In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

Lactoferrin is a nutrient classically found in mammalian milk. It binds iron and is transferred via a variety of receptors into and between cells, serum, bile, and cerebrospinal fluid. It has important immunological properties, and is both antibacterial and antiviral. In particular, there is evidence that it can bind to at least some of the receptors used by coronaviruses and thereby block their entry. Of importance are Heparan Sulfate Proteoglycans (HSPGs) and the host receptor angiotensin-converting enzyme 2 (ACE2), as based on other activities lactoferrin might prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from attaching to the host cells. Lactoferrin (and more specifically enteric-coated LF because of increased bioavailability) may consequently be of preventive and therapeutic value during the present COVID-19 pandemic.

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The Biology of Lactoferrin, an Iron-Binding Protein That Can Help Defend Against Viruses and Bacteria.

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.

Guidelines for the use and interpretatoin of assays for monitoring autophagy

Zika virus (ZIKV) has been extensively studied since it was linked to congenital malformations, and recent research has revealed that astrocytes are targets of ZIKV. However, the consequences of ZIKV infection, especially to this cell type, remain largely unknown, particularly considering integrative studies aiming to understand the crosstalk among key cellular mechanisms and fates involved in the neurotoxicity of the virus. Here, the consequences of ZIKV infection in iPSC-derived astrocytes are presented. Our results show ROS imbalance, mitochondrial defects and DNA breakage, which have been previously linked to neurological disorders. We have also detected glial reactivity, also present in mice and in post-mortem brains from infected neonates from the Northeast of Brazil. Given the role of glia in the developing brain, these findings may help to explain the observed effects in congenital Zika syndrome related to neuronal loss and motor deficit.

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Zika virus infection leads to mitochondrial failure, oxidative stress and DNA damage in human iPSC-derived astrocytes

Inhibiting cell survival and inducing cell death are the main approaches of tumor therapy. Autophagy plays an important role on intracellular metabolic homeostasis by eliminating dysfunctional or unnecessary proteins and damaged or aged cellular organelles to recycle their constituent metabolites that enable the maintenance of cell survival and genetic stability and even promotes the drug resistance, which severely limits the efficacy of chemotherapeutic drugs. Currently, targeting autophagy has a seemingly contradictory effect to suppress and promote tumor survival, which makes the effect of targeting autophagy on drug resistance more confusing and fuzzier. In the review, we summarize the regulation of autophagy by emerging ways, the action of targeting autophagy on drug resistance and some of the new therapeutic approaches to treat tumor drug resistance by interfering with autophagy-related pathways. The full-scale understanding of the tumor-associated signaling pathways and physiological functions of autophagy will hopefully open new possibilities for the treatment of tumor drug resistance and the improvement in clinical outcomes.

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Targeting autophagy to overcome drug resistance: further developments.

Virus infection induces different cellular responses in infected cells. These include cellular stress responses like autophagy and unfolded protein response (UPR). Both autophagy and UPR are connec...

https://tandfonline.com/doi/pdf/10.1080/21505594.2019.1605803

The roles of apoptosis, autophagy and unfolded protein response in arbovirus, influenza virus, and HIV infections

The first human Zika virus (ZIKV) outbreak was reported in Micronesia in 2007, followed by one in Brazil in 2015. Recent studies have reported cases in Europe, Oceania and Latin America. In 2016, ZIKV transmission was also reported in the US and the World Health Organization declared it a Public Health Emergency of International Concern. Because various neurological conditions are associated with ZIKV, such as microcephaly, Guillain-Barre syndrome, and other disorders of both the central and peripheral nervous systems, including encephalopathy, (meningo)encephalitis and myelitis, and because of the lack of reliable patient diagnosis, numerous ongoing studies seek to understand molecular mechanisms underlying ZIKV pathogenesis. Astrocytes are one of the most abundant cells in the CNS. They control axonal guidance, synaptic signaling, neurotransmitter trafficking and maintenance of neurons, and are targeted by ZIKV. In this study, we used a newly developed multiplexed aptamer-based technique (SOMAScan) to examine > 1300 human astrocyte cell proteins. We identified almost 300 astrocyte proteins significantly dysregulated by ZIKV infection that span diverse functions and signaling pathways, including protein translation, synaptic control, cell migration and differentiation.

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Zika Virus Infection Disrupts Astrocytic Proteins Involved in Synapse Control and Axon Guidance.

Zika virus (ZIKV), a neglected tropical disease until its re-emergence in 2007, causes microcephaly in infants and Guillain-Barre syndrome in adults. Its re-emergence and spread to more than 80 countries led the World Health Organization in 2016 to declare a Public Health Emergency. ZIKV is mainly transmitted by mosquitos, but can persist in infected human male semen for prolonged periods and may be sexually transmitted. Testicular Sertoli cells support ZIKV replication and may be a reservoir for persistent ZIKV infection. Electrical impedance analyses indicated ZIKV infection rapidly disrupted Vero cell monolayers but had little effect upon human Sertoli cells (HSerC). We determined ZIKV-induced proteomic changes in HSerC using an aptamer-based multiplexed technique (SOMAscan) targeting >1300 human proteins. ZIKV infection caused differential expression of 299 proteins during three different time points, including 5 days after infection. Dysregulated proteins are involved in different bio-functions, including cell death and survival, cell cycle, maintenance of cellular function, cell signaling, cellular assembly, morphology, movement, molecular transport, and immune response. Many signaling pathways important for maintenance of HSerC function and spermatogenesis were highly dysregulated. These included IL-6, IGF1, EGF, NF-κB, PPAR, ERK/MAPK, and growth hormone signaling. Down-regulation of the PPAR signaling pathway might impact cellular energy supplies. Upstream molecule analysis also indicated microRNAs involved in germ cell development were downregulated by infection. Overall, this study leads to a better understanding of Sertoli cellular mechanisms used by ZIKV during persistent infection and possible ZIKV impacts on spermatogenesis.

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Zika virus dysregulates human Sertoli cell proteins involved in spermatogenesis with little effect on tight junctions.

The re-emergence and the recent spread of the Zika virus (ZIKV) has raised significant global concerns due to lack of information in patient diagnosis and management. Thus, in addition to gaining more basic information about ZIKV biology, appropriate interventions and management strategies are being sought to control ZIKV-associated diseases and its spread. This study's objective is to identify host cell proteins that are significantly dysregulated during ZIKV infection. SOMAScan, a novel aptamer-based assay, is used to simultaneously screen >1300 host proteins to detect ZIKV-induced host protein dysregulation at multiple time points during infection. A total of 125 Vero cell host proteins, including cytokines such as CXCL11 and CCL5, interferon stimulated gene 15, and translation initiation factors EIF5A and EIF4G2, are significantly dysregulated after ZIKV infection. Bioinformatic analyses of 77 host proteins, that are significantly dysregulated ≥1.25-fold, identify several activated biological processes, including the JAK/STAT, Tec kinase, and complement cascade pathways.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/pmic.201800309

Vero Cell Proteomic Changes Induced by Zika Virus Infection

The zika virus (ZIKV) is a neurotropic virus that causes congenital abnormalities in babies when they are infected in utero. Some studies have reported these congenital abnormalities result from ZIKV attacking neural progenitor cells within the brain which differentiate into neurons, oligodendrocytes, and astrocytes. Each of these glial cells play important roles during development of the fetal brain. In addition to ZIKV-induced congenital abnormalities, infected patients experience gastrointestinal complications. There are presently no reports investigating the role of this virus at the proteomic level in gastrointestinal associated cells, so we conducted an in vitro proteomic study of ZIKV-induced changes in Caco-2, a colon-derived human cell line which is known to be permissive to ZIKV infection. We used SomaScan, a new aptamer-based proteomic tool to identify host proteins that are dysregulated during ZIKV infection at 12, 24, and 48 h post-infection. Bioinformatic analyses predicted that dysregulation of differentially-regulated host proteins results in various gastrointestinal diseases. Validation of the clinical relevance of these promising protein targets will add to the existing knowledge of ZIKV biology. These potential proteins may be useful targets towards the development of therapeutic interventions.

Kathleen K. M. Glover

Papers

The roles of apoptosis, autophagy and unfolded protein response in arbovirus, influenza virus, and HIV infections

Zika Virus Infection Disrupts Astrocytic Proteins Involved in Synapse Control and Axon Guidance.

Zika virus dysregulates human Sertoli cell proteins involved in spermatogenesis with little effect on tight junctions.

Vero Cell Proteomic Changes Induced by Zika Virus Infection

ZIKV Infection Induces DNA Damage Response and Alters the Proteome of Gastrointestinal Cells