Neural-interfaced prostheses aim to restore sensorimotor limb functions in amputees. They rely on bidirectional neural interfaces, which represent the communication bridge between nervous system and neuroprosthetic device by controlling its movements and evoking sensory feedback. Compared to extraneural electrodes , intraneural electrodes, implanted within peripheral nerves, have higher selectivity and specificity of neural signal recording and nerve stimulation. However, being implanted in the nerve, their main limitation is represented by the significant inflammatory response that the body mounts around the probe, known as Foreign Body Reaction (FBR), which may hinder their rapid clinical translation. Furthermore, the mechanical mismatch between the consistency of the device and the surrounding neural tissue may contribute to exacerbate the inflammatory state. The FBR is a non-specific reaction of the host immune system to a foreign material. It is characterized by an early inflammatory phase eventually leading to the formation of a fibrotic capsule around intraneural interfaces, which increases the electrical impedance over time and reduces the chronic interface biocompatibility and functionality. Thus, the future in the reduction and control of the FBR relies on innovative biomedical strategies for the fabrication of next-generation neural interfaces, such as the development of more suitable designs of the device with smaller size, appropriate stiffness and novel conductive and biomimetic coatings for improving their long-term stability and performance. Here, we present and critically discuss the latest biomedical approaches from material chemistry and tissue engineering for controlling and mitigating the FBR in chronic neural implants. Since scientists started to invasively study the function of the central nervous system (CNS) and peripheral nervous system (PNS), single electrodes, and later on electrode arrays, have been implanted to record neuronal activity and to stimulate single or groups of neurons to artificially induce their activation, in light of decoding their functions.Once study protocols moved from acute tests to chronic implantations and the safety of implants performed in primates suggested the possibility to move to studies in humans, a further possible application of invasive neural electrodes, beside that to investigate neuronal functions, became concrete. Electrodes started to be employed to decode subject motor intention and, bypassing neural or osteo-muscular lesions, to artificially interface the nervous system to the external environment.When this happened, neural interfaces -often named brain to computer or to machine interfaces- and the field of neuroprosthetics were born. Depending on the site and the subject receiving the implant, electrodes can also be interfaced with sensory area and fibers and, by relaying afferent streams of information, convey artificial sensory feedback.Insofar, some applications for stimulating neural electrodes, particularly deep brain stimulation (DBS) and cochlear implants, have gained the maturity to be commonly applied in clinical practice. Other applications targeting a more spatially-selective information exchange, such as cortical or peripheral nerve implants, are very-promising, yet still in a developmental phase. Their not-complete maturity is mostly due to the lack of long-lasting stability of their performance over time, mainly because of the reaction that the body mounts around them. This factor hampers to a less extent cochlear electrodes, because they do not penetrate the neural structures, and DBS, because these electrodes do not need to achieve the level of stimulation selectivity needed by information exchange. The long-term functionality and longevity of cochlear implants and deep brain stimulators have already been widely demonstrated .Contrarily, the use of invasive multichannel electrodes, implanted within stump peripheral nerves to control cybernetic hand prostheses, is an application field of neural interfaces where electrodes should achieve an intimate contact with neural fibers required to reach a reliable information transmission, and where implantable solutions seem to favor exchange selectivity.Since peripheral nerves contain both motor and sensory fibers, peripheral nerve electrodes can achieve proper bidirectional communication through the use of a single device by stimulating afferent axons .Regained sensory feedback from hand prosthesis has the potential to improve motor control , discrimUnfortunately, the standard control systems of prosthetic limbs rely on surface electromyogram, which is mainly limited by problems of high latency, as well as low specificity and robustness in long-term implants . AlthougIn the conductive surface, the dielectric constant, dissipation factor and dielectric loss factor rise with the increase of the capsule thickness. The increase of the electrical impedance is proportional to the development of the fibrotic tissue, which determines difficulties to distinguish the signal from background noise  and, eveThe immune-mediated response responsible for the capsule growth is known as Foreign Body Reaction (FBR). FBR reduction over time is probably the main challenge for future neural electrode applications in neuroprosthetics to extend the reliability of the interface .The aim of this review is to analyze the latest tissue engineering strategies and biomedical approaches for controlling and evading FBR around implantable interfaces.Although the FBR process can occur in any living tissue implanted with foreign material, such as molecularly engineered surfaces and medical devices , we willWe analyze factors supposed to be the main causes of acute and chronic neural tissue reactions, such as scarce biocompatibility, excessive size, poor flexibility, reduced electrical properties, low compliance, mechanical mismatch and micromotion.Finally, we examine the shortcomings of current electrode-producing technologies and discuss possible cutting-edge solutions for the development of promising alternatives to the present intraneural interfaces. Strategies and technologies analyzed in light of the specific application we pursue could be potentially tailored to any electrode inserted in the CNS or PNS, and interfaced with different artificial devices.In a living tissue or a nerve, any implantation of foreign material, including advanced biomaterials that surround an invasive electrode, triggers an unbalanced biological reaction  of the non-specific immune system, known as FBR, which is the natural protection mechanism of the host to the foreign body . This coThe first step (onset), which is similar to wound healing, upon foreign body implantation is the adsorption of blood and plasma proteins, such as fibrinogen, fibronectin, albumin and antibodies to the implant surface . The typvia the production of TGF-β. This multifunctional cytokine has a paramount importance as it will stimulate the fibroblast-mediated extracellular matrix (ECM) production, while reducing at the same time inflammation , Longitudinal Intra-Fascicular Electrodes (LIFEs) and Transverse Intrafascicular Multichannel Electrode (TIME) . The extIntraneural electrodes should offer a better signal-to-noise ratio during neural recording and the reduced current intensity necessary to reach the appropriate nerve stimulation . NonetheThe chronically implanted devices stimulate the aforementioned multistep cascade of foreign body response, ending in scar tissue formation and electrode encapsulation, and thus in the need of increased currents  to maintain appropriate nerve stimulation due to a progressive increase of the electrical impedance. The most frequently used metals for the fabrication of neural electrodes are gold, tungsten, platinum (Pt) and Platinum-Iridium (Pt-Ir) alloy, with Pt being considered the preferred choice for long-term neuroprosthetic applications due to its electrochemical stability, safety, resistance to corrosion and limited reactivity within a tissue environment . HoweverSo far, diverse strategies are being pursued  to creatin vivo longevity and recording performances. The importance to focus on the coating stiffness and geometric configuration , to reduce the mechanical mismatch between chronic implanted electrodes and neural tissue, has been highlighted by a recent work of The shape and topography of medical-grade polymers implanted in animal models profoundly influences the FBR at the implant surface, with the broadly accepted experimental outcome that circular and smooth surfaces, in intramuscular and percutaneous implants, minimally affect the aggressive behavior of macrophages . The usein vivo, it may activate severe immune response, and its functionalization is usually troublesome, thereby limiting its application for neural interfaces that require long-term stability  , althougtability . Likewistability  is suscetability , therebytability , b are bydrogels , as furtin vitro the neural cell adhesion  in the FBR to diverse polymer surfaces  taking advantage of microfluidic and micromachining techniques , leveraging on micro- and nano-fabrication techniques. Recently, an innovative soft robotics approach has been devised to mitigate the FBR by controlling fluid flow and shear stress perceived by the host cells . In a rachniques . Despitechniques . To overchniques , micromochniques , combinechniques , turned chniques . A new h carrier . The ele carrier . For exa carrier . Instead carrier . However carrier , or in m carrier  and as e carrier , thus revia viscous drag force due to the finely controlled liquid flow in the microfluidic channel, limiting tissue damage at a negligible extent. Such brilliant strategy could be further implemented for peripheral nerve electrodes, envisioning exciting opportunities for their chronic implants. Wireless and flexible film-based ion-selective electrodes (ISEs) have also been recently developed as miniaturized systems for performing highly sensitive and non-invasive measurements  core encased in flexible layers of parylene C with novel design features , was made through a mechanically enhanced flexible interconnection cable using a combination of parylene C and polyimide  polymer commonly used as biocompatible coating and substrate material of electrodes for soft neural implants . In a re in vivo . In this in vivo . Howeverfeatures . Interesolyimide . The forvia simple and cost-effective photolithography-free methods, such as laser micromachining and master molding of PDMS. Such versatile processes give rise to planar metal electrodes with microfluidic channel geometries  micromachining is not only cheap, and easy to realize with high parallelization, but also suitable for the fabrication of long-term neural implants that are able to produce lower inflammatory response than polyimide-based electrodes . Flexibiith PDMS , althougin vivo and in vitro . Among organic coatings, CPs have been recently investigated with the aim to improve the long-term performance of neural electrodes as they can increase their effective surface, thereby decreasing the impedance, and enhance the electrical properties of neural interfaces, thus seeming the most promising materials . In partin vitro . To datemulation . Nonethecordings . Howevercordings . Anotherin a rat . They dein vivo, thus enhancing tissue response around implanted electrodes. However, their long-term use is limited by oxidative mechanisms that partially compromised non-specific protein absorption and device performance. Therefore, recent hybrid solutions have been proposed to overcome some of the issues related to their prolonged stability and sensitivity in vivo, such as hybrid thin film photopatternable polymers, combining the properties of PEDOT with the long-term (over 10 days) moisture stability of PEG  peripheral nerve cuff electrodes for chronic implantation in animal models using two coatings of PEG or nafion, with the latter showing low interfacial impedance, together with good stability and reduced fibrotic capsule, thus justifying deeper investigation also for possible clinical applications  PEG  and PHEMy of PEG . Anothery of PEG , for poty of PEG . They dey of PEG , which cications . A diffeications . The conications , fiber sications  and micrications , is the sulation . In a resulation . The so-adhesion . To sum in vitro validation of poly(carboxybetaine) zwitterionic hydrogel coating, with a Young’s modulus in the range of the neural tissue, of a polyimide-based device to minimize the fibroblast and macrophage adhesion  and the conducting polymer PEDOT, which can be covalently attached to the electrode surface and patterned using a photolithographic process in vitro . Conductin vitro . The resin vivo studies the authors performed encapsulation of human pancreatic β-cells with chemically modified alginate formulations  to long-term protect cells from the chronic response of the immune system, without the need for broad immunosuppression. In particular, these different hydrogel formulations increased the immunoprotection of cells in immune competent mice and non-human primate models, successfully reducing FBR and preventing from pericapsular fibrotic overgrowth. Similar strategies with alginate hydrogels could therefore be translated into clinical practice to encapsulate intraneural electrodes, and exploited to overcome the challenge of foreign body rejection from the host immune system. Overall, despite the many advantages provided by conductive hydrogel coatings in terms of high electrochemical performance of the device, especially when using zwitterionic formulations, augmented quality of signal recording, reduction of the mechanical mismatch along with ultralow-fouling properties, their long-term stability and functionality in vivo still represent main limitations that need to be solved in the next future. In fact, because of their soft texture, highly hydrated jelly structure and low mechanical strength, hydrogels can be slowly degraded or damaged already during the implantation surgery, thus impairing their permanence and performance within neural tissue. However, to the best of our knowledge, at present they are by far the most promising biomimetic coatings in this context.Modulation of the FBR for intraneural interfaces can also be achieved taking inspiration from recent works in animal models of type-I diabetes . In thesA summary of the intervention strategies based on the development of advanced functional biomaterials with representative examples is reported in The aqueous characteristic of synthetic and organic hydrogel coatings, such as PEG-based and zwitterionic-based formulations, and their synthesis methods could be harnessed for therapeutic purposes. In order to modulate locally the immune response of the host tissue, various hydrogel formulations could represent a means to encapsulate or covalently incorporate growth factors, therapeutic anti-inflammatory and anti-fibrotic medications as well as small-molecule drugs . To thisin vitro attempts to control the release of dexamethasone from a conducting polymer coating of PPy on Au electrode sites was done through an electrochemically-controlled release of dexamethasone phosphate as a dopant , which is upregulated on the macrophage surface after implantation of different biomaterials, including biocompatible hydrogels, may represent a smart strategy to hamper fibrosis and capsule formation . Such thin vivo silencing of target genes involved also in chronic disease such as fibrosis, including CTGF, can be achieved through various therapeutic strategies, either via local or by means of systemic administration of viral and non-viral vectors. One of the most promising strategy is represented by the gene therapy through the selective gene knock-down mediated by the small interfering RNAs (siRNAs) or the microRNAs (miRNAs) (in vitro the anti-fibrotic activity of polyethyleneimine (PEI)-functionalized magnetic iron oxide NP-mediated delivery of siRNAs against CTGF , a key player underlying the progression of the fibrotic reaction driven by TGF-β, which is quickly induced by TGF-β in different contexts of fibrotic disease as a specific downstream effector of its activity . To date(miRNAs) . These t(miRNAs) . However(miRNAs) . The mai(miRNAs) . Among t(miRNAs) . In thisnst CTGF . The siRvia physical entrapment, as well as covalent bonding using degradable peptides or cleavable molecular linkers , SiO2 (silica), SiC (carborundum) and diamond; in addition, several organic coatings have been used, which are made of – among others - parylene C, polyimide, liquid crystal polymer (LCP), SU-8 and silicone elastomer for implantable microfabricated medical devices. Both chemical solutions, organic and inorganic, leverage on the miniaturization of the implants thanks to the material flexibility and scalability  treatment and then gradually released in vitro via electrical stimulation, retaining an elevated activity while maintaining normal electrochemical properties of the polymer surface  nanocomposite films, deposited inside a conducting PPy scaffold, to enhance the dexamethasone phosphate-loading capacity of the graphene component, by means of physical adsorption, and to elicit its electrically controlled delivery . In vitrhydrogel . As for hydrogel . Thanks in vivo survival  modifications of biomaterial surface with adhesive peptides  to partially attenuate inflammatory reaction and capsule formation ; (b) funsurvival .A summary of the intervention strategies based on the control of the interface-microenvironment interaction with representative examples is reported in To date, the design of resolutive solutions to modulate the FBR, based on the exhaustive comprehension of its molecular mechanisms, represents a major challenge for a suitable and long-lasting implantation of intraneural devices.Modern neuroprostheses may employ electrodes produced with microtechnology that, however, do not go below the size of some tens of micrometers. Various techniques of micromachining and micromolding of flexible and conductive polymer coatings may allow scientists to fine tuning the features of the electrodes to the characteristics of the host tissue, thus creating more stable devices over time. Furthermore, the integration of microfluidic ion pumps and channels into neural probes can be harnessed for extended drug delivery in the implanted tissue, so to dramatically reduce the FBR and to be much better tolerated than plain implants.Indeed, microfluidics and, most of all, nanofluidics, although promising are still quite unexplored in neuroprosthetics, and deserve further investigation.Strategies based on microfluidic, microscale and nanoscale technologies provide scalability. They can lead from the long-term and stable neurotransmission simultaneously to many tissue points, to an enhancement of spatial selectivity stimulation, through implantable microelectrode arrays and microscale actuators . Even moFor example, bioinspired cellulose nanocomposites have higher versatility and functionality than rigid silicone implants, due to their switchable stiffness characteristics, reducing neuroinflammation in chronic implants . Indeed,Moreover, nanofiber-formed nanogels and self-assembly nanoscaffold hydrogels are broadly adopted for targeted and controlled drug delivery. For instance, some antibody-drug conjugate payloads can be maintained in a target area by side chains, chemical moieties and interactions with the nanogel, thus prolonging their protective effect .Taking into consideration all of the above reviewed biomedical strategies, ultraflexible nanosized devices, coated with biocompatible and mechanically dynamic materials, may represent an optimal solution. Owing to their advantageous features of stiffness/compliance in a neural context, such devices seem to be able to significantly attenuate the intraneural invasiveness, tissue strain, micromotion stress and, in turn, the chronic inflammatory response of the tissue.It is worth noting, though, that there still remain several technical snags that must be overcome in the manufacturing of suitable nanofluidic components over the next decade. Because of its novelty, expertise in nanofluidics is not as robust as the one in microfluidics and it lacks standardized procedures for the fabrication of neural nanodevices. So, manufacturing accuracy of neural nanointerfaces still depends a lot on the ability of the single producer.However, current fabrication technologies of advanced neural electrodes that combine the employment of new CPs with complex micro- and nano-structured configurations, such as fluidic microdrives, are based on rapidly growing micro- and nano-electronics expertise. These novel methodologies permit the development and use of flexible and small-sized devices with more targeted stimulation by applying low voltages in a safe manner. Therefore, they allow researchers to obtain very good neural signals while reducing the implant invasiveness and its mechanical deformation . Furtherin vitro, such as cardiac cells. Hence, many efforts have yet to be done to achieve suitable solutions also in peripheral nerves. Despite these hurdles, we believe that the challenge of ensuring a high-resolution release of bioactive chemicals against the FBR by minimally invasive neural interfaces while, at the same time, precisely controlling neurostimulation will need nanofluidics to be fully accomplished. Indeed, the smaller the size of the invasive electrode with an associated lower stiffness, the better the response of the neural tissue and the more selective and tailored will be the control over the device functionality. Among the main advantages for the use of nanoelectrodes, there is undoubtedly the enhanced mass transport, favored by the reduced dimensions, which determines an increased flow of Faradaic currents  incorporated within conductive polymers, leveraging their tunable physicochemical properties that provide a wide versatility of intervention. A detailed investigation on how the nanotopography modifications and the chemical reactive potential of the surface can reduce plasma protein adsorption and immune cell adhesion will help control the inflammatory response. In addition, the controlled and continuous release of neurotrophic factors and the targeted delivery of therapeutic drugs to further improve the biological response to the implant and avoid the FBR will be of paramount importance. Accordingly, tailoring zwitterionic hydrogels to incorporate bioactive materials, such as ECM-derived organic components  or neural cells will be pivotal . In thisHow limiting the leakage and the exhaustion of the drug payload in the microenvironment around the electrode site? This question still remains an open issue that the implementation of surface micromachining for the synthesis of NPs as ideal drug carriers, and microfluidic technologies could likely solve in the next years. For instance, integration and modification of the electrode surface through MEMS devices, microactuators and DSRs with permeable actuating membranes, may at least help control the pharmacological release and limit the drug leakage.in vivo testing, the choice of the best biomimetic coating to be used will rely on preliminary results gained from complex in vitro co-culture systems, as Lab-on-a-Chip devices. The latter should indeed be developed so as to recapitulate more closely all biological aspects of the intricate tissue damage, vascular injury and inflammatory cascade associated with electrode implantation. Thus, leveraging on complex microfluidic and, hopefully, nanofluidic co-culture platforms for mimicking both nervous tissue microenvironment, with patient-specific cell types, and the implant-induced FBR, one could preliminarily analyze the tissue response to a certain biomaterial coating in a physiologically relevant manner (It is well known that biomaterials or tissue-engineered constructs can strongly influence the interactions between a foreign body and the host immune system. Therefore, the deposition technology of the most appropriate coating on the invasive electrode and its modification with biomimetic surfaces targeted to support tissue-specific cell functions will pave the way to the definitive solution for mitigating the FBR and for advancing the long-term use of neural-interfaced prostheses. It follows that, before t manner . Such opt manner .a priori our knowledge on the mechanisms underlying the development of the FBR for a more effective electrode engineering, comes from the use of in silico methods. In this respect, a very illuminating example showed a data-driven approach based on polynomial functions to simulate and investigate the development of scar tissue outgrowth around an implanted neural device over time (A further technical enrichment for increasing ver time . Such cover time . OverallMG conceived the idea, wrote the manuscript, and prepared figures and tables. GV supervised and revised the manuscript. SG contributed to wrote the manuscript and prepared the tables. VD supervised the manuscript preparation. GD contributed to wrote, supervised, revised, and critically discussed the manuscript. 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.


In the last decades, the demand for molecular tools for authenticating and tracing agri-food products has significantly increased. Food safety and quality have gained an increased interest for consumers, producers, and retailers, therefore, the availability of analytical methods for the determination of food authenticity and the detection of major adulterations takes on a fundamental role. Among the different molecular approaches, some techniques such as the molecular markers-based methods are well established, while some innovative approaches such as isothermal amplification-based methods and DNA metabarcoding have only recently found application in the agri-food sector. In this review, we provide an overview of the most widely used molecular techniques for fresh and processed agri-food authentication and traceability, showing their recent advances and applications and discussing their main advantages and limitations. The application of these techniques to agri-food traceability and authentication can contribute a great deal to the reassurance of consumers in terms of transparency and food safety and may allow producers and retailers to adequately promote their products. The major worries of consumers concern the origin and the safety of the food they buy. The increased awareness of the value of food quality induces the consumer to ask for transparency from food companies. At the same time, companies must be able to certify the content and origin of their products with the aim of protecting the consumer against fraud and adulterations. In this scenario, traceability and authentication are fundamental tools for reassuring consumers in terms of transparency and food safety and allowing producers to gain awareness of the value of their products. Traceability lets the tracking of the source of a food at any point in the production chain enabling the quality-control processes and cutting down the production of unsafe or poor-quality foods . Food auTraceability and authentication are integral components of the food safety and defense system and represent fundamental components of the food supply chain. A reliable authentication and traceability system can constitute an essential instrument for the protection of consumers, reducing the chance of people consuming adulterated or contaminated foods, and increasing supplier control and process safety. Consumers showed limited knowledge about the importance of authentication and traceability of food products ,4, makinA wide variety of analytical methods for food traceability and authentication have been developed and tested . Each meIn the last decades, the demand for molecular tools for food authentication and traceability has significantly increased. This is mainly due to increasingly stringent legislation in the food sector and the market strategies aiming to assess a uniform and reliable control of the whole food chain from the field to the market and to ensure that consumer choices correspond to their expectations . In thisAmong the molecular analytical methods, some techniques such as the molecular markers-based approaches are well established, while some innovative approaches such as isothermal amplification-based methods and DNA metabarcoding have only recently found application in the surveillance of agri-products. Different authors have reviewed the most commonly used molecular methods for agri-food authentication ,9,10,11,In this review, an overview on the principal analytical methods for agri-food authentication and traceability was provided, focusing in particular on the molecular approaches. We describe some of the proven and widely tested molecular approaches such as molecular markers-based methods, showing their latest applications in agri-food surveillance. Moreover, we explore the most recent technologies describing their potential and prospects in food authentication and traceability. Finally, the advantages and limits of each approach are described and discussed.In the last twenty years, an exponential growth of studies on methods for the traceability of animal- and plant-based food has been observed ,13. For Chromatography allows for the separation and quantification of macro- and microcomponents in food products. The most widely used chromatographic techniques are high-performance liquid chromatography (HPLC) and gas chromatography. Both methods have been successfully used for the identification of the geographical origin of sweet cherry cultivars . For agrImmunoassays are analytical tools based on the use of antibodies or enzymes as recognition elements to detect the presence of specific antigens. Enzyme-linked immunosorbent assay (ELISA) is the most used immunological method for food traceability. This technique is mostly used for the detection of pesticide residues in food-borne matrices ,29.Spectroscopic techniques are fast and inexpensive methods based on the use of radiated energy to analyze the properties of a specific element. They have been widely used for different purposes including agri-food traceability. Fluorescence spectroscopy is a non-invasive and relatively inexpensive technique. However, it is less used compared to other spectroscopic methods due to its low detection limit. Despite this, fluorescence spectroscopy has been successfully used to detect adulteration in edible vegetable oils . VibratiNuclear magnetic resonance (NMR) allows for the identification of the composition of complex matrices of foodstuffs. The amount of any component in a mixture can be assessed with high precision. In the last years, NMR has been widely used for geographical traceability of agri-food products. This technique has been efficiently applied to the traceability of balsamic vinegar, saffron, coffee, and tomato , and rec13C/12C and 15N/14N, influenced by climate condition and agricultural practices; 2H/1H and 18O/16O, affected by the area of origin; and 34S/32S, influenced by geology [Among the most efficient methods for food authentication are the mass spectrometry (MS) techniques. A wide array of MS applications is available for food traceability and safety purposes such as the detection of contaminants, the composition, and the origin of a product . Two MS Usually, the food traceability and authentication methods based on physico-chemical analysis are used in combination with each other in order to reach maximum sensitivity and reliability. The combined use of gas chromatography with mass spectrometry allows for accurate qualitative and quantitative analyses of complex mixtures providing noteworthy results in the surveillance of agri-products . A recenAlthough over the years these analytical methods have been proven to be highly efficient and reliable in the identification of the geographical origin and potential adulterants fraudulently added to a product, they show remarkable limitations in the detection of contaminant species and in unmasking the use of varieties not declared in the product label. Additionally, physico-chemical approaches have been shown to be highly reliable with fresh products while they tend to lose effectiveness in the analysis of processed foods. These limitations are overcome by the use of molecular methods to food traceability.DNA is a stable molecule present in all living organisms and each organism’s DNA sequence is unique, enabling the distinguishing of the species and varieties used to produce a specific food. Moreover, DNA can also be recovered in enough quality and quantity in heavily processed food matrices. Thanks to the recent advancements in molecular biology and genetics, molecular approaches have become powerful and widely used methods for the authentication of agri-food products and for tracking the raw materials across the whole industry process. Along with the most widespread and experienced molecular marker-based methods, the more recent isothermal amplification-based methods, digital PCR techniques, and NGS-based approaches appear to be very promising in the traceability of a wide range of fresh and processed agri-foods. Molecular marker-based methods are the most widely used techniques for food traceability. The main reasons are the reduced amount of template DNA required for marker detection, the chance to analyze simultaneously multiple target regions, and the possibility of obtaining both qualitative and quantitative information. In most cases, PCR-based methods are used to detect molecular marker variations . PCR is Over the last ten years, the number of works based on the use of SSR for agri-food traceability and authentication has progressively reduced, together with an increase in papers employing the more abundant and stable SNP markers , neverthRecently, SSRs have been efficiently used for the traceability of cocoa in beans and liquor , evaluatSingle nucleotide polymorphisms (SNPs) are variations in the DNA sequence involving a single base. They are the most abundant and ubiquitous markers in any living organism and their diallelic nature offers a lower error rate in allele calling compared with other molecular markers. Moreover, SNPs identification does not require DNA separation by size, and it is suitable for automation, making the analysis quick and reproducible.SNPs are widely used in the traceability of animal-based foods, especially in the genetic authentication of meat , while oSNP identification is suitable for different detection methods such as single-base primer extension, cleaved amplified polymorphic sequences assays (CAPS), HRM, and sequencing techniques. Recently, an innovative system for wine authenticity based on the use of a biosensor as the system of SNP detection was developed by Barrias et al. . DNA-basThe rapid advances of next generation sequencing technologies have allowed for the automation of SNP detection, making the analysis based on this marker more rapid and reliable . The empFor some applications, the investigation focuses on a specific and well-known target DNA region. The analysis can be performed with the purpose to amplify a DNA sequence of a specific species or variety, taking advantage of peculiar differences in that region . Conversely, PCR primers can be designed in a specific conserved region to amplify a sequence characterized by a certain polymorphism among species. This is the case of the DNA barcoding approach, representing an important tool for food traceability and authentication . IsotherStarch Synthase II (SS II) gene, coded on wheat A, B, and D genomes. The authors took advantage of some differences in the SS II-D gene to set up a quali-quantitative method for the detection of common wheat in blended flour. Silletti et al. [The presence of differences in nucleotide sequence or indels allows for the design of primers specific for a species or a variety. The detection of an amplification product makes possible the identification of adulterant species or variety in a particular food-borne sample. This approach has been widely used for the detection of common wheat in durum wheat-based products such as pasta or durum wheat bread. The identification of the presence of common wheat can be addressed by the detection of a sequence-specific of the D-genome, which is present in hexaploidy wheat but absent in durum wheat. Sonnante et al.  focused i et al.  used a ti et al.  were ablIn recent years, the development of techniques based on digital PCR (dPCR) has made the detection of a contaminant in food much faster and easier . Digitalcytochrome oxidase gene. In terrestrial plants, plastidial genes rbcL and matK, the trnH-psbA intergenic spacer and nuclear ITS2 sequence are mostly used as barcode regions [DNA barcoding was developed by Hebert et al.  and is b regions . DNA bar regions . Recentl regions  and the  regions ,96.Frequently, DNA barcoding is employed coupled with high resolution melting (HRM) analysis (Bar-HRM). It consists in the amplification of a short DNA barcoding sequence and target region detection through HRM based on the distinctive melting behavior due to differences in DNA sequence. In the last years, the Bar-HRM strategy has found a large spread in agri-food surveillance. Bosmali et al.  set up aDNA barcoding-based approaches in agri-food authentication and traceability are promising thanks to the great advances made in molecular biology techniques that allow us to combine the detection of a specific barcode sequence with modern technologies such as nanotechnologies. Based on this principle, Valentini et al.  developeThe potential of the DNA barcoding strategy can be exploited through the sequencing of amplicons. The obtained sequence can be used to differentiate and univocally identify the species present in a food sample through a comparison with specific molecular databases. Recently, Sanger sequencing of specific DNA barcode regions was efficiently used for authentication of small berries in fruit products  and the The adoption of a universal barcode shows evident limits at the cultivar level, where genetic variability is limited. To overcome these limits, the ultra-barcoding methodology was proposed  to obtaiThe use of the DNA barcoding method in the agri-food sector is supported by the availability of the Barcode of Life Database (BOLD) coordinated by the International Barcode of Life Project . This daIsothermal amplification-based techniques represent a promising alternative to classical PCR since they achieve rapid and efficient detection of a nucleic acid target without requiring the use of a thermocycler. These methods allow the amplification of a specific region in an exponential manner at a constant temperature. Over the last decade, various techniques based on isothermal amplification have been developed; although their features can vary among the different methods, they share some characteristics such as the use of a polymerase with strand-displacement activity. Some of the isothermal amplification techniques mostly used in agri-food surveillance are rolling circle amplification (RCA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA), and loop-mediated isothermal amplification (LAMP). These methods are mostly used in the detection of various micro-organisms, representing an important instrument to control food-borne diseases and safeguard food safety and quality . FurtherAmong the isothermal amplification-based methods, LAMP is the most widely used. This technique employs four to six different primers able to recognize six to eight different sequences of a target region, allowing the synthesis of large amounts of DNA in a short time. The amplification products are stem-loop DNAs with different inverted target repeats; these products can be detected with different methods including real-time assay and naked-eye detection through DNA-binding dyes or colorimetric indicators . The higDNA sequencing represents the easiest way to detect multiple species and varieties present in a specific food-borne sample. Traditional Sanger sequencing allows for the detection of a specific DNA region at a time. Although cloning may improve resolution, it requires numerous steps and is very time-consuming. Moreover, Sanger sequencing is a relatively slow method, producing reads with a length not exceeding 900 bp . Next geThe short-read sequencing approaches such as sequencing by synthesis and ion semiconductor sequencing were the first NGS techniques to be developed. Illumina is the current leader for the short-read sequencing approach. This technique is based on the peculiar bridge amplification method and the sequencing by synthesis strategy, which generates long-reads up to 300 bp . AnotherThird-generation strategies are quite recent techniques that enable overcoming many of the limitations of short-read sequencing through the sequencing of a single DNA/RNA molecule and generating reads with a length between 1 kb and 2 Mb . The maiAlthough the use of NGS technologies has spread in several diagnostics and research sectors, their use in the field of agri-food molecular traceability remains limited. A possible explanation is that NGS technologies present high costs and require extensive computational power. In addition, these strategies require high-quality DNA, which is not always possible to recover from highly processed foods. Nevertheless, a certain number of studies on agri-food traceability and authentication through NGS-based approaches have been published. There are basically two adopted strategies: whole metagenome sequencing and DNA metabarcoding.Whole metagenome sequencing (WMS) allows scanning for several species simultaneously even when these are present in a small quantity in a food matrix . This apA software pipeline, called AFS (All-Food-Seq), was developed to quantitatively measure the species composition in food-borne samples. This pipeline takes advantage of the deep sequencing of total DNA, allowing for the identification of species components through the mapping of reads to publicly available reference genome sequences and the quantification of species proportions based on a sequence read counting approach. This method has been successfully applied for the traceability and authentication of different animal- and plant-based foods .More recently, Haiminen et al.  set up aWhole metagenome sequencing has been proved to be very effective in the identification and authentication of herbal products  and the https://www.ncbi.nlm.nih.gov/bioproject/PRJNA325670/; accessed on 24 May 2021) [The whole chloroplast genome can be sequenced as an alternative to nuclear DNA for food authentication purposes. This is particularly useful in highly processed agri-foods since organellar DNA is present in high copy numbers compared to nuclear DNA, preventing degradation occurring during the production process. The sequencing of chloroplast genome produces reads that can be compared to specific databases containing complete chloroplast genome sequences such as the GenomeTrakrCP, which is publicly available at the National Center for Biotechnology Information (ay 2021) . This apay 2021) ,63.The DNA metabarcoding approach combines the high throughput sequencing strategies with DNA barcoding, allowing the analysis of multiple amplicons corresponding to different barcode regions by sequencing them in parallel. The general strategy is based on extracting the whole DNA from certain foods, amplifying a specific barcode region whose dimensions can vary from 120 up to 600 bp, sequencing the corresponding amplicon, and analyzing the sequence using specific pipelines. This strategy is particularly suitable for highly processed foods since the DNA extracted from these matrices is usually degraded, making possible only the amplification of short regions . MoreoveThe most commonly used plant barcode regions for DNA metabarcoding analysis are the nuclear ITS regions or the plastidial rbcL and psbA-trnH. In particular, the ITS1 and ITS2 regions have been used to identify plant components in herbal teas through their sequencing through two different platforms, Illumina and Ion Torrent, showing that both sequencing strategies are effective in qualitative and quantitative detection of different species . FrigeriThe effectiveness of DNA metabarcoding in the agri-food authentication and traceability sector has been widely demonstrated in the authentication of polyfloral and monofloral honey ,65,120. Recently, Gostel et al.  developeA wide variety of analytical techniques for authentication and traceability of agri-food products have been developed and tested. For a long time, chemical and biochemical approaches have been used for the detection of specific components in foodstuffs; nevertheless, in the last few decades, molecular techniques have taken the upper hand in the food surveillance sector. DNA-based methods are mostly used for the identification and quantification of species and varieties composed of fresh or processed food. Indeed, DNA is present in nearly all the cells of a given organism and its sequence remains unchanged during all production phases. Instead, proteins and secondary metabolites may be influenced by growing conditions, harvesting period, and storage environment . MoreoveA valid alternative to nuclear DNA-based analysis is the use of approaches involving the chloroplast genome, which is present in high copy numbers in vegetal cells. Indeed, heavily industrial treatments can severely affect nuclear DNA quality and quantity, while this occurs to a lesser extent with chloroplast DNA due to its abundance ,63. Despite the significant advances that have been made in molecular techniques, innovative approaches are only partially used in agri-food authentication, while traditional molecular marker-based methods, whose effectiveness have been amply demonstrated, remain the approaches of choice. Regarding molecular marker-based methods, SNPs and SSRs are largely used nowadays because of their standardized and straightforward detection systems. These approaches are used mainly in the identification of plant varieties aiming to prevent fraudulent commercial activities. SNP and SSR application for food traceability and authentication offer several advantages: they have a high level of polymorphism, high reproducibility, and can be detected on a very small portion of DNA, which in the case of fragmented DNA may constitute an important advantage . MoreoveFrequently, a food can contain several vegetal species and the availability of an instrument able to detect all the species simultaneously becomes necessary for traceability and authentication purposes. Approaches based on DNA barcoding represent an effective alternative to DNA fingerprinting methods in plant identification since they do not require the knowledge of the whole genome of an organism, being based on the exploitation of one or few genomic regions . DNA barWhole metagenome sequencing is the best strategy for authenticity, since it allows for the detection of additives, poisonous plants, allergens, and any other kind of adulterants fraudulently or accidentally added to a food-borne product. The main limitation of NGS-based methods in agri-food authentication is the obtainment of sufficiently high-quality DNA. This step is crucial to ensure that all DNA sequences present in a food-borne sample are properly identified . A largeAgri-food traceability and authentication require reliable and accurate methods for the identification of plant species and varieties in a wide collection of fresh and processed food, without ambiguity. The possibility of being aware of the composition of a food has assumed increasing importance among consumers, thanks to the action of mass communication concerning the relevance “of knowing what one is eating”. Among the different traceability techniques, molecular approaches are gaining increasing interest due to their significant advantages compared to the physico-chemical approaches. There are many various molecular methods suitable for agri-food surveillance. Some of them such as the molecular marker-based approaches have been extensively experienced and used in the agri-food sector; several authors have described their main applications in detail. Here, we presented the advances of these approaches and their most recent employment in agri-food traceability and authentication. Moreover, we provided an extensive description of the most innovative approaches such as isothermal amplification-based methods and DNA metabarcoding, which have only recently found application in agri-food surveillance. We highlighted their potential and prospects by showing the latest works on traceability and authentication based on the use of these methods. Finally, the description of the main advantages and limits of each molecular method will represent an effective prompt for anyone who wants to find the best method to authenticate or trace a specific agri-food.The wide panel of molecular techniques to traceability and authentication in the agri-food sector constitutes a powerful tool to protect both producers and consumers, ensuring consumer freedom of choice and improving the transparency of food production systems, therefore allowing honest producers to adequately promote their food products.


This Research Topic concerns bacteriocins and others RiPPs  as alternatives to antibiotics. Due to the increasing problem of antibiotic resistance globally, there is a pressing need to source such alternative antimicrobials. Here, in this editorial, we summarize the key papers that were published as part of this Research Topic, which consisted of a diverse range of research as well as review articles, pertaining to several different bacteriocins and RiPPs.in silico genome mining approaches, laboratory experimental-based approaches or a combination thereof. In one such study, using a combination of lab-based and in silico approaches, Angelopoulou et al. reported the discovery of a range of bacteriocins synthesized by strains isolated from human milk. More specifically, the authors isolated bacteriocin producers from 37 human milk samples and found 73 putative bacteriocin gene clusters, which included 16 completely novel prepeptides. Amongst the key findings of the study were the discovery of 2 novel lantibiotics, 3 class IIa bacteriocins, 4 sactibiotics, 1 novel class IIb bacteriocin, 4 new class IIc, and 2 class IId bacteriocins, highlighting that the human milk microbiota is a potentially rich source of diverse antimicrobials. In a separate in silico study, Sabino et al. found that ruminal bacteria are a potentially rich source of lasso peptides. More specifically, a genome mining approach was employed by the authors, using tools such as BAGEL4 and antiSMASH5 to screen 425 bacterial genomes from the rumen ecosystem for lasso peptide production. Overall, Sabino et al. found 23 incomplete and 11 complete putative lasso peptide gene clusters amongst the genomes analyzed. Finally, a study by Zendo et al. reported the discovery of a novel nisin variant, kunkecin A, synthesized by Apilactobacillus kunkeei FF30-6, a lactic acid bacterium isolated from honey bees. The study describes its antimicrobial activity against Melissococcus plutonius, one of the major bacterial pathogens of honeybee broods. Furthermore, the genome sequence of A. kunkeei FF30-6 revealed that the biosynthetic machinery of the novel bacteriocin resides in the plasmid pKUNFF30-6. A more in-depth analysis of the kunkecin A gene cluster also revealed that it is quite distinct from the nisin A gene cluster, and is devoid of genes corresponding to nisR, nisK, and nisI. However, both nisin A and kunkecin A are likely to share similar post-translational modification processes. The structure of kunkecin A was also proposed in the study, on the basis of observed and calculated molecular masses of the peptide. Together, these studies emphasize the growing importance of sequence mining approaches in the discovery of novel RiPPs.A number of these studies report the discovery of novel bacteriocins or RiPPs, either through Reiners et al. provided insights into the antimicrobial activity of nisin H, a natural variant of nisin, in addition to the nisin H F1I derivative. The authors expressed the peptide in a heterologous host and determined the yield, the cleavage efficiency using NisP as well as effects on the post-translational modification using mass spectrometry techniques. In addition, the authors determined the effects of nisin H and nisin H F1I on the activity of NisI and NisFEG responsible for nisin immunity, as well as the resistance determinants SaNSR and SaNsrFP from Streptococcus agalactiae. Overall, the authors found that nisin H and nisin H F1I were more potent than nisin A when tested against clinical isolates of Enterococcus faecalis, Enterococcus faecium and Staphylococcus aureus. In a separate study, Deng et al. reported that biologically active and stable forms of nisin are generated upon conjugation of synthetic hydrophobic polyproline moieties to the lipid II binding fragments of the nisin peptide. The authors used click chemistry technology to conjugate moieties of synthetic polyproline to either nisin AB (encompassing rings A and B of nisin) or to nisin ABC . Importantly, the authors found that the conjugation of the synthetic polyproline O63K with nisin ABC resulted in a 16-fold increase in bioactivity, compared to nisin ABC, highlighting the potential of semi-synthetic nisin hybrids as novel antimicrobials. Furthermore, such nisin ABC hydrids are recalcitrant to degradation by nisinase and other proteolytic enzymes at the C-terminus of nisin. These semi-synthetic hybrids, which maintain potent or enhanced antimicrobial activity, could present viable means to circumvent certain nisin resistance mechanisms. Albeit resistance to bacteriocins has been described in several studies over the years, such instances have mainly related to resistance demonstrated in in vitro studies rather than in clinical settings/food systems. Thus, bacteriocins may present viable alternatives used in clinical settings, especially in light of the widespread reports of antibiotic resistance globally. In this regard, the use of semisynthetic derivatives of lantibiotics such as nisin and other such hybrid compounds and/or non-ribosomally synthesized peptides, such as those described above, may be a possible means to circumvent issues relating to antibiotic resistance in the clinic. By gaining insights into the precise mode of action of different bacteriocins against specific clinical pathogens, the use of such hydrid compounds could be particularly important as a means to tailor-make narrow spectrum antimicrobials with a view to targeting specific pathogens in the clinic, whilst minimizing the impact on the overall host microbiome.A few studies as part of this Research Topic report the investigation of the activities of nisin variants and/or semi-synthetic hybrids of nisin. In one such interesting study, Ross et al. reported the design of synthetic peptide libraries derived from a minimal alpha helical domain of enterocin AS-48 homologs and antimicrobial activities thereof. The alpha helical region of this circular bacteriocin enterocin AS-48 is chiefly responsible for its membrane-permeating and resultant antibacterial activity. The authors conducted homology-based searches of similar domains in several bacterial genomes, used these alpha helical domains as scaffolds to design minimal peptide libraries and assessed their antimicrobial activity. An impressive total of 384 synthetic peptides were evaluated for antimicrobial activity in the study and the authors found that MICs in the low nanomolar range were obtained for the most potent peptides, with no concomitant cytotoxic effects. Thus, studies like this present a blueprint or scaffold for the rational design of semi-synthetic bacteriocin peptide libraries for further evaluation. In a separate study, Vermeulen et al. described the expression of plantaricin 423 and mundticin ST4SA, both class IIa bacteriocins in E. coli as a heterologous host, using Green Fluorescent Protein as a fusion partner. Importantly, the authors reported that His-tagged GFP fusion proteins encompassing these bacteriocins, in addition to being autofluorescent, overcame the issue of inclusion body formation, and also decreased the toxicity of these bacteriocins during heterologous expression. The autofluorescence also facilitated real-time quantification of protein yields during heterologous expression.In relation to class II bacteriocins, a very interesting study by Campelo et al. reported that a Bce-like bacitracin resistance module in Lactococcus lactis is activated upon exposure to the bacteriocin Lcn972. A further possible concern in this regard is that general MFS transporters may contribute to multidrug resistance in pathogenic bacteria, which may also include resistance to bacteriocins. These types of studies are extremely important as they highlight the potential downsides of utilizing bacteriocins, as in certain cases, resistance to other antimicrobials may inadvertently be enhanced as well. YsaDCB is a L. lactis ABC transporter that along with TCS-G, forms a detoxification module conferring protection against the cell wall biosynthesis inhibitors bacitracin and the bacteriocin Lcn972. By using a combination of heterologous expression and RT-qPCR, the authors elucidated the precise function of the ysaDCB operon. Overall, the authors found that ysaB encodes a putative Bce-like permease, whilst ysaD encodes a secreted peptide which is likely to be associated with signal relay between the YsaDCB ABC transporter and TCS-G and are together involved in largely similar but somewhat distinct mechanisms of resistance to bacitracin and Lcn972. Telhig et al. published an interesting review article relating to the use of bacteriocins to attenuate antimicrobial resistance in Gram negative bacteria. More specifically, the authors describe the potential use of unmodified and modified microcins, including lasso peptides, nucleotide peptide, siderophore peptides, and linear azole(in)e-containing peptides as a means to decrease the likelihood of antimicrobial resistance amongst Gram negative bacteria. In addition, the review described the potential of the development of cross-resistance and co-resistance in Gram negative bacteria, to various antibiotics and microcins.With regards to studies focused on the potential for bacteriocin resistance development, an interesting study by Karbalaei-Heidari and Budisa highlighted the potential of utilizing genetic code expansion strategies in lanthipeptides with a view to attenuating the likelihood of the development of antimicrobial resistance to lanthipeptides. This is because the conventional genetic code and protein engineering strategies are limited to merely 20 canonical amino acids. However, the authors in this review highlight the prospective use of non-canonical amino acids during protein translation with a view to forming semi-synthetic lantibiotics with enhanced biological and chemical properties compared to their natural variants. Another interesting review by Lagedroste et al. summarized recent studies investigating the precise roles and mechanisms of action of enzymes involved in the post-translational modification processes of lanthipeptides. Finally, a review by Rooney et al. summarized the potential of bacteriocins as “plantibiotics of the future” by reporting the details of recent studies of the antimicrobial activity of bacteriocins against Gram-negative phytopathogenic bacteria. It must be noted however that much remains to be elucidated with regards to the impact that extraneous factors such as solar radiation, alterations in light intensity, humidity and temperature have on the stability and antimicrobial activity of bacteriocins and non-ribosomal peptides when potentially being used as antimicrobial agents to control plant infections.Finally, a number of review articles covering a range of topics pertaining to bacteriocins and other RiPPs were published as part of this Research Topic. A very interesting review article by in silico mining and RiPP engineering, but also addressing some of the potential issues faced by those wishing to commercialize RiPPs for clinical, agricultural, or industrial use.In conclusion, this Research Topic has significantly added to the field of research relating to bacteriocins and RiPPs by highlighting the role of new approaches like HM wrote the initial draft of the manuscript. DF, MU, and PDC reviewed and revised the drafts. All authors have approved submission of the manuscript.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.


Normal substrate transport and signal transmission are the premise to ensure the health of biological somatic cells. Therefore, a comprehensive understanding of the molecular mechanism of intercellular substrate transport is of great significance for clinical treatment. In order to better understand the membrane protein through its interaction with receptors, to help maintain a healthy cell and the molecular mechanisms of disease, in this paper, we seek to clarify, first of all, the recognition mechanism for different types of membrane protein receptors; pathogen invasion using the transport pathway involved in the membrane; and the latest specific target sites of various kinds of membrane transport carriers; to provide an explanation and summary of the system. Secondly, the downstream receptor proteins and specific substrates of different membrane transporters were classified systematically; the functional differences of different subclasses and their relationship with intracellular transport disorders were analyzed to further explore the potential relationship between cell transport disorders and diseases. Finally, the paper summarizes the use of membrane transporter-specific targets for drug design and development from the latest research results; it points out the transporter-related results in disease treatment; the application prospects and the direction for drug development and disease treatment providing a new train of thought; also for disease-specific targeted therapy, it provides a certain reference value. With the continuous development of basic medical research and clinical treatment technology, the etiology of diseases has been further and further studied, the relationship between cell transport and diseases has been gradually clarified, and specific targeted-therapy technology has been constantly updated . The relCell membranes, including membrane carrier proteins, membrane channel proteins and ATP drive pumps, are the main transporters . MembranCells have a complicated transport process, but there are a lot of specific recognition receptors to be found on the membrane transporters which have gradually clarified the transport mechanisms underlying disease and the health status of cells, and the precision of their means of control for the development of targeting drugs on cells, and it is expected to apply basic theoretical study to clinical treatment. The latest research results on the specific targeting of the transport mechanism of plasma membrane transporters lack a systematic summary. Based on this, we have reviewed the latest research progress, including targeted recognition sites for different types of membrane transporters, specific substrate recognition and drug development. We hope to provide some ideas for the development and utilization of unconventional drugs.Membrane carrier proteins are multiple transmembrane proteins and exist in almost all types of biofilms. Each carrier protein can transport a specific solute molecule through a series of conformations that mediate the transport of solute molecules across membranes and can participate in active and passive transportation. The plasma membrane of the cell has carrier proteins for the input of nutrients . The mitochondrial intima contains carrier proteins that can input pyruvate and ADP and output ATP. Carrier proteins are highly selective in substrate transport, and usually only one class of molecules is transported. The transport of carrier proteins is similar to the process by which enzymes bind to substrates and the process becomes saturated. Some carrier proteins are pH dependent . The proThere are three types of channel proteins: ion channels, porins and aquaporins. Most of the channel proteins identified so far are ion channels . Ion cha+, K+, H+, Ca2+, and jointly maintain the homeostasis of ions inside and outside the plasma membrane. Type F is mostly found in the inner membrane of the mitochondria, the chloroplast capsule membrane and the bacterial plasma membrane. It plays an important role in energy conversion and converts ADP into ATP by proton dynamic potential, and so is also called ATP synthase [+ transport. The transport process does not involve phosphorylation intermediates, and is involved in maintaining renal acid balance, regulating apoptosis and cell cycle, nerve signal transmission and other physiological functions in animals. The structure, function and mechanism of the ABC protein have always been the focus of research [ATP-driven pumps are a family of proteins that rely on the energy released by ATP hydrolysis to transport substances across membranes, including the P-type ion pump, F-type proton pump, V-type proton pump and the ATP-binding cassette (ABC) transporter . The chasynthase . The V-tresearch . ABC traresearch .+ dependent and Na+ independent carriers, which are involved in amino acid transport in cells [Carrier proteins are important proteins in the cell membrane as they form channels for the transport of nutrients such as sugars, amino acids, nucleotides and other small molecules to enter and exit the cell. Amino acid carriers in mammalian cells can be divided into neutral, acidic, alkaline transporters, and Nain cells . Amino ain cells . Glucosein cells .AQPs exist in the form of a tetramer, and in which each monomer is involved in water transport. AQPs can participate in the absorption and secretion of water to maintain a balance of body water, electrolyte metabolism, cerebrospinal fluid secretion, urine dilution and intestinal water . AQP is Ion channel-coupled receptors are a class of macromolecules that are ion channels and have ligand-binding sites, which can act as ion channels and signal transducers. Different from ion channels controlled by potential and chemically modified ion channels, the opening and closing of these ion channels are directly controlled by ligands, which are mainly neurotransmitters . Ion-cha2) on the plasma membrane into the inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DG), and converts extracellular signals into intracellular signals. The phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway is considered to be involved in the regulation of cellular physiological processes through the activation of downstream effector factors, and is directly related to cell growth, proliferation, cancer and longevity. This pathway is involved in the occurrence of human diseases and can regulate many biological functions in the body [Cell surface transmembrane proteins are mainly composed of G-protein-coupled receptors (GPCRs), ion channels and transporters, which play an important role in neuronal signal processing and plasticity in the brain. The function of GPCRs in cell motility, growth differentiation and gene expression is closely related to tumors. The GPCRs are the largest family of cell-surface receptors and are ubiquitous on the surfaces of various eukaryotic cells. Conjugated GPCR proteins also mediate different signaling pathways . As showthe body . cGMP acthe body . Structuthe body . G protethe body .The receptors of many growth factors and cytokines have structures with single transmembrane glycoproteins, enzyme activities or involve the intracellular segment of the receptor associated with the enzyme. In contrast to the seven-fold transmembrane receptors (G protein-coupled-type receptors), these receptors are referred to as single-fold transmembrane receptors, and are also known as enzyme-linked receptors. The transmembrane regions of these receptors cross the membrane once, in contrast to the structure of seven-fold transmembrane receptors which have repeated transmembrane segments. Receptor tyrosine kinases are typical examples of enzyme-linked receptors . ImmunosThe ultimate role of the ion-channel receptor signal transduction is to cause changes in the cell membrane potential. Ion-channel receptors affect the function of cells by transforming chemical signals into electrical signals . The rapGPCRs are the largest family of cell signal transducers in the human body. GPCRs are involved in almost all physiological life activities and are responsible for regulating the responses of cells to light, odors, hormones, neurotransmitters and chemokines ,57. The Enzyme-linked receptor proteins are both receptors and enzymes. Once activated by ligands, the receptors have enzyme activities and amplify signals, and so they are also called catalytic receptors. This type of receptor transduction signal is usually related to cell growth, reproduction, differentiation and survival . Recepto+ channel plays an important role in cell conduction, which is closely related to many diseases and plays an important role in the study of tumors and cancer [+ channel protein promotes Ca2+ influx and G1 phase progression by influencing tumor cell membrane potential, or by changing cell volume, the concentration of intracellular substances related to DNA synthesis and cell cycle regulation can be affected, thus causing the rapid proliferation of tumor cells [+ channel protein expression is also associated with tumor metastasis, for example, overexpression of the G protein-gated inwardly rectifying potassium (GIRK) is associated with lymph node metastasis of breast cancer [+ channel activity by chemical reagents can inhibit the proliferation of cancer cells, indicating that the K+ channel protein can be used as a drug target.Ion-channel proteins are composed of protein complexes that allow suitably sized ions to pass along the concentration gradient. Studies have found that the Kd cancer . The K+ or cells ,64. In at cancer . Inhibit+ channel is a kind of transmembrane glycoprotein located in the plasma membrane of the cell, its main function is to maintain cell excitability and conduction. The selective Na+ permeability of voltage-gated Na+ channels is the basis of action potential generation in excitatory cells such as neurons. Bennett et al. found that prostate cancer cells with a high expression level of voltage-gate sodium channel and strong activity had better aggressiveness [The Nasiveness . In brea2+ channel is involved in intracellular regulation of almost all biological functions of the body, such as heart and muscle contraction, nerve information transmission, cell proliferation and apoptosis, cell division and differentiation, etc. [2+ entry (SOCE), as an important pathway to regulate intracellular calcium homeostasis, is closely related to tumorigenesis and plays an important role in maintaining calcium homeostasis in angiogenesis and tumor immunogenicity changes [The Caon, etc. . Studies changes .+/K+-atPase (NKA) utilizes the hydrolysis of ATP to actively transport Na+ and K+ across the membrane, against the concentration gradient to transport Na+ to the extracellular and K+ to the intracellular, which can maintain cell membrane localization and make the cell present an excited state [Naed state .Membrane transporters have different structures and physiological functions. Gene mutations usually produce defective proteins that cause abnormal transport channels. This normally results from the loss of normal function due to functional enhancement, functional loss and dominant and negative effects. Any kind of membrane transport-related protein disorder may cause abnormal physiological states in cells, resulting in cellular substrate transport disorders and cell body diseases. The cytoplasmic membrane has selective permeability, which plays an important role in cell substrate metabolism and osmotic pressure maintenance. Carrier proteins are transmembrane proteins with multiple cyclotron folds that bind specifically to the molecule being delivered to allow it to cross the plasma membrane. Their functional mutations or deletions can lead to a series of diseases related to material transport, as summarized below.GLUT1 deficiency is a metabolic disease of the brain that is caused by mutations in the SLC2A1 gene. Up to now, more than 140 different SLC2A1 variants have been found, all of which can lead to decreased expression or the complete loss of GLUT1 function, impaired glucose transport in the brain, and insufficient energy supply ,71. MutaThe dysfunction of the ATP-binding cassette (ABC) transporter proteins can lead to several human diseases. More than 20 ABC transporters are known to be associated with human diseases, ABCA4 is linked to Stargardt disease. ABCA7 is linked to Alzheimer’s disease, two-thirds of ABCB lesions lead to immune deficiency and the lesions of ABCC7 can lead to cystic fibrosis ,77. P-glPathogens invade cells by combining with specific receptors on the cell surface. Under normal circumstances, receptors on the cell surface can distinguish between pathogens and normal substances within the body. When encountering pathogens, cells stimulate autoimmunity and kill the pathogens. However, some pathogens can camouflage their surface structures, and evade cell recognition through the endocytosis pathway.In the case of HIV, HIV requires CD4 receptors to enter cells. It also requires co-receptors such as cinnamoyl-COA reductase (CCR) or CXCR. Macrophages and dendritic cells have CD4 and CCR5 receptors, and CD4T lymphocytes have CD4 and CXCR4 receptors. The entry of HIV into human cells requires the participation of CD4, CCR5 or CXCR4 receptors, otherwise it cannot cause infection . StudiesBiotherapy is a new treatment method, at present, using membrane-like micro/nano-structures, mainly composed of liposomes and nanospheres, as popular carriers for drug delivery. The accurate identification and internalization of the treatment carriers are the focus of current research, and are particularly important in improving targeted drug delivery. There are many recognition sites on the cell surface, and the ligand design based on the structural characteristics of the receptors is the main direction being taken to improve targeted drug delivery. As a natural drug delivery system, extracellular vesicles (EVs), have multiple ligand/receptor-modified lipid bilayer membranes. These can interact with targeted cells. Exosome integrin can interact with specific molecules on vascular cells mediating the exocytosis of inflammatory sites . LiposomThe cell is a complex organism that responds to different microenvironments. As a stimulus signal, drugs can also produce different effects through different targets in different microenvironments and pathological conditions. Membrane transporters enable efficient cellular metabolism, aid in nutrient sensing, and have been associated with various diseases, such as obesity and cancer . Drug trIn the treatment of malignant tumors, chemotherapy is an important method in addition to surgery. The use of small molecule antitumor drugs can effectively stop or slow the growth of malignant tumors. However, a large number of small molecule antitumor drugs do not have the ability to recognize tumor cells and will act on normal cells, leading to large adverse reactions . Based oNanodrug delivery systems have been widely studied and reported in the literature. Polymer micelles with small particle sizes can easily pass through various barriers, and be easily surface modified to increase the accumulation of drugs at the target site ,108. MicDrug-loading liposome technology has made great breakthroughs, many research studies have shown that liposome drug components can overcome the obstacles imposed by cells, in order to absorb and improve targeted distribution and other advantages, and these features ensure the drug composition of the liposome coating can be targeted for delivery to specific parts of the human body, thus improving effectiveness and minimizing side effects. At present, liposome drug delivery systems can be divided into several categories . Class APrecursor drugs have no biological activity or very low activity and become active substances after metabolism in vivo. The purpose of this process is to increase the bioavailability of drugs, strengthen targeting, and reduce the toxicity and side effects of drugs. The emergence of monoclonal antibody (mAb)-based immunotherapy has brought new ideas for targeted therapies. ADCs can selectively kill tumor cells by combining cytotoxin drugs with mAbs to deliver cytotoxin to tumor sites. The conjugation of an ADC cytotoxin, which is hydrophobic, and a mAb, which is hydrophilic, increases the possibility of drug polymerization. The biological function of mAbs is mainly determined by their structure. Antibodies are composed of antigen-binding domain (Fab) and crystallization domain (Fc). Fab specifically recognizes tumor-associated antigens and regulates downstream signaling pathways. Fc binds to the newborn IgG transporter (FcRn) and is not easily cleared in vivo, thus prolonging its half-life in the body . AntibodThe design principles of polypeptide drug coupling are similar to that of monoclonal antibody coupling, which is mainly used for drug delivery and tumor targeting. The difference is that the antibody component in ADC is replaced by polypeptide molecules that can serve as targeted ligands . PolypepRecently, aptamer drug coupling has been a focus of research. The systematic evolution of ligands by exponential enrichment (SELEX) techniques have identified single-stranded oligonucleotide libraries. Nucleic acid ligands that can bind to targets with high specificity and affinity also have many applications in the development of targeted therapies. Aptamers can form specific three-dimensional configurations  through curling and folding. They can bind to targets through van der Waals forces, hydrogen bonds, electrostatic action and base loading forces, etc. The process is similar to the binding of antibodies and antigens and so aptamers are also called “chemical antibodies” . AlthougAt present, some progress has been made in the analysis of the structure and function of molecules in cell transport. Abnormal cell transport in disease has become the focus of basic medicine research, and the development of drugs targeting the transport of diseases and other drugs based on transport structure have also made certain progress. However, cell transport is a complex and systematic process involving numerous molecular and biochemical reactions, and the current research is still insufficient. How to comprehensively understand the transport in cell health and disease is of great significance for basic science and clinical treatment.Cell transport-related proteins play an important role in the cellular and systemic levels by the regulation of energy metabolism, protein synthesis, gene expression, REDOX balance, and signal transduction pathways. Cell transportation-related proteins are associated with a wide range of pathologies, including neurodegenerative diseases, inborn metabolic errors and chronic kidney disease, as well as hormone secretion and release, and their changes in function may be related to the pathogenesis of various diseases. Interactions between membrane transport proteins and with other proteins involve the functional regulation of substrate transport in terms of transport, localization, activity and degradation levels. This is a set of fine-grained regulatory mechanisms, in-depth study of which will help to fully understand the cellular transport mechanism of regulating organisms at the cellular level, and is expected to develop targeted therapeutics for cell-transport diseases. It is hoped that scientists can further clarify the structural characteristics of different types of transporters and the domain functions of unknown residues. These findings will provide valuable insights into fine-tuned cellular transport regulatory networks, transporter interaction models, and molecular probes for diagnosing diseases.Membrane proteins play a role in cell transport and signal transduction, but can also be used as the targets for drugs. At present, the structural analysis of various membrane proteins in cells is not complete. How to find the protein differences between healthy cells and diseased cells, and how to design targeted drug therapies need further research to broaden the application range of targeted drugs. The variation of the cell membrane’s protein structure is the cause of many diseases. Targeted treatments can be achieved using gene editing, protein recombinant expression and other technologies. Similarly, rare diseases caused by abnormal cell membrane proteins are also topics that need to be focused on at present. New discoveries may be made by analyzing the etiology of various diseases.


Moreover, the reviewed studies revealed that, using fibers within the composite restorations would reduce the microleakage and improve the marginal adaptation of the restoration regardless of the fiber type. FRCs tend to strengthen the restorations of structurally compromised teeth and improve their performance compared to plain composite restorations.To reinforce extensively prepared cavities, different types of fiber reinforcement are utilized. Polyethylene and glass fibers are the most commonly used fibers in that purpose; each type has its own advantages over the other type. Therefore, the aim of this study is to review the literature to evaluate and compare the influence of different fiber reinforcement types on the performance of posterior large composite restorations. Two independent authors performed a comprehensive literature search using MEDLINE/PubMed, Google Scholar, and a manual search for cross references until July 2021. Authors selected only studies that contain comparisons between glass (continuous or short) and polyethylene (woven) fiber-reinforced composites (FRCs) in posterior cavities of human teeth, and that report the effect of fiber inclusion on fracture resistance, microleakage, and marginal adaptation of restorations. A number of 2711 potentially relevant articles were obtained from the electronic search. After extensive assessment, 2696 articles were ineligible to be included in the review, and only 15 articles met the inclusion criteria. Four out of nine studies, which tested the fracture resistance of FRC restorations, revealed similar performance of the glass and polyethylene fibers. The rest of the studies ( Extensive cavity preparation is one of the major contributing factors of tooth fragility, which could result in the partial or complete fracture of cusps or roots of posterior teeth . For insInnovative treatment solutions based on new improved materials are continuously evolving to restore the function and preserve the remaining tooth structure, with resin composite being a prime example. Resin composite can bond to tooth structure, which means, theoretically, it has the ability to regain the lost fracture resistance, and to strengthen the tooth by providing an internal splint. However, the reinforcing effect of direct composite fillings when applied alone in large cavities is highly debated . The strThe advent of fiber reinforcement has expanded the potential applications of composite restorations in restorative dentistry, as they internally strengthen the restorations and reduce the occurrence of fractures . The mosDuring the past two decades, a leno woven ultra-high molecular weight (LWUHMW) polyethylene fiber ribbon has been used to reinforce cavities . As an eContinues  glass fiber-reinforced composites have also demonstrated their ability to improve the fracture strength and to stop crack propagation in composite restorations ,19,20. MShort fiber-reinforced composites (SFRC) have also been widely used as bulk base in high stress-bearing areas to reinforce the large restorations and to mimic the stress absorbing properties of dentine . The resWith these varieties of fiber reinforcement types, the question arises as to whether polyethylene and glass FRCs similarly reinforce the cavities, or if one has a preferable performance over the other. There are limited scientific data to influence clinicians’ decisions when deciding which material to choose; therefore, the aim of this study is to conduct a review to evaluate and compare the reinforcing effect of polyethylene and glass FRCs on the performance of posterior large-composite restorations.This systemic review was achieved following the guidelines of PRISMA 2020 statement .A comprehensive electronic literature search was conducted up until July 2021 via MEDLINE/PubMed and Google Scholar; in addition, a manual search of references of the selected studies was performed to analyze all the available potential articles. The following terms were used as search keywords: fiber-reinforced composite in dentistry, polyethylene and/or glass fiber-reinforced composite, polyethylene and short fiber-reinforced composite, fiber cavity reinforcement, woven and unidirectional fibers, Ribbond fibers, everStick fibers, everStick NET, everStick C&B, everX Posterior, everX Flow, Ribbond and everStick, Ribbond and everX Posterior, Ribbond and everX Flow, fracture resistance of fiber-reinforced composite.Eligible studies for inclusion in this review are full-text studies that tested the fracture resistance, microleakage and marginal adaptation of composite restorations reinforced by both polyethylene and glass FRCs. Materials should be used only within the tooth cavity as a substructure or core build-up or filling material, but not in the root. Studies should be published in English language peer-reviewed journals; the search terms were included in either the title or abstract. Included Studies used only extracted human molars or premolars to test the materials. Articles that do not contain direct comparison, unpublished studies, personal communications, background information, and conference abstracts were excluded.Full texts of the potentially relevant articles, according to the inclusion criteria were gained and potential duplicates were carefully read before exclusion. Different reasons for exclusion were agreed between authors. The included articles were evaluated for the presence of the following factors: the two types of fibers were used in a comparable manner and tested under the same conditions.The included articles were carefully read, and data of interest were extracted and reported in Microsoft Word files. For each single article, the authors’ names, year of publication, evaluated parameters, type of control group, type and commercial name of evaluated fiber-reinforced composite, FRCs application technique, the main results and conclusions were reported. Two authors  independently assessed the risk of bias of the included studies. This was undertaken according to two previous systemic reviews ,25. The A total of 2711 relevant articles were recognized and screened for title and abstract evaluation. After assessment, 2696 were removed because they did not meet the eligibility criteria or due to duplication. Fifteen full-text articles were evaluated, and two articles were excluded due to using the tested material as an endocrown or extended it to the roots. Two more articles were found by manual search and cross references ,33,34,35In total, eight out of nine included studies evaluated the effect of FRCs on fracture resistance using endodontically treated teeth, while only one was performed using non-endodontically treated teeth . Four ouRegarding the failure mode, six out of nine studies assessed the failure mode of the restorations and divided it into favourable/reparable or unfavourable/irreparable ,28,29,32Risk of bias of the included studies is summarized in Fiber-reinforced composites are widely used to reinforce restorations of structurally weakened teeth ,37. TypeThe tooth structure left after cavity preparation is a decisive factor to determine its fracture strength . To enhaDifferent types of FRCs are among the most important examples of the modified composites due to their tooth strengthening effect . AccordiSáry et al. have compared the fracture resistance of restorations reinforced with polyethylene (Ribbond) and two types of glass FRCs (everX Posterior and everStick NET) applied using different restorative techniques . The resPrevious studies, another three of which are included in this review, compared the fracture resistance of endodontically treated teeth with MOD, MO, and class I cavities, restored with SFRC (evevX Posterior), polyethylene FRC (Ribbond) or composites without fiber reinforcement ,27,31. IKhan et al. compared the fracture resistance of root-canal-treated teeth restored with polyethylene (Ribbond) or glass (everStick C&B) FRCs under conventional composites . RestoraAll of the previous data were gained and compared, based on in vitro studies under static loading conditions, which vary from the clinical situation with dynamic loading conditions. Moreover, it is quite inequitable to compare the fracture resistance data of the laboratory studies, due to different varieties, such as the situations of the used teeth, the procedures of applying the restorations and the different study protocols; these could be considered as limitations of the included studies in the present review.Microleakage is considered as one of the major limitations of composite restorations. This phenomenon happened because of the stresses generated at the restoration-tooth interface due to different causes such as polymerization shrinkage; repetitive fatigue cycles due to masticatory forces; fluctuation of the temperature in the oral cavity . PlacingBelli et al., and Basavanna et al., evaluated the microleakage of class II composite restorations when cavities were lined with polyethylene (Ribbond) or glass (everStick NET) FRCs in combination with flowable composite, and cavity margins in enamel, dentine or root surfaces. The authors stated that both tested FRCs similarly help to reduce occlusal leakage in Class II cavities with enamel and root margins ,33. The According to Ozel and Soyman, who compared the effect of everStick NET and Ribbond FRCs on microleakage and polymerization shrinkage, both types could decrease the microleakage scores in MOD cavities. However, the results of this study stated that the volumetric polymerization shrinkage of restoration reinforced with glass FRCs was lower than that of polyethylene FRCs, although the results were not statistically significant . On the It is important to highlight that all of the previously mentioned results were obtained from short-term laboratory studies. Different results could be obtained if long-term studies were performed.Within the limitations of the present review, evidence from in vitro studies generally showed that FRCs tend to strengthen the restoration of structurally compromised teeth and improve their fracture resistance compared to composite restorations without fiber reinforcement. However, in almost all of the reviewed studies, short or continuous glass FRCs showed either the same performance or they exhibited better results than the polyethylene (woven) FRCs with regard to the fracture resistance. Moreover, using FRCs reduces the microleakage of restorations regardless of the fiber type.


Renewable energy production can kill individual birds, but little is known about how it affects avian populations. We assessed the vulnerability of populations for 23 priority bird species killed at wind and solar facilities in California, USA. Bayesian hierarchical models suggested that 48% of these species were vulnerable to population-level effects from added fatalities caused by renewables and other sources. Effects of renewables extended far beyond the location of energy production to impact bird populations in distant regions across continental migration networks. Populations of species associated with grasslands where turbines were located were most vulnerable to wind. Populations of nocturnal migrant species were most vulnerable to solar, despite not typically being associated with deserts where the solar facilities we evaluated were located. Our findings indicate that addressing declines of North American bird populations requires consideration of the effects of renewables and other anthropogenic threats on both nearby and distant populations of vulnerable species. While this magnitude of mortality is unlikely at any single renewable energy project, it is reasonable when considering cumulative effects of many renewable projects at once ,55,56, oAlthough our models were relatively sophisticated compared with analyses used in many past evaluations of renewable effects on wildlife, data limitations prevented incorporation of some demographic processes  that can affect population responses to anthropogenic stressors. Further, we assumed all anthropogenic-related fatalities are additive (i.e. not compensated for at the population level by density-dependent or other processes) , that faSome of these species-specific data issues could be addressed by increasing systematic data collection at renewable facilities. This is especially true given that rigorous fatality estimates are lacking for most species . HoweverButeo jamaicensis), horned lark and the vulnerable, aerial-foraging bank swallow [The susceptibility to fatalities at wind energy facilities of grassland-associated species, especially raptors, may be due to a suite of factors, including the prevailing habitat type at the wind facilities studied, the prolonged period of exposure of the resident birds affected, catchment area population size, and the behaviour and demography of those species present. Many vulnerable grassland raptors, including diurnally active golden eagles and white-tailed kites, and the less diurnal burrowing and barn owls, are year-round residents at the wind energy facilities we studied . All the swallow ). In con swallow ,20.The majority of current and ongoing wind energy development in the United States is in the native or converted grassland habitat of the Great Plains and Midwest  that oveIn contrast to wind facilities, for which most vulnerable species were year-round residents, the most vulnerable avian species at solar facilities were non-local migrants. Once again, this pattern of vulnerability is probably due to many of the same factors as at wind facilities, including facility placement, the size of the catchment area subpopulation, and underlying behavioural and demographic factors. The solar facilities we studied are located in low-elevation areas of the Mojave Desert and are in close proximity to important wintering and breeding grounds for songbirds and waterbirds that use the Pacific Flyway in North America. Although we did not detect vulnerability in desert-specialist species found dead at solar facilities in the region , facility placement along a constricted portion of this flyway may increase vulnerability of species engaged in long-distance migration. Other characteristics of these facilities may also increase vulnerability of migrant birds. For example, some evidence suggests reflection of polarized light from the sun, moon or artificial sources, off solar panels may attract insects or create a ‘lake effect’ mimicking a large body of water, and thereby attract water-associated species such as loons and grebes ,72. FurtDespite the patterns of vulnerability we document for birds found dead at renewable energy facilities, there are additional complexities to this problem. For example, the two energy types we considered are actually composed of multiple subtypes, with at least four generations and sizes of wind turbine technology and three different types of solar energy production (see study site in Methods). Likewise, energy facility infrastructure, including transmission lines, roads or fencing, may also contribute to site-specific fatalities. Thus, each facility embodies unique technological and infrastructural attributes, all of which may modulate the type, magnitude and seasonality of species-specific mortality . As suchDespite being the focus of massive conservation efforts –76, birdThis study illustrates, for the first time and for a large, taxonomically diverse suite of priority species, both the vulnerability of a subset of avian populations to renewable energy development, and the manner in which the demographic influence of renewable energy facilities may extend far beyond the site at which fatalities occur. Our inference is underpinned by novel integration of subpopulation size estimates into demographic models within isotopically determined catchment areas. As such, our analyses highlight the importance of incorporating spatial ecology into assessments of the demographic relevance to avian populations of anthropogenic stressors such as renewable energy. Such assessments also have important conservation implications. In the case of renewable energy, decisions about facility siting, investment and development, as well as management and mitigation actions, will be most effective if they consider both local and non-local impacts to focal species, and if their demographic frame of reference extends to breeding, wintering or stopover habitat far from where the facility is located.