A strategy to build a library of oil tracers by oleophilic Silica-encapsulated DNA nanoparticles.

Artificially synthesized DNA is involved in the construction of a library of oil tracers due to their unlimited number and no-biological toxicity. The strategy of the construction is proposed by oleophilic Silica-encapsulated DNA nanoparticles, which offers fresh thinking in developing novel tracers, sensors, and molecular machines in engineering & applied sciences based on artificially synthesized DNA blocks. .

Conventionally fractionated radiation therapy is associated with long-term survival in dogs with infiltrative lipomas.

OBJECTIVE: To describe radiotherapy outcomes for canine infiltrative lipomas and provide detailed radiotherapy planning data. ANIMALS: 24 dogs from 2000 to 2020. METHODS: In this retrospective study, dogs received 1 to 3 surgeries prior to conventionally fractionated radiotherapy for gross (18) or microscopic (8) infiltrative lipomas. Dogs received 45 to 51 Gray (Gy) in 15 to 20 daily fractions, with 71% of dogs receiving 48 Gy in daily 3-Gy fractions. RESULTS: Masses were regionally located as follows: limbs (7), trunk (13), head/neck (4). At analysis, 16/24 dogs were deceased, 5/24 were alive (median follow-up for alive dogs: 1,216 days [range, 741 to 1,870 days]), and 3/24 were lost to follow-up. One living dog had progressive disease 923 days after completing conventionally fractionated radiotherapy and received another surgery. The estimated median overall survival (OS) after completing radiotherapy was 4.8 years (1,760 days; 95% CI, 1,215 to 2,777 days; range, 23 to 3,499 days) for any cause of death, and no patients were reported to have been euthanized or died from their tumor. No statistically significant difference was found for dogs based on gross versus microscopic disease (gross OS, 4.8 years vs microscopic OS, 3.6 years; P = .45). Furthermore, the number of surgeries before radiotherapy did not impact survival (P = .96). The survival difference between females (median OS, 7.6 years; 95% CI, 963 days to not reached) versus males (median OS, 4.6 years; 95% CI, 335 to 2,245 days; P = .05) was statistically significant, although 4/5 living dogs were female. CLINICAL RELEVANCE: This study demonstrates lengthy survivals with radiotherapy, even with gross disease, for dogs with infiltrative lipomas.

Protein expression profiling identifies a prognostic model for ovarian cancer.

BACKGROUND: Owing to the high morbidity and mortality, ovarian cancer has seriously endangered female health. Development of reliable models can facilitate prognosis monitoring and help relieve the distress. METHODS: Using the data archived in the TCPA and TCGA databases, proteins having significant survival effects on ovarian cancer patients were screened by univariate Cox regression analysis. Patients with complete information concerning protein expression, survival, and clinical variables were included. A risk model was then constructed by performing multiple Cox regression analysis. After validation, the predictive power of the risk model was assessed. The prognostic effect and the biological function of the model were evaluated using co-expression analysis and enrichment analysis. RESULTS: 394 patients were included in model construction and validation. Using univariate Cox regression analysis, we identified a total of 20 proteins associated with overall survival of ovarian cancer patients (p < 0.01). Based on multiple Cox regression analysis, six proteins (GSK3α/ß, HSP70, MEK1, MTOR, BAD, and NDRG1) were used for model construction. Patients in the high-risk group had unfavorable overall survival (p < 0.001) and poor disease-specific survival (p = 0.001). All these six proteins also had survival prognostic effects. Multiple Cox regression analysis demonstrated the risk model as an independent prognostic factor (p < 0.001). In receiver operating characteristic curve analysis, the risk model displayed higher predictive power than age, tumor grade, and tumor stage, with an area under the curve value of 0.789. Analysis of co-expressed proteins and differentially expressed genes based on the risk model further revealed its prognostic implication. CONCLUSIONS: The risk model composed of GSK3α/ß, HSP70, MEK1, MTOR, BAD, and NDRG1 could predict survival prognosis of ovarian cancer patients efficiently and help disease management.

Epithelial microRNA-30a-3p targets RUNX2/HMGB1 axis to suppress airway eosinophilic inflammation in asthma.

BACKGROUND: Type 2-high asthma is a prominent endotype of asthma which is characterized by airway eosinophilic inflammation. Airway epithelial cells play a critical role in the pathogenesis of asthma. Our previous miRNA profiling data showed that miR-30a-3p was downregulated in bronchial epithelial cells from asthma patients. We hypothesize that epithelial miR-30a-3p plays a role in asthma airway inflammation. METHODS: We measured miR-30a-3p expression in bronchial brushings of asthma patients (n = 51) and healthy controls (n = 16), and analyzed the correlations between miR-30a-3p expression and airway eosinophilia. We examined whether Runt-related transcription factor 2 (RUNX2) was a target of miR-30a-3p and whether RUNX2 bound to the promoter of high mobility group box 1 (HMGB1) by using luciferase reporter assay and chromatin immunoprecipitation (ChIP)-PCR. The role of miR-30a-3p was also investigated in a murine model of allergic airway inflammation. RESULTS: We found that miR-30a-3p expression were significantly decreased in bronchial brushings of asthma patients compared to control subjects. Epithelial miR-30a-3p expression was negatively correlated with parameters reflecting airway eosinophilia including eosinophils in induced sputum and bronchial biopsies, and fraction of exhaled nitric oxide in asthma patients. We verified that RUNX2 is a target of miR-30a-3p. Furthermore, RUNX2 bound to the promoter of HMGB1 and upregulated HMGB1 expression. RUNX2 and HMGB1 expression was both enhanced in airway epithelium and was correlated with each other in asthma patients. Inhibition of miR-30a-3p enhanced RUNX2 and HMGB1 expression, and RUNX2 overexpression upregulated HMGB1 in BEAS-2B cells. Intriguingly, airway overexpression of mmu-miR-30a-3p suppressed Runx2 and Hmgb1 expression, and alleviated airway eosinophilia in a mouse model of allergic airway inflammation. CONCLUSIONS: Epithelial miR-30a-3p could possibly target RUNX2/HMGB1 axis to suppress airway eosinophilia in asthma.

Expression of AOX1 Predicts Prognosis of Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most prevalent kidney cancer worldwide, and appropriate cancer biomarkers facilitate early diagnosis, treatment, and prognosis prediction in cancer management. However, an accurate biomarker for ccRCC is lacking. This study identified 356 differentially expressed genes in ccRCC tissues compared with normal kidney tissues by integrative analysis of eight ccRCC datasets. Enrichment analysis of the differentially expressed genes unveiled improved adaptation to hypoxia and metabolic reprogramming of the tumor cells. Aldehyde oxidase 1 (AOX1) gene was identified as a biomarker for ccRCC among all the differentially expressed genes. ccRCC tissues expressed significantly lower AOX1 than normal kidney tissues, which was further validated by immunohistochemistry at the protein level and The Cancer Genome Atlas (TCGA) data mining at the mRNA level. Higher AOX1 expression predicted better overall survival in ccRCC patients. Furthermore, AOX1 DNA copy number deletion and hypermethylation were negatively correlated with AOX1 expression, which might be the potential mechanism for its dysregulation in ccRCC. Finally, we illustrated that the effect of AOX1 as a tumor suppressor gene is not restricted to ccRCC but universally exists in many other cancer types. Hence, AOX1 may act as a potential prognostic biomarker and therapeutic target for ccRCC.

Predictive value of RAD51 on the survival and drug responsiveness of ovarian cancer.

BACKGROUND: Ovarian cancer has greatly endangered and deteriorated female health conditions worldwide. Refinement of predictive biomarkers could enable patient stratification and help optimize disease management. METHODS: RAD51 expression profile, target-disease associations, and fitness scores of RAD51 were analyzed in ovarian cancer using bioinformatic analysis. To further identify its role, gene enrichment analysis was performed, and a regulatory network was constructed. Survival analysis and drug sensitivity assay were performed to evaluate the effect of RAD51 expression on ovarian cancer prognosis. The predictive value of RAD51 was then confirmed in a validation cohort immunohistochemically. RESULTS: Ovarian cancer expressed more RAD51 than normal ovary. RAD51 conferred ovarian cancer dependency and was associated with ovarian cancer. RAD51 had extensive target-disease associations with various diseases, including ovarian cancer. Genes that correlate with and interact with RAD51 were involved in DNA damage repair and drug responsiveness. High RAD51 expression indicated unfavorable survival outcomes and resistance to platinum, taxane, and PARP inhibitors in ovarian cancer. In the validation cohort (126 patients), high RAD51 expression indicated platinum resistance, and platinum-resistant patients expressed more RAD51. Patients with high RAD51 expression had shorter OS (HR = 2.968, P < 0.0001) and poorer PFS (HR = 2.838, P < 0.0001). RAD51 expression level was negatively correlated with patients' survival length. CONCLUSIONS: Ovarian cancer had pronounced RAD51 expression and RAD51 conferred ovarian cancer dependency. High RAD51 expression indicated poor survival and decreased drug sensitivity. RAD51 has predictive value in ovarian cancer and can be exploited as a predictive biomarker.

Epithelial miR-206 targets CD39/extracellular ATP to upregulate airway IL-25 and TSLP in type 2-high asthma.

The epithelial cell-derived cytokines IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) initiate type 2 inflammation in allergic diseases, including asthma. However, the signaling pathway regulating these cytokines expression remains elusive. Since microRNAs are pivotal regulators of gene expression, we profiled microRNA expression in bronchial epithelial brushings from type 2-low and type 2-high asthma patients. miR-206 was the most highly expressed epithelial microRNA in type 2-high asthma relative to type 2-low asthma but was downregulated in both subsets compared with healthy controls. CD39, an ectonucleotidase degrading ATP, was a target of miR-206 and upregulated in asthma. Allergen-induced acute extracellular ATP accumulation led to miR-206 downregulation and CD39 upregulation in human bronchial epithelial cells, forming a feedback loop to eliminate excessive ATP. Airway ATP levels were markedly elevated and strongly correlated with IL-25 and TSLP expression in asthma patients. Intriguingly, airway miR-206 antagonism increased Cd39 expression; reduced ATP accumulation; suppressed IL-25, IL-33, and Tslp expression and group 2 innate lymphoid cell expansion; and alleviated type 2 inflammation in a mouse model of allergic airway inflammation. In contrast, airway miR-206 overexpression had opposite effects. Overall, epithelial miR-206 upregulates airway IL-25 and TSLP expression by targeting the CD39-extracellular ATP axis, which represents a potentially novel therapeutic target in type 2-high asthma.

MiR-34a Inhibits Cell Proliferation and Induces Apoptosis in Human Nasopharyngeal Carcinoma by Targeting lncRNA MCM3AP-AS1.

INTRODUCTION: MCM3AP-AS1 has been characterized as an oncogenic lncRNA in several types of cancer, while its role in nasopharyngeal carcinoma (NPC) is unknown. This study aimed to investigate the role of MCM3AP-AS1 in NPC. PATIENTS AND METHODS: Paired NPC tissues and non-tumor tissues were collected from 55 NPC patients. Expression of MCM3AP-AS1 and miR-34a in paired tissues was analyzed by RT-qPCR. Interactions between MCM3AP-AS1 and miR-34a were analyzed by overexpression experiments. The roles of MCM3AP-AS1 and miR-34a in regulating NPC cell proliferation and apoptosis were explored by cell proliferation assay and cell apoptosis assay, respectively. RESULTS: Our bioinformatics analysis showed that MCM3AP-AS1 may be targeted by miR-34a, which is a well-studied tumor suppressor miRNA. In this study, we showed that miR-34a was downregulated and MCM3AP-AS1 was upregulated in NPC. An inverse correlation between the expression of MCM3AP-AS1 and miR-34a was found across NPC tissue samples. High expression level of MCM3AP-AS1 and low levels of miR-34a in NPC tissues predicted the poor survival. In NPC cells, overexpression of MCM3AP-AS1 did not affect the expression of miR34a, while overexpression of miR-34a led to downregulated MCM3AP-AS1. Cell proliferation and apoptosis assay showed that overexpression of miR-34a reduced the enhancing effects of overexpressing MCM3AP-AS1 on cell proliferation and the inhibitory effects on cell apoptosis. CONCLUSION: MiR-34a inhibits cell proliferation and induces apoptosis in human NPC by targeting MCM3AP-AS1.

microRNA-218-5p plays a protective role in eosinophilic airway inflammation via targeting δ-catenin, a novel catenin in asthma.

BACKGROUND: microRNA (miR)-218-5p is involved in cigarette smoke-induced airway inflammation. In our earlier asthma epithelial miRNA profiling data, miR-218-5p was the top 2 down-regulated miRNA. We hypothesize that miR-218-5p plays a role in asthma airway inflammation. OBJECTIVE: To unveil the role of miR-218-5p and its target gene in asthma airway inflammation. METHODS: We measured miR-218-5p expression in bronchial brushings of asthma patients (n = 50) and healthy controls (n = 15), and analysed the correlations between miR-218-5p expression and airway eosinophilia. We examined whether CTNND2 was a target of miR-218-5p, and the expression of 12 catenin family members in bronchial brushings, in cultured human bronchial epithelial (HBE) cells and BEAS-2B cells. We explored the role of miR-218-5p-CTNND2 pathway using a murine model of allergic airway inflammation. RESULTS: Epithelial miR-218-5p expression was significantly decreased and negatively correlated with eosinophils in induced sputum and bronchial biopsies, and other type 2 biomarkers in asthma patients. We verified that CTNND2 (encoding δ-catenin) was a target of miR-218-5p. Remarkably, CTNND2 was the most significantly up-regulated catenin compared with the other 11 catenin family members in bronchial brushings of asthma patients, IL-13-stimulated HBE and BEAS-2B cells. Moreover, epithelial CTNND2 expression positively correlated with airway eosinophilia in asthma. Airway mmu-miR-218-5p expression was also decreased, and Ctnnd2 expression was increased in a murine model of allergic airway inflammation. Intriguingly, mmu-miR-218-5p overexpression suppressed airway hyperresponsiveness, eosinophilic airway inflammation and Ctnnd2 up-regulation in the mouse model. Finally, perturbation of miR-218-5p or CTNND2 expression significantly altered chemokine CCL26 expression in the cell cultures and the mouse model. CONCLUSIONS AND CLINICAL RELEVANCE: Epithelial miR-218-5p plays a protective role in eosinophilic airway inflammation via targeting CTNND2, a novel catenin in asthma, and suppressing chemokine CCL26 expression.

Therapeutic effect and mechanism study of L-cysteine derivative 5P39 on LPS-induced acute lung injury in mice.

Organosulfur compounds, such as L-cysteine, allicin and other sulfur-containing organic compounds in Allium species, have been proposed to possess many important physiological and pharmacological functions. A novel L-cysteine derivative, t-Butyl S-allylthio-L-cysteinate (5P39), was designed and synthesized by combining L-cysteine derivative and allicin pharmacophore through a disulfide bond. This study aimed to explore the effects and mechanisms of 5P39 on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. At the experimental concentration (5, 10 and 20 µM), 5P39 suppressed the excessive secretion of nitric oxide (NO) and interleukin-6 (IL-6) in mice peritoneal macrophages stimulated by LPS. A mouse model of ALI was established by tracheal instillation of LPS for 2 h before 5P39 (30 and 60 mg/kg) administration. The results showed that 5P39 treatment down-regulated the wet/dry weight ratio (W/D ratio) of lungs and reduced the protein concentration, the number of total cells as well as the myeloperoxidase (MPO) activity in bronchoalveolar lavage fluid (BALF). 5P39 administration improved the histopathological changes of lungs in ALI mice with the decreased levels of pro-inflammatory cytokines in BALF. The inhibitory effects of 5P39 on the toll-like receptor 4 (TLR4) expression and macrophages accumulation in lung tissues were observed by immunohistochemistry. Additionally, 5P39 significantly attenuated the LPS-activated high expression of key proteins in TLR4/MyD88 signaling pathway. Taken together, the present study showed that 5P39 effectively alleviate the severity of ALI, and its mechanism might relate to the inhibition of LPS-activated TLR4/MyD88 signaling pathway, demonstrating a promising potential for further development into an anti-inflammatory drug candidate.

Combination of attenuated Salmonella carrying PD-1 siRNA with nifuroxazide for colon cancer therapy.

Colon cancer is a member of malignant tumors in the digestive system. Traditional treatment strategies are ineffective and improving the treatment of colon cancer is an urgent need. Targeting programmed cell death-1 (PD-1) by monoclonal antibodies has shown some therapeutic effectiveness and has advantages. Additionally, the Stat3 inhibitor nifuroxazide was employed to promote the antitumor activity. Here, we hypothesized that combining nifuroxazide with PD-1 small interfering RNA carried by attenuated Salmonella would exert a synergistic antitumor effect on colon cancer. Indeed, treatment with this combination effectively inhibited the development of colon cancer in mice and improved the survival rate. These two novel anticancer agents worked synergistically to elicit potent antitumor immunity and achieve improved therapeutic efficacy. The underlying mechanisms are mainly involved with immune regulation and cell apoptosis. This study provides a previous framework for combining this Stat3 inhibitor with RNAi designed to block immune checkpoint signaling for cancer therapy.

Epithelial SERPINB10, a novel marker of airway eosinophilia in asthma, contributes to allergic airway inflammation.

Serine peptidase inhibitor, clade B, member 10 (SERPINB10) expression is increased in IL-13-stimulated human bronchial epithelial cells and in a murine model of allergic airway inflammation. However, the role of SERPINB10 in asthma remains unknown. We examined the association between epithelial SERPINB10 expression and airway eosinophilia in subjects with asthma and the role of Serpinb10 in allergic airway inflammation in an animal model. Epithelial SERPINB10 mRNA and protein expression were markedly increased in subjects with asthma ( n = 60) compared with healthy controls ( n = 25). Epithelial SERPINB10 mRNA levels were significantly correlated with airway hyperresponsiveness (AHR) and three parameters reflecting airway eosinophilia including the percentage of sputum eosinophils, the number of eosinophils in bronchial submucosa, and fraction of exhaled nitric oxide in subjects with asthma. Moreover, epithelial SERPINB10 expression was strongly correlated with the epithelial gene signature ( CLCA1, POSTN, and SERPINB2) for type 2 status. In normal human bronchial epithelial cells cultured at air-liquid interface, knockdown of SERPINB10 suppressed IL-13-stimulated periostin (encoded by POSTN) and CCL26 (eotaxin-3) expression by inhibiting the activation of p38 MAPK. Epithelial CCL26 mRNA levels were correlated with SERPINB10 expression in subjects with asthma. Airway knockdown of Serpinb10 alleviated AHR, airway eosinophilia and the expression of periostin and Ccl26 in a murine model of allergic airway disease. Taken together, epithelial SERPINB10 is a novel marker for airway eosinophilia in asthma. Epithelial SERPINB10 contributes to allergic airway eosinophilic inflammation, at least in part, by regulating the expression of periostin and CCL26.

The enhanced antitumour response of pimozide combined with the IDO inhibitor L­MT in melanoma.

Melanoma is one of the most fatal and therapy-resistant types of cancer; therefore, identifying novel therapeutic candidates to improve patient survival is an ongoing effort. Previous studies have revealed that pimozide is not sufficient to treat melanoma; therefore, enhancing the treatment is necessary. Indoleamine 2, 3­dioxygenase (IDO) is an immunosuppressive, intracellular rate-limiting enzyme, which contributes to immune tolerance in various tumours, including melanoma, and inhibition of IDO may be considered a novel therapeutic strategy when combined with pimozide. The present study aimed to assess the antitumour activities of pimozide in vitro, and to investigate the effects of pimozide combined with L­methyl-tryptophan (L­MT) in vivo. For in vitro analyses, the B16 melanoma cell line was used. Cell cytotoxicity assay, cell viability assay, wound­healing assay and western blotting were conducted to analyse the effects of pimozide on B16 cells. Furthermore, B16 cell-bearing mice were established as the animal model. Haematoxylin and eosin staining, immunohistochemistry, terminal deoxynucleotidyl transferase dUTP nick end-labelling staining, western blotting and flow cytometry were performed to determine the effects of monotherapy and pimozide and L­MT cotreatment on melanoma. The results demonstrated that pimozide exhibited potent antitumour activity via the regulation of proliferation, apoptosis and migration. Furthermore, the antitumour effects of pimozide were enhanced when combined with L­MT, not only via regulation of proliferation, apoptosis and migration, but also via immune modulation. Notably, pimozide may regulate tumour immunity through inhibiting the activities of signal transducer and activator of transcription (Stat)3 and Stat5. In conclusion, the present study proposed the use of pimozide in combination with the IDO inhibitor, L­MT, as a potential novel therapeutic strategy for the treatment of melanoma.

Therapeutic effects of STAT3 inhibition by nifuroxazide on murine acute graft graft-vs.-host disease: Old drug, new use.

Graft­vs.­host disease (GvHD) is a major and lethal complication of allogeneic bone marrow transplantation (allo­BMT). Although great development has been made, the treatment progress of this disorder is slow. Research has illustrated that STAT3 was critical for T cell alloactivation in GvHD. In the present study, the authors hypothesized that nifuroxazide, as the STAT3 inhibitor, treatment may attenuate the development of acute GvHD (aGvHD). The results demonstrated that nifuroxazide suppressed the development of aGvHD and significantly delayed aGvHD­induced lethality. Mice receiving nifuroxazide had mostly normal­appearing skin with minimal focal ulceration, mild edema and congestion in the liver, and a less­pronounced villus injury and less inflammatory infiltrate in the small intestine. Treatment with nifuroxazide inhibited the activation of STAT3, resulting in the regulation of the CD4+ T cells and CD4+CD25+ T cells and reduction of interferon­Î³ and tumor necrosis factor­α levels. In conclusion, nifuroxazide may be efficacious for post­transplant of GvHD, providing a potent drug for use as a prophylactic or as a second­line therapy for aGvHD in clinical trials.

Nifuroxazide prompts antitumor immune response of TCL-loaded DC in mice with orthotopically-implanted hepatocarcinoma.

Hepatocellular carcinoma (HCC) is a highly aggressive malignancy with a poor prognosis and high mortality. At present, vaccination with tumor cell lysate (TCL) loaded dendritic cells (DC) has been shown to be an effective therapy against HCC. However, the ability of promoting the specific T cell immune response is rather weak, influencing the antitumor response. Thus, it is necessary to find a strategy to improve the antitumor effect of TCL-loaded DC. Activation of signal transducer and activator of transcription 3 (STAT3) significantly inhibits antitumor immune response and DC maturity. Nifuroxazide, an antidiarrheal agent, has been proved to directly inhibit STAT3 activation. Thus, we investigated whether nifuroxazide could improve the antitumor immune response in mice vaccinated with TCL-loaded DC. The study provides the theoretical and experimental basis for developing an effective adjuvant for DC vaccine to treat HCC. Our results showed that the administration of nifuroxazide and DC-loaded TCL could significantly improve the survival rate, inhibit the tumor growth, and prompt the antitumor immune responses in mice with orthotopically implanted hepatocarcinomas, thus, possibly providing a new combination strategy to treat HCC.

Adherence to ARRIVE Guidelines in Chinese Journal Reports on Neoplasms in Animals.

BACKGROUND: The Animals in Research: Reporting In Vivo Experiments (ARRIVE) guidelines were published in 2010 with the aim of improving the quality of studies involving animals. However, how well Chinese studies involving animal neoplasms adhere to these guidelines has not been assessed. OBJECTIVE: To evaluate the reporting quality of such experiments published between 2010 and 2012 in Chinese journals with support from the National Natural Science Foundation of China. METHODS: We searched the Chinese Science Citation and Chinese Journal Full-Text Databases for articles published between 2010 and 2012 involving neoplasms in animals. The data were extracted into pre-prepared forms. Reporting quality was assessed using the ARRIVE checklist-39 items plus information on blinding. RESULTS: Three hundred and ninety-six animal studies were included in the analysis: 127 studies published in 2010, 140 studies published in 2011, and 129 studies published in 2012. The range of ARRIVE score is from 12 to 27 with a maximum possible score of 40. Studies published in 2012 (P = 0.012), 2011 (P = 0.015), 2010, July~Dec (P<0.017) had a significantly larger ARRIVE checklist score than those published in Jan.~June, 2010, respectively. CONCLUSIONS: Experiments involving neoplasms in animals published in Chinese journals generally have not comprehensively reported the information recommended by the ARRIVE guidelines. We strongly recommend that researchers conducting such studies report this information.

The machinery of macroautophagy.

Autophagy is a primarily degradative pathway that takes place in all eukaryotic cells. It is used for recycling cytoplasm to generate macromolecular building blocks and energy under stress conditions, to remove superfluous and damaged organelles to adapt to changing nutrient conditions and to maintain cellular homeostasis. In addition, autophagy plays a critical role in cytoprotection by preventing the accumulation of toxic proteins and through its action in various aspects of immunity including the elimination of invasive microbes and its participation in antigen presentation. The most prevalent form of autophagy is macroautophagy, and during this process, the cell forms a double-membrane sequestering compartment termed the phagophore, which matures into an autophagosome. Following delivery to the vacuole or lysosome, the cargo is degraded and the resulting macromolecules are released back into the cytosol for reuse. The past two decades have resulted in a tremendous increase with regard to the molecular studies of autophagy being carried out in yeast and other eukaryotes. Part of the surge in interest in this topic is due to the connection of autophagy with a wide range of human pathophysiologies including cancer, myopathies, diabetes and neurodegenerative disease. However, there are still many aspects of autophagy that remain unclear, including the process of phagophore formation, the regulatory mechanisms that control its induction and the function of most of the autophagy-related proteins. In this review, we focus on macroautophagy, briefly describing the discovery of this process in mammalian cells, discussing the current views concerning the donor membrane that forms the phagophore, and characterizing the autophagy machinery including the available structural information.