We describe the use of zinc-finger nucleases (ZFNs) for somatic and germline disruption of genes in zebrafish (Danio rerio), in which targeted mutagenesis was previously intractable. ZFNs induce a targeted double-strand break in the genome that is repaired to generate small insertions and deletions. We designed ZFNs targeting the zebrafish golden and no tail/Brachyury (ntl) genes and developed a budding yeast–based assay to identify the most active ZFNs for use in vivo. Injection of ZFN-encoding mRNA into one-cell embryos yielded a high percentage of animals carrying distinct mutations at the ZFN-specified position and exhibiting expected loss-of-function phenotypes. Over half the ZFN mRNA-injected founder animals transmitted disrupted ntl alleles at frequencies averaging 20%. The frequency and precision of gene-disruption events observed suggest that this approach should be applicable to any loci in zebrafish or in other organisms that allow mRNA delivery into the fertilized egg.

Background: Rapid spread of the severe acute respiratory syndrome‐coronavirus‐2 (SARS‐CoV‐2) and concern for viral transmission by ambulatory patients with minimal to no symptoms underline the importance of identifying early or subclinical symptoms of coronavirus disease 2019 (COVID‐19) infection. Two such candidate symptoms include anecdotally reported loss of smell and taste. Understanding the timing and association of smell/taste loss in COVID‐19 may help facilitate screening and early isolation of cases.Methods: A single‐institution, cross‐sectional study evaluating patient‐reported symptoms with a focus on smell and taste was conducted using an internet‐based platform on adult subjects who underwent testing for COVID‐19. Logistic regression was employed to identify symptoms associated with COVID‐19 positivity.Results: A total of 1480 patients with influenza‐like symptoms underwent COVID‐19 testing between March 3, 2020, and March 29, 2020. Our study captured 59 of 102 (58%) COVID‐19–positive patients and 203 of 1378 (15%) COVID‐19–negative patients. Smell and taste loss were reported in 68% (40/59) and 71% (42/59) of COVID‐19–positive subjects, respectively, compared to 16% (33/203) and 17% (35/203) of COVID‐19–negative patients (p < 0.001). Smell and taste impairment were independently and strongly associated with COVID‐19 positivity (anosmia: adjusted odds ratio [aOR] 10.9; 95% CI, 5.08‐23.5; ageusia: aOR 10.2; 95% CI, 4.74‐22.1), whereas sore throat was associated with COVID‐19 negativity (aOR 0.23; 95% CI, 0.11‐0.50). Of patients who reported COVID‐19–associated loss of smell, 74% (28/38) reported resolution of anosmia with clinical resolution of illness.Conclusion: In ambulatory individuals with influenza‐like symptoms, chemosensory dysfunction was strongly associated with COVID‐19 infection and should be considered when screening symptoms. Most will recover chemosensory function within weeks, paralleling resolution of other disease‐related symptoms.

Despite the promise of immune checkpoint inhibition (ICI), therapeutic responses remain limited. This raises the possibility that standard of care treatments delivered in concert may compromise the tumor response. To address this, we employ tobacco-signature head and neck squamous cell carcinoma murine models in which we map tumor-draining lymphatics and develop models for regional lymphablation with surgery or radiation. We find that lymphablation eliminates the tumor ICI response, worsening overall survival and repolarizing the tumor- and peripheral-immune compartments. Mechanistically, within tumor-draining lymphatics, we observe an upregulation of conventional type I dendritic cells and type I interferon signaling and show that both are necessary for the ICI response and lost with lymphablation. Ultimately, we provide a mechanistic understanding of how standard oncologic therapies targeting regional lymphatics impact the tumor response to immune-oncology therapy in order to define rational, lymphatic-preserving treatment sequences that mobilize systemic antitumor immunity, achieve optimal tumor responses, control regional metastatic disease, and confer durable antitumor immunity.

Tumor cells initiate platelet activation leading to the secretion of bioactive molecules, which promote metastasis. Platelet receptors on tumors have not been well-characterized, resulting in a critical gap in knowledge concerning platelet-promoted metastasis. We identify a direct interaction between platelets and tumor CD97 that stimulates rapid bidirectional signaling. CD97, an adhesion G protein-coupled receptor (GPCR), is an overexpressed tumor antigen in several cancer types. Purified CD97 extracellular domain or tumor cell-associated CD97 stimulated platelet activation. CD97-initiated platelet activation led to granule secretion, including the release of ATP, a mediator of endothelial junction disruption. Lysophosphatidic acid (LPA) derived from platelets induced tumor invasiveness via proximal CD97-LPAR heterodimer signaling, coupling coincident tumor cell migration and vascular permeability to promote transendothelial migration. Consistent with this, CD97 was necessary for tumor cell-induced vascular permeability in vivo and metastasis formation in preclinical models. These findings support targeted blockade of tumor CD97 as an approach to ameliorate metastatic spread.