Non-invasive ventilation (NIV) has been shown to be effective in avoiding intubation and improving survival in patients with acute hypoxemic respiratory failure (ARF) when compared to conventional oxygen therapy. However, NIV is associated with high failure rates due, in most cases, to patient discomfort. Therefore, increasing attention has been paid to all those interventions aimed at enhancing patient's tolerance to NIV. Several practical aspects have been considered to improve patient adaptation. In particular, the choice of the interface and the ventilatory setting adopted for NIV play a key role in the success of respiratory assistance. Among the different NIV interfaces, tolerance is poorest for the nasal and oronasal masks, while helmet appears to be better tolerated, resulting in longer use and lower NIV failure rates. The choice of fixing system also significantly affects patient comfort due to pain and possible pressure ulcers related to the device. The ventilatory setting adopted for NIV is associated with varying degrees of patient comfort: patients are more comfortable with pressure-support ventilation (PSV) than controlled ventilation. Furthermore, the use of electrical activity of the diaphragm (EADi)-driven ventilation has been demonstrated to improve patient comfort when compared to PSV, while reducing neural drive and effort. If non-pharmacological remedies fail, sedation can be employed to improve patient's tolerance to NIV. Sedation facilitates ventilation, reduces anxiety, promotes sleep, and modulates physiological responses to stress. Judicious use of sedation may be an option to increase the chances of success in some patients at risk for intubation because of NIV intolerance consequent to pain, discomfort, claustrophobia, or agitation. During the Coronavirus Disease-19 (COVID-19) pandemic, NIV has been extensively employed to face off the massive request for ventilatory assistance. Prone positioning in non-intubated awake COVID-19 patients may improve oxygenation, reduce work of breathing, and, possibly, prevent intubation. Despite these advantages, maintaining prone position can be particularly challenging because poor comfort has been described as the main cause of prone position discontinuation. In conclusion, comfort is one of the major determinants of NIV success. All the strategies aimed to increase comfort during NIV should be pursued. In recent years, non-invasive ventilation (NIV), including non-invasive variable positive airway pressure ventilation and continuous positive airway pressure (CPAP) , has proThis has been even more true during the massive spread of severe acute respiratory syndrome-related to the novel coronavirus [severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)] pandemic, when NIV has extensively been used to cope with the massive demand for ventilatory assistance outside the intensive care unit (ICU) . In the Non-invasive ventilation has been shown to be effective in preventing intubation and improving survival of patients with ARF  when comHowever, this widespread diffusion of NIV has in turn allowed to find out the limits of its application. In this regard, NIV failure, defined as the need for endotracheal intubation, is the main issue while dealing with patients with NIV . SurprisIn patients who underwent IMV, discomfort depends on many causes, such as pain, dyspnea, sleep deprivation, anxiety, thirst, inability to communicate, and lack of control. Among these, the management of pain and dyspnea has been demonstrated to improve clinical outcomes –27. A poA list of possible factors responsible for poor comfort is shown in One of the most widely investigated aspects is related to the interface dedicated to NIV. NIV interface is a potential source of pain and claustrophobia that leads to NIV discontinuation and recourse to endotracheal intubation . When chvia the nasal mask due to the vast air loss through the mouth  oxygen therapy can be tried as an alternative in case of intolerance to the various interfaces used for NIV . The HFNThe ventilatory setting adopted for NIV is associated with varying degrees of patient comfort: patients are more comfortable with pressure-support ventilation (PSV) than volume-controlled ventilation . HoweverOn this basis, new ventilatory modalities aimed at increasing the degree of synchrony between patient and ventilator machine have been demonstrated to improve patient's comfort during NIV. Compared to pneumatically triggered and cycled-off PSV, the use of the electrical activity of the diaphragm (EADi) to drive the “neural”-pressure ventilation –69 as weThe ventilator machine is obviously important during NIV. In line with recent results , the asyOptimal patient-ventilator interaction may be of pivotal importance in NIV success. According to recent findings, high rates of asynchrony also occur during NIV. It has been demonstrated that the ability of ICU physicians to detect patient-ventilator asynchrony during NIV by inspection of flow and pressure waveforms is low. Moreover, the asynchrony detection is slightly higher with mask than with helmet and the rate of proper detection is inversely related to the prevalence of asynchrony. In patients who underwent NIV, ineffective efforts are more frequently observed with the helmet while double triggers are more recurrent with mask . RegardiSeveral strategies, such as the use of ventilators with algorithms for air-leak detection and compensation, application of leak-insensitive ventilatory modes, reduction of the applied pressure, and choice of the appropriate interface, may reduce the number of asynchronies during NIV. Moreover, the application of the neural trigger in delivering NIV has been reported to improve asynchronies, by reducing the delay from neural effort onset to inspiratory assistance initiation and reducing the incidence of ineffective efforts , 74.Inadequate humidification during NIV assistance may cause patient distress because it is associated with upper airway mucosa dryness and nasal congestion . Thus, aSurrounding noise may negatively affect patient's comfort during NIV. Recently, a “bundle of interventions” has been proposed to improve the comfort in patients undergoing NIV, such as noise reduction . Noise eThe optimization of patient's position also plays a key role in assuring comfort during NIV . The sitPatient's emotional state is a major determinant of NIV success. In the case of intolerant patients, it is suggested to try a strategic relational approach. To preserve and/or improve patient's comfort and tolerance to NIV, it is fundamental to establish a trust relationship with patients, by reassuring them during ventilatory assistance, providing information on expected benefits of NIV, and involving them in the process of care .When none of the non-pharmacological strategies listed above are successful, analgo-sedative medications schemes can be employed to manage agitation during NIV .Agitation can be caused by several factors, such as fear, pain, anxiety, sleep deprivation, fever, and hypoxia . To faceIn case of agitation due to anxiety or intolerance, the choice must fall on sedative drugs. It has been demonstrated that sedation strategy could reduce the rate of NIV failure . SedatioIn choosing the drug, the intrinsic characteristics and clinical effects of the various pharmacological categories must be considered, mainly taking into account the effects exerted by the drug on patient's own respiratory drive. Benzodiazepines should preferentially be avoided in the elderly with agitation due to the risk of paradoxical the effect and of promoting a state of delirium . In addiPropofol, thanks to its pharmacokinetic rapidity, is a particularly attractive sedative agent in NIV. However, in the choice of the propofol sedation regimen dose, it is of pivotal importance because propofol has shown to adversely affect the breathing pattern and the respiratory drive, as well as gas exchange, proportionally to the rate of its infusion ; in thisDexmedetomidine, a selective α2 agonist with intrinsic properties of sedative and analgesic effects, may be useful for sedation of NIV patients, due to its limited effect on the respiratory pattern. According to previous findings  net of tRemifentanil is a short-acting opioid proven to be safe and effective to achieve optimal sedation in case of intolerance to NIV . In keepA separate description of the advantages and disadvantages of sedative drugs in NIV is summarized in Regardless of the sedation plan adopted, sedation assessment is of pivotal importance during NIV, through subjective scales  or tool, i.e., bi-spectral index, entropy. The sedation assessment, at regular time intervals, allows to provide the desired target of sedation and to avoid hypersedation .Regarding the concern related to the respiratory drive depression by sedative medications, it is worth to remark that sedation assessment must be assured whatever the therapeutic scheme adopted. Therefore, sedative and anxiolytic drugs should be administered in the appropriate environment, staffed with well-trained personnel in the monitoring of vital signs and sedation depth and airway emergencies management .The massive spread of COVID-19 outbreak has put in crisis the surge capacity response of whole sanitary systems worldwide . In partDespite these advantages, maintaining an APP for long-lasting sessions could be very challenging. In fact, the main cause of interruption of APP has been shown to be scarce comfort .The prone position reduces the compliance of the chest wall, leading to an increase in the work of breathing, and generating discomfort . In addiHowever, during the current COVID-19 pandemic, the importance of close monitoring of the patient in NIV has clearly emerged, as despite its clear benefits, a delay in intubation turns out to be associated with worse outcomes –106.Patients with delayed onset of invasive ventilation have increased mortality and more severe pulmonary sequelae in terms of lung carbon monoxide diffusion capacity (DLCO) and radiological imaging . One posad hoc” corrective measures is mandatory to prevent NIV discontinuation related to poor patient's tolerance.In conclusion, net of the underlying pathological disease, enhancing the patient comfort, seems the best strategy to improve the NIV rate of success, especially when NIV is administered for a prolonged period of time, also in combination with APP as rescue therapy. Accordingly, a strict comfort assessment with the “GC and RS proposed the project and conducted the research. GC and RS wrote the manuscript while RS was responsible for tables and figure. ED revised the manuscript. All authors approved the final version of the manuscript.This study was financially supported by HORIZON 2020, European Commission, ENVISION–Intelligent pug-and-play digital tool for real-time surveillance of COVID-19 patients and smart decision-making in Intensive Care Units (Grant No. 101015930).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Caregivers of persons with dementia (PWD) often experience increases in depression, anxiety, and burden as the disease progresses. In fact, as the PWD’s neuropsychiatric symptoms increase and independence in ADLS decrease, caregivers psychological and physical health outcomes worsen. The literature suggests that caregiver interventions that teach specific skills are more beneficial than psychoeducational interventions, particularly regarding the amelioration of the psychological impacts of informal caregiving. However, because of caregiving demands, caregiver’s own physical limitations, and competing obligations, it can be difficult to attend caregiver support or education programs outside the home. With the emergence of the COVID-19 pandemic, arranging such interventions became more complex, Therefore, we report on preliminary qualitative outcomes of a study investigating the feasibility and acceptability of converting an in-person, group dementia caregiver education intervention, CARE, to a telehealth platform. We report the findings of two objectives: 1) lessons learned when attempting to convert an in-person group intervention to telehealth and 2) experience and perceived benefit of attending a virtual group from the perspective of the participants of our first two groups. Briefly, our findings demonstrate the strong need for technological support. Participants report positive experience regarding the convenience of attending the group from their home, the benefits of the assigned exercises, and the support they found from other group members. The COVID-19 pandemic has forced many to embrace the virtual option as they adapt to a new normal. There are undoubtedly hurdles to overcome, but there are also advantages to be leveraged.


Obesity has now reached pandemic proportions and represents a major socioeconomic and health problem in our societies. Up to now, obesity has been considered a medical issue only in high-income countries, but this disease is now dramatically on the rise in low- and middle-income countries, particularly in urban settings, affecting both adults and the pediatric population . The simplest definition of overweight and obesity is an excessive accumulation of fat due to an imbalance between the energy in and the energy out, and this adiposity represents a risk to health . But theIn the last few decades, it has been strongly demonstrated that the hypothalamus is the master regulator of energy homeostasis. The hypothalamus contains hormone- and nutrient-sensing nuclei that organize central and peripheral responses for maintaining normal body weight, food intake, energy expenditure, and nutrient partitioning. Within the hypothalamus, specialized sub-populations of neurons are connected to each other and to various extrahypothalamic regions to coordinate energy balance. Evidence has also highlighted the participation of non-neuronal populations , and even the interesting cross-talk between these types of brain cells, whose disruption leads to insulin resistance and obesity. In this Special Issue, we report on the most recent insights into the hypothalamic circuitries and pathways involving neurons ,4,5,6,7,Npgl in the mediobasal hypothalamus of mice improved glucose tolerance and attenuated insulin resistance and hyperglycemia under high-fat diet (HFD) exposure, without significant changes in food intake. The concomitant increased mRNA expression levels of galanin  suggests that both peptides could regulate each other for these actions [Regarding the importance of hypothalamic targets in obesity development, Quiñones et al.  elegantl actions .When investigating the hypothalamic regulation of obesity, in addition to the identification of potential targets mediating the homeostatic circuits, it is also important to explore the hedonic circuits mediating energy balance, identifying the salient neural, hormonal, and humoral components involved, as remarkably reviewed by Gastelum et al. . They exHypothalamic circuits controlling energy balance in response to feeding are also mediated by the gut-to-brain nutrient signaling, whose disruption leads to obesity. As extensively reported by Romani-Perez et al. , aberranAn already well-established concept in the neurobiology of obesity is that neurons are not the only characters playing a main role in the response processing and the transmission of information. This is also true in the hypothalamus, where a maladaptive interaction between neurons and their surrounding microglia and astrocytes likely contribute to the development and progression of obesity ,12. The The function of hypothalamic astrocytes and the interplay between astrocytes and neurons are also becoming very important in the study of obesity development. In this special issue, Song et al.  show theTherefore, the manuscripts published in this Special Issue provide new insights into the hypothalamic neuronal and non-neuronal pathways involved in the pathophysiology and development of obesity and related diseases such as diabetes . Via fou


Nanoparticulate systems have been extensively explored for the treatment of various diseases, including cancers. The outstanding characteristics of nanoparticles have made it possible to administer them via different routes such as intravenous or inhalation. This flexibility can improve the delivery of encapsulated drugs to the targeted cells for the treatment of lung-related diseases and cancers such as non-small cell lung cancers. The effectiveness of a treatment option needs to be validated in suitable in vitro and/or in vivo models. As the handling of in vivo models is a challenge, many researchers have turned towards in vitro models that use normal cells or specific cells from diseased tissues. This review focuses on the currently available nanoparticles for lung cancers and the type of cellular work that can be conducted to evaluate the effectiveness of a nanoparticulate system for this cancer type.Lung cancers, the number one cancer killer, can be broadly divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), with NSCLC being the most commonly diagnosed type. Anticancer agents for NSCLC suffer from various limitations that can be partly overcome by the application of nanomedicines. Nanoparticles is a branch within nanomedicine that can improve the delivery of anticancer drugs, whilst ensuring the stability and sufficient bioavailability following administration. There are many publications available in the literature exploring different types of nanoparticles from different materials. The effectiveness of a treatment option needs to be validated in suitable in vitro and/or in vivo models. This includes the developed nanoparticles, to prove their safety and efficacy. Many researchers have turned towards in vitro models that use normal cells or specific cells from diseased tissues. However, in cellular works, the physiological dynamics that is available in the body could not be mimicked entirely, and hence, there is still possible development of false positive or false negative results from the in vitro models. This article provides an overview of NSCLC, the different nanoparticles available to date, and in vitro evaluation of the nanoparticles. Different types of cells suitable for in vitro study and the important precautions to limit the development of false results are also extensively discussed. After breast cancer, lung cancer (LC) is the second most diagnosed cancer in the world, with over two million new cases in 2020 . HoweverNSCLC can be further categorized into three types, namely, adenocarcinomas, squamous cell carcinomas and large cell carcinomas. Lung adenocarcinoma occurs in the lung periphery, usually related to the surface alveolar epithelium or bronchial mucosal glands, accounting for about half of all lung cancers . AlthougCurrent treatment options for NSCLC include surgery, targeted therapy, chemotherapy, and radiation therapy . SystemiOther treatment options for patients with metastatic NSCLC who progress after platinum-based chemotherapy (second line and beyond) include pemetrexed and docetaxel  as well Nanomedicine is a nanotechnology branch that employs materials between 5–200 nm, applied to health and medicine ,22. NanoAmong the challenges for progress in NDDS is the requirement for thorough physicochemical characterization and evidence of safety and efficacy in the delivery of the encapsulated drug to cancer cells . PredictBased on the data available in the literature, NDDS has a high potential for lung delivery of active pharmaceutical ingredients (API). Due to their nanosize, these nanocarriers may be capable of effectively traversing the bronchial epithelium barrier and accumulating in deep lung regions. Herein, we present an overview of the latest nanotechnological approaches for the delivery of chemo-immunotherapeutic agents against NSCLC. Types of NDDS, use of cell evaluation, and the rationale behind their applications in permeability and cell uptake studies as well as toxicity evaluation of the NDDS are also presented and discussed.Various nanocarrier platforms are being investigated as the drug delivery systems for an arsenal treatment of NSCLC. Lipid-based  and nanostructured lipid carriers (NLCs)), polymeric-based, and metal-based nanoparticles are some of the well-studied classes of nanocarriers. They are briefly discussed in the following subsections.Doxil is the first nanoliposome formulation containing doxorubicin approved by the US FDA in 1995 to treat AIDS-related Kaposis sarcoma . LiposomSolid lipid nanoparticles (SLNs) are colloidal nanocarriers developed in the 1990s to overcome the limitations of emulsions, liposomes, and polymeric systems . SLNs arNanostructured lipid carriers (NLCs) are second-generation lipid-based nanoparticles reported in the mid-1990s to mitigate the drawback associated with SLNs, as mentioned in Polymeric nanoparticles have been extensively researched for NSCLC therapy, with polymers such as poly-lactic-acid-co-glycolic acid (PLGA), poly-lactic-acid (PLA), chitosan, and polycaprolactone ,53. PolyDendrimers are a unique class of polymeric nanoparticles first reported in the late 1970s . They arPolymeric micelles (PMs) (10–100 nm) are produced by the self-assembly of copolymers or amphiphilic surfactants in water above its critical micellar concentration f. PMs coVarious classes of metal-based nanoparticles such as gold g, silverGraphene h, a carbDelivering NDDS for lung-related diseases has been challenging, but it holds significant potential. Numerous efforts have been reported in the literature to determine the best approach, route, material, and technique to achieve the desired therapeutic outcomes. In general, lung targeted delivery may be achieved through localized  or systemic (intravenous) administration. Systemic administration has been the method of choice to ease delivery, but it causes several drawbacks such as sub-optimal concentration of drug at the site of lung cancer and toxicities on healthy cells .Localized delivery is nevertheless a preferable approach as it lowers the occurrences of adverse reactions that may happen due to systemic distribution. The inhalation route of administration is not only useful for its local effect, but may also contribute to the systemic efficacy of drugs as they may also accumulate in the lymphatic circulation following administration . As NDDSThere are specific challenges that need to be considered to ensure an efficient pulmonary delivery of NDDS. The structure of the respiratory system and lung clearance mechanism may complicate the effort to ensure a sufficient amount of drug-encapsulated NDDS are accumulated at the cancer site ,77,78. TEmerging evidence has highlighted the usefulness of nanoparticles as drug carriers, especially in cancer targeting, due to their ability to deliver various drugs with diverse characteristics in nature. However, it remains a challenge to choose the most suitable approach to closely characterize and evaluate the efficacy and safety profiles of nanomedicines in conditions that closely reflect the physiological complexity. It should be noted that the findings from preclinical studies may not represent the actual clinical outcomes of the developed nanomedicines. Herein, the cellular evaluation of nanomedicines against NSCLC lung cancer cells is reviewed based on the available data in the literature. Different types of cell lines have been reported as models to aid in the understanding of nanoformulation activity and effectiveness at the cellular level. This section will describe in detail the type of cell lines and the suitability of the cells to give the information needed in the evaluation of nanoformulations.Since the 1970s, over 200 lung cancer cell lines encompassing many of the different histological subtypes of the disease have been established. Despite the fact that they have been established many years ago, they often retain characteristics of the original tumor they were derived from . The numEpithelial lung cells are valuable tools for the study of multi-stage lung cancer pathogenesis. For research, two types of culture models are available, namely primary cultured cells and immortalized cell lines.Generally, primary cell cultures retain the morphological and biochemical characteristics of the tumor from which they were originally derived . The priPrimary cell immortalization can be performed using in vivo models, usually of mice. The microenvironment of living tissue promotes cancer cells’ growth, mainly due to the high number of epithelial components and the subsequent crosstalk of the tumor . The comIn vitro cultures of immortalized cell lines isolated from tumors have been used as model systems in cancer for at least 65 years . UnderstImmortalized cell-based models can be composed of solely immortalized cells of two primary pulmonary cell types, namely bronchial and alveolar cell lines. The commonly used bronchial cell lines include Calu-3. This cell line can be cultured under different conditions, namely air–liquid and liquid–liquid interfaces, resulting in the formation of tight epithelia in both cell cultures . HoweverThe Calu-3 cell line can be used to assess formulation transportation in the lung epithelial cell such as in the evaluation of intracellular uptake and transport capability of a liposomal powder formulation loaded with ciprofloxacin and colistin. A study showed that the co-loaded liposomes resulted in a lower transport capability of both drugs across the Calu-3 cell monolayer, resulting in an accumulation in the cell. Since the treatment was aimed at respiratory tract infections, drug retention in the cells is expected to be beneficial .In utilizing alveolar cell lines, the human lung alveolar adenocarcinoma cell line (A549) was often used as a model as it mimics the morphological and biochemical properties of human lung cells and secretes surfactant protein . MonolayCo-culture of the A549 cell line with two different types of cells to create a more complex system resembling the human airways was reported in a study by Wang and co-workers . The A54The monolayers of the single- or co-cultured cell lines can be grown under different conditions, namely liquid–liquid interface (LLI) and air–liquid interface (ALI) conditions. The former is a conventional method to evaluate nanoparticles’ dissolution rate but may not be suitable for assessing nanoparticles intended for inhalation purposes due to the extremely thin aqueous layers in the lungs . DiffereCell-based in vitro assays are routinely used to determine direct cytotoxic or antiproliferative effects of test substances on various cell lines. These assays have been used in the characterization of nanoformulations for NSCLC treatment including liposomes ,101, solA number of antiproliferative assays with different principles have been developed for the cytotoxicity screening of nanomaterials, as shown in MTT assay was first described by Mosmann  and subsIt is worthwhile to note that MTT reduction is readily affected by metabolic and other factors such as the composition of cell medium, which may, in turn, substantially affect the quantitation of cell viability . MTT forXTT, including MTS and WST-1, also presents several problems associated with high-flux drug screening, including the inability of many cell lines to metabolize the tetrazolium in the absence of an added electron transfer reagent such a phenazine methosulfate . HoweverThe WST-1 substrate offers several advantages over MTT and XTT, including water-solubility and greater stability and sensitivity . HoweverSeveral studies have highlighted misleading cell viability results when using tetrazolium salts to evaluate cell viability following treatment with nanomaterials. Wörle-Knirsch and co-workers have demonstrated that single-walled carbon nanotubes bind to MTT-formazan crystals, producing false cytotoxicity results . Data frThe measurement of ATP in cytotoxicity testing is based on the luciferin–luciferase bioluminescent reaction, which requires luciferase enzyme, luciferin and ATP, magnesium , and oxygen. ATP concentration declines rapidly when cells undergo cell death; hence, ATP levels are a reliable indicator of cytotoxicity effects . ATP in 2-25) nanoparticles are also capable of adsorbing LDH molecules, affecting the LDH assay [The LDH assay principle is typically based on cell membrane integrity and monitoring the release of lactate dehydrogenase from compromised cells . LDH assDH assay . ZnO intDH assay . AdsorptDH assay .The trypan blue exclusion assay principle is similar to the LDH assay, except that the trypan blue assay involved microscopy examination of cells. This simple assay requires the use of a hemocytometer and the calculation of cells stained with the blue dye indicating dead cells. . Hence, A recent innovative strategy to measure cell viability in real time was developed to overcome such limitations. The bioluminescent assay is based on the cellular capacity to reduce a luciferase prosubstrate to a form that is then rapidly utilized by the luciferase enzyme present in the assay . Since tIn nanoparticle-based drug delivery system, parameters such as the nanoparticle size, concentration, coating, use of GRAS excipients, agglomeration, surface corona, charge, and hydrophobicity/hydrophilicity significantly affect the biological responses at the cellular level . Thus, oCytotoxicity assays are crucial preliminary assays to determine the safety profile of nanoparticles using cell lines. In fact, the EU NanoSafety Cluster group has suggested that at least four methods of determining cytotoxicity should be used in order to obtain a reliable safety profile for novel nanomaterials . As in tIndeed, the barriers involved in the administration route must be fully understood to ensure optimal delivery of the drugs to the cancerous site. In vitro techniques are imperative to assess the fate of the formulated drug in the lung including binding, transport, uptake, metabolism, and any toxicity related to pulmonary route administration. Particularly, in vitro models for predicting the permeability of drugs or nanoparticles would offer several advantages compared to the in vivo studies, as the former offer simplicity and reproducibility, are less expensive, and do not require ethical considerations . The in Vesicular transcytosis happens in the presence of caveolae  and clathrin-coated pits  . The vesIn vitro and in vivo studies suggest that active targeting nanoparticles will increase selectivity in the cellular uptake and/or cytotoxicity over the conventional chemotherapeutic drugs and non-targeted nanoparticle platform, particularly enhancing drug efficacy and safety . Ligand For a better understanding of the uptake process, quantification of the nanoparticles in the cells would be valuable. However, the small particle size and low mass of the delivery system limit the sensitivity and resolution of certain techniques. Inductively coupled plasma (ICP)-based spectroscopic techniques have been used in the quantification of the nanoparticle uptake in the NSCLC cell lines including ICP-mass spectrometry (ICP-MS) ,174 as wAnimal models have been used in the evaluation of toxicological, allergic, pro-inflammatory, and immunological properties of pharmaceutical products or formulations in pre-clinical studies. However, utilization of ex vivo human systems has gained increasing attention for high-throughput screening of potential toxicological and allergic reactions of the lung towards pharmaceutical formulations, as they can overcome some limitations related to the animal models, including high cost and time spent. One of the most common tissue models is precision-cut lung slices (PCLS), which can be cultured from the explanted healthy or diseased lung of human or animal models, with a more mechanistic resemblance to multiple regions of the lung . This moPlacke and Fisher in 1987 had successfully developed a method to obtain PCLS, which previously imposed a technical challenge for soft tissues such as lungs . The metPCLS has been used to assess the toxicity and efficacy of biological agents or materials to the respiratory tract ,185, incBeing cultured in ALI conditions, the multi-layered monocultures were demonstrated to be compatible with testing drugs administered as a liquid aerosol by a clinical nebulizer, offering an advantage over 3D tumor spheroids . There aTo date, there is a variety of 3D cell models available that can be employed to design and evaluate the efficiency of drug nanocarriers. These models enable the researchers to study the specific aspects of the nanomedicine behavior in vivo, although none of these models can recapitulate the exact complex tumor micro-environment. Among the common 3D multicellular in vitro tissue systems are organoid and spheroid systems . They caAnother advanced 3D cell model is the microfluidics-based organ-on-a-chip (Organ Chip), which has recently received increased attention as a promising platform as this cell model accurately replicates the microenvironments of native tissues and various tissue-tissue interactions . It simuNotwithstanding the advantages of cell lines in evaluating the efficiency of delivering nanomedicine for lung cancer treatment, questions have been raised about their reliability since not all lung cancer subtypes are well represented by these cell-based experimental models . The strThe outcome of a seemingly simple cytotoxicity assay might be highly dependent upon the respective protocol and the test substance used. Various factors are known to contribute to aberrant results, such as the physical interactions between the nanomaterials used to manufacture the nanoparticles and the components in the assays , changes in cellular activities involved in redox reactions in response to the nanomaterials, and many other unknown factors that may influence the determination of the cytotoxicity profile of nano-particulate systems . PerhapsIn addition, higher concentrations of nanoparticles (>10 mg/L) appear to have a greater probability of interfering with assay function . Hence, Pharmaceutical technology has been playing a vital role in medicine, as it allows the exploration of different materials and their combinations to prepare drug carriers, and to further endow these with better properties that enable them to reach the desired target site. Many questions arise around the topic of nanocarrier-based lung drug delivery systems. Scientists have given a plethora of responses in the attempt to address all the issues raised, finding alternatives and engineering adequate systems to fulfil requirements and needs. One of the concerns is always the fate of the drug particles once they reached the target site. In pulmonary drug delivery, the objective is sometimes to retain the drug in the lung, as happens in local delivery approaches, thus minimizing the systemic absorption. In other cases, an intravenous delivery route would aim for a systemic effect, and the carriers are engineered to avoid retention at the administration site, with a specific targeting agent attached that would eventually bring the nanocarrier to the lung for the desired therapeutic effect.In vitro cell-culture-based models have been used for permeability screening and cell uptake study of drugs or formulations. Although some cell lines have limitations, continuous improvements have been made including co-culture with other cells to produce a model that can mimic the human lung epithelial barrier as closely as possible. These models are easy to exploit and do not involve ethical considerations related to animal use, as required in a general in vivo study. In a nutshell, the basis behind each decision made on an experimental model should be aligned to the research aims and relevance to human physiology and should be pertinently translated.


The role of adipose tissue (AT) inflammation in obesity and its multiple related-complications is a rapidly expanding area of scientific interest. Within the last 30 years, the role of the adipocyte as an endocrine and immunologic cell has been progressively established. Like the macrophage, the adipocyte is capable of linking the innate and adaptive immune system through the secretion of adipokines and cytokines; exosome release of lipids, hormones, and microRNAs; and contact interaction with other immune cells. Key innate immune cells in AT include adipocytes, macrophages, neutrophils, and innate lymphoid cells type 2 (ILC2s). The role of the innate immune system in promoting adipose tissue inflammation in obesity will be highlighted in this review. T cells and B cells also play important roles in contributing to AT inflammation and are discussed in this series in the chapter on adaptive immunity. Obesity is a growing healthcare problem in the United States and globally. It is a leading cause of preventable death and currently impacts more than 35% of the US population . This nuThe role of the immune system is to identify self- versus non-self to eliminate potential toxins, allergens, and pathogens without destroying the host tissue. The immune system is composed of two key functional responses, innate and adaptive immunity. The innate immune response is the initial line of defense after the host’s external barrier. This immune response is not antigen-specific, but rather recognizes molecular patterns that are inherent to the toxin, allergen, or pathogen. This allows for the rapid activation of an immune response which is followed by the development of an antigen-specific immune response. The adaptive immune system upon the first encounter of a toxin, allergen, or pathogen undergoes expansion to aid the innate immune response. Upon resolution, a subset of these adaptive immune cells persists and creates a distinct population of memory immune cells. Memory cells are faster to respond to future encounters with the same pathogen, allergen, or toxin. The cellular component of both immune responses arises from the hematopoietic stem cell. This pluripotent cell further differentiates within the bone marrow to generate either the common lymphoid progenitor or the common myeloid progenitor. The common myeloid progenitor gives rise to lineage-specific colony-forming cells which then further develop into a majority of the innate immune cells as well as megakaryocytes (platelets) and erythrocytes (red blood cells). The cellular components of the innate immune response include the granulocytes including monocytes, macrophages, neutrophils, basophils, eosinophils, mast cells, and dendritic cells, as well as adipocytes. While the adipocyte is not traditionally viewed as an immune cell, recent research has demonstrated that the adipocyte releases adipokines, microRNAs and lipids to influence the innate immune response –7. The aThe AT immune cell microenvironment in the lean state is a well-balanced crosstalk between the adipocyte and the stromal vascular fraction (SVF) or the cellular compartment of AT. In lean mice, the SVF is comprised of mesenchymal stem cells , endotheThe adipocyte, unlike most traditional immune cells, links the innate and adaptive immune systems through adipokine, lipid and exosome release and through antigen presentation. While the adipocyte is the primary site of energy storage for the body and performs multiple metabolic activities, it can assume the role of a highly functional immune cell, releasing anti- and pro-inflammatory cytokines and hormones (adipokines), as well as lipids, which also act as signaling molecules . Since iThe first adipokine described was leptin which revolutionized our understanding of the critical role that adipocytes play in whole-body energy homeostasis . Mutatiovia modulation of NADPH oxidase  on the surface of immune cells, such as macrophages, and are converted to ceramides and diacylglycerols during states of lipid overabundance as occurs in obesity. These toxic lipids enhance proinflammatory signaling . More rehandling . These ovia exosomes, which was increased in obese mice. This release of exosomes contributed to macrophage foam cell formation, suggesting that exosomes contribute to the orchestration of AT immune cells , which are a major mechanism for energy storage and release. Adipocytes not only release lipids and secrete adipokines, but they can also employ exosomes- extracellular vesicles (40-150 nm in size) of endosomal origin to participate in this process. Exosomes are increasingly recognized as a novel mechanism by which adipocytes communicate with other cells and target tissues. Their cargo contains adipokines, lipids, and microRNAs. Release is dependent on nutritional status and degrees of adiposity: increased release in obesity and decreased release with caloric restriction or lipodystrophy. Exosomes can be taken up by endocytosis, pinocytosis, or phagocytosis, and can be directed to target cells by adhesion molecules on the exosomal surface . Recentlne cells . MicroRNne cells . Howeverne cells , 56.Finally, one of the most unique features of the adipocyte is its ability to function as an antigen-presenting cell, which is described in detail in Chapter XX of this series by Deng et al. The adipocyte can present antigen to promote differentiation and activation of interferon gamma-producing CD4 Th1 cells. This activity is increased early in obesity, after only 2 weeks HFD in mice, before the AT macrophage increase, suggesting adipocytes both instigate and maintain AT inflammation . AdipocyNeutrophils are one of the initial inflammatory cells recruited to sites of host injury. As a component of the innate immune system, neutrophils have four primary activities including, phagocytosis, degranulation, reactive oxygen species (ROS) production, and neutrophil extracellular trap (NET) formation . Within Early studies in mice observed the transient infiltration of neutrophils into the AT after the start of HFD resulting in maximal levels by day 3 and undetectable levels by day 28 . Howevervia increased production of CXCL2, another known neutrophil chemoattractant . ILC2 cells are important in the maintenance of insulin sensitivity and are decreased in the setting of HFD. Innate lymphoid cells express CD4+ related cytokines, mirror Th1, Th2, and Th3 expression profiles , but difMultiple changes in the innate immune system are key contributors to inflammation in obese AT resulting in the development of obesity-related diseases as summarized in AB, AJ, and WH were involved in all aspects of writing, editing, and decision to publish. All authors contributed to the article and approved the submitted version.Funding for this manuscript includes the American Diabetes Association (1-16-ICTS-049) and The National Institutes of Health (R01HL135622).WH advises for Novo Nordisk and Intercept Pharmaceuticals.The remaining 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.


Cancer cell-derived extracellular vesicles (CEVs), a novel type of therapeutic agent in cancer treatment, can be prepared from the autocrine secretion of various cancer cells, the direct extraction of cancer cells and the combination of cancer cell-derived membranes with advanced materials. With various bioactive molecules, exosomes are produced by cells for intercellular communication. Although cancer cell-derived exosomes are known to inhibit tumor apoptosis and promote the progression of cancer, researchers have developed various innovative strategies to prepare anti-tumor vesicles from cancer cells. With current strategies for anti-tumor vesicles, four different kinds of CEVs are classified including irradiated CEVs, advanced materials combined CEVs, chemotherapeutic drugs loaded CEVs and genetically engineered CEVs. In this way, CEVs can not only be the carriers for anti-tumor drugs to the target tumor area but also act as immune-active agents. Problems raised in the strategies mainly concerned with the preparation, efficacy and application. In this review, we classified and summarized the current strategies for utilizing the anti-tumor potential of CEVs. Additionally, the challenges and the prospects of this novel agent have been discussed. Extracellular vesicles (EVs) are phospholipid bilayer membrane-coated vesicles that are generated by cells for intercellular communication , 2. EVs in vitro, in vivo, and clinical trials have been conducted using CEVs-based strategies to obtain CEVs with effective anti-tumor activity. One of the primary advantages of CEVs includes their high targetability to the tumor. Additionally, they are known to preferentially target their homologous tumor cells , which is a unique reaction triggered by irradiated cells or tissues, leading to the induction of apoptosis in unexposed cells –45. SimiIn a recent study on irradiated CEVs, the researchers irradiated the cancer cells with a single dose of 20 Gy by 6-MV X-rays to generate EVs . The resAlthough the anti-tumor efficacy of irradiated CEVs was triggered by the promotion of tumor antigen , the detThere has been an extensive increase in the use of advanced materials as biomimetic carriers in the field of cancer therapy. Nanoparticles serve as stable carriers to protect antigens from rapid clearance and degradation in the serum, achieving a stronger immune response –57. In a2Se3 and chemotherapeutic doxorubicin hydrochloride . Cisplatin-loaded CEVs were prepared from the supernatant of the mixture of A549 cells and Cisplatin . The injCEVs are the final products generated and prepared from cancer cells. Therefore, the construction of a genetically modified CEVs requires a genetically engineered cancer cell line. Thus, various design strategies were designed by transfecting therapeutic genes into cancer cells to alter the inherent characteristics of CEVs for cancer therapy –31, 33. The miRNA content of the exosomes is also known to promote the progression of cancer , such asvia transfection to boost the therapeutic efficacy of inherent proteins in CEVs. An efficient induction of anti-tumor immunity was observed after treatment with CEVs that overexpressed Rab27a (Also, CEVs are known to naturally possess the proteins that present in the corresponding cancer cells, such as immunosuppressive PD-L1  and prosd Rab27a , which id Rab27a  and antid Rab27a . It is nd Rab27a .Although gradient ultracentrifugation provides excellent purity for the preparation of CEVs, sequential ultracentrifugation is more commonly used as it is more convenient and provides a larger volume. The polymer precipitation technique is easy to perform and efficient; however, it has a complicated clean-up process. Additionally, novel strategies, such as microfluidics-based techniques and immunoaffinity capture, are also suitable for the isolation of CEVs; however, they are expensive.Thus, new techniques are required for the innovative generation of self-designed CEVs for further analysis of the anti-tumor potential of CEVs. A sequential extrusion strategy was used for the artificial synthesis of CEVs. CEVs were prepared after extrusion through 10 and 8 μm membrane filters . AdditioBefore CEVs reach the tumor area, the number of intact EVs determine the therapeutic efficacy of CEVs. The efficacy of CEVs is largely dependent on the stable construction and good targetability after preparation and preservation.via the communication of irradiated exosomes (Since the EVs derived from cancer cells can be important promotors of tumor progression, tumor metastasis, immunosuppression, and drug resistance –14, 83, exosomes . Additioexosomes , 87, altexosomes . Studiesexosomes , 61 and exosomes  can resuvia the modification of tumor-targeted-aptamers (Although the cargo carried by the EVs play a vital role in exerting a suitable therapeutic effect, their efficacy is also determined by their targetability and the effective functioning in the tumor. For the efficacious transport of CEVs to the tumor, the targetability of CEVs could be enhanced aptamers  and peptaptamers  on EVs. aptamers .CD47, on EVs in cancer patients, carries a “don’t eat me” signal ; thus, CIn the bright side, the newest researches showed the CEVs were able to be used for the trace of the early stage of neoplastic transformation  as well In the clinical experiments, the drug resistance of tumor-repopulating cells can be reversed after the application of CEVs . BesidesHowever, the low production of CEVs remains a challenge for clinical application. Compared with the CEVs from MPE, the autologous tumor cell-derived extracellular vesicles are not always available in patients with solid tumors for the preparation of CEVs. There is an urgent need for the development of an innovative strategy for the large-scale production of CEVs.Despite the predetermined notions regarding the role of CEVs based on their contents, the therapeutic role of phospholipid bilayer membrane-coated EVs prepared from cancer cells was explored. The CEVs have shown excellent potential as anti-cancer agents. The strategies employ diverse generation, application, and therapeutic mechanisms. The main strategies for CEVs generation have been summarized in four categories, including irradiated CEVs, advanced materials-combined CEVs, chemotherapeutic drug-loaded CEVs, and genetically engineered CEVs Table 1.WL was in charge of all the writing and graphics. X-DC was in charge of the design and instruction. All 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Patients with sepsis commonly suffer from coagulation dysfunction and lead to the formation of thrombus. During the development of sepsis, neutrophils migrate from the circulating blood to infected tissues and mediate the formation of neutrophil extracellular traps (NETs) that kill pathogens. However, the overactivation of neutrophils can promote the formation of immunothrombosis and even cause disseminated intravascular coagulation (DIC), which damages microcirculation. The outcome of sepsis depends on early recognition and intervention, so clinical evaluation of NETs function may be a valuable biomarker for early diagnosis of sepsis. The interaction of NETs with platelets, complement, and endothelium mediates the formation of immunothrombosis in sepsis. Inhibiting the formation of NETs is also considered to be one of the potential treatments for sepsis. In this review, we will discuss the key role of neutrophils and NETs in sepsis and septic thrombosis, in order to reveal new mechanisms for thrombosis treatment of sepsis. Sepsis is a life-threatening organ dysfunction caused by a disordered response to infection . In 2017As the most abundant white blood cell group in humans, neutrophils play an essential role in the body’s innate immunity caused by infection. An excessive activation of neutrophils can lead to the development of multiple organ dysfunction syndromes . In sepsIn summary, NETs can eliminate pathogens by mediating neutrophils, but their hyperfunction will also cause tissue damage . In thisIn 1971, Lerner first discovered the important role of neutrophils in the process of thrombosis . But it Neutrophils can activate NADPH oxidase through protein kinase C (PKC) and Raf-MEK-ERK signaling pathways to produce reactive oxygen species (ROS). ROS-activated peptidylarginine deiminase 4 (PAD4) modifies specific arginine residues on histones H3 and H4  to make The outcome of sepsis depends on early diagnosis and intervention. Thus, it has been suggested that early recognition of NETs may be a valuable biomarker of early sepsis. The degree of NETs formation is significantly related to the severity of the disease, and can independently predict the development of diffuse intravascular coagulation (DIC) and mortality . Cell frThese markers have the potential to detect NETs, although they are not directly achieved by detecting NETs. At the same time, some markers are not only related to NETs, but also related to infections caused by other mechanisms. For example, the concentration of cfDNA in plasma can activate the nucleic acid sensing pathway to generate inflammation, and participate in the formation of sepsis . In addiThe integrity of endothelial structure and function is the key to maintaining vascular homeostasis. Endothelial injury plays an important role in thrombosis. In the development of sepsis, neutrophils are recruited to the site of inflammation through rolling, activation, firm attachment, and extravasation on ECs . The rolIn septic-infected mice, the release of extracellular histones led to infiltration of neutrophils, vacuolation of ECs, intra-alveolar hemorrhage, and vascular thrombosis . When ICPlatelets play an essential role in coagulation . PlateleSepsis is usually accompanied by thrombocytopenia and occlusion of small blood vessels, called disseminated intravascular coagulation (DIC). The substances produced by NETs can either diffuse throughout the interstitium of organs, or be released into the vascular lumen and attach to the vascular wall of narrow capillaries . In the LPS can initiate the non-classical activation of platelets, and activate TLR4 on platelets which will also lead to the release of the NETs . The actTo a certain extent, NETs can trigger pro-thrombotic and pro-coagulant platelet-mediated responses by interacting with TLR4. As a key receptor, TLR4 on platelets may be an important factor in septic DIC . In septIn recent years, it has been discovered that NETs can activate three different complement pathways to form thrombosis. The complement system is stimulated by active coagulation factors, causing the immune system to recruit neutrophils . The comComplement C3 is an important mediator in the complement system and activates neutrophils during sepsis . NeutropIn recent years, researchers have proposed that there may be an effect mechanism in the link between blood coagulation and innate immune response that mediates the formation of immunothrombosis. Initially, NETs were found in patients’ arterial or venous thrombosis. Researchers gradually realized that NETs provide a scaffold to recruit red blood cells, platelets, white blood cells, as well as bind plasma proteins. The aggregation of NETs in the coronary arteries suggested that NETs might contribute to the growth and stability of thrombus . By analThe coagulation system is activated by pathogens and helps the host defend against infection by reducing the spread of pathogens and killing them . ImmunotThe presence of senescent and pro-inflammatory neutrophils in the circulation of miR-146a-deficient mice leads to increased thrombosis in the LPS-induced sepsis model . ExposurThe two-way effect of NETs and platelets represents the connection between these phenomena. Whether in infectious or non-infectious diseases, the interaction of platelets and neutrophils is crucial , and conAlthough NETs are increasingly recognized as important therapeutic targets, there is currently no clear drug that regulates NETs to guide the treatment of sepsis and sepsis-related thrombosis. Several potential targets of NETs are listed below.-/- septic mice did not enhance the thrombotic response, and circulating ICAM-1 was significantly reduced  leads to the elimination of chromatin and the release of NETs in cells  of NETs . Therefo of NETs . PAD4 in of NETs . Perdomo of NETs . PAD4 in of NETs . The PAD of NETs , and can of NETs . TherefoThe role of heparin therapy in anticoagulation is unquestionable. However, the treatment of thrombus caused by heparin in NETs is still controversial. Low-dose heparin (250U/kg) treatment can reduce the expression of NETs, histones, and pro-inflammatory factors . A largeIn septic patients, the concentration of IL-8 is closely related to the formation of NETs. IL-8 activation of mitogen-activated protein kinase is one of the main pathways for the formation of NETs in patients. Inhibition of IL-8 can significantly reduce NETosis . HistoneThere is a dynamic platelet thrombin axis in sepsis, which promotes intravascular coagulation and microvascular dysfunction. DNase can inhibit the formation of NETs by degrading DNA. DNase1 and DNase1-like 3 can be expressed independently, degrade NETs in sterile neutrophilemia and sepsis, provide double protection, and protect the host from the harmful effects of intravascular NETs . EliminaNETs have been confirmed to be related to inflammation, infection, and autoimmune diseases, and play an important role in sepsis and thrombosis caused by sepsis. NETs can not only play a role in immune defense in the inflammatory response, on the other hand, their excessive activation can also lead to a strong immune response and cause adverse consequences. In sepsis, NETs mainly through the interaction of neutrophils and platelets, leading to the functional damage of vascular ECs and the formation of immunothrombosis, further promoting the occurrence of microcirculation disorders.The existence of NETs suggests that in the treatment of sepsis, whether a new method can be used to avoid excessive activation of inflammation and prevent the further occurrence of sepsis without affecting blood coagulation. By detecting the formation potential of NETs in the peripheral circulation, it can be used clinically to identify patients at risk of DIC and poor prognosis of sepsis. Based on the evidence that NETs and platelets interact to promote intravascular coagulation and its diffusion, we believe that the early recognition of NETs in sepsis can prompt early treatment of sepsis and DIC high-risk patients, and also provide a new direction for its treatment.ZC, HZ, WC, and CM provided the conception and the structure of the article. ZC wrote the draft. All authors contributed to the article and approved the submitted version.This research was supported by the National Key Research and Development Program of China (NO. 2020YFC2008400), the National Natural Science Foundation of China , Clinical Research Plan of SHDC , Shanghai Shenkang hospital development center, clinical science and technology innovation project (NO. SHDC12018105), 2019 Fudan University Zhuo-Xue Project (NO. JIF159607); Shanghai Sailing Program (No. 20YF1418400); Shanghai Leading Talent (NO: 2019-112).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.


Amyloidosis is a group of diseases that includes Alzheimer’s disease, prion diseases, transthyretin (ATTR) amyloidosis, and immunoglobulin light chain (AL) amyloidosis. The mechanism of organ dysfunction resulting from amyloidosis has been a topic of debate. This review focuses on the ultrastructure of tissue damage resulting from amyloid deposition and therapeutic insights based on the pathophysiology of amyloidosis. Studies of nerve biopsy or cardiac autopsy specimens from patients with ATTR and AL amyloidoses show atrophy of cells near amyloid fibril aggregates. In addition to the stress or toxicity attributable to amyloid fibrils themselves, the toxicity of non-fibrillar states of amyloidogenic proteins, particularly oligomers, may also participate in the mechanisms of tissue damage. The obscuration of the basement and cytoplasmic membranes of cells near amyloid fibrils attributable to an affinity of components constituting these membranes to those of amyloid fibrils may also play an important role in tissue damage. Possible major therapeutic strategies based on pathophysiology of amyloidosis consist of the following: (1) reducing or preventing the production of causative proteins; (2) preventing the causative proteins from participating in the process of amyloid fibril formation; and/or (3) eliminating already-deposited amyloid fibrils. As the development of novel disease-modifying therapies such as short interfering RNA, antisense oligonucleotide, and monoclonal antibodies is remarkable, early diagnosis and appropriate selection of treatment is becoming more and more important for patients with amyloidosis. Amyloidosis is a group of diseases in which amyloid fibrils are deposited in tissues. According to a report from the International Society of Amyloidosis nomenclature committee in 2020, 36 proteins are listed as precursors of amyloid fibrils that may be deposited in the extracellular spaces of tissues of various organs . About hIn this review, we describe the ultrastructure of tissue damage resulting from amyloid deposition by focusing on two major systemic amyloidoses, i.e., ATTR amyloidosis and AL amyloidosis. Because novel disease-modifying therapies for ATTR and AL amyloidoses appear one after another, therapeutic insights based on the pathophysiology of amyloidosis are also described. This article is based on previously conducted studies and does not contain any studies with human participants or animals performed by any of the authors.TTR [ATTR amyloidosis and AL amyloidosis are major systemic amyloidoses that result in a fatal clinical outcome, particularly due to heart failure ,11. ATTRTTR . ATTRv aTTR , Japan [TTR , and SweTTR , while ATTR ,16,17. TTTR . In contTTR . AL amylTTR . HoweverTTR .Well-known textbook features of polyneuropathies resulting from ATTR and AL amyloidoses are progressive symmetrical sensory impairments collectively known as dissociated sensory loss, which is characterized by a loss of nociception and thermal sensation, as well as autonomic dysfunction such as diarrhea/constipation, orthostatic intolerance, dysuria, and erectile dysfunction, due to the loss of small-diameter nerve fibers ,20,21. TAccording to studies of autopsy specimens obtained from patients with ATTRv amyloidosis, the extent of the degeneration of neurons in the sensory and autonomic ganglia seems to be positively correlated with the amount of amyloid, suggesting that neurodegeneration occurs as a result of amyloid deposition ,25. ElecThe mechanisms of atrophy and subsequent degeneration of tissues near amyloid fibrils have not been fully elucidated. One of the possibilities is the stress or toxicity that is attributable to the amyloid fibrils themselves. A previous electron microscope study of nerve biopsy specimens from Portuguese ATTRv amyloidosis patients showed a long amyloid fibril penetrating the cytoplasmic membrane of a Schwann cell, suggesting direct damage of Schwann cells by amyloid fibrils . In contCaenorhabditis elegans or Drosophila also demonstrated the neurotoxicity of TTR in the absence of fibrillar amyloid deposition [Another possibility of the mechanisms of tissue damage is the toxicity of non-fibrillar states of amyloidogenic proteins ,34,35,36position . The extAnother important finding regarding tissue damage resulting from amyloid deposition is the obscuration of the basement and cytoplasmic membranes near amyloid fibrils. Electron microscope studies of nerve biopsy specimens have demonstrated that the membranes of Schwann cells become obscure when they are near amyloid fibrils in both ATTR amyloidosis and AL amyloidosis patients  21,28,2,30,31. TA previous study of cardiac amyloid deposits suggested that TTR aggregation into fibrillar structures tends to occur in association with the basement membrane because the expression of basement membrane components, such as collagen IV, laminin, and fibronectin, increases in parallel with the accumulation of amyloid fibrils . StudiesThe possible major therapeutic strategies for amyloidosis consist of the following: (1) reducing or preventing the production of causative proteins; (2) preventing the causative proteins from participating in the process of amyloid fibril formation; and/or (3) eliminating the already-deposited amyloid fibrils. As described earlier, the deposition of amyloid fibrils or non-fibrillar oligomers induces atrophy and subsequent degeneration of neighboring cells in amyloidosis. Therefore, intervention during the early stages of the disease process, before the occurrence of tissue damage by amyloid fibrils, seems to be an efficient approach.From this viewpoint, the development of therapeutic agents to reduce or prevent the production of causative proteins is remarkable in both ATTR and AL amyloidoses. Liver transplantation was established as a treatment for ATTRv amyloidosis in the 1990s to prevent the production of the variant TTR from the liver . RecentlAs for the strategy of preventing the causative proteins from participating in the process of amyloid fibril formation, TTR stabilizers, such as tafamidis and diflunisal, were demonstrated to be efficacious in patients with ATTR amyloidosis ,51. ThesRegarding the elimination of amyloid fibrils, monoclonal antibodies against the components of amyloid deposits have been considered therapeutic agent candidates. A humanized IgG1 monoclonal antibody against serum amyloid P component (SAP), which is a plasma glycoprotein found in any type of amyloid deposit, was considered a therapeutic agent for systemic amyloidosis along with the use of an agent that depletes circulating SAP . AlthougAmyloidosis is a group of diseases in which amyloid fibrils are deposited in various organs, including the nervous system, heart, lungs, liver, kidneys, gastrointestinal tract, and soft tissues. Amyloid deposits may be localized to a single organ, as in the case of Alzheimer’s disease, or systemic, such as in ATTR and AL amyloidoses.According to studies of autopsy specimens obtained from patients with ATTRv amyloidosis, the extent of the degeneration of neurons in the sensory and autonomic ganglia is positively correlated with the amount of amyloid, suggesting that neurodegeneration occurs as a result of amyloid deposition ,25. ElecAnother important finding of tissue damage resulting from amyloid deposition is the obscuration of the basement and cytoplasmic membranes of Schwann cells and cells constituting endoneurial microvessels near amyloid fibrils ,29. As aThe possible major therapeutic strategies for amyloidosis consist of the following: (1) reducing or preventing the production of causative proteins; (2) preventing the causative proteins from participating in the process of amyloid fibril formation; and/or (3) eliminating already-deposited amyloid fibrils. In addition to liver transplantation to prevent the production of variant TTR , patisir


Twenty years have passed since Brownlee and colleagues proposed a single unifying mechanism for diabetic complications, introducing a turning point in this field of research. For the first time, reactive oxygen species (ROS) were identified as the causal link between hyperglycemia and four seemingly independent pathways that are involved in the pathogenesis of diabetes-associated vascular disease. Before and after this milestone in diabetes research, hundreds of articles describe a role for ROS, but the failure of clinical trials to demonstrate antioxidant benefits and some recent experimental studies showing that ROS are dispensable for the pathogenesis of diabetic complications call for time to reflect. This twenty-year journey focuses on the most relevant literature regarding the main sources of ROS generation in diabetes and their role in the pathogenesis of cell dysfunction and diabetic complications. To identify future research directions, this review discusses the evidence in favor and against oxidative stress as an initial event in the cellular biochemical abnormalities induced by hyperglycemia. It also explores possible alternative mechanisms, including carbonyl stress and the Warburg effect, linking glucose and lipid excess, mitochondrial dysfunction, and the activation of alternative pathways of glucose metabolism leading to vascular cell injury and inflammation. Hyperglycemia is the primary etiologic factor of vascular disease associated with diabetes mellitus (DM) and accounts for most of the morbidity and mortality associated with both type 1 DM (T1DM) and type 2 DM (T2DM) , the effDuring the last century, many hypotheses have been proposed to explain the origin of diabetic complications. These include the advanced glycation end-products (AGEs)  ,7, oxidaFollowing a brief recapitulation of the biological background underlying the single unifying hypothesis proposed by Brownlee and colleagues , this reHistorically, the observation that not all cells and tissues are prone to hyperglycemia-induced damage and chronic complications brought the attention to differences about how different cells handle excess extracellular glucose. In kidney cells such as podocytes, mesangial cells, and tubular cells, as well as endothelial cells and cells of the immune system. Moreover, in cells of the neuron system, such as neurons and glia, glucose enters via facilitated diffusion through the insulin-independent glucose transporter 1 ,23. TherAt the end of the last century, the bulk of publications about the mechanisms underlying DM-induced vascular damage focused on four major pathways branching from glycolysis: increased flux of glucose and its downstream metabolites through the (1) sorbitol/polyol and (2) hexosamine pathways; (3) augmented intracellular formation of AGEs and expression of the receptor for AGE (RAGE); and (4) activation of protein kinase C (PKC) isoforms ,25. Inte2) processed in the mitochondria, the conversion to water involves the four-electron reduction of O2 by hydrogen. This is a finely coordinated process, in which the electrons from the electron donors NADH and FADH2 are transferred through Complexes I–IV of the electron chain and protons are extruded outwards into the intermembrane space, generating a proton gradient , which represents the product of superoxide dismutation catalyzed by superoxide dismutase (SOD) enzymes . So. So2O2 ialdehyde . Togethealdehyde , these Raldehyde ,73. Theraldehyde .2O2 during oxidation of hypoxanthine to xanthine and further conversion of xanthine to uric acid [Xanthine oxidase (XO) is an oxidoreductase enzyme that generates ROS such as superoxide and Hric acid . Similarric acid ,53. Therric acid .Awaiting the results of ongoing clinical trials on new drug candidates targeting specific sources and types of ROS, it should be taken the opportunity to identify and fill possible knowledge gaps and misconceptions about the role of ROS in diabetic complications. Considering that each of the relevant therapeutic targets discussed in this section probably contributes to form a ROS-signaling network underlying diabetic complications, a network pharmacology approach to explore active compounds and their pharmacological mechanisms may help to generate and apply the necessary knowledge to move the field of ROS towards mechanism-based, precision medicine .Despite the promising preclinical evidence, it remains puzzling why beneficial effects of antioxidants have not yet been translated into the clinic. Several ROS sources have been identified and intensively studied in the last two decades. Various antioxidant-based intervention strategies that target specific ROS sources appeared promising in experimental DM models, but this research has not led to any antioxidant drug entering the routine clinical practice until now. What is more, the failure of clinical trials to prove the efficacy of antioxidants against diabetic complications has contributed to challenge the view of oxidative stress as the main mediator of diabetic complications. In fact, except for a study suggesting a therapeutic potential of lipoic acid by improving endothelial dysfunction in diabetic patients , classic2O2 produced by NOX4 act as messenger molecules contributing to vasodilation, proliferation, and other important cellular functions [For long viewed as metabolic by-products or waste and simply mediators of a global redox disequilibrium in oxidative stress-related diseases, ROS also play critical roles in normal cellular function, including differentiation, proliferation, and repair . For exaunctions ,83,84. Tunctions . Togetheunctions . Furtherunctions . FinallyTo overcome the lack of specificity of classical antioxidants about ROS sources and types and the possible negative effect of inhibiting important physiological functions of ROS, new therapeutic approaches are under investigation. In addition to pharmacological inhibition of the specific ROS sources discussed in This indirect antioxidant strategy follows the suggestion that relevant therapeutic benefit may be achieved by favoring the activity of antioxidant enzymes rather than providing the antioxidant molecules. This approach is aimed at enhancing the physiological antioxidant response by providing molecules that act as agonists of transcription factors regulating the expression of antioxidant and cytoprotective genes, rather than exerting a direct antioxidant effect. These include synthetic chemicals, such as bardoxolone methyl and dimethyl fumarate (the ester derivative of fumaric acid), and natural compounds, such as sulforaphane obtained from cruciferous vegetables, which stimulate the master regulator of cell homeostasis nuclear factor (erythroid-derived 2)-like 2 (NRF2) ,88, and Finally, given the importance of subcellular localization, using targeted delivery approaches to direct antioxidants to the site of ROS generation may be a viable option to overcome the current issues with antioxidant drugs in DM see .In summary, in view of the puzzling results obtained with classical antioxidants compounds, alternative therapeutic strategies aimed at interfering directly with one or more specific ROS-producing enzymes, possibly at the site of their production, and the indirect antioxidant strategy aimed at upregulating the physiological antioxidant defense system, seem to be more promising therapeutic options for diabetic complications.An alternative explanation for the failure of clinical trials to demonstrate a protective effect of classical antioxidants on diabetic complications may be pathophysiological in nature. In this regard, an important contentious issue is whether oxidative stress occurs at an early stage in DM, preceding the onset of complications, or whether it is the result of cell and tissue damage, merely reflecting the presence of complications. Moreover, whether the activation of pathological pathways is upstream or downstream to ROS overproduction is an important point that need to be clarified. In fact, in the first case, the intervention with antioxidants could be potentially useful for managing the progression of complications, but it would have no effect on the early and intermediate steps of the pathogenic process.The hypothesis that mitochondrial superoxide generation is the master upstream mediator of diabetic complications by increasing polyol and hexosamine pathway flux, protein kinase C activation, and AGE formation is frequently regarded as a paradigm even today. However, in the last fifteen years, several results have challenged this hypothesis. Studies conducted in genetically modified mice lacking the p66 kDa isoform of the Shc adaptor molecule, a master regulator of cellular ROS status, demonstrated that hyperglycemia induces both mitochondrial and cytosolic ROS production  and thatMore direct and compelling evidence against the hypothesis of a primary role of mitochondrial ROS has been gathered recently from preclinical studies showing that T1DM and T2DM vascular complications can occur in the absence of excess superoxide, or even in the presence of reduced kidney and cardiac superoxide levels, which was associated with mitochondrial dysfunction. Consistently, enhancement of mitochondrial biogenesis and oxidative phosphorylation led to increased superoxide production with beneficial effects on kidney function and fibrosis . In addiIn summary, to identify efficient antioxidant-based therapeutic strategies, there is first a need to reassess the physiological and pathological relevance of ROS, and then to determine whether an antioxidant approach is feasible, in which situations, and if an optimal timing for treatment exists in the natural history of the disease . In geneAs discussed above, although the role of mitochondrial superoxide production remains controversial, there is convincing evidence showing that mitochondrial dysfunction and concomitant alterations in cellular energy production are critical mediators of diabetic complications. From this point of view, ROS dysregulation may be an epiphenomenon of permanent cellular bioenergetic and biochemical dysfunction, without direct therapeutic relevance to the pathogenesis of diabetic complications. To explore this hypothesis, alternative mediators of gluco- and lipotoxicity as well as mechanisms underlying the development and progression of diabetic complications have been under investigation, particularly in the past 10 years. In this section, we discuss two alternative mechanisms—namely carbonyl stress and the Warburg effect—for the pathogenesis of diabetic complications. As in Brownlee’s hypothesis, these mechanisms involve quantitative and qualitative changes in cellular glucose metabolism and abnormal mitochondrial function, but they focus on different key culprits, other than ROS, in the activation of the pathogenic pathways known to lead to vascular cell injury and inflammation.Carbonyl stress is a condition characterized by a generalized increase in the steady-state levels of reactive carbonyl compounds, namely RCS. Replacing the nature of the reactive species , the same definition also applies to oxidative stress. The carbonyl stress hypothesis is inextricably intertwined with the oxidative stress hypothesis, as some RCS require oxidative reactions for their formation .Actually, however, compared with oxidative stress, carbonyl stress is a more comprehensive term for a whole number of reasons. Firstly, RCS steady-state concentration is determined by the relative rates of their production and detoxification. Accordingly, chronic overload, exhaustion, or a decrease in the efficiency of the enzymatic pathways for RCS detoxification, including aldehyde dehydrogenases and the glyoxalase pathway, may concur to their accumulation . ImportaGlycolysis-derived RCS include the α-dicarbonyl compounds 3-deoxyglucosone (3DG) and MGO. These RCS are formed by the spontaneous degradation of triosephosphate intermediates of glycolysis . They arConvincing evidence in favor of a central role of MGO in endothelial dysfunction and the pathogenesis of vascular complications has been obtained in preclinical models. Endothelial overexpression of glyoxalase 1 (Glo1), the rate-limiting enzyme of the glyoxalase system, was demonstrated to completely prevent hyperglycemia-induced formation of the major AGEs CML and N(ε)-(carboxyethyl)lysine (CEL)  and to rRCS derived from oxidative modifications of carbohydrates and lipids  include the unsaturated trialdehyde acrolein and the dycarbonyl glyoxal (GO), originating from both glucose and lipids, and malondialdehyde and 4-hydroxynonenal, deriving only from lipids ,126 Fig. These RN-acetyl cysteine and L-carnosine, have been tested with encouraging results in preclinical studies, as comprehensively reviewed before [l-carnosine derivatives carnosinol and the enantiomer D-carnosine were shown to be effective in attenuating vascular complications of both DM [Strategies to lower RCS, including MGO, have been developed over the past years, the most important of which are RCS scavengers and glyoxalase inducers. RCS trapping agents, including hydrazine derivatives, such as aminoguanidine, vitamin B derivatives, such as pyridoxamine, and amino acid derivatives, such as d before ,131. Amo both DM ,133 and  both DM . A suita both DM ,88. TargIn summary, as in the unifying hypothesis by Brownlee and colleagues, the core of the problem seems to be the handling and disposal of excess fuel by the cell. Consistent with a critical role of excess glucose flux through the glycolytic pathway in hyperglycemia-induced carbonyl stress, increased MGO formation and accumulation was also observed in activated inflammatory cells and hypoxia, two conditions characterized by boosted aerobic glycolysis and reduced mitochondrial respiration ,137,138.Over the last two decades, a growing body of evidence has shown that diabetic complications are associated with altered mitochondrial function, as well reviewed in recent articles ,140,141.Mainly investigated in the field of cancer biology, the Warburg effect is defined as an increase in the rate of glucose uptake and preferential fermentation of glucose to lactate, even in the presence of oxygen. The Warburg effect is a feature of glycolytic cancer cells, which are so-called because they tend to favor metabolism via aerobic glycolysis rather than oxidative phosphorylation ,146,147.The Warburg-like metabolic reprogramming described by Sharma et al. in human DN  has beenTo shed some light on the possible role of the Warburg effect in diabetic complications, it is necessary to go through this complex and not completely understood bioenergetic phenomenon in a bit more detail. As mentioned above, this metabolic reprogramming has been extensively investigated in cancer cells since the 1920s, when professor Otto Warburg showed that, even under aerobic conditions, most tumor tissues continued to produce elevated levels of lactate ,150,151.Consistent with this function, glycolytic cancer cells also express high levels of the M2 isoform of the glycolytic terminal enzyme PKM, which has emerged as a key factor in the regulation of aerobic glycolysis ,157. At Interestingly, the levels of mitochondrial and glycolytic enzymes, particularly PKM2, were recently associated with the susceptibility to DN in T1DM and T2DM patients ,161. In Altogether these findings pave the way for further research into the link between carbonyl stress, mitochondrial dysfunction, and the Warburg effect in diabetic complications. They also suggest potential avenues for prevention and treatment of end-organ damage induced by DM. From a mechanistic perspective, the glycolysis by-product MGO has now been recognized as a potential driver for both the development of diabetic complications ,168 and Oxidative stress, inflammation, and the resulting tissue damage are hallmarks of chronic diseases, and DM is not an exception. Most of the literature includes oxidative stress as an important element in the pathogenesis of diabetic complications. However, because of new knowledge on the pathophysiology of ROS in health and disease, the classical view of oxidative stress as a quantitative deviation from an overall equilibrium of ROS formation and cellular antioxidant defense has changed into a more complex phenomenon that implies considering the chemical nature of ROS, subcellular or tissue location, and chemical kinetic mechanisms . HoweverRandomized controlled trials in which antioxidants were given with standard DM treatment have shown inconsistent efficacy results ,77,78,79These findings compel us to search for alternative triggers of the cellular and biochemical abnormalities that are widely recognized as the major pathological factors involved in diabetic complications: i.e., mitochondrial dysfunction, the activation of pathways of glucotoxicity (particularly RCS and AGE formation), inflammation, and redox imbalance. Similar to Brownlee’s hypothesis, two promising lines of investigation concern the changes in cellular glucose metabolism induced by hyperglycemia in target tissues of diabetic complications. Both the increased formation of the glycolytic by-products and highly reactive α-oxoaldehydes  and the establishment of a Warburg effect are closely related with intracellular metabolism of excess glucose and mitochondrial dysfunction, but independent from oxidative stress. It is intuitive and there is evidence of an interaction among quantitative/qualitative changes in cellular glucose metabolism, RCS formation, mitochondrial dysfunction, and the Warburg effect, but it is unknown whether the Warburg effect and carbonyl stress are independent processes with an underlying common cause or are linked by a cause–effect relationship. These are open questions which should be investigated to search for an alternative unifying mechanism of diabetic complications.To develop more effective preventive and therapeutic strategies, identifying the spark that makes the fire flare up and spread should be the goal of our research from tomorrow onwards.


Phantom limb pain (PLP) and phantom limb sensation (PLS) are common and distressing sequelae of amputation. Current pain management following amputation is challenging and unsatisfying. In this case study, a 74-year-old woman underwent above-knee amputation because of the rhabdomyosarcoma in the right leg. Despite several analgesics, pain was poorly controlled. The phantom limb pain and sensation were immediately reduced by the contralateral acupuncture, and abolished after the third session with no side-effects, no relapse during the next 9 months. Contralateral acupuncture showed positive effect on PLP and PLS in this case, but more robust evidence would be needed to support the efficacy of this treatment technique for indication. Most amputees complain of various distressing sensations in the amputated limb such as burning, stinging, shooting, piercing or cramping pain, known as phantom limb pain (PLP), and non-painful phantom limb sensations (PLS), , , and retained for 30 minutes , Jiexi (ST41), Zhaohai (KI6) and Taichong (LR03) on the left side were needled by stainless steel acupuncture needles . The needles were twirled after insertion and every 10 minutes thereafter to achieve  minutes .The Neuropathic Pain Symptom Inventory (NPSI), as one of the most widely used scales for characterizing neuropathic pain . Contralateral acupuncture means inserting needle on the side opposite the disease side, and is widely used in various diseases with good response, such as PLP, chronic shoulder pain, post-herpetic neuralgia, episodic cluster headache, acute traumatic pain, apoplectic hemiplegia, hemihidrosis, dizziness  for the publication of any potentially identifiable images or data included in this article.Q-aZ contributed to the conception and design of the study. ZD contributed to the treatment and analysis of data. QG contributed to drafting the text and preparing the figures. All authors read and approved the manuscript.This work was supported by the National Innovative and Core Talents Project of Traditional Chinese Medicine (grant number ZYYCY201901) and National Natural Science Foundation of China (grant number 82004463).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Nowadays, a marked, constantly increasing interest in developing functional foods aiming at the maintenance of digestive health, is witnessed. Foods with specific metabolic targets are at the spearhead of scientific research due to strong associations between the gut microbiome, oxidative stress, inflammation and metabolism .Foods aiming at digestive health constitute the second-largest positioning platform behind products tailored to general wellbeing. Such food products usually contain probiotic microorganisms and/or prebiotic dietary bioactive fibers. According to the latest definition of FAO/WHO , probiotBeyond probiotics and prebiotics, other constituents, such as bioactive compounds with antioxidant and anti-inflammatory properties and compounds associated with a targeted metabolic response, belong to the spectrum of ingredients which are used in food development aiming at the enhancement of functional properties .Currently, the functional market directed at digestive health is mainly focused on pharmaceutical products, such as pills or capsules, rather than processed foods . In thisLu et al.. Treatment of CAC mice with BO resulted in decreased tumor loads and also inhibited proliferation and metastasis of CRC cells in vitro. The study suggested that BO may provide an intervention strategy for CRC therapy, while G-protein-coupled receptor 43 (GPR43) is a potential targeting receptor during BO treatment.There is evidence that probiotics have a broad antitumor effect in colorectal cancer. However, the mechanism remains unclear. The effect of Bornlisy (BO)-cocktails of three probiotics on colitis-associated colon cancer (CAC) and the underlying mechanism were studied by Ogita et al.. In addition, SRP intake induced changes in the cecal microbiota, as observed by changes in β-diversity. SRP could be considered as a functional food component that may contribute to the maintenance of intestinal homeostasis, according to the researchers.A soybean resistant protein (SPR)-containing diet, derived from kori-tofu, a traditional Japanese food, resulted in decreased serum level of lipopolysaccharide-binding protein and higher expression of Reg3γ, a chemical barrier, which plays an essential role in the segregation of the microbiota, thereby improving the intestinal barrier function in mice, as reported by Jana et al.. HDOs constitute functional ingredients to be incorporated in food products, since they play a significant role in the modulation of gut microbiota and serve as substrates for the probiotic production of health-promoting substances, such as short-chain fatty acids (SCFAs). Butyrate generated by the selective fermentation of oligosaccharides, along with other SCFAs reduce gut inflammation. The role of HDOs in the alleviation of autoimmune diseases like inflammatory bowel disease (IBD), and others, such as, obesity, diabetes and urinary tract infection, as well as in the antimicrobial resistance, through the modulation of the gut microbiota is presented, while the mechanism of oligosaccharide utilization and disease mitigation is also discussed.The role of hemicellulose-derived oligosaccharides (HDOs) as emerging prebiotics for the alleviation of diseases is discussed by Lauridsen et al..Western diets have been associated with intestinal inflammation in contrast to plant-based diets, which often result in a reduced intake of total fats and meats and an increased intake of plant fibers that may contribute to reduced inflammation. A better understanding of the role of diet in intestinal inflammation may contribute to novel dietary interventions and to the use of specific foods or food supplements that can support the management of IBD. The influence of plant-based dietary components on the clinical disease course of IBD along with the benefits and possible limitations of plant-derived dietary components in the treatment of IBD are discussed in a review paper by Roselli et al. in a systematic review, which aims to provide the scientific literature addressing the connection between foodborne and gut microbiome and map gut microorganisms originating from fermented foods, either traditional or added with probiotics, among others. The review highlighted experimental approaches and study designs, which could be better standardized to improve comparative analysis of published datasets.Colonization ability of foodborne microbes and impact on human gut microbiota was discussed by Ma et al.. Dietary supplementation with MOA improved the health status of broilers, promoted the immune function, enhanced antioxidant enzymes, total antioxidant capacity and the expression of tight junction proteins and resulted in modulation of cecum microbiota.Apart from human health, an upsurge of interest is observed in food safety, due to the concerns associated with applying antibiotics in feed, resulting in drug resistance in pathogenic bacteria and drug residues in livestock products. Hence, the effect of dietary supplementation with mixed organic acids (MOA) on intestinal health, enzyme activity, and antioxidative characteristics in broilers was studied by Further research employing standardized and trans-disciplinary approaches is of pivotal importance for the understanding of gut microbiota and foods- probiotics- and prebiotics-interactions and their association with digestive health.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.