David J. Reiner

Bio: David J. Reiner is an academic researcher from Texas A&M Health Science Center. The author has contributed to research in topics: Caenorhabditis elegans & Cell fate determination. The author has an hindex of 17, co-authored 42 publications receiving 6538 citations. Previous affiliations of David J. Reiner include Lanzhou University & Texas A&M University.

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

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.

Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination

Abstract: Study of the nematode Caenorhabditis elegans has provided important insights in a wide range of fields in biology. The ability to precisely modify genomes is critical to fully realize the utility of model organisms. Here we report a method to edit the C. elegans genome using the clustered, regularly interspersed, short palindromic repeats (CRISPR) RNA-guided Cas9 nuclease and homologous recombination. We demonstrate that Cas9 is able to induce DNA double-strand breaks with specificity for targeted sites and that these breaks can be repaired efficiently by homologous recombination. By supplying engineered homologous repair templates, we generated gfp knock-ins and targeted mutations. Together our results outline a flexible methodology to produce essentially any desired modification in the C. elegans genome quickly and at low cost. This technology is an important addition to the array of genetic techniques already available in this experimentally tractable model organism.

Diverse behavioural defects caused by mutations in Caenorhabditis elegans unc-43 CaM Kinase II

Abstract: Calcium/calmodulin-dependent serine/threonine kinase type II (CaMKII) is one of the most abundant proteins in the mammalian brain, where it is thought to regulate synaptic plasticity and other processes. Activation of the multisubunit kinase by calcium is effectively cooperative and can persist long after transient calcium rises. Despite extensive biochemical characterization of CaMKII and identification of numerous in vitro kinase targets, little is known about its function in vivo. Here we report that unc-43 encodes the only Caenorhabditis elegans CaMKII. A gain-of-function unc-43 mutation reduces locomotory activity, alters excitation of three muscle types and lengthens the period of the motor output of a behavioural clock. Null unc-43 mutations cause phenotypes generally opposite to those of the gain-of-function mutation. Mutations in the unc-103 potassium channel gene suppress a gain-of-function phenotype of unc-43 in one tissue without affecting other tissues; thus, UNC-103 may be a tissue-specific target of CaMKII in vivo.

The RalGEF-Ral Effector Signaling Network: The Road Less Traveled for Anti-Ras Drug Discovery:

Abstract: The high frequency of RAS mutations in human cancers (33%) has stimulated intense interest in the development of anti-Ras inhibitors for cancer therapy. Currently, the major focus of these efforts is centered on inhibitors of components involved in Ras downstream effector signaling. In particular, more than 40 inhibitors of the Raf-MEK-ERK mitogen-activated protein kinase cascade and phosphoinositide 3-kinase-AKT-mTOR effector signaling networks are currently under clinical evaluation. However, these efforts are complicated by the fact that Ras can utilize at least 9 additional functionally distinct effectors, with at least 3 additional effectors with validated roles in Ras-mediated oncogenesis. Of these, the guanine nucleotide exchange factors of the Ras-like (Ral) small GTPases (RalGEFs) have emerged as important effectors of mutant Ras in pancreatic, colon, and other cancers. In this review, we summarize the evidence for the importance of this effector pathway in cancer and discuss possible directions for therapeutic inhibition of aberrant Ral activation and signaling.

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

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.

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

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.

Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012

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.

Targeting autophagy in cancer

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.

Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins

Abstract: RNA-guided engineered nucleases (RGENs) derived from the prokaryotic adaptive immune system known as CRISPR (clustered, regularly interspaced, short palindromic repeat)/Cas (CRISPR-associated) enable genome editing in human cell lines, animals, and plants, but are limited by off-target effects and unwanted integration of DNA segments derived from plasmids encoding Cas9 and guide RNA at both on-target and off-target sites in the genome. Here, we deliver purified recombinant Cas9 protein and guide RNA into cultured human cells including hard-to-transfect fibroblasts and pluripotent stem cells. RGEN ribonucleoproteins (RNPs) induce site-specific mutations at frequencies of up to 79%, while reducing off-target mutations associated with plasmid transfection at off-target sites that differ by one or two nucleotides from on-target sites. RGEN RNPs cleave chromosomal DNA almost immediately after delivery and are degraded rapidly in cells, reducing off-target effects. Furthermore, RNP delivery is less stressful to human embryonic stem cells, producing at least twofold more colonies than does plasmid transfection.