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.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-ijf2t30pbq.pdf

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

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.

https://biblio.ugent.be/publication/8555786/file/8555788.pdf

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

We describe the landscape of somatic genomic alterations based on multidimensional and comprehensive characterization of more than 500 glioblastoma tumors (GBMs). We identify several novel mutated genes as well as complex rearrangements of signature receptors, including EGFR and PDGFRA. TERT promoter mutations are shown to correlate with elevated mRNA expression, supporting a role in telomerase reactivation. Correlative analyses confirm that the survival advantage of the proneural subtype is conferred by the G-CIMP phenotype, and MGMT DNA methylation may be a predictive biomarker for treatment response only in classical subtype GBM. Integrative analysis of genomic and proteomic profiles challenges the notion of therapeutic inhibition of a pathway as an alternative to inhibition of the target itself. These data will facilitate the discovery of therapeutic and diagnostic target candidates, the validation of research and clinical observations and the generation of unanticipated hypotheses that can advance our molecular understanding of this lethal cancer.

The somatic genomic landscape of glioblastoma

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.

/pdf/molecular-definitions-of-cell-death-subroutines-q5g428rlyi.pdf

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

EVOLUTION: Of Mice . . .

The invention relates to the use of one or more cannabinoids, particularly THC and/ or CBD in combination with a non-cannabinoid chemotherapeutic agent in the manufacture of a medicament for use in the treatment of cancer. In particular the cancer to be treated is a brain tumour, more particularly a glioma, more particularly still a glioblastoma multiforme (GBM). The non-cannabinoid chemotherapeutic agent may be a selective estrogen receptor modulator or an alkylating agent.

Cannabinoids (thc:cbd ratio) in combination with an alkylating agent for use in treating brain tumour

PROBLEM TO BE SOLVED: To provide a new cancer therapeutic agent useful for cerebral tumors such as neuroglioma and glioblastoma multiforme (GBM).SOLUTION: In a method for cancer therapy, a non-cannabinoid chemotherapeutic agent and one or more cannabinoids are used in combination. For a new cancer therapeutic agent, the cannabinoid is preferably tetrahydrocannabinol (THC) and cannabidiol (CBD), the non-cannabinoid chemotherapeutic agent is preferably a selective estrogen receptor modulator such as raloxifene, tamoxifen and toremifene; and an alkylating agent such as cisplatin, temozolomide and carmustine, and as the cannabinoid, a pure compound may be used and a plant extract may be used.

Cannabinoid combined with non-cannabinoid chemotherapeutic agent (e.g., serm or alkylating agent)

. We recall that w ∈ C + p if there exist ε > 0 and C > 0 such that for any a < b < c with c − b < b − a and any measurable set E ⊂ ( a, b ) , the following holds This condition was introduced by Riveros and de la Torre [33] as a one-sided counterpart of the C p condition studied ﬁrst by Muckenhoupt and Sawyer [30, 34]. In this paper we show that given 1 < p < q < ∞ if w ∈ C + q then and conversely if such an inequality holds, then w ∈ C + p . This result is the one-sided counterpart of Yabuta’s main result in [37]. Combining this estimate with known pointwise estimates for M ♯, + in the literature we recover and extend the result for maximal one-sided singular integrals due to Riveros and de la Torre [33] obtaining counterparts a number of operators.

One-sided $C_{p}$ estimates via $M^{\sharp}$ function

Some quantitative one-sided weighted estimates

<jats:p>We recall that <jats:inline-formula>
	      <jats:alternatives>
		<jats:tex-math>$w\in C_{p}^+$</jats:tex-math>
		<jats:inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" mime-subtype="png" xlink:href="S0308210522000841_inline3.png" />
	      </jats:alternatives>
	    </jats:inline-formula> if there exist <jats:inline-formula>
	      <jats:alternatives>
		<jats:tex-math>$\varepsilon >0$</jats:tex-math>
		<jats:inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" mime-subtype="png" xlink:href="S0308210522000841_inline4.png" />
	      </jats:alternatives>
	    </jats:inline-formula> and <jats:inline-formula>
	      <jats:alternatives>
		<jats:tex-math>$C>0$</jats:tex-math>
		<jats:inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" mime-subtype="png" xlink:href="S0308210522000841_inline5.png" />
	      </jats:alternatives>
	    </jats:inline-formula> such that for any <jats:inline-formula>
	      <jats:alternatives>
		<jats:tex-math>$a< b< c$</jats:tex-math>
		<jats:inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" mime-subtype="png" xlink:href="S0308210522000841_inline6.png" />
	      </jats:alternatives>
	    </jats:inline-formula> with <jats:inline-formula>
	      <jats:alternatives>
		<jats:tex-math>$c-b< b-a$</jats:tex-math>
		<jats:inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" mime-subtype="png" xlink:href="S0308210522000841_inline7.png" />
	      </jats:alternatives>
	    </jats:inline-formula> and any measurable set <jats:inline-formula>
	      <jats:alternatives>
		<jats:tex-math>$E\subset (a,b)$</jats:tex-math>
		<jats:inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" mime-subtype="png" xlink:href="S0308210522000841_inline8.png" />
	      </jats:alternatives>
	    </jats:inline-formula>, the following holds
<jats:disp-formula>
	      <jats:alternatives>
		<jats:tex-math>\[ \int_{E}w\leq C\left(\frac{|E|}{(c-b)}\right)^{\varepsilon}\int_{\mathbb{R}}\left(M^+\chi_{(a,c)}\right)^{p}w<\infty. \]</jats:tex-math>
		<jats:graphic xmlns:xlink="http://www.w3.org/1999/xlink" mime-subtype="png" mimetype="image" position="float" xlink:href="S0308210522000841_eqnU1.png" />
	      </jats:alternatives>
	    </jats:disp-formula>This condition was introduced by Riveros and de la Torre [33] as a one-sided counterpart of the <jats:inline-formula>
	      <jats:alternatives>
		<jats:tex-math>$C_{p}$</jats:tex-math>
		<jats:inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" mime-subtype="png" xlink:href="S0308210522000841_inline9.png" />
	      </jats:alternatives>
	    </jats:inline-formula> condition studied first by Muckenhoupt and Sawyer [30, 34]. In this paper we show that given <jats:inline-formula>
	      <jats:alternatives>
		<jats:tex-math>$1< p< q<\infty$</jats:tex-math>
		<jats:inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" mime-subtype="png" xlink:href="S0308210522000841_inline10.png" />
	      </jats:alternatives>
	    </jats:inline-formula> if <jats:inline-formula>
	      <jats:alternatives>
		<jats:tex-math>$w\in C_{q}^+$</jats:tex-math>
		<jats:inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" mime-subtype="png" xlink:href="S0308210522000841_inline11.png" />
	      </jats:alternatives>
	    </jats:inline-formula> then
<jats:disp-formula>
	      <jats:alternatives>
		<jats:tex-math>\[ \|M^+f\|_{L^{p}(w)}\lesssim\|M^{\sharp,+}f\|_{L^{p}(w)} \]</jats:tex-math>
		<jats:graphic xmlns:xlink="http://www.w3.org/1999/xlink" mime-subtype="png" mimetype="image" position="float" xlink:href="S0308210522000841_eqnU2.png" />
	      </jats:alternatives>
	    </jats:disp-formula>and conversely if such an inequality holds, then <jats:inline-formula>
	      <jats:alternatives>
		<jats:tex-math>$w\in C_{p}^+$</jats:tex-math>
		<jats:inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" mime-subtype="png" xlink:href="S0308210522000841_inline12.png" />
	      </jats:alternatives>
	    </jats:inline-formula>. This result is the one-sided counterpart of Yabuta's main result in [37]. Combining this estimate with known pointwise estimates for <jats:inline-formula>
	      <jats:alternatives>
		<jats:tex-math>$M^{\sharp,+}$</jats:tex-math>
		<jats:inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" mime-subtype="png" xlink:href="S0308210522000841_inline13.png" />
	      </jats:alternatives>
	    </jats:inline-formula> in the literature we recover and extend the result for maximal one-sided singular integrals due to Riveros and de la Torre [33] obtaining counterparts a number of operators.</jats:p>

Mar Lorente

Papers

Cannabinoids (thc:cbd ratio) in combination with an alkylating agent for use in treating brain tumour

Cannabinoid combined with non-cannabinoid chemotherapeutic agent (e.g., serm or alkylating agent)

One-sided $C_{p}$ estimates via $M^{\sharp}$ function

Some quantitative one-sided weighted estimates

One-sided estimates via function