bims-exocan 2022-07-31 papers

bims-exocan

Biomed News

on Exosomes roles in cancer

Issue of 2022–07–31
sixteen papers selected by
Muhammad Rizwan, COMSATS University

Molecular mechanisms and clinical applications of exosomes in prostate cancer.
The Exosome Journey: From Biogenesis to Regulation and Function in Cancers.
Circulating Exosome Cargoes Contain Functionally Diverse Cancer Biomarkers: From Biogenesis and Function to Purification and Potential Translational Utility.
Immune-Cell-Derived Exosomes for Cancer Therapy.
Exosomes: Biogenesis, targeting, characterisation and their potential as 'Plug & Play' vaccine platforms.
Extracellular vesicle mimetics: preparation from top-down approaches and biological functions.
MiR-1246 is responsible for lung cancer cells-derived exosomes-mediated promoting effects on lung cancer stemness via targeting TRIM17.
Exosomes in Alpha-Synucleinopathies: Propagators of Pathology or Potential Candidates for Nanotherapeutics?
Engineered extracellular vesicles and their mimetics for cancer immunotherapy.
Effects of Exosomal Viral Components on the Tumor Microenvironment.
Mesenchymal Stem Cell-Derived Antimicrobial Peptides as Potential Anti-Neoplastic Agents: New Insight into Anticancer Mechanisms of Stem Cells and Exosomes.
Isolation and characterization of extracellular vesicles and future directions in diagnosis and therapy.
Extracellular Vesicles-Oral Therapeutics of the Future.
Interplay between purinergic signalling and extracellular vesicles in health and disease.
Exosome-based strategy for degenerative disease in orthopedics: Recent progress and perspectives.
Plant extracellular vesicles and their potential in human health research, the practical approach.

Biomark Res. 2022 Jul 29. 10(1): 56

Molecular mechanisms and clinical applications of exosomes in prostate cancer.

Xiaolin Cui, Qiang Fu, Xueying Wang, Pengcheng Xia, Xianglun Cui, Xiaohui Bai, Zhiming Lu.

  Prostate cancer (PC) is a common tumor in men, and the incidence rate is high worldwide. Exosomes are nanosized vesicles released by all types of cells into multiple biological fluid types. These vesicles contribute to intercellular communication by delivering both nucleic acids and proteins to recipient cells. In recent years, many studies have explored the mechanisms by which exosomes mediate the epithelial-mesenchymal transition, angiogenesis, tumor microenvironment establishment, and drug resistance acquisition in PC, and the mechanisms that have been identified and the molecules involved have provided new perspectives for the possible discovery of novel diagnostic markers in PC. Furthermore, the excellent biophysical properties of exosomes, such as their high stability, high biocompatibility and ability to cross biological barriers, have made exosomes promising candidates for use in novel targeted drug delivery system development. In this review, we summarize the roles of exosomes in the growth and signal transmission in PC and show the promising future of exosome contributions to PC diagnostics and treatment.

Keywords:  Angiogenesis; Biomarker; Drug resistance; Epithelial-mesenchymal transition; Exosome; Prostate cancer; Targeted therapy; Tumor microenvironment

DOI:  https://doi.org/10.1186/s40364-022-00398-w
J Oncol. 2022 ;2022 9356807

The Exosome Journey: From Biogenesis to Regulation and Function in Cancers.

Yuan Li, Li Meng, Bo Li, Yanxia Li, Tao Shen, Baobing Zhao.

Exosomes are a type of small endosomal-derived vesicles ranging from 30 to 150 nm, which can serve as functional mediators in cell-to-cell communication and various physiological and pathological processes. In recent years, exosomes have emerged as crucial mediators of intracellular communication among tumor cells, immune cells, and stromal cells, which can shuttle bioactive molecules, such as proteins, lipids, RNA, and DNA. Exosomes exhibit the high bioavailability, biological stability, targeting specificity, low toxicity, and immune characteristics, suggesting their potentials in the diagnosis and treatment of cancers. They can be applied as an effective tool in the diagnostics, therapeutics, and drug delivery in cancers. This review summarizes the regulation and functions of exosomes in various cancers to augment our understanding of exosomes, which paves the way for parallel advancements in the therapeutic approach of cancers. In this review, we also discuss the challenges and prospects for clinical application of exosome-based diagnostics and therapeutics for cancers.

DOI: https://doi.org/10.1155/2022/9356807
Cancers (Basel). 2022 Jul 10. pii: 3350. [Epub ahead of print]14(14):

Circulating Exosome Cargoes Contain Functionally Diverse Cancer Biomarkers: From Biogenesis and Function to Purification and Potential Translational Utility.

Megan I Mitchell, Junfeng Ma, Claire L Carter, Olivier Loudig.

  Although diagnostic and therapeutic treatments of cancer have tremendously improved over the past two decades, the indolent nature of its symptoms has made early detection challenging. Thus, inter-disciplinary (genomic, transcriptomic, proteomic, and lipidomic) research efforts have been focused on the non-invasive identification of unique "silver bullet" cancer biomarkers for the design of ultra-sensitive molecular diagnostic assays. Circulating tumor biomarkers, such as CTCs and ctDNAs, which are released by tumors in the circulation, have already demonstrated their clinical utility for the non-invasive detection of certain solid tumors. Considering that exosomes are actively produced by all cells, including tumor cells, and can be found in the circulation, they have been extensively assessed for their potential as a source of circulating cell-specific biomarkers. Exosomes are particularly appealing because they represent a stable and encapsulated reservoir of active biological compounds that may be useful for the non-invasive detection of cancer. T biogenesis of these extracellular vesicles is profoundly altered during carcinogenesis, but because they harbor unique or uniquely combined surface proteins, cancer biomarker studies have been focused on their purification from biofluids, for the analysis of their RNA, DNA, protein, and lipid cargoes. In this review, we evaluate the biogenesis of normal and cancer exosomes, provide extensive information on the state of the art, the current purification methods, and the technologies employed for genomic, transcriptomic, proteomic, and lipidomic evaluation of their cargoes. Our thorough examination of the literature highlights the current limitations and promising future of exosomes as a liquid biopsy for the identification of circulating tumor biomarkers.

Keywords:  circulating biomarkers; early cancer detection; exosomes; extracellular vesicles; lipidomics; proteomics; transcriptomics

DOI:  https://doi.org/10.3390/cancers14143350
Mol Pharm. 2022 Jul 25.

Immune-Cell-Derived Exosomes for Cancer Therapy.

Yongmei Zhao, Tianqing Liu, Mengjiao Zhou.

  Exosomes are a type of extracellular vesicles secreted by cells in normal or pathological conditions for cell-cell communication. With immunomodulatory characteristics and potential therapeutic properties, immune-cell-derived exosomes play an important role in cancer therapy. They express various antigens on their surface, which can be employed for antigen presentation, immunological activation, and metabolic regulation, leading to the killing of cancerous cells. In addition, immune-cell-derived exosomes have received extensive attention as a drug delivery platform in effective antitumor therapy due to their excellent biocompatibility, low immunogenicity, and high loading capacity. In this review, the biological and therapeutic characteristics of immune-cell-derived exosomes are comprehensively outlined. The antitumor mechanism of exosomes secreted by immune cells, including macrophages, dendritic cells, T cells, B cells, and natural killer cells, are systematically summarized. Moreover, the applications of immune-cell-derived exosomes as nanocarriers to transport antitumor agents (chemotherapeutic drugs, genes, proteins, etc.) are discussed. More importantly, the existing challenges of immune-cell-derived exosomes are pointed out, and their antitumor potentials are also discussed.

Keywords:  antitumor mechanism; cancer therapy; exosomes; immune cells; nanocarriers

DOI:  https://doi.org/10.1021/acs.molpharmaceut.2c00407
Biotechnol J. 2022 Jul 28. e2100646

Exosomes: Biogenesis, targeting, characterisation and their potential as 'Plug & Play' vaccine platforms.

Laura E Dyball, C Mark Smales.

  Exosomes are typically characterized as spherical extracellular vesicles less than 150 nm in diameter that have been released into the extracellular environment via fusion of multivesicular bodies (MVBs) to the plasma membrane. Exosomes play a key role in cell-cell communication, vary widely in their composition and potential cargo, and are reportedly involved in processes as diverse as angiogenesis, apoptosis, antigen presentation, inflammation, receptor-mediated endocytosis, cell proliferation, and differentiation, and cell-signaling. Exosomes can also act as biomarkers of health and disease and have enormous potential use as therapeutic agents. Despite this, the understanding of how exosome biogenesis can be utilized to generate exosomes carrying specific targets for particular therapeutic uses, their manufacture, detailed analytical characterization, and methods of application are yet to be fully harnessed. In this review, we describe the current understanding of these areas of exosome biology from a biotechnology and bioprocessing aspect, but also highlight the challenges that remain to be overcome to fully harness the power of exosomes as therapeutic agents, with a particular focus on their use and application as vaccine platforms. This article is protected by copyright. All rights reserved.

Keywords:  Exosomes; biogenesis; bioprocessing; extracellular vesicles; surface display; vaccine platform

DOI:  https://doi.org/10.1002/biot.202100646
Adv Healthc Mater. 2022 Jul 28. e2200142

Extracellular vesicle mimetics: preparation from top-down approaches and biological functions.

Yuan Du, Hongyi Wang, Yang Yang, Jianfeng Zhang, Yue Huang, Shunwu Fan, Chenhui Gu, Liqing Shangguan, Xianfeng Lin.

  Extracellular vesicles (EVs) have attracted attention as delivery vehicles due to their structure, composition, and unique properties in regeneration and immunomodulation. However, difficulties during production and isolation processes of EVs limit their large-scale clinical applications. Extracellular vesicle mimetics (EVMs), prepared via top-down strategies that improve the yield of nanoparticles while retaining biological properties similar to those of EVs have been used to address these limitations. Herein, we review the preparation of EVMs and summarize their characteristics in terms of structure, composition, targeting ability, cellular uptake mechanism, and immunogenicity, as well as their strengths, limitations, and future clinical application prospects as EV alternatives. This article is protected by copyright. All rights reserved.

Keywords:  biomaterials; drug delivery; extracellular vesicle mimetics; extracellular vesicles; productivity

DOI:  https://doi.org/10.1002/adhm.202200142
Environ Toxicol. 2022 Jul 27.

MiR-1246 is responsible for lung cancer cells-derived exosomes-mediated promoting effects on lung cancer stemness via targeting TRIM17.

Zhengcheng Liu, Wei Zhao, Rusong Yang.

  The stemness of lung cancer cells contributes to drug resistance, tumor occurrence, progression, and recurrence; however, the underlying mechanisms are still fragmentary. In the present study, it was found that exosomes from cisplatin-resistant cells and spheres derived from lung cancer cells enhanced the stemness of the parental lung cancer cells. Then we screened the upregulated miRNAs in spheres derived from lung cancer cells and cisplatin-resistant lung cancer cells/exosomes compared to that in the parental lung cancer cells. It was found that miR-1246 was remarkably enriched in cisplatin-resistant lung cancer cells/exosomes and spheres. Additionally, inhibition of miR-1246 attenuated the stemness of lung cancer cells induced by exosomes from cisplatin-resistant cells and spheres. Furthermore, TRIM17 was identified to the direct target of miR-1246 in lung cancer cells. Our findings suggest that exosomal miR-1246 could be as a potential target for lung cancer treatment.

Keywords:  MiR-1246; TRIM17; cancer stem cell; exosome; stemness

DOI:  https://doi.org/10.1002/tox.23625
Biomolecules. 2022 Jul 08. pii: 957. [Epub ahead of print]12(7):

Exosomes in Alpha-Synucleinopathies: Propagators of Pathology or Potential Candidates for Nanotherapeutics?

Panagiota Mavroeidi, Maria Vetsi, Dimitra Dionysopoulou, Maria Xilouri.

  The pathological accumulation of alpha-synuclein governs the pathogenesis of neurodegenerative disorders, such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, collectively termed alpha-synucleinopathies. Alpha-synuclein can be released in the extracellular space, partly via exosomes, and this extracellular protein pool may contribute to disease progression by facilitating the spread of pathological alpha-synuclein or activating immune cells. The content of exosomes depends on their origin and includes specific proteins, lipids, functional mRNAs and various non-coding RNAs. Given their ability to mediate intercellular communication via the transport of multilevel information, exosomes are considered to be transporters of toxic agents. Beyond neurons, glial cells also release exosomes, which may contain inflammatory molecules and this glia-to-neuron or neuron-to-glia transmission of exosomal alpha-synuclein may contribute to the propagation of pathology and neuroinflammation throughout the brain. In addition, as their content varies as per their originating and recipient cells, these vesicles can be utilized as a diagnostic biomarker for early disease detection, whereas targeted exosomes may be used as scaffolds to deliver therapeutic agents into the brain. This review summarizes the current knowledge regarding the role of exosomes in the progression of alpha-synuclein-related pathology and their potential use as biomarkers and nanotherapeutics in alpha-synucleinopathies.

Keywords:  alpha-synuclein; biomarkers; exosomes; glia; neurons

DOI:  https://doi.org/10.3390/biom12070957
J Control Release. 2022 Jul 22. pii: S0168-3659(22)00433-3. [Epub ahead of print]

Engineered extracellular vesicles and their mimetics for cancer immunotherapy.

Chunping Liu, Yichao Wang, Longmei Li, Dongyue He, Jiaxin Chi, Qin Li, Yixiao Wu, Yunxuan Zhao, Shihui Zhang, Lei Wang, Zhijin Fan, Yuhui Liao.

  Extracellular vesicles (EVs) are heterogeneous membranous vesicles secreted by living cells that are involved in many physiological and pathological processes as intermediaries for intercellular communication and molecular transfer. Recent studies have shown that EVs can regulate the occurrence and development of tumors by transferring proteins, lipids and nucleic acids to immune cells as signaling molecules. As a new diagnostic biomarker and drug delivery system, EVs have broad application prospects in immunotherapy. In addition, the breakthrough of nanotechnology has promoted the development and exploration of engineered EVs for immune-targeted therapy. Herein, we review the uniqueness of EVs in immune regulation and the engineering strategies used for immunotherapy and highlight the logic of their design through typical examples. The present situation and challenges of clinical transformation are discussed, and the development prospects of EVs in immunotherapy are proposed. The goal of this review is to provide new insights into the design of immune-regulatory EVs and expand their application in cancer immunotherapy.

Keywords:  Cancer immunotherapy; Exosomes; Extracellular vesicles; Immune reprogramming; Immunogenic cell death

DOI:  https://doi.org/10.1016/j.jconrel.2022.05.062
Cancers (Basel). 2022 Jul 21. pii: 3552. [Epub ahead of print]14(14):

Effects of Exosomal Viral Components on the Tumor Microenvironment.

Jing Li, Yan Zhang, Bing Luo.

  Exosomes are extracellular membrane vesicles with a diameter of 30-100 nm, produced by different eukaryotic cells that contain multitudinous lipids, nucleic acids, and proteins. They transfer membrane components and nucleic acids between cells, thereby performing an information exchange between cells. Many studies have shown that a variety of tumor-associated viruses can exert their biological functions through exosomes. The tumor microenvironment (TME) is very important in the occurrence, development, and chemoresistance of tumors. It is composed of tumor cells, fibroblasts, endothelial cells, immune cells, stromal cells, and acellular components, such as exosomes and cytokines. This review focuses on the effects of virus-related components secreted by tumor cells over the TME in several virus-associated cancers.

Keywords:  cancers; exosome; oncovirus; tumor microenvironment

DOI:  https://doi.org/10.3390/cancers14143552
Front Cell Dev Biol. 2022 ;10 900418

Mesenchymal Stem Cell-Derived Antimicrobial Peptides as Potential Anti-Neoplastic Agents: New Insight into Anticancer Mechanisms of Stem Cells and Exosomes.

Kasra Moeinabadi-Bidgoli, Malihe Rezaee, Hamidreza Rismanchi, Mohaddese Malek Mohammadi, Amirhesam Babajani.

  Mesenchymal stem cells (MSCs), as adult multipotent cells, possess considerable regenerative and anti-neoplastic effects, from inducing apoptosis in the cancer cells to reducing multidrug resistance that bring them up as an appropriate alternative for cancer treatment. These cells can alter the behavior of cancer cells, the condition of the tumor microenvironment, and the activity of immune cells that result in tumor regression. It has been observed that during inflammatory conditions, a well-known feature of the tumor microenvironment, the MSCs produce and release some molecules called "antimicrobial peptides (AMPs)" with demonstrated anti-neoplastic effects. These peptides have remarkable targeted anticancer effects by attaching to the negatively charged membrane of neoplastic cells, disrupting the membrane, and interfering with intracellular pathways. Therefore, AMPs could be considered as a part of the wide-ranging anti-neoplastic effects of MSCs. This review focuses on the possible anti-neoplastic effects of MSCs-derived AMPs and their mechanisms. It also discusses preconditioning approaches and using exosomes to enhance AMP production and delivery from MSCs to cancer cells. Besides, the clinical administration of MSCs-derived AMPs, along with their challenges in clinical practice, were debated.

Keywords:  angiogenesis; antimicrobial peptides; apoptosis; cell cycle; exosomes; mesenchymal stem cell; multidrug resistance

DOI:  https://doi.org/10.3389/fcell.2022.900418
Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2022 Jul 27. e1835

Isolation and characterization of extracellular vesicles and future directions in diagnosis and therapy.

Karina P De Sousa, Izadora Rossi, Mahamed Abdullahi, Marcel Ivan Ramirez, Dan Stratton, Jameel Malhador Inal.

  Extracellular vesicles (EVs) are a unique and heterogeneous class of lipid bilayer nanoparticles secreted by most cells. EVs are regarded as important mediators of intercellular communication in both prokaryotic and eukaryotic cells due to their ability to transfer proteins, lipids and nucleic acids to recipient cells. In addition to their physiological role, EVs are recognized as modulators in pathological processes such as cancer, infectious diseases, and neurodegenerative disorders, providing new potential targets for diagnosis and therapeutic intervention. For a complete understanding of EVs as a universal cellular biological system and its translational applications, optimal techniques for their isolation and characterization are required. Here, we review recent progress in those techniques, from isolation methods to characterization techniques. With interest in therapeutic applications of EVs growing, we address fundamental points of EV-related cell biology, such as cellular uptake mechanisms and their biodistribution in tissues as well as challenges to their application as drug carriers or biomarkers for less invasive diagnosis or as immunogens. This article is categorized under: Diagnostic Tools > Biosensing Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease.

Keywords:  EV analysis; EV characterization; EV therapeutics; extracellular vesicles; isolation methods

DOI:  https://doi.org/10.1002/wnan.1835
Int J Mol Sci. 2022 Jul 07. pii: 7554. [Epub ahead of print]23(14):

Extracellular Vesicles-Oral Therapeutics of the Future.

Martyna Cieślik, Katarzyna Nazimek, Krzysztof Bryniarski.

  Considered an artifact just after discovery, the possibility of oral delivery of extracellular vesicles (EVs) and their functional cargos has recently gained much research attention. EVs from various sources, including edible plants, milk, bacteria and mammalian cells, have emerged as a platform for miRNA and drug delivery that seem to induce the expected immune effects locally and in distant tissues after oral administration. Such a possibility greatly expands the clinical applicability of EVs. The present review summarizes research findings that either support or deny the biological/therapeutical activity of orally administered EVs and their role in cross-species and cross-kingdom signaling.

Keywords:  exosomes; extracellular vesicles; immune modulation; nanoparticles; nanovesicles; oral treatment

DOI:  https://doi.org/10.3390/ijms23147554
Biochem Pharmacol. 2022 Jul 26. pii: S0006-2952(22)00286-6. [Epub ahead of print] 115192

Interplay between purinergic signalling and extracellular vesicles in health and disease.

Valentina Carotti, Juan P Rigalli, Jenny van Asbeck-van der Wijst, Joost G J Hoenderop.

  Purinergic signalling is a receptor-mediated process characterized by the binding of extracellular nucleotides and nucleosides to purinergic receptors, which results in the activation intracellular signalling pathways, and, ultimately, leads to changes in cell physiology. Purinergic signalling has been related to the regulation of important physiological processes (e.g., renal electrolyte reabsorption; platelet aggregation; immune response). In addition, it has been associated with pathophysiological situations such as cancer and inflammation. Extracellular vesicles (EVs) are nanoparticles released by all cells of the organism, which play a key role in cell-cell communication. In this regard, EVs can mediate effects on target cells located at distant locations. Within their cargo, EVs contain molecules with the potential to affect purinergic signalling at the target cells and tissues. Here, we review the studies addressing the regulation of purinergic signalling by EVs based on the cell type or tissue where the regulation takes place. In this regard, EVs are found to play a major role in modulating the extracellular ATP levels and, specially, adenosine. This has a clear impact on, for instance, the inflammatory and immune response against cancer cells. Furthermore, we discuss the data available on the regulation of EV secretion and its cargo by purinergic signalling. Here, a major role of the purinergic receptor P2X7 and again, an impact on processes such as inflammation, immune response and cancer pathogenesis has been established. Finally, we highlight uninvestigated aspects of these two regulatory networks and address their potential as therapeutic targets.

Keywords:  ATP; Extracellular vesicles; adenosine; exosomes; purinergic receptors; purinergic signalling

DOI:  https://doi.org/10.1016/j.bcp.2022.115192
J Orthop Translat. 2022 Sep;36 8-17

Exosome-based strategy for degenerative disease in orthopedics: Recent progress and perspectives.

Rongjie Wu, Haotao Li, Chuanwei Sun, Jialin Liu, Duanyong Chen, Haiyang Yu, Zena Huang, Sien Lin, Yuanfeng Chen, Qiujian Zheng.

   Background: Degenerative diseases in orthopaedics have become a significant global public health issue with the aging of the population worldwide. The traditional medical interventions, including physical therapy, pharmacological therapy and even surgery, hardly work to modify degenerative progression. Stem cell-based therapy is widely accepted to treat degenerative orthopaedic disease effectively but possesses several limitations, such as the need for strict monitoring of production and storage and the potential risks of tumorigenicity and immune rejection in clinical translation. Furthermore, the ethical issues surrounding the acquisition of embryonic stem cells are also broadly concerned. Exosome-based therapy has rapidly grown in popularity in recent years and is regarded as an ideal alternative to stem cell-based therapy, offering a promise to achieve 'cell-free' tissue regeneration.
Methods: Traditionally, the native exosomes extracted from stem cells are directly injected into the injured site to promote tissue regeneration. Recently, several modified exosome-based strategies were developed to overcome the limitations of native exosomes, which include mainly exogenous molecule loading and exosome delivery through scaffolds. In this paper, a systematic review of the exosome-based strategy for degenerative disease in orthopaedics is presented.
Results: Treatment strategies based on the native exosomes are effective but with several disadvantages such as rapid diffusion and insufficient and fluctuating functional contents. The modified exosome-based strategies can better match the requirements of the regeneration in some complex healing processes.
Conclusion: Exosome-based strategies hold promise to manage degenerative disease in orthopaedics prior to patients reaching the advanced stage of disease in the future. The timely summary and highlights offered herein could provide a research perspective to promote the development of exosome-based therapy, facilitating the clinical translation of exosomes in orthopaedics.
Translational potential of this article: Exosome-based therapy is superior in anti-senescence and anti-inflammatory effects and possesses lower risks of tumorigenicity and immune rejection relative to stem cell-based therapy. Exosome-based therapy is regarded as an ideal alternative to stem cell-based therapy, offering a promise to achieve 'cell-free' tissue regeneration.

Keywords:  Degenerative disease; Exosome; Orthopaedics

DOI:  https://doi.org/10.1016/j.jot.2022.05.009
Physiol Res. 2022 Jul 29. 71(3): 327-339

Plant extracellular vesicles and their potential in human health research, the practical approach.

M Kocholata, J Maly, J Martinec, H Auer Malinska.

Extracellular vesicles are small membrane particles (30-1000 nm) released by Bacteria, Eukaryotes and Archaea. They have been shown to play an important role in intracellular and intercellular communication, within and between kingdoms via transport of bioactive molecules. Thus, they can be involved in altering gene expression and regulation of physiological and pathological processes of the recipient. Their unique properties make extracellular vesicles a perfect candidate vector for targeted drug delivery or a biomarker. For a long time, animal and mainly mammal extracellular vesicles have been used in research. But for plants, there had been speculations about the existence of nanovesicles due to the presence of a cell wall. Today, awareness of plant extracellular vesicles is on the rise and their research has proved they have various functions, such as protein secretion, transport of bioactive molecules or defense against pathogens. Further potential of plant extracellular vesicles is stressed in this review.