10b (SMIRs) which is overexpressed in metastatic tumors. We found that the multi-tyrosine kinase inhibitor linifanib could significantly inhibit miR-10b and reverse its oncogenic function in breast cancer and liver cancer both in vitro and in vivo. In addition, we showed that the efficacy of linifanib to inhibit tyrosine kinases was reduced by high miR-10b levels. When the level of miR-10b is high, it can "hijack" the linifanib and reduce its kinase inhibitory effects in cancer resulting in reduced anti-tumor efficacy. In conclusion, our study describes an effective strategy to screen for small molecule inhibitors of miRNAs. We further propose that miR-10b expression levels, due to the newly described "hijacking" effect, may be used as a biomarker to select patients for linifanib treatment.

MicroRNAs, Regulatory Messengers Inside and Outside Cancer Cells.

MicroRNAs (miRNAs) are a class of short non-coding RNAs (ncRNAs) with typical sequence lengths of 19-25 nucleotides and extraordinary abilities to regulate gene expression. Because miRNAs regulate multiple important biological functions of the cell (proliferation, migration, invasion, apoptosis, differentiation, and drug resistance), their expression is highly controlled. Genetic and epigenetic alterations frequently found in cancer cells can cause aberrant expression of miRNAs and, consequently, of their target genes. The tumor microenvironment can also affect miRNA expression through soluble factors (e.g., cytokines and growth factors) secreted by either tumor cells or non-tumor cells (such as immune and stromal cells). Furthermore, like hormones, miRNAs can be secreted and regulate gene expression in recipient cells. Altered expression levels of miRNAs in cancer cells determine the acquisition of fundamental biological capabilities (hallmarks of cancer) responsible for the development and progression of the disease.