Cryoprotective and cytoprotective agents (Cytoprotective Agents) are fundamental components of the cryopreservation process. This review presents the essentials of the cryopreservation process by examining its drawbacks and the role of cytoprotective agents in protecting cell physiology. Natural cryoprotective and cytoprotective agents, such as antifreeze proteins, sugars and natural deep eutectic systems, have been compared with synthetic ones, addressing their mechanisms of action and efficacy of protection. The final part of this article focuses melatonin, a hormonal substance with antioxidant properties, and its emerging role as a cytoprotective agent for somatic cells and gametes, including ovarian tissue, spermatozoa and spermatogonial stem cells. Cryopreservation techniques are routinely used in a range of medical and research applications to store biological materials, including cells, tissues and their fractions. Unfortunately, even the most efficient protocols and technologies for cryopreservation are not able to guarantee complete control of the molecular processes responsible for the spontaneous deterioration of the biological specimens, starting from lipid autoxidation, which is the first to develop by the activity of molecular oxygen .In cell and tissue samples, the cryopreservation process itself can interfere with the integrity of the stored material inducing cellular damage. The most important effects involve the cellular membrane and are the consequence of chemical and mechanical  stresses occurring during the freezing and thawing steps of cryopreservation ,3. TheseCryoprotective and cytoprotective agents (CPAs) are used to prevent these drawbacks of cryopreservation in either cellular or supracellular specimens. The ideal CPA should be able to prevent unwanted oxidation, the damaging effects of the cryopreservation process on membrane physical and chemical properties, and other cellular injuries introduced earlier and summarized in The toxicity of most common CPAs limits their application in medical protocols of cryopreservation, including those intended for cell therapy and tissue/organ transplantation of immune hematology, regenerative medicine, and the reproductive biology area of competence. These aspects have extensively been described in other recent review articles  . Better The main issues related to this process derive from the freezing of intracellular water , which results in the formation of ice crystals that destroy cells through mechanical actions and changes in the composition of the liquid phase ,32 Figu. VitrifiCryoprotective agents are classified into two main categories, namely: nonpenetrating (NPA) and penetrating agents (PA), that protect biological samples through different mechanisms . Nonpenetrating agents are small molecules  organized into polymers that increase extracellular osmolality to cause cell dehydration as a stabilization mechanism; because of this function, nonpenetrating agents require slow cooling rates. These agents are added to thawing media to slow down the influx of water in the cell, thus preventing osmotic shock and lysis ,35. In tPenetrating CPAs are small and nonionic molecules  that can cross the cell membrane to exert multiple and partially unknown mechanisms of action . This iRecently, several studies were published on the epigenetic modifications induced by CPA. It is well known that the exposure to even low concentrations (1% or less) of DMSO, can cause alterations in the epigenetic profile of embryonic stem cells. Genomic effects have been identified in the cryopreservation of gametes as well , althougStrategies to minimize CPA toxicity have been proposed, including the combination of chemical agents with different levels of toxicity (such as acetamide and formamide) with less toxic or neutralizing CPAs, and the use of more biocompatible and natural CPAs, some of which have been identified to act as toxicity-minimizing agents. These aspects are presented in the next section . The possibility to use natural CPAs in cryopreservation has stimulated interest in the scientific community, representing an alternative to synthetic counterparts and their limits in terms of toxicity and cryoprotection function. As introduced earlier in vide infra, Natural agents with cytoprotection function have also been utilized in cryopreservation, including antioxidants and cell stress preservation agents, such as ascorbic acid , vitaminDuring evolution, living organisms have developed strategies to survive in cold environments, including antifreeze proteins (AFPs) and ice nucleation agents (INAs) . Antifreeze proteins and glycopeptides AF(G)Ps are part of a family of natural CPA used by animals, such as insects, amphibians and fish, to limit ice formation and to protect against “freezing hysteresis” . Whether AFPs exert a protective role as cryoprotectants depends both on their concentration and the type of selected AFP. In Antarctic fish, the presence of AFPs protects them from freezing below −2 °C; this is also the case in insects and in freeze-tolerant plants found at sub-zero temperatures . The nonThese properties of natural AFPs have recently inspired the development of new ice-interactive cryoprotectants that have been used as an alternative to DMSO in the cryopreservation of human hematopoietic stem cells .In extreme cold environments, nature has provided additional tools to promote ice formation through ice nucleating agents. In some insect species, the presence of INAs has been associated with a seasonal adaptive elevation of their super cooling points, which has inspired use of these compounds as a CPA. These agents favor ice formation even at extreme sub-zero temperatures . SimilarSeveral organisms  live in different natural environments and efficiently use sugars  to survive in extreme climatic conditions, e.g., extreme droughts or freezing. Mammalian cell membranes are impermeable to sugars; thus, these molecules are used as extracellular additives. Different approaches have been attempted to overcome membrane permeability, such as thermotropic lipid-phase transition, transfection, poration , and eveNonreducing disaccharides  and trisaccharides (raffinose) can afford cytoprotection, and they constitute a valid alternative to standard CPA agents. According to this, Peterenko and colleagues  demonstrSugars exert their actions by inducing lipidic membrane stabilization as a result of direct interactions with polar components of the bilayer and by preventing intracellular protein aggregation. In addition, they act as osmolytes to prevent osmotic stresses . In contrast, smaller sugars such as glucose or its polymers, such as dextran, cannot provide optimal cryopreservation due to the inability to directly interact via hydrogen bond formation with the polar heads of cell membrane phospholipids . Eroglu et al.  investigCryopreservation techniques, as a final purpose, have to safely bring cells below the glass transition temperature, at which the surrounding medium vitrifies. A high glass transition temperature can facilitate the cryopreservation procedure. Sugars are effective glass formers compared to conventional cryoprotection agents, such as DMSO, ethylene glycol and 1,2-propandiol.Deep eutectic systems (DESs) are characterized by high biodegradability, cost-effectiveness and low toxicity. DESs can originate from natural primary metabolites such as sugars, amino acids, organic acids or choline derivatives, which are mixed at a specific molar ratio. These systems have been “bioinspired” by the observation that these metabolites are produced by animals and plants to adapt in extremely cold conditions. The mixtures of these components have a higher melting point depression compared to the individual constituents, such that they become liquid at room temperature . Trehalose, glucose, sorbitol and proline constitute the components of DESs, even if other compounds have recently been used to develop new solvents such as naturally occurring l-ascorbic acid (vitamin C) and choline . These molecules are successfully used in combination with DMSO to improve cell viability after thawing. The mixture of trehalose with DMSO has demonstrated to be effective in the cryopreservation of several cell lines, such as stem cells, primary hepatocytes, and HepG2 cells .NADES have been recently explored as potential CPAs for the preservation of different types of cells, including lactobacillus , mouse fAnother important consideration is that the cryoprotective effect of NADES composed of different sugars combinations  can vary in a cell-dependent manner. For example, after thawing–freezing cycles in L929 cells, NADES produced almost the same cytoprotection effect of the standard CPA DMSO, while in HaCaT, these promoted a recovery of cell viability that was significantly higher compared to DMSO . NADES represent a great alternative to CPAs in the cryobiology field, because during the thawing process, they do not need to be removed from the freezing media.In addition to its canonical function as a circadian rhythm regulator, MLT has been fully demonstrated to represent a potent CPA and cell homeostasis factor ,66. ThisMelatonin is considered to be a potent repressor of cellular oxidative stress via its activity as a direct antioxidant and free radical scavenger that depends on its hydrogen atom transfer properties , and viaThe antioxidant actions of MLT are linked to its interaction with receptors and transcriptional factors located on either the cell membrane or subcellular organelles ,72,73. TIn the mitochondria, the enzymatic dismutation of superoxide anions is facilitated by the stimulation of SIRT3, leading to the deacetylation and activation of SOD2. This process limits the damage to the organelle . At the same time, the interaction with the reactive species metabolizes MLT into cyclic-3-hydroxymelatonin, N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and N1-acetyl-5-methoxykynuramine (AMK), having similar or better detoxifying actions compared to their precursor ,74.Therefore, whereas classical antioxidants scavenge only a single ROS, MLT can detoxify multiple species via its direct scavenging activity and transcriptional effects. These properties of MLT can be important to afford cytoprotection in the cryopreservation of somatic cells. Once added to freezing solutions, MLT can easily cross the cell membrane to exert its biological function, including antioxidant and stress response effects, mitochondrial protection, and control of superoxide anion formation . These eThe most striking evidence regarding the cytoprotective function of melatonin has been reported in the case of gametes cryopreservation, which is presented in the next sections.The aim of ovarian tissue cryopreservation is the maintenance of female fertility, especially in the case of patients who undergo radiotherapy or chemotherapy that damage the ovaries, causing premature loss of ovarian functions. The preservation of ovarian tissue (OT) is the most feasible option to attempt preservation of fertility in adolescent cancer patients for whom the cryopreservation of oocytes and embryos is impossible .Because the cell types present in this specific tissue are vulnerable to severe damage, cryopreservation of OT faces different challenges. Mainly, the production of ROS causes oxidative damage and the disruption of follicular functions. ROS production has been reported to affect the vitrification process, causing cell dysfunction and a decline in oocyte survival rate by the activation of apoptotic cell death . MLT hasStudies on the cryopreservation of OT deriving from adolescent Sprague–Dawley rats confirmed this effect of MLT in protecting the integrity of follicular morphology , with a Cryopreservation is known to induce oxidation processes in spermatozoa, which are the main mechanisms causing cryodamage and reduced motility and viability in these cells . These d2O2 and O2−). In this study, MLT used at a concentration of 3 mM significantly reduced the levels of caspase-3 and the percentage of apoptotic and nonviable sperm cells, also increasing the motility and levels of AKT phosphorylation of sperm cells. Again, MLT can protect mitochondria through different mechanisms [In a recent meta-analysis, MLT was included among the antioxidants that are able to reduce ROS production and protect human sperm function during the freeze–thaw process . This mochanisms . These ichanisms . The role of MLT as a useful CPA in sperm cryopreservation has been confirmed in a series of recent studies in different species, including fish  and dog Despite the evidence of an enhanced resistance to cryodamage in spermatozoa, the available information on dosages and protocols for the use of MLT remains insufficient for a translation of this CPA into clinical guidelines of reproductive medicine for sperm cryobanking. In addition, mechanistic aspects through which MLT can positively affect human and animal spermatozoa during cryopreservation are worth investigating further.The protective effects of MLT have extensively been studied in stem cells exposed to different types of cellular stressors, including heavy metals and xenobiotics ,66,92. LSpermatogonial stem cells (SSCs) are the base ground for spermatogenesis and male fertility. They are a type of primitive spermatogonia located in the seminiferous tubules . SSCs ar−6 M concentration of this molecule was found to significantly increase cell viability, total antioxidant capacity (T-AOC) and the mitochondrial potential of these cells, while the levels of ROS and malondialdehyde (MDA) were reduced. Melatonin also improved enzyme activity and the expression of SOD, CAT and GSH-Px. At the mitochondrial level, the expression of the pro-apoptotic protein Bax was inhibited, and the expression of its anti-apoptotic counterpart Bcl-2 increased; moreover, MLT reduced both mitochondrial swelling and vacuolation, inhibiting the release of cytochrome C into the cytosol and inhibiting caspase-3 activation, which are important events in the mitochondrial pathway of SSC apoptosis. Again, MLT reversed the mechanisms of freezing-induced autophagy in SSC, regulating the expression of the autophagy-related proteins LC3-I, LC3-II, P62, Beclin1 and ATG7. When the protective effects of MLT were studied in cryopreserved goat SSCs , a 10−6 According to these findings in goat SSCs, MLT was found to promote beneficial effects in terms of cell proliferation and survival rates in mice SSCs . These studies and others in the literature suggest that the implementation of MLT in SSC cryopreservation may represent an important strategy in the field of assisted reproduction, considering either an application in future auto-transplantation, or the in vivo induction of mature sperm development .Cryopreservation can be affected by mechanical and chemical complications that influence the quality and viability of the preserved cells or tissues. Therefore, the use of CPAs is fundamental to ensure a safe process. These agents exert their cryoprotective function by increasing the total concentration of all solutes in the system, thus reducing the amount of intracellular ice formation. To exert this function, the agents should be able to penetrate cells and ensure low cytotoxicity. Still, due to DMSO’s well-proven high efficacy in cryoprotection, this remains the most used CPA in biomedical research. However, as an organic solvent, DMSO has an intrinsic toxicity and can induce cell apoptosis and uncontrolled differentiation, even if used at low dosages.New techniques of cryopreservation have been developed and implemented in recent years, including CPA combinations  and new Furthermore, as highlighted in this review article, natural CPA can represent an alternative to DMSO and other classical CPAs of synthetic origin, reducing their toxicity during cryopreservation. Due to these properties, these agents have gained increasing attention in the last several years. These include AFPs that hold great potential for an application in cryobanking . AlthougNeutralizing the toxicity of CPAs is an important aspect in cryopreservation. This can be achieved with classical CPAs that have specific neutralizing activity against other and more toxic CPAs that are used in combination, e.g., DMSO was found to partially neutralize formaldehyde toxicity . AlternaIn perspective, genetic studies have suggested the possibility of monitoring the susceptibility of the cryopreserved material to cryoprotectant toxicity . This in


RPN), signifying a need for immediate control action. Additionally, a nanosafety control-banding tool allowed hazard classification and the identification of control actions required for mitigation of occupation risks due to the released airborne silver nanoparticles.The COVID-19 pandemic instigated massive production of critical medical supplies and personal protective equipment. Injection moulding (IM) is considered the most prominent thermoplastic part manufacturing technique, offering the use of a large variety of feedstocks and rapid production capacity. Within the context of the European Commission-funded imPURE project, the benefits of IM have been exploited in repurposed IM lines to accommodate the use of nanocomposites and introduce the unique properties of nanomaterials. However, these amendments in the manufacturing lines highlighted the need for targeted and thorough occupational risk analysis due to the potential exposure of workers to airborne nanomaterials and fumes, as well as the introduction of additional occupational hazards. In this work, a safety-oriented failure mode and effects analysis (FMEA) was implemented to evaluate the main hazards in repurposed IM lines using acrylonitrile butadiene styrene (ABS) matrix and silver nanoparticles (AgNPs) as additives. Twenty-eight failure modes were identified, with the upper quartile including the seven failure modes presenting the highest risk priority numbers ( Specific studies dedicated to volatile organic compound (VOC) release analysis are not available for IM to the best of our knowledge. However, within this context, the HSE has highlighted the potential for release of several hazardous fumes from ABS plastic thermal processing while also emphasizing that heating temperature is a critical determinant of fume production . SubstanZitting et al.  exposed Based on the Hierarchy of Controls scheme, as described in ISO 45001:2018 Occupational health and safety management systems—Requirements with guidance for use , a multiRecommended best practices and administrative controls, critical in exposure mitigation and consequently in operators’ safety, include proper signage of the workplace area about nanohazards, isolation of the workplace area from non-nanoparticle-related activities, allowing access to the area to authorized and trained persons only, and performing regular maintenance testing of the extraction ventilation equipment. Concerning PPE, the use of a FFP3 respirator for the whole duration of the process is considered necessary. An upgrade to full-/half-face respirators with P3 particulate filters and appropriate filters for organic toxic fumes and volatile organic compounds must be considered depending on emitted UFPs, ENMs, and VOCs. In addition, according to the EC recommendations, operators and workers involved in nanomaterial processes should be face-fit tested for the respirators they use as personal protective equipment via a quantitative fit testing procedure. Quantitative respirator fit testing ensures that maximum protection is offered by the respirators, particularly against nanoparticles, the high mobility and low mass of which renders them capable of passing through microscopic gaps left by an improperly fitting respirator . Double Concerning FM11 (eruption), administrative controls, such as a periodic inspection and maintenance program for the compressor, tubing, and related pressure valves, are important in order to reduce risk and prevent a possible eruption. In the case of FM23 (electric shock), all electrical equipment of the pilot line must be properly insulated by design. Furthermore, the cooling water bath for the extruded material should not be close to exposed cables and sockets. In this way, protection is ensured against electrocution due to a short-circuit caused by water spillages. In addition, engineering controls and appropriate safety measures, such as spill-absorbent pads beneath the equipment/machinery and adjustable guards on the sides of the water tank bath, further reduce the risk. The use of special insulated gloves should also be taken into consideration.For the failure mode concerning cuts, bruises, and injuries (FM1), administrative controls, such as training of personnel for proper use of the machinery/equipment, labelling of the equipment for any physical hazards arising during the operation of the pilot line, and developing written operating procedures for the convenience of the operators, can reduce the existing risk. In addition, concerning PPE, operators must be equipped with cut- and heat-resistant work gloves (comfort-grip gloves) when the equipment of the pilot line is under load, as well as with mandatory safety goggles for ocular protection. Further discussing FM1, the intake area of the feeding system’s opening should be protected by design by a fixed hopper or another fixed guard.RPN scores in RPN was presented for the electrical-type hazards related to the use of the equipment , given that equipment was CE-marked and appropriately installed and set up. Dust emissions that may be produced from pelletizer use  and the last step of the injection process  can be adequately controlled through the emission control and safety measures described for nanoparticle and nanomaterial exposure , such as ventilation, respiratory protection, and safety glasses. Various parts of the production line entail high-temperature hazards , which can be mitigated through the use of heat-resistant gloves for any necessary direct interference with hot parts of the equipment as well as proper risk perception and following the standard operating procedures. It is noted that the definition of and strict adherence to SOPs constitute a fundamental safety measure for many of the FMs, indicating the importance of administrative controls within a comprehensive safety system. FM9, which displayed an RPN score of 15, concerned the possibility of hearing damage as a result of the compressor use, which can be controlled through placing of the noise-inducing equipment in a remote room. Isolation of the noise sources can be applied on a priority basis as a noise exposure control, and the application of engineering controls such as acoustic screens and barriers is also recommended before personal protection is implemented. For occasional attendance in noisy areas, a noise refuge may be considered, which consists of an employee-occupied acoustic enclosure aimed to keep noise out [The other failure modes, although presenting lower oise out . Given tThe intrinsic characteristics of the methodologies applied in the present study entailed several limitations. It should be noted that exposure, as described within the tool input in On-site exposure assessments based on airborne (nano)particle release measurements constitute a robust method to quantify the potential occupational risks posed by handling nanomaterials. These exposure studies often involve several visits and process walkthroughs in the workplace under investigation, using a diverse set of instruments to get airborne particle concentration readings or workplace air samples, and they may use tiered or “step-wise” approaches . SupportSeeing that one of the primary alterations in the process was the introduction of nanomaterials, aside from safety aspects, the environmental impact of such a process modification would be important to study as well . This woRPN because of high respiratory system penetration leading to adverse health effects. For a more detailed assessment of the process steps involving ENMs, the activities were evaluated through the Stoffenmanager Nano tool via a control-banding classification of AgNP handling, showing that the use of LEV, source containment, and FFP3 respirators would lower the task- and time-weighted exposure to moderate risk bands. Based on the Hierarchy of Controls scheme, a multitude of engineering controls, best practices, administrative actions, and case-specific PPE were proposed for each of the emerged threats. Administrative actions such as SOPs, signage, and periodic maintenance would mitigate the risks from eruption, cuts, and bruises, while the appropriate ventilation flow rate, LEV and HEPA filtration, and FFP3 respirators and P3 filters would minimize UFP, ENM and VOC exposure. This work provides the necessary guidelines for risk assessment and occupational health and safety management of repurposed industrial production lines of critical medical devices during the pandemic era. Extended studies focusing on the safety and sustainability aspects of such repurposing setups would be valuable for establishing and optimizing the long-term viability of manufacturing line repurposing approaches.This study presents an innovative safety-oriented FMEA regarding a repurposed injection moulding manufacturing line as part of the imPURE project. A low concentration of silver (Ag) nanoparticles was added in a polymer matrix because of their antimicrobial properties. Twenty-eight failure modes were identified in the four nodes of the repurposed manufacturing process , and seven of these were selected for further analysis. These seven represented the riskiest identified threats, including cuts, bruises, and injuries during operation; electric shock; eruption of high-pressure vessels; hearing damage due to high noise levels; inhalation of hazardous fumes; and exposure to ENMs. The FM concerning inhalation of UFPs and ENMs presented the highest


Nanodiamonds (NDs) are a class of carbon nanomaterials with sizes ranging from a few nm to micrometres. Due to their excellent physical, chemical and optical properties, they have recently attracted much attention in biomedicine. In addition, their exceptional biocompatibility and the possibility of precise surface functionalisation offer promising opportunities for biological applications such as cell labelling and imaging, as well as targeted drug delivery. However, using NDs for selective targeting of desired biomolecules within a complex biological system remains challenging. Urinary bladder cancer and bacterial cystitis are major diseases of the bladder with high incidence and poor treatment options. In this review, we present: (i) the synthesis, properties and functionalisation of NDs; (ii) recent advances in the study of various NDs used for better treatment of bladder cancer and (iii) bacterial cystitis; and (iv) the use of NDs in theranostics of these diseases. Nanodiamonds (NDs) are carbon-based materials that have attracted considerable interest in biomedical research due to their outstanding properties, particularly their biocompatibility and surface functionalisation capabilities. Nanodiamonds were first synthesised in 1963 by detonation of carbon-based explosives ,3. For vA unique set of outstanding properties of NDs makes them attractive for targeted drug delivery systems, as well as for theranostics and imaging applications. The broad spectrum of key advances includes exceptional biocompatibility, large carrier capacity, versatile surface chemical properties that enhance drug binding and enable sustained drug release, and stable bright fluorescence based on crystallographic defects. Compared to other forms of nanoparticles, NDs also impress with their exceptional physical and chemical stability and inertness, efficacy, biosafety and low cost.Mass production of NDs began in the early 80s , with thDNDs typically have a round to oval shape and measure about 5 nm in diameter . Their s0) and negative (NV¯), with ZPL (zero phonon line) emissions of 575 nm and 637 nm, respectively [S), with a concentration of up to 300 ppm (for HPHT ND type Ib). ND, made from natural diamond type Ia (NS concentration up to 3000 ppm), is also available. High energy particle irradiation of type Ib ND followed by annealing leads to the formation of NV centres with red emission, while the ND of diamond Ia show green luminescence resulting from the formation of N-V-N (H3) centres after irradiation and annealing [Monocrystalline NDs obtained by processing synthetic HPHT diamond are currently commercially available, with the smallest average particle size ranging from 10 nm to up to micrometres. In the HPHT method, large diamonds are first produced, which are later irradiated with high-energy electrons, protons or helium ions to create NV centres. They exist in two charge states, neutral  or lithium aluminium hydride (LiAlH4); (ii) by a Fentone reaction (a solution of H2O2 with iron (II) sulphate (FeSo4)); (iii) by a mechanochemical treatment; (iv) by a photochemical reaction [2 groups on the surface of NDs (amination), including ultraviolet (UV) irradiation of hydrogenated diamonds in the presence of chlorine gas, treatment of chlorinated NDs with ammonia at elevated temperatures [Most of the available DNDs have an oxygenated surface. This is due, for example, to the application of water or ice for cooling in the detonation process, which leads to a reaction with highly reactive hydroxyl species . Only NDreaction ,26,27,28reaction ,29,30. Seratures , and treeratures .Most approaches to ND surface modification have been developed for DNDs, as synthesis method for DNDs was developed early on and DNDs are widely available commercially . AlthougBladder cancer is among most common cancers worldwide, with nearly 600,000 new cases in 2020 ,50. It iIn general, the treatment and diagnosis of all types of cancer is currently one of the most pressing problems in medicine. The targeted administration of drugs that directly target cancer cells significantly reduces the risk of harming normal cells of the neighbouring tissue, thus reducing the side effects of the drug ,56. A moOther mechanisms for the delivery and accumulation of nanoparticles in the tumour are also known. For example, passive tumour targeting, co-called the EPR effect (enhanced permeability and retention) . The EPROnce near the surface of the cancer cells, NDs are internalised by endocytosis ,55. EndoSince retinoids have the potential to reduce the incidence and recurrence of bladder cancer and improve therapy , the tra- ion concentration, and the NDs were found to be able to efficiently transport the drug into living cells [One of the most commonly used and studied chemotherapeutic agents is doxorubicin (DOX), an apoptosis-inducing drug that can be conjugated to the surface of NDs. Such ND-DOX has been applied to numerous cancer cells such as colorectal carcinoma cells, tetracycline-resistant leukaemia cells, brain tumour cells, liver carcinoma, breast adenocarcinoma and also in vivo mouse models of liver and breast cancer ,82,83,84ng cells .On the other hand, the pH also influences the release of the DOX from the NDs. In an alkaline environment (pH = 8), the carboxyl group of ND together with the amino group of DOX form a stable and non-covalent bond on the surface of ND, which served as a simple physical adsorption. In an acidic environment of an endocytic compartment such as the early or late endosome, the NDs carboxyl group is ionised, effectively releasing DOX. Thereafter, DOX is affirmatively absorbed into the cytoplasm and later into the nucleus . InteresThe search for newer, innovative and more effective treatments could lead to NDs being used in the treatment of bladder cancer in the future. In addition, NDs could also be used to treat bacterial cystitis due to their antibacterial and anti-inflammatory properties.Escherichia coli (UPEC). Patients with UTI are most often prescribed antibiotic treatment. Despite antibiotic treatment, the probability of a female patient having a second UTI within six months is 25%. It was found that in most cases the initial infection and recurrence are caused by the genetically identical strain of UPEC [Urinary tract infections (UTI) are the second most common infection in women, causing over 8 million UTI cases annually in the United States . The cau of UPEC . This suUPEC penetrate superficial, highly differentiated, so-called umbrella cells and the underlying less differentiated intermediate and basal urothelial cells by two different mechanisms. The umbrella cells are infected by direct penetration of UPEC through the apical plasma membrane. This membrane is highly specialised and organised into urothelial plaques composed of four major transmembrane proteins, the uroplakins. Uroplakin Ia presents a high level of terminally exposed mannose residues, which serve as the attachment point for UPEC adhesion  90,91,9,991,92. Since interaction between pathogenic bacteria and host cells is the critical step prior to host colonisation, prevention of such contact has been used as a promising strategy to treat bacterial bladder infections. Inhibition of FimH-mediated adhesion to umbrella cells is generally used as an effective antibiotic alternative or supportive strategy to reduce UPEC-related infections. Unfortunately, monovalent mannose suspensions have very little inhibitory effect in preventing FimH-mediated bacterial adhesion to urothelial cells . ND partIn addition to using NDs as carriers for glycan groups, they have been successfully used as attachment particles that are bacteriostatic or bactericidal for both extracellular and intracellular UPEC colonies . In thisAll diseases of the urinary bladder share the extreme difficulty of reaching the affected cells with conventional treatment methods, both in the case of cancer transformation and bacterial infections. The main obstacle is the exceptionally impermeable apical plasma membrane of the umbrella cells, which prevents the penetration of cytostatics to cancer cells nested in lower layers of urothelium  and antiDetection and specific labelling of cancer cells in the bladder is an important step prior to transurethral resection of tumours, allowing surgeons to efficiently remove the cancerous tissue. Although white light cystoscopy remains the diagnostic standard for the detection of bladder tumours, flat cancerous lesions can often be missed . For thiTo the FNDs with a functionalised surface , variousLien et al. suggested that fluorescent and magnetic NDs (FMNDs) could be used to label and track bladder cancer cells . In theihttps://clinicaltrials.gov). Quantum dots are also becoming increasingly popular for fluorescent labelling of cells and as labels for antibodies. In particular, the inorganic QDs are known for their long fluorescence lifetime, high fluorescence quantum yield and photostability [In recent decades, various types of nanoparticles (NPs) have been developed as contrast agents for cell tracking in biomedical examinations, in addition to NDs, as described by Jen-Shyang Ni et al. in 2020 . The mostability . Howevertability . Comparetability  and thusThe treatment of bladder cancer and bacterial cystitis has not improved significantly in the last decade, although the social and economic burden of both diseases is high, especially in developed countries. In view of this problem, the search for innovative approaches is crucial, and NDs are one of the promising tools. The great biocompatibility, low toxicity and wide range of possible specific functionalisation of NDs are their greatest advantages. In addition, DNDs and FNDs could also be used for theranostics, as they allow visualisation of cancer urothelial cells as well as UPEC-infected urothelial cells, and also show potential for the treatment of bladder cancer and bacterial cystitis. However, in vivo studies show that NDs accumulate in animal organs and there are no confirmed studies on the long-term effects of their release from organs. This is a real problem, especially as many patients require repeated therapies. Intravesical application of therapeutic NDs can be used for bladder cancer and bacterial cystitis. Such application allows direct contact of the drug conjugated to the NDs with the diseased urothelium and immediate excretion of the NDs with micturition. Therefore, in the case of the bladder, there is no concern about accumulation of NDs in other organs of the body. However, in our opinion, the main problem is the small number of studies using NDs to treat bladder cancer and bacterial cystitis. In addition, standardised ND materials and drug delivery approaches are needed to enable clinical-level applications.


This paper points out the design, development and deployment of the AutoInflammatory Disease Alliance (AIDA) International Registry dedicated to pediatric and adult patients affected by Undifferentiated Systemic AutoInflammatory Diseases (USAIDs).This is an electronic registry employed for real-world data collection about demographics, clinical, laboratory, instrumental and socioeconomic data of USAIDs patients. Data recruitment, based on the Research Electronic Data Capture (REDCap) tool, is designed to obtain standardized information for real-life research. The instrument is endowed with flexibility, and it could change over time according to the scientific acquisitions and potentially communicate with other similar tools; this platform ensures security, data quality and data governance.The focus of the AIDA project is connecting physicians and researchers from all over the world to shed a new light on heterogeneous rare diseases. Since its birth, 110 centers from 23 countries and 4 continents have joined the AIDA project. Fifty-four centers have already obtained the approval from their local Ethics Committees. Currently, the platform counts 290 users . The Registry is collecting baseline and follow-up data using 3,769 fields organized into 23 instruments, which include demographics, history, symptoms, trigger/risk factors, therapies, and healthcare information access for USAIDs patients.https://clinicaltrials.gov/.The development of the AIDA International Registry for USAIDs patients will facilitate the online collection of real standardized data, connecting a worldwide group of researchers: the Registry constitutes an international multicentre observational groundwork aimed at increasing the patient cohort of USAIDs in order to improve our knowledge of this peculiar cluster of autoinflammatory diseases. NCT 05200715 available at  Undifferentiated Systemic AutoInflammatory Diseases (USAIDs) represent a group of undefined medical conditions increasingly reported in medical literature. The acronym USAIDs identifies a subset of patients characterized by self-limiting episodes of inflammation that fail to meet criteria for the established monogenic or multifactorial autoinflammatory diseases, but display features of autoinflammatory disorders. Patients with USAIDs do not carry confirming pathogenic mutations in genes associated with monogenic autoinflammatory diseases and do not fulfill any of the diagnostic or classification criteria currently available for multifactorial autoinflammatory disorders. There are neither definite diagnostic criteria available, nor specific laboratory investigations for identifying USAIDs , 2. USAIPriority of the AIDA project is the development and maintenance of international registries for patients affected by monogenic and multifactorial autoinflammatory disorders and ocular inflammatory diseases. To date, nine international registries have been launched, including Behçet's disease (BD), monogenic autoinflammatory diseases, Still's disease, Schnitzler's syndrome, Periodic Fever, Aphthous stomatitis, Pharyngitis and cervical Adenitis (or PFAPA) syndrome, non-infectious uveitis, non-infectious scleritis, vacuoles, E1 enzyme/X-linked autoinflammatory somatic (or VEXAS) syndrome and undifferentiated autoinflammatory diseases. The use of these registries will allow sharing of knowledge, experience, and different perceptions on the clinical, therapeutic and research approaches in the field of rare diseases , 4.This paper purposes to point out the design, development and deployment of the AIDA International Registry dedicated to patients with USAIDs, which corresponds to a multicentre, non-interventional, population- and electronic-based observational clinical study. The registry will include patients with unexplained systemic inflammation suggested by laboratory, clinical or therapeutic clues which will be widely clarified in the methods section.The AIDA Registry for USAIDs patients has been conceived to collect both retrospective and prospective data. In particular, retrospective phase refers to demographic, clinical, laboratory, instrumental and therapeutic information available at the time of enrollment into the Registry; prospective phase includes clinical, therapeutic and socioeconomic data acquired thereafter.Variables included in the Registry have been selected on the basis of clinical and laboratory features generally described in patients with autoinflammatory diseases; evidence currently available in literature; therapeutic details required to comprehensively describe treatment options proposed for monogenic and multifactorial autoinflammatory diseases, and information required during the follow-up visits according with the best standard of care.Some information may be repeated and, therefore, recorded in both the retrospective and prospective phases, as for example data about the cardiovascular risk and information about fertility, pregnancy and breastfeeding period. The retrospective assessment includes clinical and laboratory data referring to the start of symptoms, the time at the diagnosis, and the time at the enrollment into the Registry; for each treatment performed during the patient's history, clinical and laboratory data would be required referring to the start of the treatment, the 3-, 6- and 12-month visits and at the last assessment while on the treatment. Conversely, the follow-up visits will be added at the patient re-evaluations following the inclusion in the AIDA Registry; a follow-up re-evaluation should take place at least every year and at any change in the treatment strategy, as for the introduction of new drugs and posology changes.Since only data related to the standard routine management are recorded and no additional specific investigations are required, no funds are provided for patient's enrollment and no further impact on national healthcare will be determined by the participation in the AIDA project. Similarly, treatments administered prior or after the enrollment in the AIDA Registry are drawn by the best standard of care and are not influenced by the study protocol.support@aidaregistry.org or using a specific form at the bottom of the following page: https://aidanetwork.org/en/aida.Any center managing USAIDs may participate in the project without limitations regarding the location, medical specialty, or type of practice setting. The centers that would like to participate, can make a request by contacting the Promoter directly or by sending an email from the web page contact the AIDA Team by writing to The only required prerequisite is obtaining approval from the local Ethics Committee and appointing a Principal Investigator whose function is coordinating the study locally, and Site Investigators responsible for the documentation and data entry for that site.RUNX1, BCOR, WTI or TP53 genes); (d) increased inflammatory markers during attacks ; (e) laboratory-proved evidence of NLRP3 inflammasome activation; (f) increased serum levels of interleukin (IL)-1 and/or IL-6 during clinical manifestations; (g) effectiveness of colchicine or corticosteroid administration; (h) response to IL-1 or IL-6 inhibitors; (i) absence of cyclic neutropenia, immunodeficiency, chronic infections, inflammatory bowel diseases, autoimmune diseases or neoplasms explaining the systemic inflammatory picture.Among the objectives of the USAIDs Registry, the primary one is to enroll the largest number of patients with systemic inflammation that potentially involves all organs and tissues due to dysfunction of the innate immunity, which cannot be framed in the field of monogenic or multifactorial autoinflammatory diseases. At current, criteria for enrolling patients in the basket of USAIDs include: (a) presence of recurrent stereotyped clinical manifestations with no symptoms between episodes; (b) positive family history despite lack of genetic mutations; (c) hematological disorders associated with somatic mutations  the identification of new genetic syndromes in addition to those currently included among monogenic autoinflammatory diseases; (b) the categorization into subgroups characterized by similar signs and symptoms and response to therapies according to a_clustering method; (c) the search for new diagnostic or classification criteria; (d) the assessment of disease complications and life-threatening sequelae; (e) the identification of predisposing factors for a worse outcome and systemic amyloidosis development; (f) the detection of biomarkers and predictive factors for disease monitoring; (g) the identification of variables capable of identifying patients more likely responsive to different therapeutic approaches; (h) the description of the socioeconomic impact of these diseases in association with the epidemiologic burden in different geographic contexts; (i) the evaluation of current clinimetric tools in different diseases and contexts alongside with assessment of new clinimetric instruments specific for USAIDs patients; (j) the behavior of disease during pregnancy and post-partum period; (k) the impact of chronic disease inflammation on the cardiovascular risk. When a substantially high number of patients is enrolled, more specific and more cutting-edge studies will be proposed in the context of the AIDA network.Primary inclusion criterion is the presence of systemic inflammation driven by innate immunity  with theInfectious, neoplastic and autoimmune diseases have to be firstly excluded according to the best standard of care for any of those clinical conditions.Patients included in the USAIDs Registry should have been preliminarily assessed in order to exclude monogenic autoinflammatory diseases through a Next Generation Sequencing (NGS) approach, when available, or using other methods of genetic sequencing based on the specific patient's clinical framework. In addition, patients fulfilling clinical diagnostic criteria for Familiar Mediterranean Fever –16, haveThe first national regulatory approval of the AIDA project has been obtained in June 2019 by the Ethics Committee of Azienda Ospedaliera Universitaria Senese, Siena, Italy (Ref. N. 14951). Later, expert centers for the diagnosis, clinical management and treatment of autoinflammatory diseases have approved the project across Europe, the Middle East, North Africa and the North America and the South America and actively participate in the AIDA International Registry.This project, already registered at ClinicalTrials.gov (ID: NCT05200715), follows Declaration of Helsinki recommendations. All patients enrolled have to provide their written informed consent after having been carefully informed about the project and its aims, long-term purposes, lack of any impact on their clinical and therapeutic course. The possibility to refuse entering or withdrawing from the study at any time with no impact on the clinical management is well-cited and patients have to be informed about the personal data privacy and security in accordance with the local and/or European regulations. As far as adolescents are concerned, their parents  have to comply with the study requirements during the whole study.Both patients and Principal Investigators may withdraw their consent to use of data for statistical analyses at any time. If a patient withdraws the consent, no further data for that patient will be entered into the Registry and, if requested by the patient, all prior data will be deleted soon after her/his request to the Promoter. Patients' data are collected and stored in accordance with the EU General Data Protection Regulations (GDPR) on the processing of personal data and protection of privacy (2016/679/EU) .”Data are collected through Research Electronic Data Capture (REDCap), which is an electronic data capture tool developed at the Vanderbilt University Medical Center. It is hosted at Virginia Commonwealth University (Award Number UL1TR002649) and can be also used to develop patients' registries. The software is distributed at no costs and currently about over 5,700 Institutions in 145 Countries have already joined this online opportunity .Investigators included into the AIDA project can log in the Registry through the REDCap web-interface and later insert data on the Instruments (pages) of the Registry. None of the recruited Principal Investigators and Site Investigators are allowed to see information inserted by other Centers. The electronic data entry system of the Registry is in English.https://aidanetwork.org/en/) may be accessed by anyone who wants to learn about this Project, the Registry website (https://sitbio.med.unisi.it/redcap/redcap_v12.2.1/index.php?pid=40) is hosted separately and requires credential to meet data privacy regulations.While public website of the AIDA Network , 110 centers around the world corresponding to 290 users  have already joined the network  in 4 Continents have already joined the Project sharing their knowledge and experience about autoinflammatory diseases. At current  have been created and organized into 23 instruments (forms corresponding to different pages) to constitute the USAIDs Registry.The common data elements refer to patient's demographics, medical history, laboratory features, genetic characteristics, comorbidities, symptoms at disease onset, symptoms developed over time, cardiovascular risk, work-up examinations, pregnancies after symptoms onset, disease complications, long-term clinical outcomes, treatments administered, short- and long-term responses to treatments, management of different therapeutic strategies in terms of posology changes and drug combinations, and impact on national healthcare. Specific fields appear only if required based on patient's clinical history, thanks to a branching system of questions. Therefore, only a small part of the 3,769 fields appears to the Investigators for each patient, and the number of questions to be answered depends exclusively on the complexity of the patient's clinical history. With regard to the prospective phase of the AIDA project, longitudinal data are acquired using a specific follow-up instrument, which includes details about clinical and laboratory features and treatments update.Data elements from AIDA Registries for other autoinflammatory diseases are shared, while specific data elements for USAIDs have been added to describe the specific field of these entities.Furthermore, a specific instrument includes items possibly setting up an early form of clinical diagnostic scores and criteria; this instrument describes the reasons leading to the enrolment of the patient among USAIDs. In addition, the Eurofever scores and diagnostic/classification criteria for other multifactorial autoinflammatory diseases have been included in this instrument , 15, 19.The Instruments constituting the Registry and their reference time-points are listed in Associazione Italiana Febbri Periodiche), the National Association of People with Rheumatic and Rare Diseases  and the National Association of Rheumatic Patients . More in detail, the associations of patients were asked to provide their opinion since the very beginning of the AIDA project; patients' point of view and impressions were required before starting the project, while concerns, especially regarding the protection of personal data, were carefully answered. Patients did not desire a registry mainly focused on therapeutic aspects; in fact, we expanded the focus to all the different issues of the disease. To date patient organizations are of Italian origin only; however, other international centers have been invited to facilitate the participation of other organizations worldwide by diffusing the project among patients.In the last few years, the role of patients has progressively been increasing to become central in stimulating the research effort and quality of clinical management ; furtherIn the last years, the use of online networks has gradually increased, facilitating the spreading of knowledge and establishing worldwide research collaboration between researchers, clinicians, pharmaceutical companies, patients' advocatory groups, and patients with their families. This constitutes a great revolution in the field of rare diseases, where the small number of cases and the difficult-to-reach diagnosis are barriers to translational research and the identification of a large patient cohorts. In this context, the AIDA network and the corresponding registries dedicated to rare diseases have been created as a unifying agent in the field of autoinflammatory diseases to gather forces currently spread across the various referring centers. This highly concerns USAIDs, which correspond to a cluster of medical conditions lacking a widely shared classification and definition along with an internationally accepted protocol for management and treatment. Indeed, the concept of USAIDs itself is at an early stage of development and the knowledge on this clinical group of diseases is embryonic at current.The AIDA network has been also created to facilitate the international consultation and involvement of all medical figures dealing with the management of autoinflammatory diseases in general, and USAIDs in particular. Actually, the AIDA network already includes and enhances the communication between different specialties, such as rheumatologists, immunologists, gastroenterologists, dermatologists, ophthalmologists, internal medicine physicians, geneticists and pediatric rheumatologists for patients with childhood onset disease. This project represents a demonstration of how well a web-based worldwide collaboration can overcome the fragmentation of clinical and research experiences, expanding and improving our knowledge in the field of rare and complex diseases.The AIDA project has met the expectations for the development of an online platform dedicated to patients with autoinflammatory diseases, including those characterized by a clear but undifferentiated clinical picture. This represents the first fundamental step to overcome the limitations related to the poor number of patients included in case-series and small individual studies currently available . This isThough an International Registry enables the recruitment of a high number of patients that is required to assess response to treatment and prognostic variables among specific subgroups of patients. A long-term observational Registry could allow assessing any change in the natural history of USAIDs as a consequence of the new therapeutic approaches available at current. Furthermore, the efficacy of a therapeutic strategy might suggest the involvement of a particular molecular pathway in disease pathogenesis of specific patient subgroups.The Registry may also be a precious source of real-life data regarding the role of chronic inflammation on the cardiovascular risk as well as disease behavior during pregnancy and postpartum. The prospective phase will also provide data about the socioeconomic impact of these diseases and benefits that national healthcare could obtain from different therapeutic strategies.As a whole, achieving all the goals aspired by this Registry will improve the knowledge about this peculiar basket of inflammatory disorders and facilitate their early diagnosis, with a consequent decrease of long-term or life-threatening complications, such as amyloidosis, and a positive impact on quality of life and life expectations . NoteworThe AIDA Registry for USAIDs patients has the usual shortcomings typically present in observational studies, especially selection biases deriving from the number of missing data and any non-consecutive enrolment of patients. In addition, entering data into the Registry requires time and attention, especially when patient's medical history is particularly complex, as for patients with long-term disease course, multiple treatment approaches attempted over time and many posology changes to report. Nevertheless, entering retrospective data requires 2–3 h, while completing the form at follow-up visits takes a maximum of 10–15 min. Despite its limitations, this Registry has the potential, given also its geographical extension, to eventually achieve all the objectives thus shedding light on a quite unknown field of autoinflammation.Of note, a branch of the AIDA project defined as “AIDA for patients” is under development. “AIDA for patients” is an online tool based on the REDCap technology primarily aim at involving patients in the collection of the data, especially regarding the impact of the disease on the quality of life and on socio-economic aspects and the current status of the disease with specifically built patients reporting outcome (PROs). Moreover, “AIDA for patients” was born to enhance patients' participation in the decision-making process when establishing the lines of research and the strategies to follow, in order to seek the growth of the AIDA project.In conclusion, the AIDA International Registry for USAIDs patients has been developed and activated to facilitate the collection of standardized data and enable international multicentre collaborative research. Data sharing, implementation, and optimisation of research about autoinflammatory diseases, along with international consultation, and dissemination of knowledge represent pivotal goals that may be easier to achieve via this international effort offered by the AIDA network.The studies involving human participants were reviewed and approved by Azienda Ospedaliera Universitaria Senese, Siena, Italy (Ref. No. 14951). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.FD wrote the first draft of the manuscript. AV conceived and designed the study and revised the draft of the manuscript. DR revised the draft of the manuscript. LC conceived and designed the study and accounts for AIDA Registries Coordinator. AB is the bioengineer involved in the technical management of the platform and registries. GL, PR, FCi, GE, MC, EW-S, MM, BO, PS, AT, SA-M, ED, EA, FL, JS, SC, ID, DI, IM, KJ-R, RJ, KL, CG, AAb, PC, GRo, and AAl were involved in data recruitment in the Registry dedicated to patients with USAIDs. DY, MT, GRa, FR, FCa, MK, MF, VC, MS, RP, VB, SGe, SGu, TG, VG, FI, HG, IA, RK, DA-I, BF, and CF included in the authorship as investigators from the top contributor centers for any of the other seven AIDA Registries. Authorship has been established based on the number of data recruited in the AIDA Registries on February 7th, 2022. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Autism spectrum disorder (ASD) is considered a complicated neurodevelopment disorder with rising prevalence globally. ASD is characterized by a series of events including varying degrees of defects in communication, learning, and social interaction which is accompanied by stereotypical behavioral patterns. Despite extensive research, the current diagnosis for ASD is complex and almost solely based on the behavioral assessments of the suspected individuals. The multifactorial etiopathology of this disease along with the diversity of symptoms among different individuals adds to the current intricacies for accurate prognosis of ASD. Hence, there exists a dire need for biologically relevant biomarkers for an early diagnosis and for tracking the efficacy of therapeutic interventions. Until recently, among various biofluids, saliva has gained increasing interest for biomarker identification, the advantages include the non-invasive nature and ease of sample handling. This mini-review aims to provide a succinct summary of recent literature on saliva-based diagnostic modalities for ASD, examine various studies that highlight the potential use of proteomic and/or RNA-based biomarkers. Finally, some conclusive perspectives of using the salivary system for ASD mechanistic details and diagnosis are also discussed. Autism spectrum disorder (ASD), generally referred to as autism, is a widespread, genetically determined, and diverse neurological illness having fundamental mental abnormalities along with autism’s habits, abilities, and problems . The intBiomarkers represent premonitory signals that allow either early identification or the determination of the desired result during a relatively primordial phase of illness. The scientific evidence for the diagnosis comes from blood, urine, and cerebral spinal fluid. Biomarkers are employed as an indication of a physiological element that indicates a preclinical symptom, degree of the ailment, or counterfeit symptom of the illness in certain situations . One sucThe pathogenesis of ASD is multifaceted, with genetic, environmental, parent aging, and medical issue all playing a role. Demonstrating a validated marker biomarker is difficult because the etiology of ASD is still mostly unclear. Researchers around the globe are seeking these indicators mostly in the bloodstream, amniotic fluid, or saliva . Saliva via biases inside the composition of the bacterial cavity induced by favoring certain foods whilst avoiding others  are single-stranded RNA sequences that interact with the expression of genes by binding to target mRNA and controlling protein translation. In ASD, a variety of miRNA expression patterns have been discovered, the majority of which are associated with central nervous formation and functioning . A few oTannerella quantity was the most important of the identified miRNA/bacteria correlations (In earlier investigations, differential expression patterns of miRNAs in patients with ASD have been discovered in research, suggesting that miRNAs might be used as biomarkers for ASD screenings. Brain tissue, blood, and cultured peripheral lymphoblast were utilized to identify miRNA in ASD patients . Howeverelations . The preelations . These felations . As a reThe benefits of salivary fluid for autism investigation as well as its possible therapeutic use cannot be overstated. Numerous elements must be addressed, or a plan for saliva studies and salivary utilization should be devised. Generally, the technological and operational characteristics of the studied marker should be considered. To limit the possible diversity in the realm of neurodevelopmental disorders, particularly ASD, homogeneous sets outlining specific categories, solely focusing on age group and intestinal and dietary history, would be proposed. The use of a salivary system inside the diagnosing procedure is contingent on the discovery of potential ASD biomarkers. The finding of these biomarkers, nevertheless, was not the sole purpose of the salivary study; it also necessitates broad investigation on several biological materials. Thereafter, saliva can be used as a non-invasive additional method with all of its benefits. The recent discovery of efficient isolation protocols from salivary exosomes has aid in the hope of an improved and timely diagnosis of neurodegenerative diseases , similarVS and FN conceptualized the work. VS and SC wrote the manuscript. SK, VS, and FN reviewed the manuscript. All authors contributed to the article and approved the submitted version.


Frequent transfer of rural labor to cities in developing countries significantly impacts agricultural production. However, whether off-farm employment can promote chemical fertilizer (CF) reduction is still controversial. This study incorporates business scale (BS) and fragmentation degree of arable land (FDAL) into the theoretical analysis framework, shedding light on regulating effects of arable land resource endowment in the process of off-farm employment which influences CF application under different BS and FDAL scenarios. It also empirically tests the theoretical framework by employing the survey data of 318 rice farmers in Jilin Province. The results indicate that: (1) off-farm employment, in general, promotes the adoption behavior of machinery by farmers, and mechanical tillage can significantly reduce the intensity of CF application. (2) If farmers have large BS and non-dispersed farmland parcels, contiguous cultivation will meet the scale threshold for mechanical farming and obtain economies of scale to reduce the intensity of CF application. (3) If farmers have small BS and dispersed farmland parcels, the scale threshold of mechanical farming cannot be met. In order to stabilize agricultural production, farmers will increase the intensity of CF application. According to the empirical results, we put forward some suggestions. By 2050, the global population will exceed 9 billion, resulting in greater demand for food, cultivated land, water, and other resources. At the same time, it is faced with enormous challenges such as environmental degradation, food safety, and climate change, which are difficult to maintain food’s quantity and quality safety. "Food insecurity" has become one of the most critical and complicated problems in Asia and even the world , 2. DiffFood system includes agricultural development, environmental sustainability, food supply, food consumption and nutrition access. Therefore, climate change, smallholder sustainability, labor transfer, environmental degradation and non-genetic diseases and their global solutions are all related to the food system , 6. In dWith the acceleration of global urbanization and industrialization, most developing countries are experiencing two phenomena: population urbanization and rural hollowing out –14. ChinSome scholars have focused on the impact of off-farm employment on agricultural chemicals investment. As two basic factors in agricultural production, off-farm employment will affect CF application . TherefoHowever, other scholars believe that whether off-farm employment can promote CF reduction depends on the situation. Mathenge and Smale  estimateAll of the above studies suggest that whether off-farm employment affects CF application may be influenced by differences in the duration of off-farm industry, different crops grown, and differences in sources of income earned from engaging in different off-farm activities, resulting in different outcomes. This paper holds that the reason for the disagreement is that existing studies have mainly followed the single analysis logic of "off-farm employment—factor substitution—CF application", which leads to the simplification of factor allocation caused by off-farm employment, and thus ignores the differences in factor allocation and the potential of CF reduction under the conditions of different arable land resource endowment. The optimal factor selection behavior differs under different arable land resource endowment scenarios. A larger BS and lower FDAL cannot overcome the disadvantages of small plot size, therefore farmers usually use CF instead of labor, which increases the intensity of CF application. On the contrary, the economies of scale generated by contiguous cultivation induce long-term investment behavior of farmers, resulting in CF reduction effects. It can be inferred that the reallocation of production factors due to off-farm employment in different arable land resource endowment scenarios may lead to an obvious contrast in CF application.Therefore, the contribution of this paper mainly has two points: firstly, we discuss the regulatory role of arable land resource endowment in the process of off-farm employment affecting CF application. Although studies have explored the regional heterogeneity of the impact of off-farm employment on carbon fiber application intensity under different terrain conditions, they have not revealed the reasons for the differences in farmers’ fertilizer application behavior under the same geographical environment. In fact, as the main body of CF application, farmers with different arable land resource endowments have different fertilization behaviors. However, the existing research has not been paid attention to.Secondly, we further analyzed the intermediary role of mechanical farming in the impact of off-farm employment on CF application and revealed the internal mechanism of off-farm employment leading to the difference in CF application.This paper reveals the relationship among off-farm employment, factor allocation, and CF reduction under different arable land resource endowment scenarios, so as to breaking through the one-way thinking that off-farm employment affects CF input and explaining the internal mechanism of off-farm employment that leads to different allocations of production factors that affect CF application. In general, this paper is divided into seven parts. Following the introduction, the second part is literature review. the third part proposes a research hypothesis based on the theoretical analysis, the fourth part introduces the research area and data sources, and describes the econometric methods, the fifth part analyzes the empirical results, the sixth part provides a discussion, the seventh part draws a conclusion and policy implication.Off-farm employment plays an important role in the livelihood of most households in developing countries. Family members engaged in agricultural production are not constrained by any time or place and earn off-farm remuneration by participating in productive activities other than agricultural production, thereby alleviating household mobility constraints. At present, many studies have made important contributions to understanding the reasons –53 and rThis paper argues that off-farm employment brings about both a household labor shortage and an easing of liquidity constraints. In the context of the increasing prevalence of off-farm employment, in order to maintain agricultural production as much as possible, farm households tend to invest their off-farm income in agriculture to compensate for the impact of labor shortage. Thus, how farmers choose factors to replace labor shortage is the focus of this paper. In contrast, off-farm work is able to transform the labor factors with which agricultural production competes into other capital factors and use them in agricultural production as a way to mitigate the labor loss effect and maintain household livelihoods.The literature on the role and impact of off-farm employment is divided into two main parts. The first part is the impact of off-farm employment on agricultural production. For example, Lisa Pfeiffer et al.  studied The second part of the literature focuses on the impact of off-farm employment on changes in the allocation of factors of production in agriculture. Off-farm employment relaxes the liquidity constraints on household agricultural production and seeks to increase the purchase of other production alternatives to household labor, leading to an increase in other factors of agricultural production inputs . WanglinThe new economics of labor mobility (NELM) , 67 theoRural land in China is collectively owned and is characterized by two main features: small scale and high fragmentation. As a result, clarifying land property rights, improving the land exchange market, and promoting the centralized circulation of farmland contract management rights are considered critical measures if agricultural production is aiming to gain economies of scale , 71. In The existence of factor allocation effect in agriculture leads to a significant substitution relationship between material factor input and labor force. The increase of off-farm employment leads to a decline in the quality of labor force and an increase in the relative price of agricultural labor force. To avoid losses in agricultural output and efficiency, farmers adjust the structure of factor inputs in response to changes in the relative price of factors. On the one hand, the productive service outsourcing organizations have induced a strong demand for machinery services to replace labor force, on the other hand, the low price of CF promotes the substitution of CF factors for labor factors. Limited by the high investment threshold and asset specificity of agricultural machinery and reduction technologies, farmers generally have little incentive to invest in the short term . OutsourThe land transfer caused by off-farm employment does not directly act on CF inputs, but provides the basis and prerequisite for the adjustment of the factor structure. When the remittance effect caused by off-farm employment is greater than the labor shortage effect, that is, the income from engaging in off-farm activities is larger than the loss from abandoning agricultural production, farmers tend to retain part-time status and seek to reallocate production factors to stabilize agricultural production. In discerning with the relationship between off-farm employment and CF inputs under different arable land resource endowments, two main types of scenarios can be summarized. First, the BS is small and the FDAL is high. Smaller plot scales cannot reach the scale threshold for mechanical intervention, so CF is a better substitute for labor shortages than machinery, and farmers tend to apply more CF. Second, the BS is large and the FDAL is low. With the continuous increase of labor and leisure costs, the substitution of CF for labor shortage will lead to higher production costs under a large plot scale. Meanwhile, the larger plot scale provides conditions for continuous planting and market capacity for socialized service outsourcing. Therefore, machinery is easy to intervene in agricultural production and produces the scale effect of CF reduction. At this time, farmers tend to get involved in the division of labor system by purchasing contiguous mechanical farming services. CF reduction depends not only on Farmers’ awareness of green production but also on the adoption of CF application machinery with the progress of agricultural reduction technology. Not only do the development of agricultural machinery and the progress of CF application technology improve labor productivity, but also reduce the intensity of CF application. The CF reduction effect of mechanical fertilization is mainly reflected in two aspects: The first is the round-tripping investment effect of reduction machinery. Farmers can effectively overcome the high investment threshold and strong asset specificity of mechanical equipment by purchasing mechanized deep fertilization and controlled release service. The second is the roundabout introduction effect of reduction technologies. By purchasing technology-intensive products and services, farmers apply fertilizer reduction technology directly to agricultural production , which lH1: Off-farm employment in general can reduce the intensity of CF application through mechanical substitution.H2: If farmers have a small BS and dispersed farmland parcels, off-farm employment will increase the intensity of CF application.H3: If farmers have a large BS and non-dispersed farmland parcels, Off-farm employment will increase the machinery use behavior of farmers, which in turn reduces the intensity of CF application.The study area of this paper is Jilin Province, which is located in the middle of Northeast China and is one of the three cold black soil belts in the world . There aThe study applied a multi-stage sampling technique to select respondents among rice farmers. Interviews were conducted in 11 county-level cities, counties and districts and 22 villages and towns under six prefecture-level cities in Jilin Province, namely, Baicheng City (Taonan City), Changchun City , Jilin City , Tonghua City , Yanbian City , and Songyuan City  from September to November 2020  for a total of 331 questionnaire-based interviews with rice farmers . These aDuring the field investigation, all ethical issues were properly addressed. First, The Jilin Agricultural University Graduate Research Ethics Committee in China authorized this investigation. Secondly, in the preparation stage of the interview, we first obtained the consent authorization from the Zone Executive Officer (ZEO). On this basis, the exact information in the consent form was communicated to each target respondent by phone or SMS, and their verbal consent was obtained. Subsequently, during the field research, we invited the target respondents to fill out an informed consent form to certify their consent to the survey. All interviewers were informed that they could withdraw from the survey activities at any time. Our research activities do not involve minors. In addition, the research group is composed of graduate students, teachers and other researchers, and does not belong to government organizations and foreign-related surveys, so it does not need the approval of statistical departments.How off-farm employment, BS, and FDAL affect CF application is the focus of this paper, which explores the differences in the intensity of CF application affected by off-farm employment under different arable resource endowment scenarios. This paper draws on the modeling approach of Liang et al.  to constThe variables in Formulas , 2) and and2) ani represents the intensity of CF application by farm households, measured by the average application amount of CF per unit of farmland (ha) by farm household i. Maci represents the use of machinery by farm households, measured by whether farmer i used machinery in fertilization.The predicted variable. Cfertilizeri stands for off-farm employment, which is expressed by the proportion of off-farm income of farmer i in total household income. Bscalei represents the BS of farmer i, which is reflected by the total scale of rice planting. Nplotsi represents the FDAL of farmer i, which is reflected by the number of farmland plots.The explanatory variable. Naemployi is a control variable that includes personal characteristics , household characteristics , production and business characteristics , and external environment characteristics  of agricultural decision-makers. During the investigation, we found that three cooperation modes may generate in the mode of production organization and operation, like "family self-employment", "Cooperation between enterprises and farmers" and "Cooperation between cooperatives and farmers". Among them, "family self-employment" is manifested as self-management of agricultural production by farmers, self-production, and self-sold. "Cooperation between enterprises and farmers" is an economic contract signed between enterprises and farmers to carry out integrated management, but farmers are more restricted by enterprises and have unequal market status. Under this model, farmers transfer land to enterprises. Receiving regular salary payments from the enterprise that takes planting income, farmers are not liable for operating costs. Unlike the first two, "Cooperation between cooperatives and farmers" is voluntarily united by the same kind of agricultural products operators and sets up a democratic management of mutual economic organizations. Having no constraints and greater rights, farmers take the production income in the mode of cooperative unified management and production and operation.The control variables. Xi is a dummy variable to control for regional fixed effects such as climate, pests and diseases; εi is the stochastic disturbance team; β0 is the intercept term; and β1、β2、β3、β4、β4j、β5j are the parameters to be estimated. In this study, the predicted variable Cfertilizeri has to be processed with logarithm to weaken the potential heteroscedasticity of the model [The dummy variables. Dhe model .The formula containing the interaction terms is constructed as follows:i×Bscalei and Naemployi×Nplotsi respectively represent the interaction term between off-farm employment and BS, and the interaction term between off-farm employment and FDAL. In this paper, the interaction terms are centralized to overcome potential multicollinearity.NaemployEndogeneity is a common problem in studying farmers’ behavioral decisions and the factor that influences them . It is mIn the literature related to the study of urban and rural labor transfer employment, migration networks are often used as an instrumental variable to examine individual or household participation in out-of-home employment , 82. RurThe meanings of the variables and a descriptive statistical analysis are shown in Regression (2) and (3) show that there was a significant negative effect of BS on CF application intensity both before and after controlling for the FDAL. This suggests that the expansion of rice BS reduces the intensity of CF application. When the FDAL is certain, the larger the scale of business, the more likely it is to meet the scale effect threshold of rice production, which makes the reduction of labor factors increase the marginal output rate of mechanical input and reduce the CF application intensity. On the contrary, the smaller the BS, the more the farmers tend to apply more CF to replace the labor factor and increase the intensity of CF application.Regression (3) and (4) indicate that the FDAL positively affects the intensity of CF application before and after controlling for the BS. This shows that the deepening of the FDAL will lead to an increase in the application of CF. When farmers business at a certain scale, a higher FDAL means that farmers may not be able to capture the scale effect of rice planting, which in turn induces the substitution effect of reduced labor factors to take effect. Therefore, when the rural labor factor decreases, the diseconomy of plot scale will directly lead to farmers’ preference to apply CF instead of labor, thus increasing the intensity of CF application. While the lower the FDAL, it may meet the scale effect threshold and lead to farmers’ demand for mechanical farming, and reduce the intensity of CF application. Regression (5) and (6) represent the regression results of machinery use affecting CF application intensity after controlling the BS and FDAL, respectively. The results indicate that arable land resource endowment can influence CF application intensity by affecting machinery use.The estimation results of Regression (8) and (10) indicate that the expansion of BS can promote the use of agricultural machinery before and after controlling the FDAL. This suggests that the larger BS can reach the scale threshold for mechanical farming and enhance the incentive of farmers to use machinery. Similarly, the estimation results of Regression (9) and (10) show that the deepening of FDAL has a significant negative effect on the use of farm machinery under the condition of controlled and uncontrolled BS, which indicates that the reduction of arable land fragmentation level favors farmers’ machinery selection behavior.In summary, off-farm employment, in general, promotes the machinery use behavior of farmers, thus reducing the intensity of CF application. Hypothesis H1 is confirmed.In this paper, a two-stage regression of the instrumental variables was conducted by using the 2SLS method and the estimation results for the instrumental variables are reported in The results of the second stage estimation show that the effects of off-farm employment, scale of business, FDAL, and their corresponding interaction terms on CF application intensity are in the same direction as the benchmark model and generally consistent with the benchmark model at the significance level.The survey population includes 69 farm households that do not participate in off-farm employment. To confirm the robustness of the benchmark model results, the reduced sample size method is used to conduct robustness tests by excluding the sample data that do not participate in off-farm employment and selecting the remaining sub-samples as the new total sample. Compared with the existing studies that mainly focus on the impact of off-farm employment on farmers’ overall CF application behavior and the difference of CF application intensity between regions, we found that off-farm employment has different effects on CF application intensity under different arable land resource endowments. Firstly, off-farm employment in general promotes the adoption of agricultural machinery, which achieves the economies of scale in rice planting and leads to a CF reduction effect. Affected by the small scale and scattered farmland plot, Chinese agricultural production modes are mainly represented by family management with small farmers as the main body. In recent years, the Chinese government has introduced corresponding measures to encourage farmers to transfer land. However, due to the limitation of arable land topography, land contract management right transfer is mainly concentrated in the north. Jilin Province, with a vast territory, is a main grain-producing area in China. Land transfer further expands the land scale, providing conditions for the popularization of machinery and the development of socialized service organizations. When the farmland plot scale is large, mechanical farming is a tool to improve the efficiency of resource utilization and achieve economies of scale. At this time, machinery is the optimal choice for factor reallocation by off-farm employment under the conditions of the large-scale farmland plot. Currently, although the fact that a large amount of small-scale and scattered farmland plot still exist, the arable land in Jilin Province shows a centralized trend. The improvement in farmland plot scale reduces the threshold for mechanical intervention, which provides an indirect pathway for off-farm employment to reduce the intensity of CF application.Secondly, the difference in farm resource endowments regulates the relationship between off-farm employment and CF application. To compensate for the labor shortage effect caused by off-farm employment and stabilizing agricultural production, farmers tend to apply more CF on arable land with small BS and high fragmentation degree, which leads to the increase of CF application intensity That is because the smaller plot scale cannot meet the scale threshold for mechanical farming resulting in the inability of farmers to reconfigure the ideal production factors (machinery). Therefore, CF becomes the optimal choice for the allocation of production factor. Although the marginal return of successive increases in CF factor inputs is decreasing, the marginal return is positive. Meanwhile, the low price of CF factors makes the farmer increase CF input until the marginal return is zero.Thirdly, off-farm employment provides opportunities for machinery selection, which significantly promotes farmers’ machinery adoption behavior as well as indirectly reduces CF application intensity in the reason of arable land with large BS and low fragmentation degree. As mentioned earlier, when the arable land endowment resource meets the threshold for mechanical farming, the optimal substitution factor for farmers is machinery. Farmers can improve resource utilization efficiency and obtain economies of scale through the round-tripping effect of mechanical farming and reduction technology, so as to reduce CF application intensity. This suggests that, unlike the case of direct substitution of CF, which is affected by the regulation effect, off-farm employment does not directly affect CF application in the large farmland scale scenario, but indirectly reduces CF application intensity by directly influencing the adoption of agricultural machinery.This study enhances the current understanding of the role of off-farm employment in influencing CF application. Firstly, we discussed the differences in the role of off-farm employment on fertilizer application intensity in different arable land resource endowments. Existing micro studies mainly focus on the analysis of off-farm employment affecting the farmers’ overall CF application behavior from a single perspective –86. The In addition to contributing to theory, our study provides some managerial insights for government administrators to seek to promote the reduction and efficiency of CF under the background of off-farm employment. Firstly, in recent years, the farmland in northeastern China has been gradually concentrated on specialized households, which not only improves the economies of scale of rice planting, but also has a positive impact on reducing CF application intensity. However, the arable land has not changed the essence of small-scale and fragmentation. Therefore, government administrators should actively guide farmers to participate in land replacement and integration in order to expand the BS as well as reduce the FDAL through continuous land transfer especially in flat areas, to obtain scale economy of CF reduction. Secondly, we point out that the use of machinery plays an important role in reducing the intensity of CF application. Limited by the specificity of agricultural machinery assets and high investment threshold, farmers generally have insufficient long-term investment motivation, but the contiguous planting of land transfer provides market capacity for agricultural machinery socialized service. As a result, agricultural machinery socialized service organizations come into being. However, the development of socialized service organizations depends on the contiguous planting of large-scale farmland plots and the low-cost purchase price of agricultural machinery. Therefore, the government should formulate different subsidy policies for agricultural machinery procurement according to the types and used times of agricultural machinery to improve the motivation of farmers and socialized service organizations. By increasing the round-tripping investment effect of farmers, we can obtain the economies of scale of mechanical farming and reduce the intensity of CF application. Thirdly, for small plots of arable land that cannot be transferred due to topographic restrictions, we should introduce social service organizations with accurate reduction technology, cultivate crops with high economic value and low CF demand and reduce the intensity of CF application through intensive cultivation.Off-farm employment includes full-time off-farm employment and part-time off-farm employment. Part-time off-farm employment may be "off-farm work in leisure time and farm in busy time", which will not lead to the reduction of labor factors in agricultural production while causing the return of remittance effect. Due to the strong mobility of farm households involved in off-farm employment, it is difficult for us to obtain comprehensive information on part-time off-farm employment of farm households other than to extract data from the survey questionnaire. The estimation results of full-time off-farm employment and part-time off-farm employment will differ due to differences in household livelihood strategies. Therefore, an in-depth analysis is necessary to further investigate differences between the two different off-farm employment patterns affecting CF application.In addition, this study uses cross-sectional data, which may lead to biased estimation results by ignoring differences in cropland quality across regions. Therefore, it is necessary to conduct a fixed-point tracking investigation to obtain panel data in the future to overcome the bias of model results.Off-farm employment not only improves the livelihood of families in developing countries but also has a significant impact on agricultural production. In this paper, we analyzed the impact of off-farm employment on the mechanical use and CF input intensity of rice growers in Jilin Province, China. Descriptive statistical analysis and econometric results show that off-farm employment affects CF application intensity through two mechanisms. That is, off-farm employment affects CF application with situational dependence. Based on the viewpoint of factor allocation and NELM theory, this study proved the difference in the regulating effect of arable land resource endowment in the process of off-farm employment affecting CF application intensity. The results of the study show that the arable land resource endowment is mainly reflected in two aspects: the BS and the FDAL. There are differences in factor substitution behavior and CF reduction potential expressed by different arable land resource endowments. In general, off-farm employment increases farmers’ demand for mechanical farming, and the use of machinery is an effective means of reducing the intensity of CF application. If farmers have a small BS and dispersed farmland parcels, it can’t meet the scale economy of rice planting and even hinder the substitution of machinery for labor force. Therefore, the reduction effect of CF brought by off-farm employment may be limited and even increase the intensity of CF application. If farmers have a large BS and non-dispersed farmland parcels, the arable land endowment meets the scale threshold for mechanical farming to obtain the CF reduction effect of economies of scale. Therefore, off-farm employment will significantly contribute to CF reduction. Based on theoretical analysis, this study makes an empirical test conducted by using the sample data of 318 rice growers in Jilin Province. After overcoming the potential endogeneity in the econometric model, the robustness test based on sub samples also supports the above conclusions.Scholars generally believe that off-farm employment is an important measure to improve family livelihood, and it will also have a significant impact on agricultural production. However, there is still controversy about how off-farm employment affects CF application. They argue that off-farm employment positively or negatively influences CF application or is influenced by differences in the timing of off-farm employment, type of off-farm employment, and crop species, resulting in differences in CF application. However, this paper holds that the fundamental reason why off-farm employment affects the application of CF lies in the difference in arable land resource endowment of farmers. After incorporating arable land resource endowment into the theoretical analysis framework and empirically analyzing the moderating role of arable land resource endowment, it is found that off-farm employment affects CF application through two mechanisms and produces different results. The findings of this study are not only applicable to understanding the impact of off-farm employment on machinery adoption and CF application, but can also enlighten how to influence inputs of other factors such as pesticides, herbicides, and water resources. At present, the confirmation of farmland property rights in China can accelerate the transfer and merger of land to a certain extent, thus contributing to the improvement of production efficiency and the reduction of CF application intensity. For the government, our findings provide a basis for decision-making on how to promote land management and reduce the intensity of CF application. Under the background of increasing off-farm employment, it affirmed the implementation direction of farmland confirmation and farmland transfer policy and the effectiveness of cultivating agricultural social service organizations. For farmers, when off-farm employment leads to labor shortage, increasing the BS and reducing the number of plots in the form of land transfer can reach the scale threshold of mechanical farming, and through the purchase of agricultural production services can obtain the CF reduction effect of large-scale operation and reduce the cost of CF inputs. Our research also shows that the scale of land can provide conditions for the use of modern equipment, reduce the input of agricultural chemicals, improve soil quality and agricultural non-point source pollution. Finally, it provides general policy suggestions for solving the agricultural non-point source pollution problem in developing countries with the background of the continuous outflow of the rural labor force.Given the importance of off-farm employment in improving household livelihoods and increasing agricultural factor inputs, and the difference of the influence of cultivated land resource endowment on the intensity of CF application. The policy formulation should focus on reducing the restrictive factors in the process of land circulation concentration, especially in flat plain areas. For scattered plots that are severely restricted by topography, green organic agriculture should be encouraged to improve the ecological added value of products. In addition, due to the specificity of agricultural machinery assets and the high silent cost, the government should strengthen the support and cultivation of agricultural production service outsourcing organizations, give certain subsidies to agricultural machinery buyers, increase the scale effect of agricultural production and reduce the intensity of CF application through scale.S1 File(DO)Click here for additional data file.S1 Data(DTA)Click here for additional data file.