Arousal may be understood on a spectrum, with excessive sleepiness, cognitive dysfunction, and inattention on one side, a wakeful state in the middle, and hypervigilance, panic, and psychosis on the other side. However, historically, the concepts of arousal and stress have been challenging to define as measurable experimental variables. Divergent efforts to study these subjects have given rise to several disciplines, including neurobiology, neuroendocrinology, and cognitive neuroscience. We discuss technological advancements that chronologically led to our current understanding of the arousal system, focusing on the multifaceted nucleus locus coeruleus. We share our contemporary perspective and the hypotheses of others in the context of our current technological capabilities and future developments that will be required to move forward in this area of research. The concept of arousal is one that is best defined on a spectrum. Through a clinical lens, behaviors relating to inattention or cognitive dysfunction associated with under-stimulation may reflect mild to moderate levels of an arousal deficiency, with excessive daytime sleepiness and sedation being symptoms of the most extreme cases. On the other hand, behavior associated with hypervigilance, insomnia, overstimulation, and fear or panic may indicate a mild or moderate level of excessive arousal, with psychosis and hallucinations being symptoms in the most extreme cases. In the center of the spectrum, arousal is a state of wakefulness in which an individual is awake, alert, able to create and problem solve . HoweverCognitive psychology, neurobiology, neuroendocrinology, and cognitive neuroscience have shared technological advancements expanding our understanding of the behavioral, network-, circuit- and synaptic-level responses to stimuli. However, silos of advancements within each discipline persist, continuing to hinder the potential for interdisciplinary synergism. In perspective shared by Pessoa, Pessoa describes a significant modern challenge in brain-behavior research embodied by a granular focus on “causal explanations” at the expense of theoretical and conceptual descriptions that may be shared and provide deeper understanding across disciplines .In the sections that follow, we discuss the Locus Coeruleus (LC)- Norepinephrine (NE) system in the context of this quandary as it illustrates a system with interdisciplinary interest that has made significant strides in recent years and yet lacks a common vernacular to facilitate cross-disciplinary communication. The LC-NE system is involved in various neurobiological processes, including the modulation of sleep-wake cycles, facilitation of attention, responses to stress, initiation memory formation, and retrieval. It modulates blood flow, metabolism, and the distribution of oxygen and glucose throughout the brain. Thus, it is evident that the LC is a brain region that directly or indirectly has been investigated or described across several scientific disciplines. Though scarce, we discuss works in the literature that integrate the findings of these efforts across multiple levels of neurobiological function –8.We start by providing historical context, first by the recollection of the neuroanatomical and physiological studies that led to our current understanding of the LC-NE system and subsequently discussing the evolution of Arousal Theory. Because the broader fields of cognitive psychology and neurobiology have historically advanced our understanding of arousal, primarily independently of each other, we first discuss them separately. We then transition to a synthesis of recent literature that brings to light a critical intersection of these fields by discussing current theories that directly or indirectly implicate the LC-NE system in large-scale network dynamics under various arousal states. Finally, we discuss pathology related to the LC-NE system dysfunction, emphasizing hyper-arousal in stress-related disorders. Along the way, we discuss technological advancements that preceded each milestone of conceptual evolution and the past and current day challenges that have emerged over time.Early scientific observations on how the body adapts to a life-threatening situation, aging, or disease, are the foundations of stress-related research as we know it today . The earIn the decades that followed the initial conception of “stress,” scientists across disciplines debated how to define the stress response and measure it under experimental conditions . Some toMeanwhile, other investigators refuted the idea of a unified explanation of such responses. They emphasized the diversity of stress effects , noting Technological advancements facilitated continued growth in neurobiology and neuroendocrinology, particularly the monumental discovery of microelectrodes . AlthougEmanating from Cannon's work, and during Harris's time, the endocrinologist Selye became known for conceptualizing the first stress model, known as General Adaptation Syndrome. Selye published widely on General Adaptation Syndrome, using his work and his peers  to suppoThe foundation of neuroendocrinology provided by Harris' work was significantly advanced when a trainee of Selye, Guillemin, and Schally independently demonstrated that a hypothalamic factor whose chemical structure was not yet defined stimulated the release of adrenocorticotropin releasing hormone (ACTH) from the pituitary. Thus, emerged the first description of the corticotropin-releasing factor (CRF). The scientific lineage of Selye's work, from his trainee, Guillemin, and later, Guillemin's trainee, Wylie Vale, would define the adrenal-hypothalamic-pituitary response to stress as we know it today .The initial conception in 1931 by Ernst Ruska, and the later application of the transmission electron microscope (TEM) to biological samples in the 1940s, enabled scientists to observe the subcellular structure of neurons for the first time . The useThe work of Coons et al., who first used fluorescein-labeled antibodies to localize antigens at the light microscopic level, inspired increasingly advanced antigen-detection methods , 44. TheA line of neuroanatomical studies employed indirect labeling techniques to visualize Tyrosine Hydroxylase (TH), the first and rate-limiting enzyme of catecholamine biosynthesis . These sWylie Vale et al. seminal work characterized a 41-residue peptide found to induce corticotropin-like and endorphin-like immunoreactivity in the anterior pituitary cells. Subsequent studies by this group characterized CRF-immunoreactive nerve fibers in the hypothalamus , its effAt this time, scientists believed CRF could mediate stress-induced LC activation based on anatomical evidence that demonstrated dense CRF-immunoreactive fibers within the dendritic pericoerulear regions surround the LC core . The refLater physiological studies supported earlier neuroanatomical studies by confirming that CRF engages the LC during acute and chronic cognitive and physical stressors , 87, 88.Initially described in 1929, Berger described an instrument capable of measuring electrical activity waves that send pulses across the brain . Today, 2), and the amount of synchronization across neurons . These are the essential elements of modern neural dynamics and set the foundation for the rapidly growing field of Neural Field Theory. Five EEG frequency bands are defined by the frequency ranges in which they occur and are associated with diverse cognitive processes. The delta band (1–4 Hz) is the slowest but has the highest amplitude and is related to non-REM slow-wave sleep (SWS) (The EEG provides information about the speed of an oscillation (frequency), the amount of energy in a frequency band  . The theep (SWS) . Meanwhiep (SWS) . In contep (SWS) , and whiep (SWS) , abnormaep (SWS) .The use of the EEG in research observing brain states provided the first evidence for the involvement of the LC and other brainstem structures in arousal. The pivotal work of Moruzzi and Magoun revealed that stimulation of the reticular formation elicited an excitation that ascended from the lower bulbar region of the brainstem, through the pons, midbrain tegmentum, and into the caudal diencephalon . MoreoveSubsequent lesion studies implicated a mid-pontine, pre-trigeminal area of the brainstem in behavioral state-dependent EEG activity that was varied when the animal was awake and alert but synchronized during sleep . Subsequin vitro and were demonstrated to have an essential role in mediating transitions of sleep to wake in vivo  on surrounding neurons and glial cells , 143. ThAmidst the progress of charting the involvement of CRF in stimulating the LC-NE system in the physiological response to stress, Sterling and Schulkin and Sterling introduced the concept of allostasis as a process of reestablishing stability in response to a challenge , 153. ShThe emergence of brain imaging technologies, first Positron Emission Topography (PET) in 1974, and subsequently Blood Oxygen Level Dependent (BOLD) functional magnetic resonance imaging (fMRI) in 1990, enabled scientists across the spectrum of neurobehavioral research to collect and analyze precise spatial information about the brain regions that gave rise to cognitive processes [; revieweA pivotal advancement came with the discovery that fMRI and EEG could be used in combination to derive high-resolution spatio-temporal information about neuronal events at the timescale of tens of milliseconds . This adIn their synthesis of Arousal Theory with PET and BOLD fMRI data available at the time, Corbetta and Shulman build on the concept of top-down and bottom-up processing of information during the orienting reflex, suggesting that attention is controlled by two neuroanatomically defined systems . The DorThe continued investigation of cognitive processes using combined fMRI and EEG led to the discovery of the first resting-state large-scale brain network. Studies in non-human primates and humans converged to identify the Default Mode Network (DMN), defined by core regions that were reliably activated during internally focused tasks such as forming perceptions of others or retrieving memories –172. TheRegions of the VAN are often combined with several limbic structures to collectively form the Salience Network (SN), which detects, integrates, and filters incoming sensory, autonomic, and emotional information to determine the relative importance of a stimulus , 8. ThesIn terms of network analysis, the strength and direction of a relationship between large scale networks are represented as edges weighted by between-network correlation coefficients. Importantly, in addition to positive correlations (excitatory/engaged), there can also be negative or anti-correlation (inhibitory/disengaged) between edges . Anti-coThese factors become important when considering how large-scale networks interact with each other. Based on their specialized functions, it is advantageous for large-scale networks to operate independently, in synchrony, or in opposition, under various circumstances. The DMN, known to be engaged during stimulus-independent tasks or those related to internal thought, is usually suppressed during CEN activation –184. WhiA recent study employed hierarchal cluster analysis and spectral dynamic causal modeling to demonstrate that core regions of the SN and DAN that terminate in core regions of the DMN were negative (inhibitory), while connections arising in the core of DMN that terminate in SN or DAN regions were weakly positive (excitatory) . MoreoveIn discussing their physiological study on LC responses under different stages of arousal in 1981, Aston-Jones and Bloom proposed that the LC-NE “system may serve to facilitate transitions between global behavioral states” . At the Dalley et al. proposed that an influx of NE to the PFC for novel stimuli may signal a mismatch between action and reward to promote behavioral modification . An actiIn a severe departure from the heightening complexity of LC-NE functional theories at the time, Bouret and Sara put forth the elegant notion that the evolved LC-NE system operates analogously to a small number of synchronized neuromodulatory cells observed in crustacean . The cruAs previously discussed, during task states that do not require focused attention, the LC-NE system is characterized by a high tonic baseline, and investigators have proposed that this state is linked with the generation of spontaneous thoughts . In contper se but instead asserts that the anterior insula, a critical component of the SN, initiates the switch in behavioral states through large-scale network dynamics. This side argues that effective switching between brain states is at least partially dependent on the function of the SN, consisting of anterior insular and dorsal anterior cingulate regions  timescales , 206. A An excellent, recent review  highlighEmerging information on the LC-NE system indicates a growing need for computational tools. Until recently, the LC-NE system had been considered a homogenous nucleus of noradrenergic cells that fire synchronously and serve as a reactionary system that both centrally alerts and peripherally prepares for a behavioral response in the face of environmental threats to survival. However, accumulating data suggest that the LC may operate in a modular fashion, with specialized sub-regions that facilitate specific aspects of behavioral responses and cognition. The data are convincing, presenting anatomical, molecular, and electrophysiological evidence that at least two distinct populations of LC neurons exist that project to the prefrontal vs. motor cortex and are electrophysiologically and biochemically distinct . In supp42; , likely under phasic conditions , 252. ThIn line with our hypothesis, a recently published theory presents a compelling argument for the impaired phasic discharge of LC neurons in neurodegenerative disease. Janitzky proposes that the presence of persistent high tonic discharge may impair the function and protective actions of phasic discharge . First, 42 plaques and hyperphosphorylated tau pathology in several mouse models of AD (In the context of Mather and Harley's GANE hypothesis, the role of GABAergic interneurons is of particular interest. In this regard, the LC-NE system utilizes interneurons as lateral agents of inhibition to heighten the signal of salient information while minimizing the signal of irrelevant information. A study by the Mucke and Palop group  suggestsls of AD , 261. Th42 plaques are activated—an event thought to exacerbate cognitive deficits in AD patients. Nuntagij et al. utilized 3D reconstruction EM in both 3 × Tg mice and naturally aged dogs to extensively describe the close spatial relationship between Aβ42 deposits and the neutrophil, revealing entangled and branched plaques engulf soma, and apical dendrites (42 aggregation may disrupt intracellular transport, leading to the dysfunction of mitochondria, potentially resulting in autophagic degeneration (Aside from accelerating the advancement of our understanding of the physiological properties of the brain, computational neuroscience is also a leading edge of modern neuroanatomy. The emergence of 3D EM reconstruction has allowed for the in-depth spatial, and morphological analysis of the brain microenvironment of healthy and pathological disease states that are complex and varied, such as in AD. One facet of AD pathology is an inflammatory state in which microglia and astrocytes surrounding affected neurons and Aβendrites . The abiendrites . Thus, 3neration .A recent study used structural equation modeling on an open-access dataset of magnetization transfer images from the Cambridge Center for Aging and Neuroscience cohort to test the hypothesis that LC signal intensity values would be more closely related to NE-dependent functions in older adults compared to younger adults. The investigators concluded that age-related reduction of LC structural integrity is associated with cognitive and behavioral impairments . In lineIn light of the computational advancements discussed throughout this review, the expansive field of single-cell transcriptomics warrants mention here. Recent studies from the Allen Institute and others unveil a comprehensive view of genomic cortical neuron heterogeneity that cannot be overstated. The results predict the existence of at least 37 distinct peptidergic neuron types that are characterized by transcript abundance and taxonomic profiling, and compose cortical neuromodulatory networks . As we pAs we have moved forward, technological advancements in the evolving field of computational neuroscience have afforded scientists unprecedented resolution in the study of neural dynamics across spatial and temporal scales. In reference to the LC-NE system, this evolution has occurred across disciplines for decades. Today, with instruments of ever-increasing spatial and temporal resolution and data-driven analytics, the decades to come promise to reveal LC-NE system mechanics as they progressively unfold in space over time. The continued study of this multifaceted system in the context of stress- or arousal-related psychiatric disorders, including neurodegeneration, may facilitate a deeper understanding of within-network and between-network LC-NE dynamics. In time, undoubtedly, this will directly or indirectly lead to the discovery of novel therapeutics to treat the underlying systemic brain imbalances that drive the symptoms and progression of a wide array of such disorders.EV and JR conceived the idea. JR wrote the manuscript. EV checked the manuscript for accuracy. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


It is now widely appreciated that gastrointestinal function is central to the regulation of metabolic homeostasis. Following meal ingestion, the delivery of nutrients from the stomach into the small intestine  is tightly controlled to optimise their subsequent digestion and absorption. The complex interaction of intraluminal nutrients  with the small and large intestine induces the release of an array of gastrointestinal hormones from specialised enteroendocrine cells (EECs) distributed in various regions of the gut, which in turn to regulate gastric emptying, appetite and postprandial glucose metabolism. Stimulation of gastrointestinal hormone secretion, therefore, represents a promising strategy for the management of metabolic disorders, particularly obesity and type 2 diabetes mellitus (T2DM). That EECs are distributed distinctively between the proximal and distal gut suggests that the region of the gut exposed to intraluminal stimuli is of major relevance to the secretion profile of gastrointestinal hormones and associated metabolic responses. This review discusses the process of intestinal digestion and absorption and their impacts on the release of gastrointestinal hormones and the regulation of postprandial metabolism, with an emphasis on the differences between the proximal and distal gut, and implications for the management of obesity and T2DM. As the key interface between ingested nutrients and the body, the gastrointestinal tract is now recognised to play a central role in regulating postprandial metabolism. During the fasting state, ghrelin is released from the gastric Gr-cells to drive food intake . After mFollowing meal ingestion, the stomach stores the ingested content and grinds digestible solids into small particles prior to delivering them into the small intestine. The latter occurs at a relatively constant caloric rate , driven by antral and duodenal contractions against tonic and phasic pyloric pressures , to optiThe digestive products are transported by peristalsis and absorbed by passive diffusion and/or active transport via distinctive transporters at specific regions of the gut. For example, absorption of glucose involves both active transport from the lumen into the enterocytes via sodium-glucose cotransporter 1 (SGLT-1) and facilitated diffusion across the basolateral side of enterocytes through the glucose transporter 2 (GLUT2), taking place predominantly in the upper small intestine ,8,9,10. While intestinal EECs maintain a low secretory profile during fasting, the interaction between intraluminal contents and EECs during the digestive process represents the main driver of the secretion of gastrointestinal hormones. The latter, including GLP-1, GIP, CCK and PYY released from distinctive EECs in various regions of the gastrointestinal tract , are nowEECs are equipped with a variety of chemo-sensors linking the sensing of intraluminal stimuli to the secretion of gastrointestinal hormones. For example, carbohydrates can be detected by both their sweet taste through sweet taste receptors (STRs) ,23,24, aGLP-1 is secreted from the enteroendocrine L-cells located mainly in the ileum and colon in response to each of the macronutrients, although fat, relative to glucose and protein, is generally more potent at stimulating GLP-1 secretion when administered into the duodenum in humans ,45. HoweGLP-1 binds to its receptor, expressed in a variety of metabolic tissues, to regulate glucose, lipid and energy metabolism. Within the pancreas, GLP-1 stimulates insulin secretion from the pancreatic β-cells and suppresses glucagon secretion from the α-cells in a glucose-dependent manner . For thiEffects of GLP-1 signalling on lipid metabolism have been noted in both preclinical and clinical studies. GLP-1 has been shown to inhibit the production of lipid proteins ) that are involved in the synthesis and transport of chylomicrons in the enterocytes, thereby improving lipid metabolism in rodents ,63. SimiGLP-1 also has the capacity to regulate energy intake and expenditure. Both exogenous GLP-1 and the GLP-1 receptor agonists suppress energy intake ,67, and GIP is released from the enteroendocrine K-cells, distributed predominantly in the upper small intestine . GIP is In health, GIP stimulates insulin secretion in a glucose-dependent manner by binding to the GIP receptor expressed on the pancreatic β-cells , which cCCK is secreted from the enteroendocrine I-cells located in the duodenum and upper jejunum . In miceExogenous CCK is reported to attenuate the postprandial glycaemic excursion in humans. This effect is secondary to the slowing of gastric emptying, rather than a direct effect on glucose metabolism; intravenous administration of CCK at physiological doses diminishes the glycaemic response to an oral, but not an intraduodenal, glucose load in healthy males . SimilarPYY is a 36-amino-acid peptide co-released with GLP-1 from L-cells . Like otPYY participates in the regulation of appetite and energy intake. PYY-null mice exhibit increased daily food intake and weight gain when compared with wild type mice, and this phenotype is reversed with PYY3-36 administration . PYY binPYY may be also involved in the regulation of postprandial glycaemia, given its effect to slow gastric emptying in both rodents and humans ,126,127.The upper small intestine  represents a major site of nutrient absorption. Given that the delivery of nutrients into the small intestine is controlled by gastric emptying, it is not surprising that the rate of nutrient absorption (such as glucose) is related directly to the rate of gastric emptying . In the EECs secreting GIP, CCK, GLP-1 and PYY exhibit high regional specificity of distribution along the gastrointestinal tract. Their secretory profiles are, therefore, largely dependent on the region of the gut exposed to intraluminal stimuli. In response to meal ingestion, increments in plasma GIP and CCK usually occur earlier than those of GLP-1 and PYY , consistThe extent to which nutrients are delivered to the more distal regions of the gut is dependent not only on their rate of entry to the small intestine but also on their digestion and absorption in the upper gut. For example, ablation or inhibition of SGLT-1 that reduces proximal intestinal glucose absorption augments the GLP-1 and PYY responses to oral glucose in rodents ,168. In Compared with intraduodenal infusion, administration of nutrients directly into the jejunum or ileum is more effective at stimulating GLP-1 and PYY secretion ,185,186.As discussed, the upper gut coordinates the delivery of nutrients for intestinal digestion and absorption, and is primarily responsible for the release of GIP and CCK after meals, whereas the interaction of intraluminal nutrients and bioactive compounds with the distal gut gives rise to the secretion of both GLP-1 and PYY. These variations in nutrient absorption and gastrointestinal hormone secretion along the gastrointestinal tract are of major relevance to the regulation of postprandial glycaemia and appetite.It is now widely recognised that the rate of gastric emptying represents a major determinant of the glycaemic profile in response to carbohydrates in both health and diabetes ,199. WhiIn health, GIP contributes to approximately 50% of the incretin effect  and may However, when nutrients are administered intraduodenally, a threshold of the caloric load is required to achieve suppression of appetite , indicatIn recognition that the gut microbiota are an essential regulator of the host energy metabolism , and thaThe gastrointestinal tract serves not only as the site of nutrient digestion and absorption but also as an endocrine organ secreting a variety of gastrointestinal hormones as a result of the complex interaction between ingested nutrients, bioactive compounds and EECs to regulate postprandial glucose metabolism and energy intake. Given the major difference in the distribution of EECs between the upper and lower gut, the load and delivery of nutrients, as well as the digestive processes in the gastrointestinal tract, have major implications on how these EECs are stimulated. Exposure of the upper gut to nutrients is associated with predominantly GIP and CCK release, whereas increasing the delivery of nutrients to the distal small intestine and colon is associated with augmented secretion of GLP-1 and PPY. These distal gut hormones appear more potent in mediating postprandial glucose metabolism and suppressing energy intake than those secreted from the proximal gut. Accordingly, the distal gut is becoming an appealing target for the management of T2DM and obesity, using nutritional, pharmacological or surgical approaches to increase its exposure to nutrients and other bioactive compounds. Future development in this area is likely to yield novel therapies for T2DM and obesity of high efficacy without the need for surgical procedures.


Infrared spectroscopy has long been used to characterize chemical compounds, but the applicability of this technique to the analysis of biological materials containing highly complex chemical components is arguable. However, recent advances in the development of infrared spectroscopy have significantly enhanced the capacity of this technique in analyzing various types of biological specimens. Consequently, there is an increased number of studies investigating the application of infrared spectroscopy in screening and diagnosis of various diseases. The lack of highly sensitive and specific methods for early detection of cancer has warranted the search for novel approaches. Being more simple, rapid, accurate, inexpensive, non-destructive and suitable for automation compared to existing screening, diagnosis, management and monitoring methods, Fourier transform infrared spectroscopy can potentially improve clinical decision-making and patient outcomes by detecting biochemical changes in cancer patients at the molecular level. Besides the commonly analyzed blood and tissue samples, extracellular vesicle-based method has been gaining popularity as a non-invasive approach. Therefore, infrared spectroscopic analysis of extracellular vesicles could be a useful technique in the future for biomedical applications. In this review, we discuss the potential clinical applications of Fourier transform infrared spectroscopic analysis using various types of biological materials for cancer. Additionally, the rationale and advantages of using extracellular vesicles in the spectroscopic analysis for cancer diagnostics are discussed. Furthermore, we highlight the challenges and future directions of clinical translation of the technique for cancer. Cancer is a major cause of death worldwide, accounting for a staggering estimated 9.6 million deaths in 2018 [To date, the gold standard for most cancer diagnosis is still the microscopic evaluation of stained tissue samples by pathologists, which is performed when cancerous or pre-cancerous lesions are observable and already contain significant genetic changes. Moreover, the use of the histopathological diagnosis is invasive, time consuming and has limited sensitivity as it depends heavily on the subjective judgement of pathologists which leads to intra- and inter-observer variations. Therefore, misdiagnosis with high false negative and false positive rates is common in tissue assessment . In factBiomarkers, which are defined as disease-related molecular changes in body fluids and tissues , are essTraditionally, FTIR spectroscopy has been used by chemists for the characterization of molecular structures. Nonetheless, the potential of the technique to analyze biological specimens as a cancer diagnostic tool has been recognized for decades . The relVarious types of biological materials including blood ,17,18,19In this review, we discuss on the clinical applications of FTIR spectroscopy for cancer using biological materials ranging from the most commonly used blood and tissue specimens to the infrequently used urine and sputum samples. Additionally, the rationale and advantages of using EVs for FTIR spectroscopic analysis in cancer screening and diagnosis are discussed. Furthermore, we highlight the challenges and future directions in clinical translation of the technique for cancer.−1 of the electromagnetic spectrum, the spectral regions typically measured for examining these specimens are the fingerprint region (1450–600 cm−1) as well as the amide I and II region (1700–1500 cm−1). Higher-wavenumber region (3500–2550 cm−1) is correlated to stretching vibrations including C–H, O–H, N–H and S–H, while lower-wavenumber regions are usually associated with bending and carbon skeletal fingerprint vibrations [FTIR spectroscopy detects biochemical compositions including nucleic acids, proteins, lipids and carbohydrates within biological samples by precisely identifying molecular conformations, bonding types, functional groups and intermolecular interactions of which the specimen is composed. As each molecule has a unique spectrum depending on the wavelength and quantity of infrared radiation being absorbed, IR spectroscopy produces a signature spectral fingerprint of absorbance peaks for multiplex parameters of genome, lipidome, proteome and metabolome in the examined sample. Essentially, the biochemical fingerprint changes are unique to the molecular alterations in specific diseases, providing valuable diagnostic information for each patient’s health status. As biological materials absorb energy in the mid-IR region  in patient cases just as other diagnostic tests, the technique shows promising results for many different types of cancer when compared to existing diagnostic tests. Moreover, studies comparing different classification, stages and grades of cancer through FTIR analysis have been performed and are discussed in the following section. Although some studies may be preliminary and the sample size may be limited, these investigations demonstrated the strong potential of FTIR spectroscopy as a highly sensitive, specific and accurate cancer diagnostic tool that is valuable for further investigation and development.Effective cancer management requires accurate staging and grading of the disease to establish suitable treatments, predict clinical behavior of malignancies and facilitate interchange of precise information between clinicians. Cancer staging denotes the anatomic extent of the disease spread. The internationally accepted staging criteria for cancer, the tumor-node-metastasis (TNM) system, comprises: (1) tumor size and local growth (T); (2) extent of lymph node metastases (N); and 3) occurrence of distant metastases (M) . The TNM occurrenLima et al. [An essential potential application of FTIR spectroscopy is its use in accurate cancer classification, staging and grading. a et al.  have acha et al. . On the a et al.  investiga et al. ,55, witha et al. .While the current gold standard for most cancer diagnosis remains as the invasive histopathological diagnosis, this subjective and time-consuming method warrants novel diagnostic techniques with improved accuracy in classification, staging and grading for cancer management. Due to the unique anatomical spread patterns of cancer, each cancer type may require distinct classification system. FTIR spectroscopy may represent a better technique as it could provide specific spectral signatures for each unique disease, including each different stage and grade of each cancer type. Moreover, many investigations on the application of FTIR spectroscopy have demonstrated its strong potential in this important clinical need, which may substantially enhance cancer management and improve patient care.In this era of technological advancement, automation has arisen in many different fields, including the diagnosis of cancer. Following the reduced cost of electronic components, computers with enhanced processing capabilities and memory capacity are built, leading to the rise of computer aided/assisted diagnosis (CAD) which combines algorithms or methods from pattern recognition and digital image processing . As histImportantly, the application of automated FTIR spectroscopy in clinical diagnostic settings has been investigated to improve accuracy and reproducibility of cancer diagnosis, while omitting the need for complex and time-consuming clinical processing of tissue biopsy samples. Automated marker-free histopathological annotation of lung tumor classes and subtypes of adenocarcinoma without further treatment of the tissue samples have been conducted by Großerueschkamp et al.  using FTMany of the current automated image analysis systems aim to detect certain objects of interest, such as glands, lymphocytes, mitosis and nuclei, among the various elements found in a histopathology image. However, challenges including out-of-focus objects, missing or broken boundaries of objects, variations in shape and size of objects, differences of intensity levels within objects, similarity between objects of interest and other artefacts as well as overlapping structures, may hinder this task . AdditioChemotherapy, radiotherapy and surgery are the most common types of cancer treatments nowadays, with surgery being the basis for solid tumor treatment. To ensure complete removal of tumor during surgery, visible tumor is resected with a fringe of normal tissue. Adequacy of resection margins in surgery is of utmost importance to prevent under- or over-treatment as the range of surgical removal is directly correlated to long-term survival and post-operative recovery of patients. Overly small resection range may cause tumor recurrence after surgery and affect the long-term survival, while a beyond range of resection could result in longer duration of postoperative recovery or even surgical complications . HoweverDepciuch et al. demonstrated both Raman spectroscopy and IR spectroscopy could detect changes of various biomolecules in breast cancer tissues in comparison with the normal samples . FurtherApproximately 40 min are needed for intraoperative pathological examination of frozen section, which is the most common method used to assess the cutting edge . MultiplBesides surgery, chemotherapy and radiotherapy which have been the most widely used treatments, immunotherapy has become an essential therapeutic alternative in recent years and is now the treatment of choice in many cases. On the other hand, nanotechnology has lately offered nanostructures as novel therapeutic options with functions to enhance treatment outcome, such as controlled drug delivery, directed target therapy and treatment combination . SubsequAn important feature of FTIR spectroscopy is its ability to detect the presence of relapse as well as to monitor therapeutic efficacy in patients. Kaznowska et al.  investigNevertheless, given that blood samples can be obtained less invasively, the use of these specimens may be more favorable compared to tissue samples for the monitoring of therapeutic response and disease progression. Zelig et al.  have utiFrom the perspectives of both cancer patients and physicians, the main purpose in follow-up is the early detection of recurrences . HoweverExtracellular vesicles (EVs) are cell-derived membrane nanovesicles that are released to the extracellular space and circulation. They contribute to intercellular communication and reflect the physiological as well as pathological conditions in the body . EVs havRecent studies suggest that EVs are the transport form for various molecules such as mRNAs, miRNAs, cytokines, hormones, autoantigens, surface receptors and tissue coagulation factors, which might be paracrine regulators of target cell function and metabolism from their parental cells ,78,79. F−1 from liquid but not in dry phases of the serum and plasma samples were considered irrelevant [Despite numerous studies demonstrating the potential use of EVs in disease diagnosis, FTIR spectroscopic analysis of EVs for cancer diagnosis has not been widely investigated. One of such investigation is from Krafft et al.  who haverelevant .In another study, Zlotogorski-Hurvitz et al.  assessedThe ability of FTIR spectroscopy to provide accurate signature of biomolecular content of the analyte with small-scale preparation and high-speed detection, allows the development of a label-free disease-specific EV biomarker which is both time and cost-effective. However, the current most commonly used method in EV studies, differential centrifugation, only allows isolation of enriched fraction of the EV subtypes. Therefore, development of novel methodology for complete purification of each EV subtype would be required to aid in cancer biomarker identification as well as for the study of EVs and their role in cancer, which are still poorly understood. Furthermore, it is important to note that the promising findings of FTIR spectroscopy warrants further research for validation in larger patient cohorts with varying stages and different grades of cancer for clinical translation of the methodology.As more studies show promising results of FTIR spectroscopic analysis using biological specimens in cancer screening, diagnosis, management and monitoring, the challenge now is to translate these methods to routine clinical practice. Nonetheless, most of these studies have been performed on rather small sample size. Hence, large scale clinical trials are required to prove its utility in actual clinical environment and illuminate barriers in implementation that need to be overcome. Large patient cohorts with varying stages and different grades of cancer will be useful in validating the technique. Appropriate selection of patients and control subjects is of utmost importance to reduce the risk of false positives as unmatched comparison groups in sex, age, and physical conditions including hormonal status or pathologies besides the disease of interest, may lead to biased results and differences between the groups could be caused by these confounding factors instead of the disease of interest .Furthermore, standardization of sample collection and storage is vital to not only achieve experimental reproducibility in an individual laboratory but among different laboratories as well . To dateAlthough FTIR spectroscopic analyses of many types of biological materials have been shown to be possible methods for clinical applications in cancer, there are limitations of using these complex biological specimens. Firstly, the use of the current commonly analyzed blood and tissue samples are collected invasively from patients and requires the need for trained medical personnel which would greatly increase the cost and time required for diagnosis. Additionally, contamination during sample preparation such as paraffin in tissue specimens may produce background and interfere with the spectral information . AlthougAnother factor to be considered for the implementation of FTIR spectroscopy for clinical use is that instruments from different manufacturers may have distinct responses and spectral distortions. Moreover, backgrounds have to be addressed using pre-processing algorithms in order to compare results between different studies. Besides that, considerations on the method of sample preparation as well as the optical substrate and acquisition mode used should be taken into account in pre- and post-processing procedures . AdditioTaken together, FTIR spectroscopy holds promise for use as a novel clinical tool for cancer. Importantly, this is evident through the numerous investigations using the technique on multiple cancer types . The tecEven though FTIR spectroscopy may not be able to identify specific molecules when compared to molecular tests, it allows the qualitative and quantitative analyses of different classes of molecules simultaneously such as nucleic acids, proteins and lipids. Therefore, the technique provides the overall status of the analyzed specimen and is ideal for complicated diseases like cancer, which are multifactorial and examining isolated molecules alone may not give complete information . Hence,


Plant Synthetic Biology, Bioengineering, and Biotechnology covers broad aspects of plant synthetic biology, ranging from tools to the design of predictable functions and precision genome editing, to re-designed photosynthesis, plant bioengineering techniques, and rational design of novel plant traits.Plant synthetic biology combines engineering principles with plant biology toward the design, construction, and testing of plants with new traits  is presented in the context of network cross-talk through omics data mining to unravel the underlying complexity required for stress sensing and signaling. Transgene-free genome editing and utilization of nanoparticles as gene delivery tools are discussed as tools to introduce the relevant traits.Engineering stress tolerance in plants has huge potential to accelerate and keep pace with the effects of climate change. In the review by Peng et al., summarize recent efforts in obtaining plant-derived biosynthetic and the extracellular matrix protein, collagen. These products have potential use as scaffold proteins for biomatrices. Spider silk analogs and collagen are composed of a large number of tandem block repeats, which together with the need for post-translational modifications makes production in bacterial hosts challenging leaving plants as an alternative production host.HVS, NP, and PEJ co-edited the Research Topic. All authors wrote, edited, and approved the final version of the Editorial.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Elder abuse is mistreatment of an older adult by a caregiver or another person in a position with an expectation of trust. Adversely affecting as many as 10% of community-dwelling older adults in the US, it may include physical abuse, sexual abuse, psychological abuse, financial exploitation and neglect. Mental illness and substance use by caregivers, family members and victims themselves have been described as risk factors for multiple forms of elder abuse in prior cross-sectional analyses but the impact on these cases is poorly understood. To explore this association we conducted a focus group using a semi-structured format involving an inter-disciplinary group of elder abuse professionals that are part of the New York City Elder Abuse Center enhanced multi-disciplinary team (EMDT) in Staten Island, New York. Focus group participants reported that opioid, cocaine, cannabis and alcohol use is common among perpetrators of elder abuse, especially in cases of financial mistreatment, verbal and physical abuse. Other potential consequences included eviction of the older adult victim, co-dependency and involvement of the older adult in the procurement of illicit substances, and substance use by the older adult. Respondents specifically expressed concerns that the opioid epidemic, including rising heroin use, may be changing the frequency and nature of elder abuse, and that case investigations offer an opportunity to facilitate referrals for formal substance use disorder assessment and treatment. Future work includes additional focus groups and quantitative analysis to clarify the intersection between substance use and elder abuse and inform intervention and prevention strategies.


Rapid outbreaks and the spread of African swine fever across China and other countries have resulted in the huge loss of pig populations and sustainability of the global food supply chain.Because the most vulnerable and affected segment will be the small holders of the pig population, transformation of the pig industry to medium- and large-scale farms, together with better standardized production systems and biosecurity, will facilitate future survival of the industry and can contribute substantially to food security.During the pork shortage, a looming food crisis changed protein consumption behavior from meat to alternative protein sources.The ASF outbreaks in Asia might render the virus deleterious to the global pig industry associated with globalization. Travelers and international trading are the major carriers of a human-driven disease such as the ASF virus.World demand for food is expected to increase due to population growth and change in consumption patterns. Available food supply at affordable prices is of importance for the sustainability of global food security. Agricultural sector plays a major role in food security by providing enough food for society. Agricultural productivity growth is necessary for both domestic use and exportation. However, the fluctuations of production volume in major exporting and importing countries can directly impact the world food balance. Normally, agricultural products in many regions are primarily for domestic consumption. However, there are a few countries that provide the large scale of the agricultural production to support exports.Several factors affect the success of livestock production systems including sustainable resource management, evolving agricultural technology, financial management, and animal disease control . ClimateASF) has caused a major impact on pig production in China, and later in other neighboring countries, causing the worst economic crisis for the swine industry and the food security in the regions  . It was  regions .During the past three decades, pig production has changed rapidly from smallholders to an intensive industry in many developing countries. In the developed countries, large-scale production systems have achieved high levels of production performance and become the main type of pig farming systems. Therefore, only a few traditional forms of pig production survive in the developed countries. In developing countries, large- and medium-scale production systems have also been practiced. However, the largest group of pig population is still found in the traditional small-scale production systems or backyard farming . Type ofThe development of technology, knowledge, and innovation has resulted in opportunities to improve management, housing, breeding, and feeding methods. In addition, the performance parameters of sows and growing pigs have been available on a computerized recording system to maximize the potential of sow and herd productivity . These fIn recent decades, massive growth in pig production in the Asia-Pacific region has been recognized, whereas pig numbers in the United States and European Union are increasing slowly or holding steady. Major pig production areas are in Asia (mainly in China possessing over 50% of the world pig population), the European Union, the United States, Brazil, and Russia . China hASF is one of the economically important viral diseases in the swine industry. Originating in Africa, ASF did spread across Europe through Georgia in 2007 . The disStarting from 2018 to early 2020, the outbreak of ASF has been reported in 21 countries in different regions around the world. In the Asia-Pacific region, the ASF outbreak has been found in at least 14 countries including Mongolia, Vietnam, Cambodia, Hong Kong (SAR-PRC), Korea (Dem. People’s Rep.), Laos, the Philippines, Myanmar, Republic of Korea, Timor-Leste, Indonesia, Papua New Guinea, and India .The Chinese government reported the outbreak of ASF in August 2018. Since the first ASF outbreak, more than 1 million pigs have been culled across China due to disease outbreaks in an attempt to stop ASF from spreading . NeverthDuring the past 10 yr, the fluctuations of pork prices depended on market forces. However, the ASF outbreak in China had a dramatic change in the fluctuation of pork prices. China’s pork production was massively disrupted, resulting in the highest pork prices ever and impacting to the market prices of other protein sources such as chicken meat and aquaculture products. Due to the increasing demand for pork in China, the control of live pig and pig product movement between regions in China is very difficult. Pork prices in China surged 47% in August 2019 . In addiIn February 2019, ASF outbreak was first confirmed in a northern province of Vietnam by Vietnam Department of Livestock Production, Ministry of Agriculture and Rural Development . The ASFImmediately after penetrating the Vietnamese pig herds, the spreading occurred quite rapidly. After a few months, the epidemic reached its peak, having spread to more than 8,200 communes in 63 provinces and cities within 9 mo . After 1Crisis shortages of pork were experienced when the ASF epidemic was at its peak. Directly infected pigs and pigs in the infected zones are culled to stop the spreading. The very low pork price differences in the epidemic zone compared with the higher pork prices in the disease-free areas are a great motivation for smuggling. Later, due to the shortage of pork throughout the country, price of pork were doubled or tripled. Many solutions have been applied such as restructuring the livestock sector, pork production model, centralized and controlled local supply chain and enhanced disease prevention by improving biosecurity at various levels to reduce ASF losses and reproduce pork supply capacity. Along with strengthening ASF control measures, ensuring food supply is promoted, such as shifting husbandry models and encouraging pig repopulation toward strict biosecurity, and at the same time increasing development of poultry flocks and ruminant herds. In the long-term solution, necessary conditions for re-stocking of pigs with high biosecurity poses a difficulty to the small to medium farms on finding the areas of disease free together with huge investment in qualified imported breeders and well-equipped farm facilities.Repopulation is an early implementation strategy to prepare for the scenario of pork shortage. The success of restocking is questionable on proper disease control continuing to occur when latent circulation of the ASF virus still exists in the infected areas due to the virus persistence in the environment. Currently, ASF has become endemic in several pig production areas in Vietnam. Eliminating ASF virus from the pig herds or from the country in general is absolutely a big challenge and it may take decades. ASF prevalence and contamination of the virus in the environment could have threatened the repopulation of the small to medium farms and even the big industry farms with good biosecurity. In addition to ASF, the Vietnamese pig production has also been burdened with several major pathogens .Practical researches are needed to clarify several major issues including suitable farming models in the ASF virus contaminated areas, proper procedures to test disease-free pigs for repopulation, risk analysis on the supply chain within and between countries, and inactivation procedures on contaminated water and feed. It should be noted that wild boars have not yet played a major role in the ASF transmission in Asian countries. However, it is worth finding if wild boars would be a part of the reinfection later in the infected countries. The establishment and implementation of technical training and education programs provided by FAO and local authorities to improve the stake holders’ knowledge and farming practice on ASF control are essential. Specialized technical teams should be established and deeply trained to be ready to guide, respond, and promptly handle disease consequences to ensure successful and sustainable restocking. Ultimately, research on vaccine and antiviral medicine development are more proactive strategy and expectations for the current disease control for the farmers in the infected countries.The spreading and persistence of ASF is often associated with traditional small-scale production systems, and usually the absence of disease prevention and control strategies . The traThe Vietnamese ecosystem links people, animals, and the environment very closely, not only in terms of habitats but also in terms of health and emerging diseases. Vietnam is the world’s 15th most populous country. The density of people and terrestrial and underwater animals is high, particularly in the outskirt area of big cities. Vietnam is experiencing rapid growth of livestock production. In 2018, the total pig herd was officially estimated at 28 million, the 6th largest producer of pork in the world. The pig production increased in not only in large-scale farms but also in diverse farming models varying from household, backyard farm, concentrated farms (medium and large), and cooperatives. However, the actual estimated number of pigs in the whole country had actually eclipsed over 40 million in 2016 due to the rapid increase of small farms which were not yet fully determined. The small and scattered pig production still accounts for a high proportion, with a total of 2.9 million pigs estimated over 50% of pigs nationwide. This scenario creates a diverse and complex ecosystem for animals, people, and pathogens.The spread trajectory of ASF in Vietnam is noted to be somewhat complicated. Earlier, the disease occurred in backyard and/or small farms because pigs are often fed or contact with salvaged food and leftovers from households and restaurants. If those pigs were not handled strictly and quickly diagnosed for quarantine and stamping out, the sick pigs were then sold to the local slaughterhouses and later the virus would widely spread into the environment and supply chain. Then, the local outbreaks began to spread through small farms, particularly, the opened-door farms having poor biosecurity and farming practices from villages to villages. Later, widespread contamination including the presence of ASF virus in the environment creating many possible vectors would eventually gain its presence into the larger farms. In addition, the long-standing tradition of purchasing fresh meat from Vietnamese wet markets or directly from the local slaughterhouses could facilitate the spreading of the virus locally. Additionally, there are huge numbers of pig farming combining with human houses to reuse the leftovers and food waste for feeding pigs in order to save cost of pig feed, leading to the higher risk of getting infected . Thus, tASF virus is characterized by slow and not high spread in nature when the infected pigs are strictly monitored, isolated, and fully quarantined , but theA high risk of ASF introduction into Thailand is considered since the country is surrounded by the ASF-infected neighboring countries. Based on the risk assessment of ASF introduction into Thailand without emergency response plans, it could cause severe economic losses at least 125 billion baht (USD 4.045 billion) . It shouThe Thai government approved 150 million baht (USD 4.7 billion) on the preparation for emergencies on national level. Accordingly, the cooperation between the Livestock Department with relevant agencies, swine farmers, and the private sectors has been involved for ASF contingency plans. This plan is comprised of three phases: pre-outbreak, outbreak, and postoutbreak and spending mainly for the risk factor control associated with the ASF introduction including (1) illegal movement of pigs and pork products along the border areas; (2) tourists and visitors from the ASF affected countries; and (3) vehicles, tools, equipment, pigs, food, and feed from the ASF risk areas. The development of disease diagnosis, establishment of laboratory networks, and increased public awareness is included. In addition, on-farm biosecurity standard has greatly been improved  by increAfter ASF outbreak, the global food security will be challenged by a growing demand for agricultural products and the efficacy of productivity varied by regions. This current shortage in pork availability has impacted to the pork prices and changed in meat consumption behavior to other alternative protein sources. Rabobank showed that the global animal protein production was slow in 2019. It might be because of a dramatic decline in Chinese pork production while the other meat production was rising . EuropeaFurthermore, the impact of rising feed ingredient prices on costs of livestock production would impact livestock producers because 60% to 70% of pig production cost are feed cost. Therefore, the cost has a dramatic effect in small-scale farms comparing to the large-scale farms since the ability to organize the feed supply operations serving stores and the opportunity cost of labor and medicine do favor the big operation. Many traditional small scale and backyard farming would gradually disappear.However, the ASF crisis would often lead to the potential investors to be major players in the future pig industry. ASF free herds or countries would gain greater benefit for exportation of pork. Therefore, the transformation of the pig industry from backyard and small scale to medium- and large-scale farms together with better standardized production systems and biosecurity would survive in the future.COVID-19) situation (At present, the pig production trend has changed to the medium- to large-scale production systems in many countries. However, the stakeholder network society of the pig industry in the region is still unable to control the pig movements within or between countries due to high demand in restocking and trading. The highly interconnectivity of worldwide pork production and globalization due to heavy trafficking of people could increase the risk of foreign pathogens introduction into other countries . During ituation , limitatRapid outbreaks and the spread of ASF across China and other countries, resulting in the huge loss of pig populations and reflecting sustainability in the food supply chain. ASF outbreak has significant impact on the global protein supply, with concerning the balance of pork production and consumption. The soaring pork prices due to the shortage of pork have been increased in Asia. A looming food crisis has changed meat consumption behavior to other alternative protein sources. In any scenario, the most affected and most vulnerable segments of the pig population will be the small holders. Transformation of the pig industry to medium- and large-scale farms together with better standardized production systems and biosecurity would survive in the future. Additionally, improving productivity can also contribute substantially to food security. Therefore, the disease control strategies of various pig production systems in Asia are challenging. Since ASF has been known as a human-driven disease, the development of disease control measures and the collaboration with the relevant agencies and farmers could still be of importance in the ASF control strategies.Conflict of interest statement. Authors have no conflicts of interest.


Plastic, usually derived from non-renewable sources, is among the most used materials in food packaging. Despite its barrier properties, plastic packaging has a recycling rate below the ideal and its accumulation in the environment leads to environmental issues. One of the solutions approached to minimize this impact is the development of food packaging materials made from polymers from renewable sources that, in addition to being biodegradable, can also be edible. Different biopolymers from agricultural renewable sources such as gelatin, whey protein, starch, chitosan, alginate and pectin, among other, have been analyzed for the development of biodegradable films. Moreover, these films can serve as vehicles for transporting bioactive compounds, extending their applicability as bioactive, edible, compostable and biodegradable films. Biopolymer films incorporated with plant-derived bioactive compounds have become an interesting area of research. The interaction between environment-friendly biopolymers and bioactive compounds improves functionality. In addition to interfering with thermal, mechanical and barrier properties of films, depending on the properties of the bioactive compounds, new characteristics are attributed to films, such as antimicrobial and antioxidant properties, color and innovative flavors. This review compiles information on agro-based biopolymers and plant-derived bioactive compounds used in the production of bioactive films. Particular emphasis has been given to the methods used for incorporating bioactive compounds from plant-derived into films and their influence on the functional properties of biopolymer films. Some limitations to be overcome for future advances are also briefly summarized. This review will benefit future prospects for exploring innovative methods of incorporating plant-derived bioactive compounds into films made from agricultural polymers. Most food packaging is produced from synthetic materials from non-renewable sources, which, despite having excellent barrier and resistance properties, are causing serious environmental problems due to the generation of high amounts of non-degradable solid waste . HoweverThis factor, along with environmental concerns, in combination with consumer demands for high quality eco-friendly products that are related to those found in nature  have driven the development of new technologies, such as the production of biodegradable films from polymers from renewable sources ,2,4, for2), water (H2O) and biomass under aerobic conditions and hydrocarbons, methane and biomass under anaerobic conditions [The biodegradability of plastic materials is defined as their ability to decompose through the enzymatic action of microorganisms . The polnditions .Polysaccharides, such as starch ,8,9, celIn addition to biopolymers, plant-derived bioactive compounds such as essential oils, vitamins, minerals, polyphenols, carotenoids, among others, are widely distributed in nature. Different parts of plants, such as leaves, flowers, seeds and roots can potentially be used in the production of environment-friendly films with functional properties due to their biological nature . Some biThe combination of biopolymers with natural bioactive compounds has enabled the development of bioactive films with new and/or improved properties, i.e., antioxidant ,27 and aThe incorporation of bioactive compounds into biodegradable films has been extensively studied ,29,31,32The application of bioactive films in packaging as a strategy for extending the shelf life, stability and safety of food products has shown great potential ,37,38. I2) emission reduction. Several studies involving biodegradable polymers of agricultural origin ), is a biodegradable cationic polymer derived from chitin, a polysaccharide of animal origin. Chitosan shows antimicrobial properties [Alginate and pectin are anionic polysaccharides. Pectin is derived from the plant cell wall and it is obtained commercially by aqueous extraction of pectic material from some fruits, being found in higher amounts in citrus fruits and apples . It conswn algae . Both biwn algae . The comwn algae . The addwn algae . Chitosaoperties  and the operties . Accordioperties , due to operties , biocompoperties , biodegrThe most widely used proteins in the production of edible films are gelatin ,23, wheyBovine milk whey is a by-product of the dairy industry. It represents the aqueous portion of milk that separates from the clot during cheese making or casein production and consists of a complex mixture of globular proteins (~0.6%), lipids, minerals and lactose, in water (93%) . Drying Whey protein isolate (WPI) has the ability to form films with a wide range of functional properties depending on their structural cohesion . Native 2) behavior in comparison to films based on low-density polyethylene, methylcellulose, starch and pectin. According to a study by Giacomelli [Soy protein isolate (SPI) is a byproduct of the soybean oil industry, it comprises a set of macromolecules of varied sizes and structures formed from 18 different amino acid residues, being constituted by the soy storage proteins . Due to acomelli  on thermacomelli .Despite the promising characteristics of edibility, biocompatibility and biodegradability presented by polymer from agricultural sources, it is still evident that there are limitations to be overcome for the commercial success of these films , especially when compared to synthetic plastics . In geneIn addition, most films made from polymers of agricultural origin are obtained on a laboratory scale, using the casting method. This method is based on the dispersion of macromolecules into a suitable solvent or mixture of solvents, thus obtaining the film-forming suspension that is subjected to thermal gelatinization. The resulting solution is deposited in molds of relatively small sizes ; thyme abica L. ; Ziziphotial oil ; orange tial oil ; cinnamotial oil ; black ptial oil ; rosemartial oil ; Saturejtial oil ;Syzygium cumini) [fruit pulps, purees, juices and extracts as a source of phenolic compounds and vitamins: guabiroba ; blackbe cumini) ; mulberr cumini) ; papaya  cumini) ; mango a cumini) ; acerola cumini) .Citrus sinensis), passion fruit (Passiflora edulis) and watermelon (Citrullus lanatus), whereas the vegetables were zucchini (Cucurbita pepo), lettuce (Lactuca sativa), carrot (Daucus carota), spinach (Spinacea oleracea), mint (Menthas p.), yams (Colocasia esculenta), cucumber (Cucumis sativus) and arugula (Eruca sativa) [Acca sellowiana waste by-product  ; pomelo  sativa) , Acca sel flour, ); roastel flour, ; pine nul flour, ; ethanoll flour, .Leaves, flowers , fruits,Plant-derived bioactive compounds are being considered interesting ingredients for the production of biodegradable and bioactive films due to their natural origin and functionality . StudiesR. officinalis, A. herba alba Asso, O. basilicum L., M. pulegium L. [Acca sellowiana waste by-product (feijoa peel flour) [There is a wide and growing list of bioactive compounds that have been or are being incorporated into films, and phenolic compounds  are the most common ones. The plant-derived bioactive compounds are incorporated also to contribute to the general quality ,93,103, l flour) , for exaThere is still a wide range of plant-derived bioactive compounds that have not been characterized in terms of physical–chemical properties, toxicity and edibility. This fact implies that extensive investigations, including tests that assess the in vitro and in vivo (food product) efficiency of plant-derived bioactive compounds, are still needed for improving food safety. The choice of specific plant-derived bioactive compounds for application depends on their bioactive properties , availability and cost–benefit ratio .One of the great challenges to be overcome for future advances and insertion of bioactive and biopolymer films into the market is the standardization of the functional, barrier and mechanical properties presented by them. It is known that the amount of biopolymers , as well as bioactive compounds  present in agricultural products and their byproducts  vary in composition and quantity, depending on the conditions adopted in agricultural practices: planting ; harvest (maturity degree); post-harvest (processing and storage conditions); part of the plant; and, mainly for bioactive compounds, how much the plant responds to environmental aggressions ,108,109.Glechoma longituba, a plant widely spread in China that has long been used as a beneficial health ingredient in human diet. In addition to the great variations in the levels of extracts soluble in ethanol, total flavonoids, chlorogenic acid, caffeic acid, rosmarinic acid, oleanolic acid and ursolic acid proven among different populations of Glechoma longituba, it was observed that the chemical property of the soil and the climatic condition exerted significant influences on the concentrations of bioactive compounds present in the plant [A recent study showed the influence of different factors  on the bioactive compounds of he plant . More stOne of the ways to decrease the variability of bioactive compounds is to extract or purify them. Recent reviews have reported the use of compressed fluids, mainly under sub- and supercritical conditions, for the extraction of bioactive components from natural matrices ,111. HowMoreover, when the bioactive compound is extracted and purified, it is more exposed to external adverse conditions , which facilitates its volatilization and oxidation during the production of films due to processing conditions  ,35,82, wCurrently, several studies have investigated the incorporation of bioactive compounds into biopolymer-based films for application such as active food packaging ,78,80,94The incorporation of bioactive compounds into films must be in accordance with the characteristics of the desired packaging system, the bioactive compound used, as well as the food to which these films will be applied. As a result, various ways of incorporating plant-derived bioactive compounds into film-forming solutions produced by agro-based polymers have been reported, and the most commonly used are:(a) producing the film using inherently bioactive biopolymeric materials from agricultural products and their byproducts ,112,113;(b) directly mixing bioactive compounds with biopolymers during the film producing process ,74,75,79(c) encapsulating the bioactive compounds, keeping them trapped inside a wall material, forming micro or nanoparticles. Then, performing the incorporation by physical and direct mixing of micro or nanoparticles with the biopolymer in the film producing process ,115,116;(d) encapsulating the bioactive compounds, keeping them trapped inside a wall material, forming micro or nanoparticles. Then, carrying out the incorporation by spraying micro or nanoparticles onto the biopolymer during in the film producing process ,117,118.In recent years, researches have focused on the sustainable use of agricultural and agro-industrial products and byproducts, which include bagasse, barks, stalks, seeds, among others, as raw materials for the production of bran, flour, fibers, starch extraction ,120,121 Films produced with saffron flour obtained from the extraction residue of saffron dye showed antioxidant activity due to the presence of curcuminoids in the flour . Light bManiglia et al.  used babelimination of the need for incorporating materials that are sources of bioactive compounds or isolated bioactive compounds during their preparation, which can lead to a reduction in production costs;development of films with special functional properties, such as antioxidants and antimicrobials, and with added value, since they carry bioactive compounds capable of delaying the discoloration and oxidation of other compounds, preventing bacterial growth, as well as prolonging the shelf life of food products .Utilization of agricultural and agro-industrial products and byproducts as raw material can contribute to minimizing environmental issues caused by their disposal.The production of bioactive films with inherently bioactive biopolymer-based materials, i.e., those that naturally contain bioactive compounds in their composition, presents the following advantages:Therefore, it is necessary to explore the potential of biopolymer-based materials obtained mainly from agricultural products and byproducts, derived from the processing of fruits and vegetables, which are rich sources of low-cost bioactive biomaterials .Instead of using inherently bioactive products, bioactive films can also be developed by incorporating natural raw materials that are sources of bioactive compounds or isolated bioactive compounds into the biopolymer film-forming solution during preparation. Some studies incorporate the bioactive compounds into the biopolymer during solubilization ,32, whilR. officinalis L., A. herba alba Asso, O. basilicum L. and M. pulegium L. [Acca sellowiana waste byproduct  exertedel flour  and pectel flour , respectel flour .Brito et al.  producedSome natural bioactive compounds show instability when exposed to high temperatures, light, pH, oxygen, among other processing conditions , which cThe encapsulation of bioactive compounds is one of the ways to stabilize and maintain their viability and extend their shelf life. Encapsulation refers to the process of trapping the bioactive compound into microsystems (microparticles) or nano-systems (nanoparticles) of structural engineering in order to develop a thermodynamic and physical barrier that protects against adverse conditions , releasing it when necessary .The strategy of incorporating encapsulated bioactive compounds into the film has been used for: (i) maintaining the stability and viability of active compounds when exposed to unfavorable conditions; (ii) improving compatibility between packaging polymer and bioactive compound ; (iii) increasing the availability of the bioactive compound; (iv) controlling the release of these compounds by specific stimuli .Helichrysum italicum) extract [The encapsulation of some bioactive compounds enables their use in packaging applications, which is the case of 2-phenyl ethanol, for example. The encapsulation of 2-phenyl ethanol was essential to reduce losses due to volatilization or degradation during the film production process. Zarandona et al.  observed extract .Becerril et al.  describeOnce the bioactive compound is encapsulated, it can be incorporated into the film-forming solution directly ,115,127 The final functionality of films is related to their bioactive properties , their ability to serve as barrier to water vapor, oxygen, carbon dioxide and ultraviolet (UV–vis) light, water vapor permeability; mechanical properties  and other physical properties (such as opacity and color) . The preThe use of plant-derived bioactive compounds with antioxidant and antimicrobial properties in biodegradable films as a sustainable strategy for maintaining and/or extending the shelf life of food products has been promising , since mThe major concern regarding microbial growth, in addition to microbial deterioration (deteriorating microorganisms), which reduces the shelf life of various food products ,131, is Escherichia coli and Staphylococcus aureus around the films. Films without tea polyphenol did not show antimicrobial effect, proving that it was the tea that generated this property [Therefore, the use of natural antimicrobial and antioxidant compounds in biodegradable films tends to contribute to the preservation of food quality and safety. An example of this was observed when the incorporation of tea polyphenol into a film made with pomelo peel flour caused an antimicrobial effect on the film; as its concentration was increased from 5 to 20%, there was an increase in the inhibition zone for property .In another study, films incorporated with essential oils from medicinal plants showed the ability to kill and inhibit the growth of Gram-positive and Gram-negative bacteria, with varying activity regarding the inhibition zone (from 18.50 to 38.83 mm) for each bacterium [The incorporation of blueberry extract into films containing isolated soy protein also transferred antioxidant capacity to the film, delaying the oxidation and hydrolysis of the pork fat packed with it during storage  . MalherbMalpighia glabra L.) or seriguela (Spendias purpurea) pulps caused an antioxidant effect on dendê oil during 40 days of storage and can be applied to control product oxidation. A reduction of 50%, 49% and 56% in the total phenolic compounds of the films incorporated with mango, acerola and seriguela pulps was observed during storage of dendê oil. This suggests the migration of these compounds from the films to the dendê oil during storage [Films incorporated with mango, acerola ; swelling induced release ; disintegration induced release  . The chaPiñeros-Hernandez et al.  studied Kevij et al.  came acrThe curcumin released from the composite films into different food simulants is favored by lipophilic substances and can be controlled by reducing the polarity of the starch nanovehicle by acetylation . The relIn general, the highest release rates of bioactive compounds to food or food simulants occur when the film-forming matrix is porous or soluble in the medium, or even when the bioactive compound has a high affinity with the surrounding medium. The longer the contact time between the film and the food or the food simulator, the greater the release rate of the compound. The strategy of using encapsulated bioactive compounds in films tends to generate greater protection from encapsulated compounds  and a better-controlled release.The encapsulation of marjoram essential oil with pickering emulsions provided a significantly slower release profile in pectin film samples in comparison to essential oil-loaded nanoemulsions . Nieto-SSo, it is clear that the solubility of the encapsulating agent in the polymeric matrix and in the food simulants significantly influence the release rate of the bioactive compound from the film to the surrounding environment. This resource has great potential for controlling its release. In addition to the type of encapsulating agent used, the technique used for the incorporation of the encapsulated bioactive compounds into the film-forming solution also seems to influence its release rate.Nogueira et al. ,117 obseThe production of multilayer films has also been used as a strategy to modify the location of bioactive compounds in the polymer matrix and to improve the control of the release of bioactive compounds from the film to foods or food simulants. Layer-by-layer (LbL) deposition is one of the methods used in this production. The layers are formed one by one, being physically or chemically bonded together to form the final multilayer film ,139. BasIn addition to the location of bioactive compounds in the film-forming matrix, the way in which they are distributed also appears to affect the bioactivity of the film. Another major challenge is to be able to homogenously incorporate bioactive compounds into the film-forming biopolymer matrix. Studies have shown regions with particles of bioactive compounds agglomerated within the biopolymer matrices ,90,117, The more homogeneous the distribution of the bioactive compound in the film, the more efficient it will act on the food surface. For example, films containing allyl isocyanate (AIT) microemulsions showed stronger antimicrobial activity and were more homogeneous than those containing conventional emulsions. According to Guo , the facDespite the promising results, more studies should be carried out to evaluate the relationship between the location and the distribution of bioactive compounds in the film with their release rate in food. The vast majority of works perform release tests using food simulants to understand the migration of bioactive compounds from films to different types of food. Migration is measured using chemical tests that are specific to quantify the bioactive compounds or group of bioactive compounds in question. This is because it is extremely difficult to measure the direct migration of bioactive compounds from a film to a food due to complex compositions  found in most foods . GeneralIn addition, these types of tests are especially difficult due to the large number of external variables that must be evaluated, such as time and temperature conditions, quantity, and space between the film and the food. All of these variables can lead to different types of results, which is why the standardization of the conditions adopted in the release tests is so necessary. Advances in the specificity, sensitivity and selectivity of the appropriate analytical techniques to determine the amount of bioactive compounds present in films and in food will allow quantification of their migration to real foods. These advances in analytical techniques will also enable the achievement of more accurate results. All of this will help to fill some of the gaps that still exist in searches, such as: the lack of knowledge on the ideal concentration of each bioactive compound for each type of food; the release kinetics of bioactive compounds for food products ; and mainly how to control the unintended migration of bioactive compounds to food.The knowledge on the functional properties of films is of fundamental importance and scientific and technological interest due to the requirements that the different films must present to be used as food packaging, since many industrial applications depend on these properties. Among the main functions of films from agro-based polymers that will be used as food packaging are the mass transfer control (to avoid gain or loss of solutes and volatiles), mechanical protection (protecting the integrity of food and packaging), protection barrier (controlling the migration of water vapor and gases between the product and the environment), low water solubility (protecting from solubilization in water). The incorporation of plant-derived bioactive compounds in the film-forming solution, besides modifying the bioactive properties of resulting films, can concomitantly modify their structure and, as a result, modify their functionality, such as their ability to serve as a water vapor barrier; water vapor permeability; tensile strength; elongation at break; and their physical properties such as opacity, color and thickness ,74.2·day·kPa), water solubility (from 14.18 to 25.46%), and reduced tensile strength (from 22.71 to 3.97 MPa) [The increasing incorporation from 0 to 40% (mass/mass of biopolymer) of blackberry pulp into the arrowroot starch film-forming solution, apart from generating color with tones ranging from violet to magenta, typical of blackberry pulp, also promoted an increase in thickness (from 0.065 to 0.133 mm), elongation (from 3.18% to 13.59%), water vapor permeability (from 3.62 to 4.60 g.mm/m.97 MPa) . The inc.97 MPa) . The inc.97 MPa) ,103.In general, bioactive compounds with hydrophilic characteristics tend to increase the water vapor permeability of the film, since the water vapor transfer is generally caused by the hydrophilic composition of the film and depends on the composition of hydrophilic to hydrophobic compounds in the film . FurtherAs for films made with fruit pulps, the discontinuity of the polymeric matrix may be due, not only to the incorporation of bioactive compounds, but also to other structures present in their composition, such as proteins, lipids, fibers, vitamins, minerals and sugars that fit the biopolymer matrix through different types of intermolecular interactions . AnotherPlasticizers are used in films to improve both their physical and mechanical properties, joining possible cracks in the biopolymer matrix. When added to the film-forming solution, they modify the molecular organization of the network by increasing the free volume between the molecules. This action usually causes a reduction in intermolecular interactions between the adjacent chains of the biopolymer, increasing the mobility of these chains . ConsequGamma-aminobutyric acid, a nutritive bioactive compound, in addition to generating antioxidant activity, also acted as a plasticizer when incorporated into the pectin film, proving to be an alternative to glycerol . HydroalIn general, plasticizers increase flexibility and reduce the water vapor barrier. These are undesirable characteristics for films that will be applied to foods with intermediate humidity, which are characterized by high water activity (from 0.65 to 0.90) and an overall soft texture .The incorporation of encapsulated plant-derived bioactive compounds into films made of agro-based polymers has also proved to be a viable alternative for improving the mechanical and barrier properties of films based on biodegradable biopolymers.Wang et al.  reported9 g·Pa−1·s−1·m−1) and water solubility . These results demonstrate that the properties presented by the films highly depend on the characteristics of the encapsulated bioactive compound, the characteristics of the encapsulating agent, and the compatibility between the encapsulating materials, bioactive compounds and the film-forming material [The increasing incorporation of curcumin-loaded native and acetylated starch nanoparticles (0.01 to 0.1%) into films made with banana starch and Aloe Vera gel also increased their tensile strength from 3.74 to 5.01 MPa and 4.80 MPa, respectively. This improvement in tensile strength was attributed to the strong interaction between the native starch nanoparticles and the biopolymer matrix. The small size and the high presence of hydroxyl groups on the surfaces of native starch nanoparticles allowed the formation of many hydrogen bonds with the biopolymer matrix, resulting in an increase in tensile strength. Lower tensile strength values found in films with curcumin-loaded acetylated starch nanoparticles in comparison to native starch nanoparticles were attributed to fewer hydroxyl groups present on the surface of the nanoparticles, due to the starch acetylation process, which consequently generated a weaker interaction with the biopolymer matrix. Nevertheless, the increase in film stiffness, due to the incorporation of nanoparticles, reduced elasticity (elongation at break from 56.3 to 45.4%). The presence of highly hydrophobic curcumin molecules limited the interaction between water and the film matrix, leading to a decrease in water vapor permeability , the good interaction between soy protein concentrate, a material used for forming the film, and the alginate used as a wall material in the microencapsulation of the oil resulted in a more continuous film matrix with few cavities, which led to improvements to them . The incFor Almasi et al. , the appLi et al.  observedThe location of the particles of bioactive compounds in the films, within the polymeric matrix or on its surface, also influence the properties presented by them. Films incorporated directly into encapsulated blackberry pulp were less soluble in water than films incorporated with blackberry powder by spraying and the location of these film particles influenced this behavior. The fact that blackberry dust particles remained on the film surface when incorporated by spraying allowed its direct contact with water leading to solubilization, given its porous and hydrophilic characteristic, which consequently generated holes on the film; as a result, the water molecules entered the starch matrix and led to its solubilization. Water vapor permeability was also affected. The increase in blackberry particles in the film led to their aggregation. The aggregation of particles reduces the interaction between the active surface area and the polymeric matrix, decreasing its cohesiveness and consequently increasing water vapor permeability .Agro-based polymers incorporated with plant-derived bioactive compounds have great advantages in relation to synthetic plastic, as they are environmentally friendly and have characteristics of edibility, biocompatibility and natural bioactive properties. However, despite their many advantages, their use as bioactive food packaging on an industrial scale is still limited by several factors. The main factors that limit its commercial success are listed and described in this section.With the exception of starch, raw materials of agricultural origin, such as cellulose, alginate sodium, pectin, chitosan, gums and proteins, such as whey, soy, gluten and gelatin, among others, the isolation process of these polymers can require high energy consumption and expensive chemicals, which affect the economic and environmental values of the films produced with them. The great variability regarding the physical–chemical composition of the origin of both the agricultural polymers and the plant-derived bioactive compounds also hinder the standardization of the bioactive functional properties, water vapor permeability, tensile strength, elongation at break, as well as of the physical properties of films produced with them. The current laboratory-scale production of agro-based films incorporated with plant-derived bioactive compounds has problems such as the inability to make continuous films, long drying times and imprecise thickness control. Thus, to overcome these issues, better and cheaper production methods are needed along with investments in scaling machines so that the films produced can compete with the current ones, not only in terms of efficiency, but also in terms of price .In addition, as agro-based polymers are mostly hydrophilic, films produced with them have lower properties than plastics derived from petroleum, especially in terms of mechanical strength, such as insufficient stretching, and poor water vapor barrier properties .These properties can still be impaired by the heterogeneous dispersion ,90,117 oAgro-based polymers, i.e., starch, sodium alginate, pectin, chitosan, cellulose, whey protein, gelatin, soy and gluten proteins have been extensively used for the production of environment-friendly food films or packaging. In addition, the combination of agro-based polymers and derived-plant bioactive compounds  allows the development of bioactive films with antioxidants, antimicrobial activity and innovative colors. Different methods have been used for the production of bioactive films, such as the use of inherently bioactive biopolymer materials, the direct incorporation of free or encapsulated bioactive compounds into the film-forming solution and the spraying of free or encapsulated bioactive compounds onto the film-forming solution already on the support plates, among others. The functional, barrier and resistance properties presented by the films are directly influenced by the interactions between the bioactive compounds and the film-forming solution. In general, films produced with agro-based polymers still have limited mechanical and barrier properties when compared to synthetic plastic films. The use of bioactive films to increase the shelf life, stability and safety of foods has proved to be promising. In addition, the strategic use of bioactive films as vehicles and targeted delivery of bioactive compounds to food has proved to be useful for the stability and safety of foods that have been packaged with them. The improved stability and controlled release rate provided by the films allow the bioactive compounds to be transferred from the film to the surface of the food, where they start to act, preventing the growth of microorganisms and the oxidation of compounds, such as lipids. However, information on the mechanisms involved in this process is still limited especially regarding the controlled release of the bioactive compound into the food. Although films have several positive characteristics such as biodegradability and bioactive properties, they are still produced on a laboratory scale, and issues related to processing difficulties, high cost, and difficulties in standardizing film properties need to be resolved so that their production can move onto an industrial scale.