Author
Nazzy Pakpour
Other affiliations: University of Pennsylvania, University of California, Davis
Bio: Nazzy Pakpour is an academic researcher from California State University, East Bay. The author has contributed to research in topics: Plasmodium falciparum & Anopheles stephensi. The author has an hindex of 21, co-authored 33 publications receiving 6261 citations. Previous affiliations of Nazzy Pakpour include University of Pennsylvania & University of California, Davis.
Papers
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TL;DR: In this paper, the authors present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macro-autophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes.
Abstract: 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.
For example, 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 versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase 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. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.
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. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. 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 assays, we hope to encourage technical innovation in the field.
5,187 citations
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Broad Institute1, Tehran University of Medical Sciences2, George Washington University3, European Bioinformatics Institute4, Sapienza University of Rome5, Temple University6, Tomsk State University7, University of Notre Dame8, Centre national de la recherche scientifique9, French Institute of Health and Medical Research10, Imperial College London11, James Cook University12, Massachusetts Institute of Technology13, Simon Fraser University14, University of California, Davis15, Institut de recherche pour le développement16, Kansas State University17, Foundation for Research & Technology – Hellas18, University of Perugia19, Virginia Tech20, University of Nevada, Las Vegas21, Baylor College of Medicine22, Boston College23, Harvard University24, University of Manchester25, University of California, San Francisco26, University of Cyprus27, National Health Laboratory Service28, University of Crete29, Kenya Medical Research Institute30, University of Arizona31, University of Pennsylvania32, Indian Council of Medical Research33, New Mexico State University34, Liverpool School of Tropical Medicine35, Vanderbilt University Medical Center36, Vanderbilt University37, University of Geneva38, Swiss Institute of Bioinformatics39, Texas A&M University40, Chiang Mai University41, Oswaldo Cruz Foundation42, Rio de Janeiro State University43, Indiana University44, University of Santiago de Compostela45, Wellcome Trust Sanger Institute46, Liverpool John Moores University47, University of Georgia48, Harvey Mudd College49, University of California, Irvine50, University of Groningen51, Centers for Disease Control and Prevention52, Biogen Idec53
TL;DR: The authors investigated the genomic basis of vectorial capacity and explore new avenues for vector control, sequenced the genomes of 16 anopheline mosquito species from diverse locations spanning ~100 million years of evolution Comparative analyses show faster rates of gene gain and loss, elevated gene shuffling on the X chromosome, and more intron losses, relative to Drosophila.
Abstract: Variation in vectorial capacity for human malaria among Anopheles mosquito species is determined by many factors, including behavior, immunity, and life history To investigate the genomic basis of vectorial capacity and explore new avenues for vector control, we sequenced the genomes of 16 anopheline mosquito species from diverse locations spanning ~100 million years of evolution Comparative analyses show faster rates of gene gain and loss, elevated gene shuffling on the X chromosome, and more intron losses, relative to Drosophila Some determinants of vectorial capacity, such as chemosensory genes, do not show elevated turnover but instead diversify through protein-sequence changes This dynamism of anopheline genes and genomes may contribute to their flexible capacity to take advantage of new ecological niches, including adapting to humans as primary hosts
476 citations
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TL;DR: It is demonstrated that increased Akt signaling in the mosquito midgut disrupts parasite development and concurrently reduces the duration that mosquitoes are infective to humans.
Abstract: Malaria (Plasmodium spp.) kills nearly one million people annually and this number will likely increase as drug and insecticide resistance reduces the effectiveness of current control strategies. The most important human malaria parasite, Plasmodium falciparum, undergoes a complex developmental cycle in the mosquito that takes approximately two weeks and begins with the invasion of the mosquito midgut. Here, we demonstrate that increased Akt signaling in the mosquito midgut disrupts parasite development and concurrently reduces the duration that mosquitoes are infective to humans. Specifically, we found that increased Akt signaling in the midgut of heterozygous Anopheles stephensi reduced the number of infected mosquitoes by 60–99%. Of those mosquitoes that were infected, we observed a 75–99% reduction in parasite load. In homozygous mosquitoes with increased Akt signaling parasite infection was completely blocked. The increase in midgut-specific Akt signaling also led to an 18–20% reduction in the average mosquito lifespan. Thus, activation of Akt signaling reduced the number of infected mosquitoes, the number of malaria parasites per infected mosquito, and the duration of mosquito infectivity.
185 citations
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Virginia Tech1, Southern Medical University2, Russian Academy of Sciences3, Saint Petersburg State University4, University of Minnesota5, Savitribai Phule Pune University6, European Bioinformatics Institute7, University of California, Davis8, California State Polytechnic University, Pomona9, Kansas State University10, Centre national de la recherche scientifique11, Pasteur Institute12, University of Notre Dame13, University of California, San Francisco14, Virginia Bioinformatics Institute15, University of Arizona16, University of Santiago de Compostela17, Wellcome Trust Sanger Institute18, University of California, Irvine19
TL;DR: The genome sequence and annotation of the Indian strain of the type form of An.
Abstract: Anopheles stephensi is the key vector of malaria throughout the Indian subcontinent and Middle East and an emerging model for molecular and genetic studies of mosquito-parasite interactions The type form of the species is responsible for the majority of urban malaria transmission across its range Here, we report the genome sequence and annotation of the Indian strain of the type form of An stephensi The 221 Mb genome assembly represents more than 92% of the entire genome and was produced using a combination of 454, Illumina, and PacBio sequencing Physical mapping assigned 62% of the genome onto chromosomes, enabling chromosome-based analysis Comparisons between An stephensi and An gambiae reveal that the rate of gene order reshuffling on the X chromosome was three times higher than that on the autosomes An stephensi has more heterochromatin in pericentric regions but less repetitive DNA in chromosome arms than An gambiae We also identify a number of Y-chromosome contigs and BACs Interspersed repeats constitute 71% of the assembled genome while LTR retrotransposons alone comprise more than 49% of the Y contigs RNA-seq analyses provide new insights into mosquito innate immunity, development, and sexual dimorphism The genome analysis described in this manuscript provides a resource and platform for fundamental and translational research into a major urban malaria vector Chromosome-based investigations provide unique perspectives on Anopheles chromosome evolution RNA-seq analysis and studies of immunity genes offer new insights into mosquito biology and mosquito-parasite interactions
115 citations
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TL;DR: It is concluded that spontaneous reactivation of infectious HSV-1 occurs in the mouse TG and is likely the principle cause of viral protein expression in these tissues, and a better model of human disease than existing models.
Abstract: Careful studies of mouse trigeminal ganglia (TG) latently infected with herpes simplex virus type 1 (HSV-1) indicate the presence of productive cycle viral gene products and persistent immune response, suggesting ongoing spontaneous viral reactivation in these tissues. In the present study we set out to determine whether infectious virus is present in murine TG latently infected with HSV-1 (KOS). At 37 days after ocular inoculation we found a small amount of infectious virus in ca. 6% of latently infected murine TG. Furthermore, the amount of infectious virus that we detected (PFU per viral antigen-positive neuron) was similar to that detected in acutely infected ganglia. We conclude that spontaneous reactivation of infectious HSV-1 occurs in the mouse TG and is likely the principle cause of viral protein expression in these tissues. We next examined the role of latency-associated transcript (LAT) in spontaneous ganglionic reactivation by examining ganglia latently infected with KOS dlLAT1.8, a LAT deletion virus. Through the use of immunocytochemistry we found that KOS dlLAT1.8 had a rate of spontaneous ganglionic reactivation very similar to that of HSV-1 (KOS). Studying spontaneous ganglionic reactivation of HSV in the mouse TG allows a direct study of viral reactivation from latently infected neurons without the potential confounders and complicating downstream events that accompany the study of viral reactivation by explantation or peripheral viral shedding. Since most cases of human viral shedding and reactivation are not associated with a known trigger, spontaneous ganglionic reactivation of HSV-1 may be a better model of human disease than existing models.
77 citations
Cited by
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Lorenzo Galluzzi1, Lorenzo Galluzzi2, Ilio Vitale3, Stuart A. Aaronson4 +183 more•Institutions (111)
TL;DR: The Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives.
Abstract: Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.
3,301 citations
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French Institute of Health and Medical Research1, University of Paris-Sud2, Institut Gustave Roussy3, University of Texas Southwestern Medical Center4, Thomas Jefferson University5, University of Massachusetts Medical School6, Roswell Park Cancer Institute7, Johns Hopkins University School of Medicine8, Penn State Milton S. Hershey Medical Center9, Goethe University Frankfurt10, St. Jude Children's Research Hospital11, University of Zurich12, University College London13, South Australia Pathology14, University of Adelaide15, Ludwig Institute for Cancer Research16, University of Graz17, Istituto Superiore di Sanità18, University of Michigan19, Northwestern University20, University of Rome Tor Vergata21, University of Cambridge22, University of Bern23, Ghent University24, Harvard University25, Karolinska Institutet26, University of Leicester27
TL;DR: A functional classification of cell death subroutines is proposed that applies to both in vitro and in vivo settings and includes extrinsic apoptosis, caspase-dependent or -independent intrinsic programmed cell death, regulated necrosis, autophagic cell death and mitotic catastrophe.
Abstract: In 2009, the Nomenclature Committee on Cell Death (NCCD) proposed a set of recommendations for the definition of distinct cell death morphologies and for the appropriate use of cell death-related terminology, including 'apoptosis', 'necrosis' and 'mitotic catastrophe'. In view of the substantial progress in the biochemical and genetic exploration of cell death, time has come to switch from morphological to molecular definitions of cell death modalities. Here we propose a functional classification of cell death subroutines that applies to both in vitro and in vivo settings and includes extrinsic apoptosis, caspase-dependent or -independent intrinsic apoptosis, regulated necrosis, autophagic cell death and mitotic catastrophe. Moreover, we discuss the utility of expressions indicating additional cell death modalities. On the basis of the new, revised NCCD classification, cell death subroutines are defined by a series of precise, measurable biochemical features.
2,238 citations
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TL;DR: A way forward is suggested for the effective targeting of autophagy by understanding the context-dependent roles of autophile and by capitalizing on modern approaches to clinical trial design.
Abstract: Autophagy is a mechanism by which cellular material is delivered to lysosomes for degradation, leading to the basal turnover of cell components and providing energy and macromolecular precursors. Autophagy has opposing, context-dependent roles in cancer, and interventions to both stimulate and inhibit autophagy have been proposed as cancer therapies. This has led to the therapeutic targeting of autophagy in cancer to be sometimes viewed as controversial. In this Review, we suggest a way forwards for the effective targeting of autophagy by understanding the context-dependent roles of autophagy and by capitalizing on modern approaches to clinical trial design.
1,606 citations
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TL;DR: It is shown that CQ mainly inhibits autophagy by impairing autophagosome fusion with lysosomes rather than by affecting the acidity and/or degradative activity of this organelle.
Abstract: Macroautophagy/autophagy is a conserved transport pathway where targeted structures are sequestered by phagophores, which mature into autophagosomes, and then delivered into lysosomes for degradati...
1,178 citations
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Daniel J. Klionsky1, Amal Kamal Abdel-Aziz2, Sara Abdelfatah3, Mahmoud Abdellatif4 +2980 more•Institutions (777)
TL;DR: In this article, the authors 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.
Abstract: 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.
1,129 citations