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)

Interest in the topic of tumour metabolism has waxed and waned over the past century of cancer research. The early observations of Warburg and his contemporaries established that there are fundamental differences in the central metabolic pathways operating in malignant tissue. However, the initial hypotheses that were based on these observations proved inadequate to explain tumorigenesis, and the oncogene revolution pushed tumour metabolism to the margins of cancer research. In recent years, interest has been renewed as it has become clear that many of the signalling pathways that are affected by genetic mutations and the tumour microenvironment have a profound effect on core metabolism, making this topic once again one of the most intense areas of research in cancer biology.

/pdf/regulation-of-cancer-cell-metabolism-1dhy51t2fl.pdf

Regulation of cancer cell metabolism

Epigenetic mechanisms are essential for normal development and maintenance of tissue-specific gene expression patterns in mammals. Disruption of epigenetic processes can lead to altered gene function and malignant cellular transformation. Global changes in the epigenetic landscape are a hallmark of cancer. The initiation and progression of cancer, traditionally seen as a genetic disease, is now realized to involve epigenetic abnormalities along with genetic alterations. Recent advancements in the rapidly evolving field of cancer epigenetics have shown extensive reprogramming of every component of the epigenetic machinery in cancer including DNA methylation, histone modifications, nucleosome positioning and non-coding RNAs, specifically microRNA expression. The reversible nature of epigenetic aberrations has led to the emergence of the promising field of epigenetic therapy, which is already making progress with the recent FDA approval of three epigenetic drugs for cancer treatment. In this review, we discuss the current understanding of alterations in the epigenetic landscape that occur in cancer compared with normal cells, the roles of these changes in cancer initiation and progression, including the cancer stem cell model, and the potential use of this knowledge in designing more effective treatment strategies.

/pdf/epigenetics-in-cancer-53kplki7aw.pdf

Epigenetics in Cancer

Irrespective of the morphological features of end-stage cell death (that may be apoptotic, necrotic, autophagic, or mitotic), mitochondrial membrane permeabilization (MMP) is frequently the decisive event that delimits the frontier between survival and death. Thus mitochondrial membranes constitute the battleground on which opposing signals combat to seal the cell's fate. Local players that determine the propensity to MMP include the pro- and antiapoptotic members of the Bcl-2 family, proteins from the mitochondrialpermeability transition pore complex, as well as a plethora of interacting partners including mitochondrial lipids. Intermediate metabolites, redox processes, sphingolipids, ion gradients, transcription factors, as well as kinases and phosphatases link lethal and vital signals emanating from distinct subcellular compartments to mitochondria. Thus mitochondria integrate a variety of proapoptotic signals. Once MMP has been induced, it causes the release of catabolic hydrolases and activators of such enzymes (including those of caspases) from mitochondria. These catabolic enzymes as well as the cessation of the bioenergetic and redox functions of mitochondria finally lead to cell death, meaning that mitochondria coordinate the late stage of cellular demise. Pathological cell death induced by ischemia/reperfusion, intoxication with xenobiotics, neurodegenerative diseases, or viral infection also relies on MMP as a critical event. The inhibition of MMP constitutes an important strategy for the pharmaceutical prevention of unwarranted cell death. Conversely, induction of MMP in tumor cells constitutes the goal of anticancer chemotherapy.

Mitochondrial Membrane Permeabilization in Cell Death

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.

To the editor: PAX5 gene encodes a member of the paired box (PAX) family of transcription factors. This gene encodes the B-cell lineage specific activator protein [1]. PAX5 gene deletion in mice leads to dedifferentiation or transdifferentiation of B-cells into other hematopoietic cell lineages [1,2], indicating that PAX5 is involved in both maintenance of B-cell and control of terminal B-cell differentiation. Alterations of differentiation factors are frequently found in hematologic malignancies and contribute to their pathogenesis. PAX5 gene alterations by translocation are recurrent in small plasmacytoid B-cell lymphocytic lymphomas and diffuse large B-cell lymphomas (DLBCL) [3]. Also, PAX5 gene is somatically mutated in both childhood and adult precursor B-cell lymphoblastic leukaemia (B-cell ALL), being altered approximately in one third of the B-cell ALL [4,5]. Also, somatic mutation of PAX5 gene has been detected in diffuse large cell lymphomas as a consequence of aberrant hypermutation [6]. Functionally, the PAX5 mutants inhibited transcriptional activity of PAX5 [4]. Although PAX5 gene alterations have been identified in many B-cell lineage neoplasia, there has been no report on PAX5 mutation in multiple myelomas. To see whether PAX5 gene is somatically mutated in multiple myelomas and non-Hodgkin lymphomas (NHL) of Korean patients, we analysed the entire coding region (exon 1–10) and 50-untranslated region of PAX5 gene (GenBank accession number AF386790) in fresh bone marrow aspirates of 47 multiple myelomas by polymerase chain reaction (PCR)-based single-strand conformation polymorphism (SSCP) assay. Bone marrow aspirates from multiple myeloma patients were used in this study only when the myeloma cells were more than 70% of the cells in the bone marrow smear. Also, we analysed 146 NHL, including 100 DLBCL. Approval was obtained from the Catholic University of Korea, College of Medicine’s institutional review board for this study. Genomic DNA, each from tumour cells and normal cells of the same patients, was amplified by PCR with 13 primer pairs. Radioisotope ([P]dCTP) was incorporated into the PCR products for detection by autoradiogram. Other procedures of the PCR-SSCP and DNA sequencing were described in our previous studies [7]. In the PCR-SSCP, we were not able to find aberrantly migrating bands that may represent somatic mutation on the SSCP from the multiple myelomas. In the NHL, we

PAX5 somatic mutation is rare in multiple myelomas and non-Hodgkin lymphomas of Korean patients.

Targeted sequencing of burn scar-related squamous cell carcinomas identified PIK3CA amplification

To the Editor: Alteration in the cellular metabolism is one of the cancer hallmarks. Fructose-1,6-bisphosphate aldolase A (ALDOA) gene encodes an enzyme ALDOA that converts fructose 1,6-bisphosphate into G3P and DHAP, which in glycolysis have proven benefits for cancer cells [1]. High ALDOA expression has been demonstrated in many cancers and is associated with overall survival of the patients, suggesting its functions as a candidate oncogene [2]. However, genetic alterations of ALDOA in the coding sequences are reported to be rare. A recent genome-wide sequecning study for promoters in breast cancers discovered somatic promoter mutations in many genes, including ALDOA (3.3% of breast cancers) [3]. The ALDOA promoter mutation was found at a hotspot (chr16: 30077131). It is wellknown that alterations in promoter play a role in the cancer pathogenesis [4], suggesting that ALDOA promoter mutation might reside in other cancers as well as in breast cancer. In this study, human tumor tissues from 1834 patients from diverse organs were analyzed (Table 1). Approval for this study was obtained from the Catholic University of Korea, College of Medicine’s institutional review board. Since the ALDOA promoter mutation has been found in the hotspot [3], we amplified a region encompassing the hotspot with a primer pair by polymerase chain reaction (PCR) (forward: 5-TCGTAAAG GAAAAAGCTCGGC-3, reverse: 5-GATTCAAG GAGAGAACGCGG-3, size: 174 bps) and subsequently displayed in single-strand conformation polymorphism (SSCP) and DNA sequencing [5]. Overall, we detected ALDOA somatic promoter mutations in two cases: one (chr16: 30,077,035 G > A) in a childhood acute lymphoblastic leukemia (ALL) and the other (chr16: 30,077,051 C > T) in a colon carcinoma. Neither of them (Table 1) overlapped with the hotspot [3]. In this study, the prevalence of ALDOA somatic promoter mutations (ALL (0.3%) and colon carcinoma (0.3%)) is significantly lower (P < 0.001) than that in breast cancers (1.7%) [3]. Also, the mutation sites were different from that in breast cancers [3], indicating that the ALDOA mutations might be specific to breast cancer or might be very rare in other tumors, if any. Our results suggest that ALDOA promote mutation may not be clinically available for cancer patients due to the low incidence.

Promoter Mutation Analysis of ALDOA Gene in Solid Tumors and Acute Leukemias.

To the Editor: Janus kinases (JAK) play important roles in normal hematopoiesis. Also, mounting evidence indicates that deregulations of JAKs are important in hematopoietic tumor development (1). Fusion of JAK2 and TEL genes to generate TEL-JAK2 kinase has been detected in patients with acute lymphoblastic leukemias (ALL) (1). JAK2 p.V617F mutation is an important underlying mechanism of polycythemia vera development (1). Recently, two types of somatic mutations of JAK1 (p.T478S in exon 10 and p.V623A in exon 13) have been reported in bone marrows of acute myelogenous leukemia (AML) patients (2). These JAK1 mutations were associated with activation of STAT signaling in response to type I interferon (2). As JAK1 signaling is important in both myeloidand lymphoid-lineage development (1), it could be hypothesized that the JAK1 mutations in exon 10 and 13 might occur in other types of leukemias besides AML. To see whether exon 10 and 13 of JAK1 are somatically mutated in acute leukemias and multiple myelomas (MM), we analyzed the mutations in 106 AMLs, 74 ALLs (60 B-ALL and 14 T-ALL) and 31 MMs. The leukemia and MM DNAs were extracted from bone marrows of the patients (age range 20–80). All of the patients were Korean. Approval was obtained from the Catholic University of Korea, College of Medicine’s institutional review board for this study. JAK1 p.T478S and the JAK1 p.V623A mutations have been detected within exon 10 and exon 13, respectively (2). To detect somatic mutations in these exons, genomic DNAs each from tumor cells and normal cells were amplified with two primer pairs covering the exon 10 and the exon 13. Radioisotope ((P)dCTP) was incorporated into the polymerase chain reaction (PCR) products for the detection by single-strand conformation polymorphism (SSCP) autoradiogram.as described previously (3). On SSCP autoradiogram, all of the PCR products were clearly seen. However, the SSCP from the acute leukemias and MM did not reveal any aberrantly migrating band compared to the wild-type bands from normal tissues, indicating there was no evidence of JAK1 exon 10 nor exon 13 mutation in the cancer specimens. We repeated the experiments twice, including PCR and SSCP, and found that the data was consistent. In leukemia pathogenesis, activation of cytokine signaling is crucial (1). As JAK1 transmits and activates cytokine signaling pathways (1), activating mutations of JAK1 gene could be an important mechanism of leukemogenesis. Based on an earlier report on activating JAK1 mutations in AMLs (2), we expected to detect some somatic mutations of JAK1 exon 10 and 13 in our set of AML specimens. However, we detected no somatic mutation of JAK1 in the exon 10 and 13. Statistically, there is no significant difference in the JAK1 mutation frequencies between the previous study (2) and our study (Fisher’s exact test, P = 0.22), suggesting that a racial difference between the two studies does not exist. In addition, neither ALL nor MM harbored the JAK1 mutations. Recently, Tomasson et al. (4) analyzed JAK1, JAK2, JAK3 and TYK2 genes simultaneously in AMLs and found no additional somatic mutations of JAK genes, indicating somatic mutations of JAK genes are rare in AML. In a recent study, Flex et al. (5) found frequent somatic mutations of JAK1 in T-ALL and B-ALL, most of which were detected outside of exon 10 and 13. Also, our recent study showed that T-ALL harbored JAK1 mutation in exon 14 and 17 (6). The data presented here and these two documents suggest that JAK1 mutations may be common in ALL, but their incidence in exon 10 and 13 may be low in ALL. As for MM, mutational analysis of the entire coding sequences of JAK1 has not been performed yet, and the incidence of JAK1 mutation outside of exon 10 and 13 remains unknown.

Absence of JAK1 exon 10 and 13 mutations in acute leukemias and multiple myelomas.

This paper investigates the analytical results of the seismic response of multi-span prestressed concrete (PSC) I-girder bridges under seismic loads. To perform numerical analyses, a three-span PSC I-girder bridge with a width of 12 m, a total length of 100 m, and a maximum span length of 40 m was modeled, and a virtual location was selected to consider the soil properties of the area where the bridge was constructed. The seismic load acting on the PSC I-girder bridge was applied in consideration of the soil properties around the pier and the wave passage effect of the bedrock in the artificial seismic load generated, according to the U.S. Nuclear Regulatory Commission (NRC) standard. The analysis results confirmed that the seismic load, with consideration of the soil properties and wave passage effect, generated the maximum response acceleration and bending moment at the deck of the bridge—152% and 232% greater than without considering them, respectively. Therefore, in order to ensure the earthquake resistance of the bridge, the soil properties of the area where the bridge will be built and the wave passage effect of the bedrock must be considered.

https://www.mdpi.com/2076-3417/12/3/1415/pdf?version=1643373488

Sug Hyung Lee

Papers

PAX5 somatic mutation is rare in multiple myelomas and non-Hodgkin lymphomas of Korean patients.

Targeted sequencing of burn scar-related squamous cell carcinomas identified PIK3CA amplification

Promoter Mutation Analysis of ALDOA Gene in Solid Tumors and Acute Leukemias.

Absence of JAK1 exon 10 and 13 mutations in acute leukemias and multiple myelomas.

Influence of Seismic Loads Considering Soil Properties and Wave Passage Effect on the Seismic Response of a Multi-Span PSC Girder Bridge