Sreejayan Nair

Bio: Sreejayan Nair is an academic researcher from University of Wyoming. The author has contributed to research in topics: Autophagy & Insulin resistance. The author has an hindex of 24, co-authored 53 publications receiving 6562 citations. Previous affiliations of Sreejayan Nair include New York Medical College & College of Health Sciences, Bahrain.

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.

Targeted therapy for high-grade glioma with the TGF-β2 inhibitor trabedersen: results of a randomized and controlled phase IIb study

Abstract: This randomized, open-label, active-controlled, dose-finding phase IIb study evaluated the efficacy and safety of trabedersen (AP 12009) administered intratumorally by convection-enhanced delivery compared with standard chemotherapy in patients with recurrent/refractory high-grade glioma. One hundred and forty-five patients with central reference histopathology of recurrent/refractory glioblastoma multiforme (GBM) or anaplastic astrocytoma (AA) were randomly assigned to receive trabedersen at doses of 10 or 80 µM or standard chemotherapy (temozolomide or procarbazine/lomustine/vincristine). Primary endpoint was 6-month tumor control rate, and secondary endpoints included response at further timepoints, survival, and safety. Six-month tumor control rates were not significantly different in the entire study population (AA and GBM). Prespecified AA subgroup analysis showed a significant benefit regarding the 14-month tumor control rate for 10 µM trabedersen vs chemotherapy (p= .0032). The 2-year survival rate had a trend for superiority for 10 µM trabedersen vs chemotherapy (p = .10). Median survival for 10 µM trabedersen was 39.1 months compared with 35.2 months for 80 µM trabedersen and 21.7 months for chemotherapy (not significant). In GBM patients, response and survival results were comparable among the 3 arms. Exploratory analysis on GBM patients aged ≤55 years with Karnofsky performance status >80% at baseline indicated a 3-fold survival at 2 and 3 years for 10 µM trabedersen vs chemotherapy. The frequency of patients with related or possibly drug-related adverse events was higher with standard chemotherapy (64%) than with 80 µM trabedersen (43%) and 10 µM trabedersen (27%). Superior efficacy and safety for 10 µM trabedersen over 80 µM trabedersen and chemotherapy and positive risk-benefit assessment suggest it as the optimal dose for further clinical development in high-grade glioma.

Akt2 knockout preserves cardiac function in high-fat diet-induced obesity by rescuing cardiac autophagosome maturation

Abstract: Dear Editor, Accumulating studies have demonstrated that the autophagy–lysosome pathway, a major pathway governing protein and organelle degradation and recycling, is a house keeper in cardiomyocytes under physiological conditions (Mizushima and Klionsky, 2007). However, the role of autophagy in the heart under pathological conditions is still controversial (Nemchenko et al., 2011). In vivo studies depicted that inhibition of mammalian target of rapamycin (mTOR), a primary inhibitory regulator of autophagy, is capable of attenuating pressure overload-induced cardiac dysfunction (McMullen et al., 2004). To the contrary, recent studies have also indicated that suppressing autophagy is beneficial for cardiac hypertrophy (Cao et al., 2011). Along the same line, activated autophagy has been proved detrimental for pressure overload-induced heart failure (Zhu et al., 2007). However, the role of autophagy in the heart in high-fat diet (HFD)-induced obesity is poorly understood. To date, there is little evidence suggesting a role of autophagy in heart anomalies associated with diet-induced obesity, although a number of upstream regulators of autophagy have been identified to play a role in HFD-induced obesity. For example, the primary inhibitor of autophagy mTOR may be hyperactivated by an HFD and contribute to the development of cardiac dysfunction (Birse et al., 2010). As the major activator of mTOR, the Akt family of serine–threonine kinases is also activated by an HFD in the heart. However, the precise role of Akt2, one of the three Akt isoforms predominantly found in the heart, in autophagy regulation in HFD-induced obesity still remains elusive. To this end, the present study was designed to evaluate the role of autophagy and autophagy flux in HFD feeding-induced cardiac geometric and functional changes with a special focus on Akt2 signaling. HFD intake significantly increased body and organ (heart, liver, kidney and adipose tissue) weights compared with low-fat diet (LFD) feeding (Supplementary Table S2). Western blot analysis confirmed the absence of Akt2 in hearts from Akt2 mice (Supplementary Figure S1A and B). Interestingly, HFD feeding upregulated cardiac expression of Akt2 (Supplementary Figure S1A and B) but not that of Akt1 (Supplementary Figure S7A and B) and Akt3 (Supplementary Figure S7A and C). Akt2 knockout did not affect body or organ weight in LFD-fed mice (Supplementary Table S2). However, Akt2 knockout effectively nullified HFD-induced gain in body and organ/tissue weights, in particular the heart (Supplementary Table S2). Accumulating studies have demonstrated that Akt regulates cell growth and lipid biosynthesis through mTORC1. Accordingly, we found that an HFD-activated Akt (Supplementary Figure S6A and B) and mTORC1 (Supplementary Figure S7A and I) in the heart, both of which were mitigated by Akt2 knockout. These data depict a beneficial effect of Akt2 knockout against HFD-induced weight gain possibly through the inhibition of Akt-mTORC1 activation. In addition, HFD feeding significantly increased the level of triglyceride, the effect of which was ablated by Akt2 knockout (Supplementary Figure S1C). Further scrutiny of glucose metabolism using intraperitoneal glucose tolerance test revealed overt glucose intolerance following HFD intake in the wild type (WT) which was partially attenuated in the Akt2 mice (Supplementary Figure S1D and E). HFD feeding significantly compromised myocardial geometry and function as evidenced by overtly increased LV ESD, LV EDD and LV mass, as well as decreased fractional shortening associated with unchanged septum and posterior wall thickness. Interestingly, Akt2 knockout ameliorated HFD feeding-induced cardiac geometric and contractile anomalies (Figure 1A and B and Supplementary Figure S2A–E). Further assessment of cardiomyocyte contractile function revealed consistent findings. HFD feeding dampened cardiomyocyte contractile capacity (decreased peak shortening and maximal velocity of shortening/re-lengthening) associated with unchanged duration of shortening and re-lengthening, which was recovered by Akt2 knockout (Figure 1C and D and Supplementary Figures S2F–I and S3A–C). Besides, Akt2 knockout significantly ameliorated intracellular Ca2+ handling dysfunction induced by an HFD in the WT mice (Supplementary Figure S2J–O). Additionally, HFD feeding induced cardiac hypertrophy (Supplementary Figure S4A–H), interstitial fibrosis (Supplementary Figure S5A and B), and activated cardiac protein synthesis pathway (Supplementary Figures S6A–K and S7A, J, and K), which were obliterated by Akt2 knockout. Taken together, these results supported that Akt2 knockout protected murine hearts against HFD-induced cardiac pathological hypertrophy. Interestingly, our data revealed that the expression of LC3B I (microtubule-associated protein light chain 3 I, type B) was dramatically increased following HFD feeding in both the WT and Akt2 mice, indicating that HFD feeding may trigger the initial autophagy steps (Figure 1E and Supplementary Figure S8A, C–G, J, and L– O). LC3B II integrates onto the autophagosomal membrane, and is widely used as a marker of autophagosomes. Nonetheless, an increase in LC3B II may represent either an increase in autophagosome formation (initiation of autophagy) or a doi:10.1093/jmcb/mjs055 Journal of Molecular Cell Biology (2013), 5, 61–63 | 61 Published online December 19, 2012

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.

Absence of S6K1 protects against age- and diet-induced obesity while enhancing insulin sensitivity. [Erratum: 2004 Sept. 23, v. 431, no. 7007, p. 485.]

Abstract: Elucidating the signalling mechanisms by which obesity leads to impaired insulin action is critical in the development of therapeutic strategies for the treatment of diabetes. Recently, mice deficient for S6 Kinase 1 (S6K1), an effector of the mammalian target of rapamycin (mTOR) that acts to integrate nutrient and insulin signals, were shown to be hypoinsulinaemic, glucose intolerant and have reduced β-cell mass. However, S6K1-deficient mice maintain normal glucose levels during fasting, suggesting hypersensitivity to insulin, raising the question of their metabolic fate as a function of age and diet. Here, we report that S6K1-deficient mice are protected against obesity owing to enhanced β-oxidation. However on a high fat diet, levels of glucose and free fatty acids still rise in S6K1-deficient mice, resulting in insulin receptor desensitization. Nevertheless, S6K1-deficient mice remain sensitive to insulin owing to the apparent loss of a negative feedback loop from S6K1 to insulin receptor substrate 1 (IRS1), which blunts S307 and S636/S639 phosphorylation; sites involved in insulin resistance. Moreover, wild-type mice on a high fat diet as well as K/K Ay and ob/ob (also known as Lep/Lep) micetwo genetic models of obesityhave markedly elevated S6K1 activity and, unlike S6K1-deficient mice, increased phosphorylation of IRS1 S307 and S636/S639. Thus under conditions of nutrient satiation S6K1 negatively regulates insulin signalling.

Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion.

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...