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Therapeutic and reproductive cloning--implications and recommendations

Ng SC, Ng Y, Liow SL, Chen N

Med J Malaysia, 2003 Mar;58 Suppl A:102-10.
PMID: 14556357

The announcement of Dolly's birth took the world by storm, mainly because what was thought impossible has become possible. Optimism that new approaches in agriculture and medicine abound, as much as fear and imagination leading to Frankenstein-like scenarios. Scientifically, cloning refers to replicating an animal with the same nuclear genetic material; whilst it may refer to embryos as the source material, the current storm refers to differentiated ("somatic") cells. Cloning technology is useful in the following areas: agriculture, to produce animals of superior or specific qualities; endangered animals, to increase genetic diversity through widening the gene pool; understanding fundamental questions in developmental embryology, through the use of laboratory animals; and in human therapy, to produce cells and possibly tissues for repair and regeneration. In the first 2 instances, it is reproductive cloning. In the last instance, it is therapeutic cloning, as no individuals are "produced". Human reproductive cloning is not allowed by all governments that have deliberated on it, and therapeutic cloning is allowed by some under certain circumstances. As therapeutic cloning has great potential in cures of many diseases, it should be allowed but with safeguards to prevent abuse and reproductive cloning in the human.
Fulltext Assessors for communication skills: SPs or healthcare professionals?

Liew SC, Dutta S, Sidhu JK, De-Alwis R, Chen N, Sow CF, et al.

Med Teach, 2014 Jul;36(7):626-31.
PMID: 24787534 DOI: 10.3109/0142159X.2014.899689

The complexity of modern medicine creates more challenges for teaching and assessment of communication skills in undergraduate medical programme. This research was conducted to study the level of communication skills among undergraduate medical students and to determine the difference between simulated patients and clinical instructors' assessment of communication skills.
Phosphoinositide 3-kinase δ inactivation prevents vitreous-induced activation of AKT/MDM2/p53 and migration of retinal pigment epithelial cells

Han H, Chen N, Huang X, Liu B, Tian J, Lei H

J Biol Chem, 2019 10 18;294(42):15408-15417.
PMID: 31467081 DOI: 10.1074/jbc.RA119.010130

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that play a critical role in transmitting signals from cell-surface molecules to intracellular protein effectors. Key PI3Ks include PI3Kα, PI3Kβ, and PI3Kδ, which are regulated by receptors. The signaling pathway comprising the PI3Ks, along with a Ser/Thr kinase (AKT), a proto-oncogene product (mouse double minute (MDM)2), and a tumor suppressor protein (p53), plays an essential role in experimental proliferative vitreoretinopathy (PVR), which is a fibrotic blinding eye disorder. However, which PI3K isoforms are involved in PVR is unknown. A major characteristic of PVR is the formation of epi (or sub)-retinal membranes that consist of extracellular matrix and cells, including retinal pigment epithelium (RPE) cells, glial cells, and macrophages. RPE cells are considered key players in PVR pathogenesis. Using immunoblotting and immunofluorescence analyses, we herein provide the evidence that PI3Kδ is highly expressed in human RPEs when it is primarily expressed in leukocytes. We also found that PI3Kδ inactivation through two approaches, CRISPR/Cas9-mediated depletion and a PI3Kδ-specific inhibitor (idelalisib), not only blocks vitreous-induced activation of AKT and MDM2 but also abrogates a vitreous-stimulated decrease in p53. Furthermore, we demonstrate that PI3Kδ inactivation prevents vitreous-induced proliferation, migration, and contraction of human RPEs. These results suggest that PI3Kδ may represent a potential therapeutic target for RPE-related eye diseases, including PVR.
Antipyretic and antitumor effects of a purified polysaccharide from aerial parts of Tetrastigma hemsleyanum

Zhu B, Qian C, Zhou F, Guo J, Chen N, Gao C, et al.

J Ethnopharmacol, 2020 May 10;253:112663.
PMID: 32045682 DOI: 10.1016/j.jep.2020.112663

ETHNOPHARMACOLOGICAL RELEVANCE: Tetrastigma hemsleyanum Diels et Gilg (Sanyeqing) is traditionally used as a folk medicine for the treatments of inflammation, high fever, hepatitis and cancer, and can improve the immune function of the patient. It belongs to the family of Vitaceae, and is mainly distributed in southeast China (Yunnan province) and can be found in India (Andaman Islands), Myanmar, Thailand, Vietnam, Malaysia and Indonesia in the valleys with 1100-1300 m above the sea level.
AIM OF THE STUDY: The present study aimed to characterize the chemical properties of a purified polysaccharide extracted from the aerial part of Tetrastigma hemsleyanum (SYQP) and investigate its antipyretic and antitumor effects in mice models.
MATERIALS AND METHODS: Water-soluble crude polysaccharides from the aerial parts of Tetrastigma hemsleyanum were extracted and fractionated by DEAE and gel permeation chromatography. Homogeneity, molecular weight, monosaccharide composition, and FTIR analysis were performed to characterize the SYQP. Antipyretic effect of SYQP was examined using Brewer's yeast induced hyperthermia test. Antitumor effect was investigated using H22 tumor bearing mice. The serum cytokines were determined to evaluated the biological activities of SYQP.
RESULTS: SYQP was composed of galacturonic acid (GalA), glucose (Glc), mannose (Man), arabinose (Ara), galactose (Gal), and rhamnose (Rha) with a molar ratio of 11.3:7.1:2.5:1.0:0.9:0.5 and it had an average molecular weight of 66.2 kDa. The oral administration of SYQP at 200 and 400 mg/kg could markedly suppress the hyperthermia of mice induced by Brewer's yeast and decrease the production of cytokines especially prostaglandin E2 (PGE2) in the serum of mice. SYQP inhibited the growth of H22 tumor in mice with inhibitory rate of 39.9% at the administration dose of 200 mg/kg and increased the production of cytokines such as tumor necrosis factor-alpha (TNF-a) and interferon γ (IFN-γ). Experimental results showed that the preventive administration of SYQP before lipopolysaccharide (LPS) reduced the high cytokine levels such as IL-6, IL-10 and IFN-γ, indicating that SYQP might act as a competitor with LPS to interact with toll like receptor 4 (TLR4), which further regulated the secretion of cytokines.
CONCLUSION: The anti-inflammatory and antitumor activities of SYQP might be related to its regulation of host immune function by controlling the secretion of cytokines.
An interdigitated aptasensor to detect interleukin-6 for diagnosing rheumatoid arthritis in serum

Chen N, Yang H, Li Q, Song L, Gopinath SCB, Wu D

Biotechnol Appl Biochem, 2021 Dec;68(6):1479-1485.
PMID: 33244818 DOI: 10.1002/bab.2068

Rheumatoid arthritis (RA) is an autoimmune disorder causing chronic inflammation in the small joints of the articular bone and destruction of articular cartilage. RA causes stiffness, pain, joint destruction, substantial comorbidity, and functional disability. Early-stage diagnosis of RA can help in the treatment of the disease and expand the patient life span. Interleukins are a group of inflammatory cytokines; in particular, an abundance of interleukin-6 (IL-6) was found in the synovial fluid and serum. In RA patients, the levels of IL-6 have been found to be correlated with the disease, and this work focused on detecting IL-6 by its aptamer with the help of a biotin-streptavidin strategy on an interdigitated electrode. A sensitivity of 1 fM (0.021 pg/mL) and a limit of detection of 10 fM (0.21 pg/mL) were found by a linear regression [y = 0.6413x - 0.6249; R² = 0.952] of the linear range from 1 fM to 100 pM. This method enhanced the immobilization of higher aptamer molecules for recognizing RA in serum-containing samples and is applicable to other diseases.
Fulltext Profile of Microbial Keratitis

Lim Wen Siang J, Wu Zhuan O, Yi Chen N, Sok Lin N

Cureus, 2021 Dec;13(12):e20663.
PMID: 35103213 DOI: 10.7759/cureus.20663

PURPOSE: To study the demographic characteristics, predisposing factors, and latest trends of causative organisms and to analyze the prognostic factors of visual outcome in microbial keratitis.
METHODS: A retrospective study of patients diagnosed with microbial keratitis who required hospital admission in the period between January 2018 and December 2020 in Taiping Hospital, Perak, Malaysia.
RESULTS: A total of 75 eyes of 74 patients who were admitted to the hospital were studied. The male to female ratio was 13.8:1. Seventy percent of patients in this study were within the productive age group between 20 and 59 years old, with a mean age of 48 years old, and 51.4% of them were labourers. Cornea foreign bodies (42, 56%) were the most common predisposing factors and were associated with good visual outcomes (P<0.005). Other significant predictors for the final visual outcome were: presenting visual acuity, size of ulcer, duration of hospitalization, and duration of resolution. The mean duration of hospitalization was seven days. Corneal scrapings were done in all cases where 44 eyes (58.7%) were found to be positive for growth. Ten eyes (13.3%) that ended up with evisceration yielded a positive result. Gram-negative bacteria was the most prevalent causative organism of infective keratitis in the local/this region. Pseudomonas sp (20, 26.7%) being the most common bacterial isolate, was seen in all four contact lens-related cases and was associated with poor visual outcome and a high rate of evisceration. Patients who developed complications such as cornea melting (9, 12%), cornea perforation (11, 14.7%) and endophthalmitis (7, 9.3%) were associated with poor visual outcomes. Likewise, patients who required therapeutic interventions such as corneal gluing, tarsorrhaphy, and penetrating keratoplasty generally had poor visual outcomes (P<0.005; P=0.000008).
CONCLUSION: Microbial keratitis is a major cause of ocular morbidity globally. Understanding the demographic and epidemiological characteristics of microbial keratitis of the region is important in the initial prompt treatment of the patients and may eventually improve the visual outcome.
Structural properties and hydrolysability of recycled poplar residues (Populus L.): Effects of two-step acetic acid and sodium sulphite pre-treatment

Chu J, Li S, Chen N, Wen P, Sonne C, Ma NL

Chemosphere, 2022 Mar;291(Pt 1):132679.
PMID: 34718007 DOI: 10.1016/j.chemosphere.2021.132679

Poplar trees rapidly yield wood and are therefore suitable as a biofuel feedstock; however, the quality of poplar is modest, and the profitability of poplar cultivation depends on the efficiency of the harvesting process. This study offers a simple and sustainable technique to harvest lignocellulosic resources from poplar for bioethanol production. The proposed two-step pretreatment method increased the surface lignin content and decreased the surface polysaccharide content. The cellulose content increased to 54.9% and the xylan content decreased to 6.7% at 5% AC. The cellulose yield of poplar residues (Populus L.) reached 65.5% by this two-step acetic acid (AC) and sodium sulphite (SS) treatment method. Two-step pretreatment using 5% AC and 4% SS obtained a recovery of nearly 80% of the total available fermentable sugar. The surface characterization showed a higher porosity in treated samples, which improved their hydrolysability. This method decreased the amount of lignin in plant biomass, making it applicable for further wood resource recovery or waste recycling for biorefinery purposes at very low costs.
Fulltext Physicochemical and In Vitro Digestion Properties of Curcumin-Loaded Solid Lipid Nanoparticles with Different Solid Lipids and Emulsifiers

Yu Y, Chen D, Lee YY, Chen N, Wang Y, Qiu C

Foods, 2023 May 18;12(10).
PMID: 37238863 DOI: 10.3390/foods12102045

Curcumin-loaded solid lipid nanoparticles (Cur-SLN) were prepared using medium- and long chain diacylglycerol (MLCD) or glycerol tripalmitate (TP) as lipid matrix and three kinds of surfactants including Tween 20 (T20), quillaja saponin (SQ) and rhamnolipid (Rha). The MLCD-based SLNs had a smaller size and lower surface charge than TP-SLNs with a Cur encapsulation efficiency of 87.54-95.32% and the Rha-based SLNs exhibited a small size but low stability to pH decreases and ionic strength. Thermal analysis and X-ray diffraction results confirmed that the SLNs with different lipid cores showed varying structures, melting and crystallization profiles. The emulsifiers slightly impacted the crystal polymorphism of MLCD-SLNs but largely influenced that of TP-SLNs. Meanwhile, the polymorphism transition was less significant for MLCD-SLNs, which accounted for the better stabilization of particle size and higher encapsulation efficiency of MLCD-SLNs during storage. In vitro studies showed that emulsifier formulation greatly impacted on the Cur bioavailability, whereby T20-SLNs showed much higher digestibility and bioavailability than that of SQ- and Rha-SLNs possibly due to the difference in the interfacial composition. Mathematical modeling analysis of the membrane release further confirmed that Cur was mainly released from the intestinal phase and T20-SLNs showed a faster release rate compared with other formulations. This work contributes to a better understanding of the performance of MLCD in lipophilic compound-loaded SLNs and has important implications for the rational design of lipid nanocarriers and in instructing their application in functional food products.
Extensive intragenomic variations of the 18S rDNA V4 region in the toxigenic diatom species Pseudo-nitzschia multistriata revealed through high-throughput sequencing

Wang H, Liu K, He Z, Chen Y, Hu Z, Chen W, et al.

Mar Pollut Bull, 2024 Apr;201:116198.
PMID: 38428045 DOI: 10.1016/j.marpolbul.2024.116198

Metabarcoding analysis is an effective technique for monitoring the domoic acid-producing Pseudo-nitzschia species in marine environments, uncovering high-levels of molecular diversity. However, such efforts may result in the overinterpretation of Pseudo-nitzschia species diversity, as molecular diversity not only encompasses interspecies and intraspecies diversities but also exhibits extensive intragenomic variations (IGVs). In this study, we analyzed the V4 region of the 18S rDNA of 30 strains of Pseudo-nitzschia multistriata collected from the coasts of China. The results showed that each P. multistriata strain harbored about a hundred of unique 18S rDNA V4 sequence varieties, of which each represented by a unique amplicon sequence variant (ASV). This study demonstrated the extensive degree of IGVs in P. multistriata strains, suggesting that IGVs may also present in other Pseudo-nitzschia species and other phytoplankton species. Understanding the scope and levels of IGVs is crucial for accurately interpreting the results of metabarcoding analysis.
Synthesis, Antiphospholipase A₂, Antiprotease, Antibacterial Evaluation and Molecular Docking Analysis of Certain Novel Hydrazones

El-Sayed NN, Alafeefy AM, Bakht MA, Masand VH, Aldalbahi A, Chen N, et al.

Molecules, 2016 Dec 02;21(12).
PMID: 27918459

Some novel hydrazone derivatives 6a-o were synthesized from the key intermediate 4-Chloro-N-(2-hydrazinocarbonyl-phenyl)-benzamide 5 and characterized using IR, ¹H-NMR, 13C-NMR, mass spectroscopy and elemental analysis. The inhibitory potential against two secretory phospholipase A₂ (sPLA₂), three protease enzymes and eleven bacterial strains were evaluated. The results revealed that all compounds showed preferential inhibition towards hGIIA isoform of sPLA₂ rather than DrG-IB with compounds 6l and 6e being the most active. The tested compounds exhibited excellent antiprotease activity against proteinase K and protease from Bacillus sp. with compound 6l being the most active against both enzymes. Furthermore, the maximum zones of inhibition against bacterial growth were exhibited by compounds; 6a, 6m, and 6o against P. aeruginosa; 6a, 6b, 6d, 6f, 6l, 6m, 6n, and 6o against Serratia; 6k against S. mutans; and compounds 6a, 6d, 6e, 6m, and 6n against E. feacalis. The docking simulations of hydrazones 6a-o with GIIA sPLA₂, proteinase K and hydrazones 6a-e with glutamine-fructose-6-phosphate transaminase were performed to obtain information regarding the mechanism of action.
Biochar for cadmium pollution mitigation and stress resistance in tobacco growth

Ren T, Chen N, Wan Mahari WA, Xu C, Feng H, Ji X, et al.

Environ Res, 2021 01;192:110273.
PMID: 33002505 DOI: 10.1016/j.envres.2020.110273

Pot experiments were conducted to investigate the influence of biochar addition and the mechanisms that alleviate Cd stress in the growth of tobacco plant. Cadmium showed an inhibitory effect on tobacco growth at different post-transplantation times, and this increased with the increase in soil Cd concentration. The growth index decreased by more than 10%, and the photosynthetic pigment and photosynthetic characteristics of the tobacco leaf were significantly reduced, and the antioxidant enzyme activity was enhanced. Application of biochar effectively alleviated the inhibitory effect of Cd on tobacco growth, and the alleviation effect of treatments is more significant to the plants with a higher Cd concentration. The contents of chlorophyll a, chlorophyll b, and carotenoids in the leaves of tobacco plants treated with biochar increased by 9.99%, 12.58%, and 10.32%, respectively, after 60 days of transplantation. The photosynthetic characteristics index of the net photosynthetic rate increased by 11.48%, stomatal conductance increased by 11.44%, and intercellular carbon dioxide concentration decreased to 0.92. Based on the treatments, during the growth period, the antioxidant enzyme activities of tobacco leaves comprising catalase, peroxidase, superoxide dismutase, and malondialdehyde increased by 7.62%, 10.41%, 10.58%, and 12.57%, respectively, after the application of biochar. Our results show that biochar containing functional groups can effectively reduce the effect of Cd stress by intensifying the adsorption or passivation of Cd in the soil, thereby, significantly reducing the Cd content in plant leaves, and providing a theoretical basis and method to alleviate soil Cd pollution and effect soil remediation.
Predictors of inadequate bowel preparation in older patients undergoing colonoscopy: A systematic review and meta-analysis

Zhang Y, Wang L, Wu W, Zhang S, Zhang M, She W, et al.

Int J Nurs Stud, 2024 Jan;149:104631.
PMID: 37963423 DOI: 10.1016/j.ijnurstu.2023.104631

BACKGROUND: Factors that predict the quality of bowel preparation in older adults are unclear, and current guidelines do not provide specific recommendations.
OBJECTIVE: This meta-analysis aimed to identify risk factors for inadequate bowel preparation in older patients.
METHODS: PubMed, Embase, Web of Science, China National Knowledge Infrastructure (CNKI), Wanfang, and VIP databases were searched from their inception to February 2023. Cohort and cross-sectional studies exploring the risk factors for inadequate bowel preparation were included in this systematic review. Odds ratio (OR) values from individual studies were pooled using fixed-effects and random-effects models. In addition, a sensitivity analysis and assessment of publication bias were performed.
RESULTS: This meta-analysis included six studies (n = 1553) on previous abdominal surgery, six studies (n = 1494) on constipation, seven studies (n = 1505) on diabetes, eight studies (n = 2093) on non-compliance with the diet regimen, seven studies (n = 1350) on incomplete intake of laxative, and nine studies (n = 2163) on inadequate exercise during preparation. The pooled analysis showed that history of abdominal surgery (OR = 2.72; 95 % confidence interval, CI: 2.07 to 3.56), constipation (OR = 3.56, 95 % CI: 2.41 to 5.25), diabetes (OR = 2.54, 95 % CI: 1.81 to 3.57), non-compliance with the diet regimen (OR = 2.51, 95 % CI: 1.96 to 3.21), incomplete intake of laxative (OR = 2.43, 95 % CI: 1.60 to 3.67), and inadequate exercise during preparation (OR = 3.13, 95 % CI: 2.39 to 4.11) were independent risk factors for inadequate bowel preparation in older patients undergoing colonoscopy.
CONCLUSIONS: Three comorbid factors and three behavioral factors were significantly associated with inadequate bowel preparation in older adults. This meta-analysis provides valuable information for developing predictive models of poor bowel preparation.
Fulltext Genetic Mapping, Candidate Gene Identification and Marker Validation for Host Plant Resistance to the Race 4 of Fusarium oxysporum f. sp. cubense Using Musa acuminata ssp. malaccensis

Chen A, Sun J, Viljoen A, Mostert D, Xie Y, Mangila L, et al.

Pathogens, 2023 Jun 09;12(6).
PMID: 37375510 DOI: 10.3390/pathogens12060820

Fusarium wilt of banana is a devastating disease that has decimated banana production worldwide. Host resistance to Fusarium oxysporum f. sp. Cubense (Foc), the causal agent of this disease, is genetically dissected in this study using two Musa acuminata ssp. Malaccensis segregating populations, segregating for Foc Tropical (TR4) and Subtropical (STR4) race 4 resistance. Marker loci and trait association using 11 SNP-based PCR markers allowed the candidate region to be delimited to a 12.9 cM genetic interval corresponding to a 959 kb region on chromosome 3 of 'DH-Pahang' reference assembly v4. Within this region, there was a cluster of pattern recognition receptors, namely leucine-rich repeat ectodomain containing receptor-like protein kinases, cysteine-rich cell-wall-associated protein kinases, and leaf rust 10 disease-resistance locus receptor-like proteins, positioned in an interspersed arrangement. Their transcript levels were rapidly upregulated in the resistant progenies but not in the susceptible F2 progenies at the onset of infection. This suggests that one or several of these genes may control resistance at this locus. To confirm the segregation of single-gene resistance, we generated an inter-cross between the resistant parent 'Ma850' and a susceptible line 'Ma848', to show that the STR4 resistance co-segregated with marker '28820' at this locus. Finally, an informative SNP marker 29730 allowed the locus-specific resistance to be assessed in a collection of diploid and polyploid banana plants. Of the 60 lines screened, 22 lines were predicted to carry resistance at this locus, including lines known to be TR4-resistant, such as 'Pahang', 'SH-3362', 'SH-3217', 'Ma-ITC0250', and 'DH-Pahang/CIRAD 930'. Additional screening in the International Institute for Tropical Agriculture's collection suggests that the dominant allele is common among the elite 'Matooke' NARITA hybrids, as well as in other triploid or tetraploid hybrids derived from East African highland bananas. Fine mapping and candidate gene identification will allow characterization of molecular mechanisms underlying the TR4 resistance. The markers developed in this study can now aid the marker-assisted selection of TR4 resistance in breeding programs around the world.
Fulltext Author Correction: Dense sampling of bird diversity increases power of comparative genomics

Feng S, Stiller J, Deng Y, Armstrong J, Fang Q, Reeve AH, et al.

Nature, 2021 Apr;592(7856):E24.
PMID: 33833441 DOI: 10.1038/s41586-021-03473-8
Fulltext Dense sampling of bird diversity increases power of comparative genomics

Feng S, Stiller J, Deng Y, Armstrong J, Fang Q, Reeve AH, et al.

Nature, 2020 11;587(7833):252-257.
PMID: 33177665 DOI: 10.1038/s41586-020-2873-9

Whole-genome sequencing projects are increasingly populating the tree of life and characterizing biodiversity1-4. Sparse taxon sampling has previously been proposed to confound phylogenetic inference5, and captures only a fraction of the genomic diversity. Here we report a substantial step towards the dense representation of avian phylogenetic and molecular diversity, by analysing 363 genomes from 92.4% of bird families-including 267 newly sequenced genomes produced for phase II of the Bird 10,000 Genomes (B10K) Project. We use this comparative genome dataset in combination with a pipeline that leverages a reference-free whole-genome alignment to identify orthologous regions in greater numbers than has previously been possible and to recognize genomic novelties in particular bird lineages. The densely sampled alignment provides a single-base-pair map of selection, has more than doubled the fraction of bases that are confidently predicted to be under conservation and reveals extensive patterns of weak selection in predominantly non-coding DNA. Our results demonstrate that increasing the diversity of genomes used in comparative studies can reveal more shared and lineage-specific variation, and improve the investigation of genomic characteristics. We anticipate that this genomic resource will offer new perspectives on evolutionary processes in cross-species comparative analyses and assist in efforts to conserve species.
Fulltext Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1

Klionsky DJ, Abdel-Aziz AK, Abdelfatah S, Abdellatif M, Abdoli A, Abel S, et al.

Autophagy, 2021 Jan;17(1):1-382.
PMID: 33634751 DOI: 10.1080/15548627.2020.1797280

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.

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