In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

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We describe a conditional in vivo protein trap mutagenesis system that reveals spatio-temporal protein expression dynamics and assesses gene function in the vertebrate Danio rerio. Integration of pGBT-RP2 (RP2), a gene-breaking transposon containing a protein trap, efficiently disrupts gene expression with >97% knockdown of normal transcript levels while simultaneously reporting protein expression of each locus. The mutant alleles are revertible in somatic tissues via Cre recombinase or splice-site blocking morpholinos, thus representing the first systematic conditional mutant alleles outside the mouse model. We report a collection of 350 zebrafish lines including a diverse array of molecular loci. RP2 integrations reveal the complexity of genomic architecture and gene function in a living organism and can provide information on protein subcellular localization. The RP2 mutagenesis system is a step towards a unified codex of protein expression and direct functional annotation of the vertebrate genome.

To the editor Coronavirus disease 2019 (COVID-19), which emerged in Wuhan, China in December 2019, is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has become a major global public health concern [1]. Positive detection of SARS-CoV-2 RNA in nasopharyngeal swab samples, sputum samples or bronchoalveolar lavage samples by reverse transcriptase polymerase chain reaction (RT-PCR) has been used to confirm SARS-CoV-2 infection [2]. Recently, positive detection of IgM and IgG antibodies specific to SARS-CoV-2 has also been recognized as deterministic evidence for confirmed SARS-CoV-2 infection [3,4]. However, the antibody response to SARS-CoV-2 currently remains inadequately understood in COVID-19 patients. In the present study, we investigated the patterns of antibody response to SARS-CoV-2 in patients with COVID-19, aiming to better clarify the humoral immunological response during SARS-CoV-2 infection.

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The zebrafish embryo is transparent and can tolerate absence of blood flow because its oxygen is delivered by diffusion rather than by the cardiovascular system. It is therefore possible to attribute cardiac failure directly to particular genes by ruling out the possibility that it is due to a secondary effect of hypoxia. We focus here on pickwickm171 (pikm171), a recessive lethal mutation discovered in a large-scale genetic screen. There are three other alleles in the pik complementation group with this phenotype (pikm242, pikm740, pikm186; ref. 3) and one allele (pikmVO62H) with additional skeletal paralysis. The pik heart develops normally but is poorly contractile from the first beat. Aside from the edema that inevitably accompanies cardiac dysfunction, development is normal during the first three days. We show by positional cloning that the 'causative' mutation is in an alternatively-spliced exon of the gene (ttn) encoding Titin. Titin is the biggest known protein and spans the half-sarcomere from Z-disc to M-line in heart and skeletal muscle. It has been proposed to provide a scaffold for the assembly of thick and thin filaments and to provide elastic recoil engendered by stretch during diastole. We found that nascent myofibrils form in pik mutants, but normal sarcomeres are absent. Mutant cells transplanted to wildtype hearts remain thin and bulge outwards as individual cell aneurysms without affecting nearby wildtype cardiomyocytes, indicating that the contractile deficiency is cell-autonomous. Absence of Titin function thus results in blockage of sarcomere assembly and causes a functional disorder resembling human dilated cardiomyopathies, one form of which is described in another paper in this issue.

SUMMARYCilia are essential for normal development. The composition and assembly of cilia has been well characterized, but the signaling and transcriptional pathways that govern ciliogenesis remain poorly studied. Here, we report that Wnt/-catenin signaling directly regulates ciliogenic transcription factor foxj1a expression and ciliogenesis in zebrafish Kupffer's vesicle (KV). We show that Wnt signaling acts temporally and KV cell-autonomously to control left-right (LR) axis determination and ciliogenesis. Specifically, reduction of Wnt signaling leads to a disruption of LR patterning, shorter and fewer cilia, a loss of cilia motility and a downregulation of foxj1a expression. However, these phenotypes can be rescued by KV-targeted overexpression of foxj1a. In comparison to the FGF pathway that has been previously implicated in the control of ciliogenesis, our epistatic studies suggest a more downstream function of Wnt signaling in the regulation of foxj1a expression and ciliogenesis in KV. Importantly, enhancer analysis reveals that KV-specific expression of foxj1a requires the presence of putative Lef1/Tcf binding sites, indicating that Wnt signaling activates foxj1a transcription directly. We also find that impaired Wnt signaling leads to kidney cysts and otolith disorganization, which can be attributed to a loss of foxj1 expression and disrupted ciliogenesis in the developing pronephric ducts and otic vesicles. Together, our data reveal a novel role of Wnt/-catenin signaling upstream of ciliogenesis, which might be a general developmental mechanism beyond KV. Moreover, our results also prompt a hypothesis that certain developmental effects of the Wnt/-catenin pathway are due to the activation of Foxj1 and cilia formation. KEY WORDS: Wnt/-catenin signaling, Ciliogenesis, Foxj1, Kupffer's vesicle ZebrafishWnt/-catenin signaling directly regulates Foxj1 expression and ciliogenesis in zebrafish Kupffer's vesicle

Rationale Although a cardioprotective function of target of rapamycin (TOR) signaling inhibition has been suggested by pharmacological studies using rapamycin, genetic evidences are still lacking. Here, we explored adult zebrafish as a novel vertebrate model for dissecting signaling pathways in cardiomyopathy. Objective We generate the second adult zebrafish cardiomyopathy model induced by doxorubicin (DOX). By genetically analyzing both the DOX and our previous established anemia-induced cardiomyopathy models, we aim to decipher the functions of TOR signaling in cardiomyopathies of different etiology. Methods and Results Along the progression of both cardiomyopathy models, we detected dynamic TOR activity at different stages of pathogenesis as well as distinct effects of TOR signaling inhibition. Nevertheless, cardiac enlargement in both models can be effectively attenuated by inhibition of TOR signaling via short-term rapamycin treatment. To assess the long term effects of TOR reduction, we utilized a zebrafish target of rapamycin (ztor) mutant identified from an insertional mutagenesis screen. We show that TOR haploinsufficiency in the ztor heterozygous fish improved cardiac function, prevented pathological remodeling events, and ultimately reduced mortality in both adult fish models of cardiomyopathy. Mechanistically, these cardioprotective effects are conveyed by the anti-hypertrophy, anti-apoptosis, and proautophagy function of TOR signaling inhibition. Conclusions Our results prove adult zebrafish as a conserved novel vertebrate model for human cardiomyopathies. Moreover, we provide the first genetic evidence to demonstrate a long-term cardioprotective effect of TOR signaling inhibition on at least two cardiomyopathies of distinct etiology, despite dynamic TOR activities during their pathogenesis.