The dire need for the assessment of human and environmental endangerments of nanoparticulate material has motivated the formulation of novel scientific tools and techniques to detect, quantify, and characterize these nanomaterials. Several of these paradigms possess enormous possibilities for applications in many of the realms of nanotoxicology. Furthermore, in a large number of cases, the limited capabilities to assess the environmental and human toxicological outcomes of customized and tailored multifunctional nanoparticles used for drug delivery have hindered their full exploitation in preclinical and clinical settings. With the ever-compounded availability of nanoparticulate materials in commercialized settings, an ever-arising popular debate has been egressing on whether the social, human, and environmental costs associated with the risks of nanomaterials outweigh their profits. Here we briefly review the various health, pharmaceutical, and regulatory aspects of nanotoxicology of engineered multifunctional nanoparticles in vitro and in vivo. Several aspects and issues encountered during the safety and toxicity assessments of these drug-delivery nanocarriers have also been summarized. Furthermore, recent trends implicated in the nanotoxicological evaluations of nanoparticulate matter in vitro and in vivo have also been discussed. Due to the absence of robust and rigid regulatory guidelines, researchers currently frequently encounter a larger number of challenges in the toxicology assessment of nanocarriers, which have also been briefly discussed here. Nanotoxicology has an appreciable and significant part in the clinical translational development as well as commercialization potential of nanocarriers; hence these aspects have also been touched upon. Finally, a brief overview has been provided regarding some of the nanocarrier-based medicines that are currently undergoing clinical trials, and some of those which have recently been commercialized and are available for patients. It is expected that this review will instigate an appreciable interest in the research community working in the arena of pharmaceutical drug development and nanoformulation-based drug delivery. The urgent necessity for the assessment of human as well as environmental hazards of nanoscaled material has motivated the evolution of newer measurement techniques for detecting, quantifying, and characterizing the risks associated with these nanomaterials. Many of these methodologies have enormous capabilities to be applied in diverse arenas of nanotoxicology. In several other events, the restricted dialogues among environmental specialists and human toxicity analysts have confined the fullest utilization of this resource. Vast spectrums of progress have been made in developing the methodologies for nanoparticle analyses in the context of the applications of these methodologies for human or environmental toxicological paradigms ,2. MultiIn these recent times, various types of nanoparticle-based platforms have been formulated and exhaustively utilized in a wide spectrum of manufacturing procedures for healthcare-based and other products, viz. drugs, cosmetics, insulations, paints, filters, semiconductor-based devices, cosmetics, and biomedical devices . In the Particularly, silver-based nanoparticulate systems exhibit enhanced antimicrobial activities and are among the majorly employed metal-driven nanoformulations that have been detected in various consumer-based goods, viz. cosmetic products, clothes, household items, biomedical tools, and food packages. Because of this preponderance in these consumer goods, silver-based nanoformulations can also be released into environmental surroundings and pose a menace with regard to human health and well-being. In fact, silver nanoparticles are reported to exhibit late toxicities in various beings . SeveralVarious types of nanoparticles could well be distinguished from each other by their distinct physicochemical characteristic features. One of the obvious methods for the categorization of nanoparticles can also be based on their biocompatibility and toxicity, which can impart a primary component to a nanoparticle’s identity. However, various nanoparticles are composed of many components including the sore and shell made up of different types of materials, the primary contacting portion with the nanoparticle’s external biological environment is often done not by the inner core but by the nanoparticles’ outer coating materials. Therefore, nanoparticles are mostly well-defined in the context of the outer surface of the nanoparticle, which is often composed of a distinct material from that in the core position . The bioZhang et al. formulated the nucleic acid functionalized gold nanoparticles (DNA-modified Au nanoparticles for investigating the transportation purpose) and checked the biocompatibility with the help of analyzing the RT-PCR of the messenger RNA transcript levels . LikewisSchooneveld and coworkers in their report suggest that hybrid organic and inorganic nanoparticulate carriers composed of polymers and some of the inorganic materials and natural biomolecules can extend a concourse of benefits in nanophotonic paradigms, nanoformulations mediated catalysis, and nanomedicine-related fields. When employed as multifunctional carriers for biomedical approaches, higher biocompatibility has been reported to be achieved, and possibilities of the integration of various (contrast-generating) materials in the same formulation can also further allow their detectability in the multimodal paradigms . SchooneTherefore, there have been very valid and genuine concerns regarding the nanoparticles’ toxicities, and not much has been known of as how nanosized particles and entities can act in human patients or healthy volunteers. The hydrodynamic diameter or particle size of the nanoparticles, as well as their surface or interfacial characteristics, could provide them access to such localizations which may sometimes not be accessible to large-sized nanocarriers. Surface characteristic features can further impact the biodistribution via various phenomena, viz. non-specific adsorption of various protein molecules from the serum, their removal by the macrophage cells, and bringing about the localized perturbations in biological barriers that can otherwise restrict their accession . A recenFurther, anticancer nanomedicines impart improvised therapeutic characteristics as well as diagnostic platforms to overcome multidrug resistance in tumor cells. Additionally, these anticancer nanomedicines can also surpass the disadvantages of present-day therapeutic modalities which include poor aqueous solubilities of hydrophobic pharmacological compounds, nanoparticle agglomeration, and culminate into improved biocompatibility as well as biodistribution ,34,35,36Likewise, nanoemulsions have also been used as excellent drug-delivery nanovehicles and other pharmaceutical applications due to their optimum size, shape, and biocompatibility profiles ,49. One The exponential increase in the usage and production of nanoparticles (NP) raised perilous effects on human health as well as the environment. They are highly vulnerable to biomedical applications and several studies are going on for nanotoxicity assessment . Among aAs per previously published articles, it was summarized that elevated reactive oxygen and nitrogen species production, inflammation, and cytokine overproduction results in apoptosis, autophagy, and necrosis cell death mechanisms . These rThe factual advancement of research and explorations can be demonstrated by its clinical translation capability and this backdrop could well be proved in recent times by the handiness and accessibility of almost 50 types of nanoparticulate formulations in the cosmopolitan markets which flourished in markets, raising around USD 140 billion from 2015–2016 from almost USD 50 billion from 2008–2009, where anticancer medicinal approaches have contributed the major portion. Somewhere around 2019, human serum albumin nanoparticles encapsulating the paclitaxel with the brand name Abraxane reached an estimation of around USD 970 million in overall revenue ,80. OnceThe nanoparticles may induce dose-dependent as well as time-dependent cytotoxicity in various cell lines in which their in vitro safety and toxicity are being assessed. This cytotoxicity is assessed in terms of the decrease in cell viability by employing various cytotoxicity assays, the chief among those being the MTT cell viability assay. Recent epidemiologic explorations on nanomedicinal formulations and their carcinogenicities have so far been indeterminate. Databases required for assessing the carcinogenic potential of nanoparticles have similarly been quite deficient. Evaluations of carcinogenicities and their significance with respect to humans have often been unsettled in terms of quantitative and qualitative outcomes. In qualitative contexts, smaller sizes, absorptive capacities, capabilities, times, and durations of retention; their biodistribution overcoming most of the biological hindrances; and subcellular or molecular-level interactions have all been found of having major influences . CompareIn the context of various nanoformulations, various characteristic features such as structures, sizes (distributions), shapes, and specific surface characteristics (viz. coatings) have been preferred for pharmaceutical applications and are non-toxic. Regarding the structures, nanoparticles either made up of organic polymers or metallic inorganic nanoparticles coated with organic, natural, or nature-mimicking polymers have often been reported to be non-toxic and highly biocompatible for pharmaceutical applications ,102,103.It has generally been observed that nanoparticles lesser than 300 nm have been found to be the least toxic and are biocompatible with most of the cell lines as well as blood cells. Furthermore, the toxicity of nanoparticles is dependent upon their size but majorly depends upon the dose in which nanoparticles have been administered and cells or tissues have been exposed to the enhanced concentrations of nanoparticles ,104,105.With respect to the shape of nanoparticles, various shapes have been formulated and applied for pharmaceutical applications and nanoparticle shapes include nanospheres, spherical polymeric micelles, hollow polymeric nanovesicles, nanoworms, nanorods, and nanostars. Hinde and coworkers have demonstrated that it is actually the shape-derived aspect ratio of nanoparticles, rather than the nanoparticles which decides the toxicological features of nanoparticles. The nanoparticle shapes possessing higher aspect ratios exhibit higher toxicities in comparison to the nanoparticle shapes with lower aspect ratios .In the context of the surface coating of nanoparticles, surface-coating procedures using polymers, lipid bilayers, and environmentally responsive molecular coats have been formulated for the loading, retaining, protecting, and releasing loaded therapeutic drug cargo, and imparting stability, hemocompatibility, and biocompatibility in physiological media and blood microenvironment ,111,112.The nature and interactions of nanocarriers help in understanding the assimilation pathways followed to enter into the organisms. As it became essential to assess the potential hazard of nanocarriers, the risk assessment of the nanocarrier’s chemical moiety has been the relevant approach to determine the possible risks. Nanocarrier risk assessment is an important process where scientific principles are applied to designate hazards linked with environmental and human exposure to chemicals . To asseSystemically administered nanoparticles have the capability to cause the infusion reaction in clinical subjects, an untoward reaction that generally either prolongs or stops the clinical translation potentials of these nanomedicines. This reaction can exhibit itself in terms of rash, chest pain, chills, fever, rigors, etc., or sometimes trouble with respiratory ventilation, while in some rare cases, these could also prove to be lethal. Identification of the risks of infusive reaction in the earlier stages of the drug developmental phases can aid in the mitigation of the vital safety detrainment when nanoformulations are translated into clinical stages, and hence can save both money and time of developers, and protecting the patients from significantly harmful consequences ,119. HenIn comparison to in vitro assessment, in vivo results remain more relatable to human beings. It could be explained with the help of various crucial factors such as hormonal alterations and cell–matrix interaction. Additionally, chronic and long-term toxicity effects cannot be measured in in vitro studies . During Hence, these studies help in understanding the pharmacokinetics and pharmacodynamics of nanocarriers. The research articles presented in vivo testing showed variable data depending upon the animal species, analyzed selected end points, and study duration. Furthermore, studies reported important endpoints such as clinical signs of infection, altered serum albumin/creatinine ratios, variation in organ weights, and histopathological lesions in various organs . In receToday, applied science has flourished. However, concerns about potential health effects have arisen for untested materials . In factBecause of the chemical or structural ramifications of customized multifunctional drug-delivery nanoparticles, conventional regulative methods could sometimes not be suitable to assess and evaluate their toxicological as well as safety implications. Furthermore, the necessity for placebo-controlled clinical trials as well as therapeutic regimens also requires further reconsideration. The US-FDA also inducted the Nanotechnology Regulatory Science Research Plan to address some of the bigger scientific lacunae in terms of the comprehension, methodologies, and techniques needed for making the regulatory evaluations of nanomaterial-based particles and various other nanoformulations . This inAn inclusive characterization and assessment of nanoparticles and their intractability with cellular and tissue components are necessary and significant for their applicability in drug-delivering paradigms. While efficacy and safety assessments have become vital for the clinical translation and development of nanoparticles, in-depth comprehension of the nanoparticles’ mechanism of action can lead to the opening of newer disciplines in drug carriers’ functionalization and customization. Identifying the vital dimensions which can prove enough for achieving the prolonged targeting become critical for the mitigation of the risk associated with the higher complexities of these nanosystems . HoweverFurther, scientists must ensure the reproducibility of nanocarriers which helps in relying on ultimate results. Moreover, skilled professionals and highly sophisticated instruments are required to characterize the nanocarriers, and the physicochemical properties of nanocarriers are imperfectly understood . On accoThere have been very many claims that drug-delivery nanoparticles can cause harm to the environment and to humans as well; therefore, more studies and explorations are required to be undertaken for a clearer comprehension of the untoward effects of these nanoparticles. Regulatory authorities of various regions and countries along with international assemblies need to elevate their regulatory guidelines in the context of safety, toxicity, and good manufacturing as well as laboratory procedures. Large amounts of investment funds previously caused the advancements of various research institutions, professional and regulatory agencies, pharmaceutical firms, and testing labs for the creation of standardized operation protocols for assessments and evaluations . The strRegulating and standardization authorities  have been seriously committed to developing and validating the methodologies to characterize various intrinsically defined characteristics and media-dependent external features of nanoparticles and to describe their routes of exposures and various related hazards exhibited by nanoparticles to environments as well as human health. Questions including “which testing parameters are dependable for the identification of the possible health implications of nanoparticles” and “how the knowledge and comprehension can be translated in the context of regulatory requirements” need to be elucidated to avoid false positives and negatives and misrepresentations of safety or toxicology data for the nanoparticle safety research .Although there have been unfathomed stakes among the policy-making agencies and scientific communities for moving from animal-oriented individualized toxicity evaluations towards structured hazard assessment and screening schemes, the deficiency of pragmatic steering regarding the accorded applications of non-animal assessment methodologies in the regulatory settings has culminated in lower regulatory and industrial adoption . The genFor regulation proposals, biological nanoparticles have been under the models set by EMA. These frameworks are the regulatory plans and schemes for following-on the nanoparticles, which also undertake testimonials for the relative qualities, preclinical and clinical cases, and reports . The indAllowing for comfortable and easy-going accession and admittance to the information and data;Generation of confirmable, coherent, logical, and high-quality data;Fosterage of cross-disciplinary and co-operative research and explorations;Development of the working and practical relations among decision-making authorities, regulatory agencies, as well as several other significant partners;Increase the openness and receptiveness of regulatory agencies and authorities for the newer data, information, tools, and techniques .Freshly gained data and information could be employed for regulatory projects when fundamental policy formulators as well as lawmakers can fully interpret, translate, and can draw inferences from it. Therefore, the central feature for the successful consolidation of newer data and information in the context of regulatory frames and models and deciding procedures is to clearly exhibit the dependability and significance of their effects and consequences for regulation objectives ,152. ForAlthough customized drug-delivery multifunctional nanoparticles have exhibited appreciable remedial benefits for a wide spectrum of pharmacological and theragnostic applicability, their clinical translational paradigms have not advanced as speedily as the profuseness of encouraging preclinical outcomes could have hinted. For moving any of the functional nanoparticles from the bench side to the bed, various respective experimental and observational demanding situations are required to be answered . From thFurthermore, nanomedicine’s clinical translation has always been a costly and time-taking procedure. Nanoparticle technologies often demonstrate quite larger complexities as compared to conventional or traditional formulation strategies which implicate the dispersion of free drugs in bases . Various important issues in the context of the clinical translational developments of nanoparticles have been mentioned in Recently, some scientific advancement achieved as an answer to COVID-19 disease has efficiently streamlined the several years and decades of the challenging situations and obstacles in clinical translation pathways confronted by nanoparticle-based medicines. Indeed, rapidly growing and quick approvals, which uphold the highest measures for safety and toxicity assessments and also need lesser bureaucratic hurdles, have yielded appreciable hopes in terms of the future of nanoparticle-based medicines . Back in 2005, pharmaceutical technologies and industries functioned along with the regulatory agencies for setting up the European Technology Platform on Nanomedicine (ETPN), which was the first step in addressing the applicability of nanotechnology-based formulations in health and disease. The ETPN aimed at the creation of circumstances for flourishing and productive translations of nanomedicine-based products by forming and affirming the public funds and finances in the bright areas of nanoparticle-based formulation, research and development, and planning and formulating the unique proficient infrastructures called the Nanomedicine Translational Hubs. ETPN Nano Medicinal Translation hubs offer customized instructing by the Translation Advisory Board, nanomedicine characterizations by NCL, as well as GMP manufacturing via pilot-based original platforms ,162.For multifunctional drug-delivery nanoparticles which integrate presently existing traditional or conventional medicines with newer carrier-based technologies, or which integrate existing medicines with conventional or traditional carrier-based technologies for some newer biomedical, diagnostic, or therapeutic applications, intellectual property situations have become further perplexing with more complicated drug delivering platforms, e.g., those that can integrate commercial targeting moieties (viz. antibodies) or coating materials (viz. Eudragit) which were possessed by various other firms and industries. Intellectual property strategy can further involve several patents of any technological platform and require cross-licensing placements ,164. TheFrom the commercialization viewpoint, the essential infrastructure, in-depth comprehension of nanoparticles, and a set of capabilities needed for the business development of nanoparticles have currently not been constituted in most of the pharmaceutical industries. Such elements are also needed to be taken into consideration while performing the assessments and final cost-effectiveness of nanomaterials as compared to present conventional or traditional therapeutic approaches . AdditioFor improving the opportunities for academic nanomedicine-based designs to be translated into the clinics and to be carried forward towards the formulation and product developmental stages, it requires the advocacy for some more active attitudes for the isolation and endorsements of those platforms and settings which actually possess quite viable and higher commercial as well as clinical potentials. Further, there are key development challenges initiated from wider commercialization feasibilities, and taking into account several other core clinical consequences and ultimately directing the preclinical as well as key pharmaceutical prospects needs to be considered prior to engaging in clinical translational objectives . These cThe initial and foremost challenge for the consideration prior to any of the nanoparticle-based medicines being undertaken for the developmental process can correlate with the commercialization as well as practical feasibilities from the viewpoint of their elementary or chief targeted indications. In this regard, both the capability of improved patients’ welfare and the sample size of the ultimate patient populations are significant. Considerably amended patients’ benefits could also include increased pharmacological effectiveness, lesser toxicological implications, or merely by nanoparticle-based medicinal formulations necessitating lesser frequency doses or enabling a better commodious administrative route than comparative drug products (and hence encouraging the patients’ compliance) . In thesFurther, a number of approaches can be undertaken to maximize the safety and minimize the toxicological impacts of nanoparticles on health. This can start with the “safety by design” strategy which is considered one of the initial points when novel nanoparticles are engineered, and rigorously adopting the principles and practices of drug discovery and development throughout the entire processes of products’ formulation and development (F&D) ,176,177.Furthermore, there is the combination of nanotherapeutics with photothermal therapy (PTT) a light-dependent therapy formulated for the eradication of cancers through the converting of light energy into heat for the optical absorption phenomena. In this context, the photothermal absorbers should possess the optimized absorbances within initial 650–850 nm or subsequent 950–1350 nm biological windows, when the light will interpenetrate intensely inside the cancerous tissues and will cause minimal toxicity to the adjacent healthy tissues . ComputaNanomedicine presents new health systems, paradigms, and chances, and several clinical products have already come into the market. Precise observational records and analysis or characterization have become quite crucial for all the scientific platforms and it becomes significant to not amplify the possible consequences for “nanotechnological” research and explorations; e.g., in its guiding written document the “US-FDA id not unconditionally judging the nanomaterial-based platforms or their applicability as such benignant or calumniatory.” The necessity for rich and string assessment has become quite evident, but cases for exceptional attention and care may not be that apparent ,185. TheIn the recent past, explorations and developments in nanomedicine with regard to the interdisciplinary biological and nanotechnological arena have developed rapidly. The establishment of analysis and describing the standards can heighten the qualities as well as the wholeness of published explorations and research, promoting the reutilization and improving the outcomes, and can well modify the comparative assessments throughout various kinds of nanomedicines. Nano as a technology could well be seen as a solution to various problems in biomedical developments but has not been endorsed as the “performing nano for interest of the nano”. From these prospects, it could well be thought of as problem-derived nanomedicine or biomedicine conception, and has become a more significant requirement in bio and nano research for the improvement of success rates of nanomedicine translation into patient settings . A succeThere has been no doubt that nanomedicine therapy with continuously enhancing multifunctionalities will continue to exist in the coming times. As novel and more complicated nanoparticulate designs are appearing, appreciably improved tools, techniques, and methods for defining toxicity and biocompatibility will be required to be produced, specifically for those which could evaluate cellular, intracellular, and tissue and organ biocompatibility. While the contingents of matters concerning the scaleup of production and good manufacturing practices have often not been talked about, advanced and convoluted nanoparticles demonstrate that attempts towards surpassing good manufacturing and regulation constraints are under constant progress. Although several demanding situations have been existing for the translational potentials of multifunctional drug-delivery nanoparticles which have currently been employed for research and exploration purposes and as instruments for the accepted formulations for clinical medicines, their possible rewards should act as the driving force for their productive advancements and the continuous emergence of newer classes of nanoparticle-based therapies. The safety and toxicity assessment of medicinal nanoparticles need stimulation from several origins and subjects. The productive adaption and adjustments of the risk evaluation protocols to nanoparticles rely directly on the capability of individuals in industries, materials sciences, toxicology, and regulatory agencies understanding how their corresponding expertness accompanies the other aspects. There have been many clear acknowledgments of the values and significance of these multidisciplinary collaborative attempts for the improvements of nanoparticles’ toxicity assessment procedures. However, only limited approximations have been put into the practices yet, since regulatory authorities, the scientific community, and pharmaceutical industries perform from several distinct effronteries and have been quite engaged in their respective viewpoints.In these newer times, evolving tools and techniques confront a more skeptical and postulating world—their advantages and gains to the societies need to be crystal clear, but also the scientific community including engineers needs to predict and qualify the probable risks related to their applications. In the case of nanobiomedicine and nanotechnologies, these hazards beleaguer the probable environmental entailments of widespread applications of customized and engendered multifunctional nanoparticles. Presently, nanoparticle exposure and health and safety concerns may not pose any considerable risks to public health and safety, since the most prevailing route of their exposure restricts the appraisal of their employability. However, with the increase in their quantities and kinds of customizations of drug-delivery nanoparticles applied in human populations, their potential regarding accidental environmental effects can further multiply correspondingly. Although it has become challenging to evaluate the hazards of engineered and customized nanoparticles before these may be well defined as commercial products, active research and explorations have become crucial for assuring a sustainable nanomedicine field. Overall, the applications of nanoparticles in biomedicine have the capability to create major effects on human safety. Therefore, it is advised to facilitate the advancements of personalized nanomedicine for particular patient groups, where nanotherapy can be well oriented by the patient’s specific genetic makeup as well as illness profiles. For example, illness-specific features and characteristics, viz. capillary permeabilities, expression levels of various cell surface receptors, and biomolecular pathways activity, can be assessed and applied for designing personalized medicine-based approaches. The physicochemical characteristics (viz. hydrodynamic diameter or nanoparticle size or their structures) of drug-delivery systems could further be tailored and customized in the context of the severity of illness for optimized remedial advantages. These concepts will considerably improve the ways in which patients are treated. However, for these phenomena to happen, there have still been several concerns which are required to be covered which include the basic comprehension of the pathophysiology of specific illnesses and nanobiological interaction of these multifunctional and customized nanoparticles in the patients, and several commercialization setbacks and roadblocks associated with manufacturing processes, costs, economics, and regulation criteria. Lastly, researcher communities are required to undertake the minimizations of the various complexities of nanoparticles and consider the ultimate dosage form for clinical applications, for the nanoformulation to possess the capability of translational potential into patient-based nanotherapeutic paradigms. Reduction and minimization of the complexities are needed for the pathophysiological or medicinal requirements and become paramount in the designing of nanoparticles and their synthetic schemes for the generation of clinically applicable nanosized therapeutic regimens.The application of several analytical tools and methods which can use the complementarity, statistically strong, and significant data in the context of engineered nanoparticles characterization as well as biological localization or bioaccumulation is doubtlessly an assuring scheme to exploit these in both patients and environmental toxicity paradigms. Some of these tools and methodologies are employed for both environmental as well as patient-based samplings, whereas other techniques lie within specific domains of the nanobiomedical explorations. Clearly, as described above, a methodological cross-assessment and evaluation is quite obvious and needs to be immediately undertaken by researchers in nanomedicine disciplines. Beyond these obvious steps, a wider and fundamental intractability among the human’s nanomedicinal and environmental arenas must happen, since nanotoxicological data procured in these fields of explorations can be of considerable relevance to other fields as well. As the requirements for the development and advancements of specialized tools and techniques for the detection, characterization, and quantification of these customized and engineered nanoparticles in complicated systems can be shared by various fields, it has become imperative that these processes are channelized by collaborative attempts of scientific communities, by focusing on the analytic plannings and schemes adjustable for all arenas of nanoparticle and nanomedicine research.


Despite the importance of intervention fidelity in interpreting the outcomes of complex public health interventions, there is a lack of both reporting fidelity trial protocols and uniformity. In evaluating complex, adaptable/pragmatic interventions in resource-strapped settings with systemic issues, unique challenges to intervention adherence and monitoring are introduced, increasing the importance of a fidelity protocol. We aim to describe the intervention fidelity and monitoring protocol for the Healthy Life Trajectories Initiative (HeLTI) South Africa, a complex four-phase intervention set in urban Soweto, starting preconceptionally and continuing through to pregnancy, infancy, and early childhood to improve the health of young women and reduce the intergenerational risk of obesity.The HeLTI SA fidelity protocol was based on the NIH Behaviour Change Consortium (NIH BCC) Treatment Fidelity Framework, outlining the following components of intervention fidelity: study design, provider training, intervention delivery, intervention receipt, and intervention enactment. Context-specific fidelity challenges were identified. The intervention fidelity components and associated monitoring strategies were developed to align with HeLTI SA. Strategies for fidelity monitoring include, amongst others, qualitative process evaluation methods, reviewing observed and recorded intervention sessions, monitoring of activity logs, standardized training, and intervention session checklists. Possible challenges to fidelity and fidelity monitoring include high provider turnover, lack of qualification amongst providers, difficulty tracing participants for follow-up sessions, participant health literacy levels, and the need to prioritize participants’ non-health-related challenges. Solutions proposed include adapting intervention delivery methods, recruitment methods, and provider training methods.The NIH BCC Treatment Fidelity Framework provided a solid foundation for reporting intervention fidelity across settings to improve intervention validity, ability to assess intervention effectiveness, and transparency. However, context-specific challenges to fidelity (monitoring) were identified, and transparency around such challenges and possible solutions in low- and middle-income settings could help foster solutions to improve adherence, reporting, and monitoring of intervention fidelity in this setting.PACTR201903750173871. Registered on 27 March 2019Pan African Clinical Trials Registry The online version contains supplementary material available at 10.1186/s13063-022-06696-w. Bukhali) TrialAdministrative information for HeLTI SA  South Africa (SA) is one site of the multinational HeLTI consortium, with complementary trials ongoing in Canada, India, and China, in collaboration with the World Health Organization. HeLTI aims to evaluate the cumulative effect of a complex package of interventions starting in the preconception period, through pregnancy and early childhood on childhood obesity as the primary outcome , 11. ObeDue to the multifaceted nature of the HeLTI SA intervention , fidelitWe aim to describe the intervention fidelity protocol for HeLTI SA, based on the NIH Behaviour Change Consortium Fidelity framework . This prHeLTI SA is an individual randomized controlled trial, recruiting 6800 young women between 18 and 28 years old through community-based recruitment methods. Recruitment started in October 2019 and is expected to conclude by the end of March 2022 . Particihttps://pactr.samrc.ac.za; identifier: PACTR201903750173871).Ethical approval for the HeLTI SA trial and process evaluation was obtained from the Human Research Ethics Committee  (HREC) of the University of the Witwatersrand, South Africa, with reference numbers M1811111 and M190449. The trial is registered with the Pan African Clinical Trials Registry  on health, development, and disease risk throughout an individual’s life course . The preThe behaviour change components of HeLTI SA are grounded in three main theories that, together, reflect the combined importance of context, the individual, and support for behaviour change. Firstly, the theory of planned behaviour suggests that intentions predicate behaviours and that these are determined by factors including personal attitudes, subjective norms, and perceived control; secondly, control theory proposes that behaviour results from minimizing deviation from goals, standards, and ideals; lastly, social cognitive theory suggests an interaction between goal-directed behaviour, personal factors, and environment or social context . HeLTI SThe HCS approach is applied during each of the HeLTI SA intervention sessions and is incorporated into the intervention manual for each phase. To evaluate whether participant sessions reflect the theory that HCS is based on, its use is monitored through the completion of a form by health helpers, which includes self-reflection on the use of HCS . This information is monitored by project coordinators through REDCap, the data management system used by HeLTI SA . This toThe intervention dose for HeLTI SA consists of three main components: the health resources, the in-person and telephonic intervention sessions with health helpers, and the micronutrient supplements. The optimal dose is described in detail in the intervention manual/protocol for each intervention phase, and although the number and frequency of each dose component are pre-determined, the duration of individual participant sessions is based on participant needs and is expected to be diverse. The duration of each phase is outlined in the intervention protocol and is fixed, except for the preconception phase which varies depending on the timing of the participant’s potential pregnancy. Moreover, the monthly contact sessions and level of content are consistent across the intervention phases. The study design incorporates methods for electronic monitoring of the length, frequency, and number of contact sessions on REDCap. An overview of the strategies and monitoring plan is provided in Table There are some potential challenges to ensuring fidelity to study the design in our setting. Reaching participants through in-person sessions may be more difficult than telephonic sessions, an issue amplified by reduced in-person contact due to the ongoing COVID-19 pandemic. As a result, the dose of in-person sessions may be disproportionally lower than the telephonic sessions, potentially affecting the effectiveness of HCS and SMARTER planning strategies as discussed in more detail in the “To what extent and how was training standardized across health helpers? How will skill acquisition be measured? How will skills be trained across health helpers with different backgrounds, training, or skill levels? How will loss or change in skills be minimized within and across health helpers? [To improve uniformity and quality of intervention delivery, it is essential to train the intervention providers  in terms of the study protocol, content to delivery to the participants, and new skillsets and to maintain these skills for the duration of the study. The following questions help evaluate the study’s fidelity to provider training: helpers?   and with as few as one health helper per training group. Where possible, the same instructor is used for each group of health helpers. A pilot study describing the implementation of the training method and intervention delivery has been evaluated and reported elsewhere . An offiDue to the extensive duration of the complete intervention (up to 87 months), maintaining the health helpers’ skills throughout the intervention is particularly important. Refresher training is provided when required, as determined by continuous process evaluation of intervention sessions. Continued adherence by health helpers to the trained components of HCS is monitored as described under the “A potential challenge to fidelity for behavioural interventions in general is that there are unavoidable differences between providers, including personal communication style, experience, and interpersonal conduct. In pilot work, health helpers were mostly young women (aged 20–30) having completed secondary education, recruited for their ability to engage and work with the target participants (young women from varying socioeconomic circumstances). For HeLTI SA, as in the South African real-life setting for community health workers, health helpers will not be required to have an official tertiary or health qualification. While past experience within (community) healthcare is an asset, it is not a prerequisite for the health helper position. While these factors reflect a real-life situation relevant to implementation and potential scale-up of the intervention, they may result in greater variation between health helpers in terms of educational background, health literacy, training, or professional experience. The standardized training, training material, and adherence to the study protocol can help to mitigate the differences between individual health helpers.A second potential challenge to provide training in our setting, as mentioned under the “How will the study measure and control for external or non-specific intervention effects? How can delivery of the intended intervention by providers be ensured? How can differences within intervention groups be reduced? How can adherence to the intervention protocol be ensured? , whether supplements were delivered, and whether resource materials are received by the participant. This also captures data on the assessment of six areas of health  and the relevant actions that are undertaken  throughout the intervention. The implementation fidelity criteria in Additional file Road to Health booklet, a parent-held birth and development medical record given to each child born in South Africa), the delivery of these by trained health helpers is still unique to the intervention arm. The providers of the control components, trained call centre agents, are also trained on the distinction between the intervention vs control arms of the trial during their training sessions.Women in HeLTI SA are individually randomized, since cluster randomization proved to be unfeasible in the pilot trial . The intAs described in the “Since a tertiary degree or specific past experience is not required, the delivery may be impacted by limited health literacy amongst health helpers. However, as described in the “How can the participant’s understanding of the provided information be verified? How can the participant’s ability to use cognitive and behavioural skills taught during the intervention be verified? How can issues that interfere with receipt be addressed?  changes resulting from the intervention.Fidelity to both receipt and enactment of the intervention is dependent on participant circumstances, needs, and background. Low health literacy is a concern in low- or middle-income settings , 25, andParticipants in our setting have been found to live with pressing social and economic challenges, which may overshadow the importance of a health-related intervention (receipt) and reduce participants’ agency and autonomy to enact change (enactment) , 26. In Based on the NIH BCC conceptual guidelines for fidelity intervention improvement and monitoring, this paper describes the intervention fidelity protocol and monitoring plan for HeLTI SA, the ongoing randomized controlled trial investigating the impact of a complex, multi-phase intervention on maternal and child health in Soweto, South Africa. The aim of establishing and standardizing a fidelity protocol is to increase the ability to reliably draw conclusions about the effectiveness of the intervention in question and its underlying theory. However, the development of the protocol for our complex behaviour change intervention has also highlighted the challenges in our setting.In low- and middle-income settings, where reporting of fidelity has been found to be inconsistent , transpaSince HeLTI SA has been designed to pragmatically inform the local South African healthcare landscape, these challenges to fidelity are also important to consider in terms of the feasibility and scalability of the intervention, and other similar interventions, in real-life settings. Some proposed or implemented solutions include encouraging health behaviour change in the context of the participant’s pressing life circumstances and adapting health helper training strategies to be feasible yet thorough in the context of high turnover and limited resources. Fidelity to the intervention protocol versus the adaptability needed to implement such solutions may seem mutually exclusive. However, adaptations based on pilot findings to improve delivery, adherence, and participant engagement can be made while upholding the key functional components of the intervention. This concept of “functional fidelity” has been described for the implementation of complex behaviour change interventions , 28, andIn conclusion, the five NIH BCC fidelity components were used to develop a fidelity protocol and monitoring plan for HeLTI SA and to improve the ability of investigators to establish whether the intervention is being implemented as intended. In addition, recognizing challenges to both ensuring and monitoring intervention fidelity allows researchers to transparently find solutions that prevent compromising the study’s ability to draw reliable conclusions. The development and sharing of fidelity protocols and associated challenges, particularly in low- and middle-income settings, can help future researchers to develop fidelity strategies and monitoring plans despite the presence of context-specific challenges.Bukhali) started in October 2019 and is expected to conclude by the end of August 2022.Fidelity protocol version: 1.0; Date: December 2021. Recruitment for HeLTI SA (Additional file 1: Fig. S1. Criteria checklist to assess fidelity of the implementation components for the intervention arm of the trial. Fig. S2. Criteria checklist to assess fidelity of the implementation components for the control arm of the trial.


In 1872, George Huntington described an illness that is “confined to certain families… in whose veins the seeds of the disease are known to exist.” That seed was later identified as the expansion of the cytosine-adenine-guanine (CAG) repeats within the huntingtin gene that leads to an expanded glutamine repeat tract in the N-terminal end of the huntingtin protein  functioning through endogenously encoded calcium indicators and a miniaturized microscope or miniscope during the progression of the disease. This technology will provide new insights into the progressive functional changes in cortical and striatal neurons, interneurons, and astrocyte microcircuits and its correlation with the development of phenotypical signs in transgenic mouse models of Huntington's disease.In the brain, the huntingtin mutation affects all cell types, including interneurons, which, based on early  Harper, . Then, t Harper, . Voelkl Dickson et al. evaluated hypothalamic transcriptome profiles of overexpression of the wild-type huntingtin gene, mutant huntingtin, or full-length human mutant huntingtin in the BACHD model. Although the transcription profiles are different between the models used, their results indicated alterations in sterol and cholesterol metabolism in the over-expressing wild-type huntingtin gene and mutant huntingtin; in the case of full-length mutant huntingtin, changes in the hypothalamic gene expression differ between young and old BACHD mice, which might be related to the phenotype of this transgenic model. Impaired cholesterol and sterol metabolism emerges as a key alteration in Huntington's disease, as evidenced by the evaluation of postmortem brain samples and transgenic models  underlies depression in Huntington's disease, which is improved with bupropion treatment and when combined with serotonin reuptake inhibitors elicited a better efficacy to alleviate depression in Huntington's disease (McLauchlan et al., Finally, as mentioned above, Huntington's disease patients present psychiatric disturbances, which often occur before the motor signs. Among these alterations, depression, anhedonia, apathy, anxiety, obsessions-compulsions, and irritability are the most commonly reported (Paoli et al., Overall, the research articles and the minireview that comprise this Research Topic expand our knowledge on previous topics and open new venues to deepen our understanding of the etiology of Huntington's disease in the hope of identifying new therapeutic targets and future treatments that help patients beyond just alleviating symptoms of the disease.AME-S wrote the original draft. GVR and LG reviewed and edited it. All authors approved the final manuscript.


Graphene and its derivatives show great potential for biosensing due to their extraordinary optical, electrical and physical properties. In particular, graphene and its derivatives have excellent optical properties such as broadband and tunable absorption, fluorescence bursts, and strong polarization-related effects. Optical biosensors based on graphene and its derivatives make nondestructive detection of biomolecules possible. The focus of this paper is to review the preparation of graphene and its derivatives, as well as recent advances in optical biosensors based on graphene and its derivatives. The working principle of face plasmon resonance (SPR), surface-enhanced Raman spectroscopy (SERS), fluorescence resonance energy transfer (FRET) and colorimetric sensors are summarized, and the advantages and disadvantages of graphene and its derivatives applicable to various types of sensors are analyzed, and the methods of surface functionalization of graphene and its derivatives are introduced; these optical biosensors can be used for the detection of a range of biomolecules such as single cells, cellular secretions, proteins, nucleic acids, and antigen-antibodies; these new high-performance optical sensors are capable of detecting changes in surface structure and biomolecular interactions with the advantages of ultra-fast detection, high sensitivity, label-free, specific recognition, and the ability to respond in real-time. Problems in the current stage of application are discussed, as well as future prospects for graphene and its biosensors. Achieving the applicability, reusability and low cost of novel optical biosensors for a variety of complex environments and achieving scale-up production, which still faces serious challenges. Since the isolation of graphene in 2004, two-dimensional (2D) layered materials have attracted great attention . CompareOptical biosensors offer significant advantages over traditional analytical sensing technologies, enabling direct, real-time and label-free detection of biological and chemical substances. Along with the advances in emerging science and technology, a variety of cross-disciplinary and advanced approaches  are applied to novel optical biosensors. Optical detection is achieved by using the interaction between the optical field and the biometric element. There are two main modes of optical biosensing: label-free mode and label-based mode. In the label-free mode, biosensing is achieved based on signals generated directly from the interaction of the analytic material with the transducer. Label-based mode sensing achieves biosensing by using a label and then generating an optical signal using methods such as colorimetry, fluorimetry, or luminescence. Biomolecular detection is widely used in food safety, environmental monitoring, disease prevention and clinical treatment . In partIn the past decade, biosensing applications based on two-dimensional materials have developed rapidly ,16. In tThe preparation method of 2D materials plays an important role in whether they are suitable for biosensors, biomolecular detection and biomedical therapy. Graphene and its derivatives mainly include four forms, which are graphene itself, GO, rGO and GQDs. In this section, we focus on the synthesis of these 2D nanomaterials.2 hybridized carbon atoms . T. T2 V−1 n plasma b 63]. W. W2 V−1 n plasma c 64]..2 V−1 S−3) and potassium chlorate (KClO3) to form graphite oxide . . 109]. HThe bottom-up approach is to assemble GQDs from molecular precursors, and the diversity of carbon precursors determines the size and performance of the synthesized GQDs. Among the bottom-up methods, cage opening of fullerenes methods, stepwise organic methods and microwave-assisted methods are commonly used.Loh et al. reported an open-cage synthesis of GQDs by metal-catalyzed fullerenes; they used fullerene as the reactant, and through the strong interaction between carbon atoms of fullerene on the ruthenium surface, vacancies appeared on ruthenium surface, which contributed to the embedment of fullerene molecules, carbon clusters occurred at high temperature, and GQDs with different shapes were formed through diffusion and aggregation . Chua etStepwise organic synthesis is the synthesis of GQDs from small carbon precursors by organic reactions, and the structure of the synthesized product can be controlled. Yan et al. prepared GQDs by stepwise organic synthesis, and their quantum dots were composed of 168, 132 and 137 conjugated carbons formed by the oxidation of polystyrene dendritic precursors. The synthesized GQDs are very stable, and the method can control the properties of the synthesized GQDs according to the requirements a 113,11. Tian etHydrothermal synthesis of GQDs is time-consuming and not suitable for large-scale industrial production. The method of microwave-assisted synthesis of GQDs can accelerate the synthesis speed, heat evenly, and the size distribution of precursor is uniform. The main advantage is that GQDs can be synthesized without any passivating agent. Using aspartic acid and ammonium bicarbonate as raw materials, Zhang et al. prepared blue luminescent GQDs by microwave-assisted synthesis at 560 W working power b 116]. . 116]. ZIn this section, we summarize and discuss the common methods for the preparation of graphene and its derivatives and analyze the advantages and disadvantages of each method. We can decide the method to prepare the samples based on the desired target properties and the advantages and disadvantages of various preparation methods, and present them in The methods for functionalizing graphene-based nanomaterials with biomolecules can be divided into two categories, non-covalent modification and covalent functionalization, according to the principle of interaction. Both methods have their advantages and disadvantages. For example, non-covalent crosslinking does not affect the intrinsic properties and structure of nanomaterials, nor does it affect the activity of biomolecules . The preUsually, the simplest and fastest way to prepare biomolecule-functionalized graphene and its derivative complexes is the direct physical adsorption of biomolecules on the surface of graphene and its derivative materials without the aid of external conditions. The electrostatic, π-π stacking, hydrogen bonding and hydrophobic forces between biomolecules and graphene and its derivative materials play an important role in facilitating their interactions. Due to the large two-dimensional aromatic surface, graphene materials can be tightly bound to molecules containing aromatic rings by π-π superposition . TherefoGiven the high stability of covalent bonding, covalent modification methods are of great importance for functionalized graphene nanomaterials of biomolecules. Since the surfaces of GO and rGO contain oxygen-containing functional groups such as carboxyl and hydroxyl groups, they can effectively immobilize biomolecules containing a variety of functional groups . Most biOptical biosensors have high sensitivity and can even detect single molecules. Currently commonly used optical biosensors are mainly based on the following methods, surface plasmon resonance (SPR), surface-enhanced Raman spectroscopy (SERS), fluorescence resonance energy transfer (FRET) and colorimetric  133]. S. S133]. The process when a donor fluorophore in the excited state non-radiatively transfers its own energy to a neighboring acceptor fluorophore so that the acceptor emits fluorescence at its characteristic wavelength is known as FRET. The spectral overlap, distance and relative orientation between the donor and the receptor all affect the efficiency of FRET. The distance and relative orientation between the donor and receptor are particularly important for the construction of highly sensitive FRET biosensors . The FRES. aureus-specific gene sequence detection based on GQDs and gold nanoparticle (AuNPs) pairs  by binding to GO by Zhu et al.  pairs a 145]; ; S. aureu et al. c 147]. . S. aurecreening d 151]. . S. aure13–1015 times . . 3O4 nanAmong optical biosensors, colorimetric biosensors are capable of producing signals visible to the naked eye that can be observed without the use of other tools. Colorimetric sensing is closely related to the materials used to make the sensors. Depending on the sensing material, the sensing mechanism of the colorimetric sensor varies . Colorim−1 [2O2 and cholesterol. The sensor exhibited good sensitivity and selectivity for cholesterol (LOD of 78 pM) and H2O2 (LOD of 6.88 μM) with high stability at different temperatures. The biocompatibility of the composite CuO:GNS is superior to that of CuO  of graphene and its derivatives . We introduce optical biosensors for biomolecule detection: FRET biosensors, SERS biosensors, SPR biosensors, colorimetric biosensors, and summarize the research progress of graphene and its derivatives in the field of sensing. Graphene and its derivatives are two-dimensional layered materials composed of spNovel optical biosensors and other applications using graphene and its derivatives as sensing elements have been developed, which can be used for the detection of a range of biomolecules such as single cells, cellular secretions, proteins, nucleic acids, and antigen-antibodies; these new high-performance optical sensors are capable of detecting changes in surface structure and biomolecular interactions with the advantages of ultra-fast detection, high sensitivity, label-free, specific recognition, and the ability to respond in real-time. However, the application areas of graphene and its derivatives-based biosensors are limited, and many applications are restricted to biomolecule detection, which needs to be extended to other medical fields. In addition, efforts should be made to achieve the applicability, reusability, and low cost of new biosensors to adapt to various complex environments, not limited to the laboratory, and to achieve scale-up production, which still faces serious challenges.(1)Understanding the interaction mechanism between biomolecules and graphene and its derivative materials. The analytical performance of optical biosensors based on graphene and its derivatives is linked to the number and arrangement of biomolecules on the surface of graphene and its derivative materials.(2)The homogeneity of graphene and its derivatives in terms of size, thickness, biocompatibility, and the number of surface functional groups is difficult to ensure; this inhomogeneity has a great impact on the performance evaluation and reproducibility of the constructed optical biosensors. Optimization of production methods and improved control of the size and morphology of the produced particles and standardization of the manufacturing process.(3)The type and functionalization of precursors during the preparation of graphene and its derivatives often introduce different impurities, and further research should be conducted to simplify effective sample preparation methods to avoid the interference of other substrates and to ensure that their biosensing performance as signal nanoprobes is not affected.(4)It is also a serious challenge to develop a biofunctionalization method that maintains high stability without changing the structure and function of biomolecules and graphene and its derivative materials, to develop biosensor devices with high stability and reproducibility, and to expand the practical applications of biosensors based on graphene and its derivative materials.(5)Emphasis should be placed on miniaturization, standardization, and multiple readouts of biosensors to reduce the incidence of false negative/positive results. Further miniaturization, integration with fluidic systems, and integration of multi-chip systems are important challenges for the next generation of graphene and its derivatives optical biosensors.(6)The combination of graphene and its derivative optical biosensors with smartphones, in particular, has great potential to enable low-cost, reliable and versatile biosensing platforms that can be widely used in the field and provide excellent convenience for on-site detection of pathogens in remote areas.


One of the most intriguing aspects of aging is the heterogeneity of the older adult population. Some age rapidly and lose their independence, while others remain physically active and cognitively preserved despite their age and number of comorbidities . AlthougThe World Health Organization (WHO) advocates moving away from a disease-focused model of aging and frailty, and toward a more positive model of healthy aging  that focHowever, to date, the tools that geriatricians can use to measure the contribution of each domain to the IC model, as well as the factors and mechanisms that contribute to sustainability and physical reserve, are not fully understood. This highlights the need for more studies to understand the inter-dependent yet distinct contribution of IC and resilience vis-à-vis frailty toward healthy aging , 9. ThisJung, Baek, Kwon, et al. describe their experience using the Clinical Frailty Scale (CFS) to evaluate patients in the emergency department of a busy Asian hospital. They demonstrated that CFS administered in the emergency department could predict adverse events, such as the development of pressure sores, delirium, falls, repeated hospitalizations, and placement in long-term care institutions. Thus, the choice of a tool for screening and measuring frailty is determined largely by the clinical purpose of frailty identification. Another article by Jung, Baek, Jang, et al. compared the original classification and culturally modified classification of the CFS by considering the culturally-sensitive items of food preparation and household chores in instrumental activities of daily living (IADL) which may be less applicable to older men in Korea. The main implication is the reclassification of CFS 5 (impairment in IADL) to CFS 4 in the affected men. The results demonstrate that the modified CFS had better construct validity and comparable predictive validity for the composite outcome of institutionalization and death, alluding to the salience of cultural adaptation of selected items for accurate frailty assessment in older persons. In addition, it is important to consider the perception of older persons toward their functional capabilities, especially in the context of concomitant cognitive impairment  in community dwelling older adults ≥60 years with falls or risk of falls. Of note, the presence of 3 out of 6 impaired IC denotes a >80% probability of belonging to the low/moderate participation class. The identified IC risk factors of physical functioning, cognitive status, hearing impairment and malnutrition may thus be potential intervention targets to improve participation of older adults with falls or at risk of falls.Three articles in the Research Topic touch upon the psychosocial aspects of IC. The cognitive and psycho-emotional domains of IC are determined not only by the individual characteristics of each person, but also by the state of one's social environment , 20. FanZhang et al. developed a new composite measure of aging that integrated phenotypic and functional aging with potential for assessment of geroprotective programs. This composite measure better predicted mortality risk compared with each aging measure in isolation, and was responsive to modifiable lifestyle factors including smoking status, body mass index, alcohol consumption, and leisure-time physical activity. Tan et al. demonstrated preliminary evidence of a novel technology-enabled autonomous multi-domain community-based interventions with exercise, nutrition, and polypharmacy components in improving frailty status, physical performance and strength in pre-frail older adults. Using the Senior Technology Acceptance and Adoption Model, the study also explicated user experience insights which can affect the usability and enjoyment of technological interventions for older persons. In their non-randomized controlled study of a multi-domain exercise and nutrition intervention in pre-frail older persons, Tay et al. reported that reversal to robustness at 1-year was similar between intervention and control groups, suggesting that focusing only on the locomotion and vitality domains may not adequately address component domain losses to optimize pre-frailty reversal. Furthermore, the intriguing result that IC rather than intervention exposure influences reversal to robustness suggest that an IC-guided approach to target identified domain declines may be more effective in preventing frailty progression. Lastly, in the systematic review of multi-domain and lifestyle interventions to support IC, Bevilacqua et al. reported that the majority of successful interventions are based on a goal setting approach whereby older persons are actively and involved in defining the intervention goals. The observation that there were no studies which utilized the IC framework to design the intervention, highlights a significant gap which can inform the future research agenda.The next major theme in our Research Topic revolves around the area of interventions to regress the process of pathological aging and to restore healthy aging. In the area of outcome measures of multidimensional aging, As guest editors, the 14 papers presented in this Research Topic provide a valuable compendium of fresh insights and perspectives in the rapidly-growing field of IC, frailty, and healthy aging. It is our sincere hope that this Research Topic will spur further conversations and explorations to advance this exciting field of research in geroscience and geriatrics.EF, BA, and WL contributed to conception of the editorial. EF wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.


Circular RNAs (circRNAs) are a new series of noncoding RNAs (ncRNAs) that have been reported to be expressed in eukaryotic cells and have a variety of biological functions in the regulation of cancer pathogenesis and progression. The TME, as a microscopic ecological environment, consists of a variety of cells, including tumor cells, immune cells and other normal cells, ECM and a large number of signaling molecules. The crosstalk between circRNAs and the TME plays a complicated role in affecting the malignant behaviors of digestive system cancers. Herein, we summarize the mechanisms underlying aberrant circRNA expression in the TME of the digestive system cancers, including immune surveillance, angiogenesis, EMT, and ECM remodelling. The regulation of the TME by circRNA is expected to be a new therapeutic method. Cancer of the digestive system (DSC) has the highest mortality rate among invasive cancers worldwide. Although current treatments, including surgery, radiotherapy, and immunotherapy are improving, the average survival time of patients with advanced DSC remains low due to the cryptic, rapid, and aggressive nature of early symptoms –4. In reRecently, many circRNAs in eukaryotic cells have been characterized by high-throughput RNA sequencing and new bioinformatics algorithms, and have crucial roles in different types of cancers –15. In tAt present, the crosstalk between circRNAs and various components in the TME has attracted great attention. The role of circRNAs in the TME may become another emerging direction for cancer treatment. Recent studies on circRNAs mainly focus on their impact on cancer biological behavior, but lack a systematic summary of the tumor microenvironment, especially in digestive system tumors.In this review, we summarize the clinical features and biological functions of DSCs affected by circRNAs and the regulatory mechanisms in the TME. CircRNAs will be a potentially useful tool for the diagnosis and therapeutic targeting of DSC. Finally, we also discussed the messenger role of exosomal circRNAs in the TME of digestive system cancers, and revealed the vital role of exosomal circRNAs in signal transmission.It was once thought that the translation mechanism of eukaryotic cells could not translate circRNAs due to the circRNA’s unique ring structure . HoweverMost current studies have reported that circRNAs are the products of back-splicing of the precursor mRNA of the exon, and its downstream 5’ splice site is connected with the upstream 3’ splice site by a 3’-5’ phosphodiester bond at the junction site , 38. TheThe second model is intron pairing-driven circularization. In this biogenesis mechanism, two introns flanking the pre-mRNA exon/exon have one structure capable of interlinking. Flanking introns are close to each other, forming a secondary conformation that allows reverse splicing of the splice site. Most intron pairing patterns are promoted by ALU repeats. The longer the intron length is, the greater the chance that they will have more ALU elements, thereby enhancing exon circularization.Third, the main mechanism of this mode is through RNA-binding proteins (RBPs). RBPs are able to bind to pre-mRNA and link flanking introns together, this process is induced by protein dimerization, which creates an RNA loop.The expression level of homeostatic circRNAs can be regulated at three stages. First, the biogenesis of circRNA begins with the transcription and binding of Pol II to the pre-mRNA that produces circRNA ; second,via the YTHDF2-HRSP12-RNase P/MRP axis , to promote protein complex formation and to allow interactions between different proteins . First tvia specialized sequences in the 5’ noncoding region and then produce small proteins and micropeptides  ,and N6-mpeptides .via exon skipping can be seen as a passive function of circular transcripts , oral squamous cell carcinoma (OSCC), and tongue squamous cell carcinoma (TSCC). For instance, circOGDH is upregulated in ESCC and its overexpression is closely related to tumor size and poor prognosis. In terms of cell activities, circOGDH can promote tumor growth by promoting proliferation, metastasis, invasion, and glutamine metabolism . As a tuThe deterioration of cancers relies on the recruitment and reprogramming of tumor cells to the surrounding normal cells . TherefoMacrophages are released into the blood as immature monocyte precursors from bone marrow and migrate to different tissues for corresponding differentiation . Differein vitro  transporters to squeeze out chemotherapeutic drugs and block their killing effects by efflux of drugs, leading to the development of drug-resistant phenotypes . There iAutophagy is a conserved self-degrading system and a tightly coordinated process. On the one hand, autophagy can suppress tumor progression by removing misfolded proteins and dysfunctional organelles –271 On tin vivo by targeting the miR-129-5p/MMP11 axis , Leading Talents of Zhongyuan Science and Technology Innovation (214200510027), Henan Provincial Medical Science and Technology Research Plan (SBGJ202102117 and SBGJ2018002), Henan Medical Science and Technology Joint Building Program (LHGJ20210324), Science and Technology Innovation Talents in Henan Universities (19HASTIT003), Outstanding Foreign Scientist Studio in Henan Province (GZS2020004), and the Gandan Xiangzhao Research Fund (GDXZ2022002).All five figures in this manuscript were created with BioRender.com.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.


Sc) from the prototypical TSE form that afflicts ovines. PrPSc misfolding from its physiological counterpart, cellular prion protein (PrPC), is the unifying pathogenic trait of all TSEs. PrPSc is resistant to intracellular turnover and undergoes amyloid-like fibrillation passing through the formation of soluble dimers and oligomers, which are likely the effective neurotoxic entities. The failure of PrPSc removal is a key pathogenic event that defines TSEs as proteopathies, likewise other neurodegenerative disorders, including Alzheimer's, Parkinson's, and Huntington's disease, characterized by alteration of proteostasis. Under physiological conditions, protein quality control, led by the ubiquitin-proteasome system, and macroautophagy clears cytoplasm from improperly folded, redundant, or aggregation-prone proteins. There is evidence that both of these crucial homeostatic pathways are impaired during the development of TSEs, although it is still unclear whether proteostasis alteration facilitates prion protein misfolding or, rather, PrPSc protease resistance hampers cytoplasmic protein quality control. This review is aimed to critically analyze the most recent advancements in the cause-effect correlation between PrPC misfolding and proteostasis alterations and to discuss the possibility that pharmacological restoring of ubiquitin-proteasomal competence and stimulation of autophagy could reduce the intracellular burden of PrPSc and ameliorate the severity of prion-associated neurodegeneration.Transmissible spongiform encephalopathies (TSEs), or prion diseases, are progressive neurodegenerative disorders of the central nervous system that affect humans and animals as sporadic, inherited, and infectious forms. Similarly to Alzheimer's disease and other neurodegenerative disorders, any attempt to reduce TSEs' lethality or increase the life expectancy of affected individuals has been unsuccessful. Typically, the onset of symptoms anticipates the fatal outcome of less than 1 year, although it is believed to be the consequence of a decades-long process of neuronal death. The duration of the symptoms-free period represents by itself a major obstacle to carry out effective neuroprotective therapies. Prions, the infectious entities of TSEs, are composed of a protease-resistant protein named prion protein scrapie (PrP Such semantic unification is particularly useful because comprehends, under a common basic alteration, neurodegenerative diseases otherwise heterogeneous for etiology, histopathology, and clinical presentation. Although sporadic onset represents the most prevalent occurrence of all neurodegenerative disorders, significant signs of progress in the characterization of the mechanisms of pathogenesis have been obtained by studying inherited forms of proteopathies. Noteworthy, pathogenic mutations have been described not only in genes encoding for disease-specific proteins but also in genes involved in the mechanisms of protein quality control and turnover . PrPSc is derived from the post-translational rearrangement of the glycoprotein cellular prion protein (PrPC) , and autophagy (ALP) has been also described in association with other neurodegenerative disorders characterized by protein misfolding  or the autophagic pathway. There is compelling evidence that cellular and pathological prion proteins are crucial clients of the mechanisms of protein quality control undergoing chaperone-assisted refolding , to degradation or disaggregation in the cytosol. All these pathways provide vital contributions to proteostasis and are also joined in a complex network of mutually compensatory activities  and autophagy. Heath shock proteins (HSPs) are chaperones activated by a variety of cell stresses, including heat, oxidative, and inflammatory insults; they comprehend different subgroups . Accessory proteins, named co-chaperones, are also often recruited to the complexes with substrate proteins and act as adaptors to guide ubiquitination and interaction with proteasomes  play a crucial role in the control of proteostasis.Aggrephagy, cooperates with UPS, being mainly involved in the removal of larger or aggregation-prone proteins that cannot enter the narrow pore of the proteasome , in which client substrates bind with chaperone and co-chaperone complexes and are translocated within lysosomes by lysosome-associated protein 2A (LAMP2A)  showed a consistent association with prion diseases in humans, animals, and in experimentally infected rodents and cells. Indeed, the analysis of brains from sporadic and familial CJD patients showed that Hsp73 expression is inversely proportional to neuronal loss indicating that differences of vulnerability to PrPSc in different brain areas may depend on the neuroprotective activity of chaperones , whose overexpression upregulates Hsc70 and LAMP2A, which, in turn, translocate PrPSc in the lysosomal lumen  can be transported from affected areas to distant neurons. In particular, misfolded forms of b-amyloid or α-synuclein can be secreted into the extracellular milieu, possibly via exosomes, thereby affecting in a prion-like manner the normal physiology of the neighboring cells. This hypothesis was proposed as a plausible reason for the time course of idiopathic AD and PD, which only become symptomatic in middle to late life. For a review, see Yoshida and Hasegawa .Sc from the periphery to the brain in infectious TSEs , neuronal death results from the toxic activity of PrP soluble oligomers generated during the amyloidogenic process occurring after its misfolding (Bucciantini et al., Sc deposits in brain parenchyma. Amyloid plaques containing PrPSc are not invariantly present, being detected in a minor part of variant and sporadic CJD cases, and their ultrastructural aspect ranges from multicentric, typical of familiar GSS, florid in most cases of variant CJD or unicentric, typical of kuru (Ghetti et al., Neuronal plasticity and long-term survival rely on a complex network of sensors that detect perturbation of proteostasis and effectors that put in action a series of countersteps, including chaperone-assisted protein refolding and disaggregation, UPS, and autophagy (Weibezahn et al., The increase in vacuolar structures with the features of lysosomes, containing ubiquitinylated proteins, and of large autophagosomes, containing ribosomes and other cytoplasmic constituents, has been often described by post-mortem analysis of brains from animals and humans affected by prion diseases and in experimentally prion-infected rodent brains (Boellaard et al., Relevant insight regarding such issue also comes from studies about the pathogenic consequence of autophagy impairment in Alzheimer-related neuronal death. Immunohistochemical analysis of cortical dystrophic neurites of patients affected by Alzheimer's disease shows a significant increase in electron-dense autophagosomes compared with age-matched healthy subjects (Boland et al., in vitro and preclinical studies, which demonstrated that the stimulation of autophagy prevents or reverts the aggregation, and the subsequent toxicity of several misfolded proteins including PrPSc or derived peptides (Cortes et al., Sc in mice led to the deposition of ubiquitin-tagged proteins in brain areas interested by vacuolation and the deposition of amyloid plaques containing PrPSc (Lowe et al., Sc and tissue degeneration (Lowe et al., Given the involvement of UPS and autophagy impairment in a broad spectrum of human proteopathies (Bence et al., Sc for degradation (Direct links between pathological prion proteins and the impairment of specific proteostatic effectors are reported by relevant papers and comprehend mainly the downregulation of chaperones in prion-infected cell lines and animals, and the impairment of the capacity of the proteasome to accept the aggregated form of PrPradation .PRNP mutations produce modifications in structure, topology, and metabolism of mature PrP and favor spontaneous conversion in protease-resistant PrPSc (Petersen et al., Sc accumulation in some late-onset inherited forms of prion diseases since it has been demonstrated that disease-associated mutations of prion protein downregulates chaperones and hamper UPS-mediated proteolysis (Zanusso et al., PRNP gene, produces a truncated form of PrP lacking the C-terminal GPI, which retains its N-terminal signal peptide and is not properly exposed in the plasma membrane. Such form of PrP is rapidly degraded by intact UPS, but accumulates in the cytoplasm as PK-resistant protein if UPS is pharmacologically blocked (Zanusso et al., Sc extracted from scrapie-infected mouse brain and recombinant β-refolded prion protein inhibit the proteolytic activity of 26S proteasome due to the stabilization of the 20S core in a closed conformation and the prevention of its opening by ubiquitinylated substrates (Kristiansen et al., Sc, and it was restored if PrPSc or β-refolded recombinant PrP are incubated with antibodies directed against its oligomeric aggregation state (Kristiansen et al., Studies performed in transgenic mice and transfected cell lines harboring mutant forms of PrP have demonstrated that Sc oligomers exert neurotoxicity (Amici et al., Sc itself through the impairment of proteasome-mediated protein degradation and that this mechanism represent could represent a major cause of neurotoxicity induced by misfolded oligomers in a wide range of neurodegenerative diseases.UPS blockade, which mainly affects chymotrypsin and caspase-like proteolytic activity of the proteasome, leads to the intracellular accumulation of otherwise short-living proteins and has been proposed as a possible mechanism by which PrPper 1,000,000 persons per year, poses objective obstacles toward the pursuit of a cure. The scarcity of recruitable patients and the rapid progression of neurological and cognitive decline after diagnosis are the reason of very few clinical trials performed, which were also characterized by the frustrating lack of efficacy of all the therapeutic approaches that have been so far tested. Noteworthy, most of these trials have been carried out using structurally and clinically unrelated compounds with the main goal to prevent PrPC-PrPSc conversion and PrPSc aggregation as observed in preclinical studies (Forloni et al., Except for the vCJD outbreak in the mid of 1990s that attracted a significant interest in the scientific community on prion biology, the very low incidence of human TSEs, with the most frequent sCJD affecting 1–2 individuals However, the growing evidence that the impairment of proteostasis is a shared pathogenic trait between prion diseases and other protein misfolding diseases suggests the utility of changing therapeutic strategy to adopt a common approach for all neurodegenerative disorders of CNS, both the more prevalent and rarest ones (Butler et al., Sc molecules in persistently infected cells and counteracts PrPSc accumulation in the brain of mice intracerebrally inoculated with two prion strains. Thus, the pharmacological inhibition of mTOR could find rational use also in the treatment of non-inherited forms of prion disease (Nakagaki et al., Newer approaches able to boost the self-defensive proteostatic competence of neurons are presently investigated (Thellung et al., Sc and other aggregation-prone polypeptides linked to brain proteopathies. Moreover, the possibility to stimulate autophagy without interfering with the function of immune systems, as is the case of rapamycin analogs, would be highly desirable in virtue of a potentially milder toxicity in chronic treatment. Indeed, besides the direct mTOR inhibition, other mechanisms that can stimulate autophagy and produce inhibition of PrPSc intracellular accumulation have been described.The proof-of-principle of a possible anti-prion activity of the autophagy-stimulating molecules, acting through the inhibition of mTOR, prompted the screening of many other compounds, heterogeneous for structure, origin, mechanism of action, and possible original therapeutic indications, for the capacity to stimulate autophagy and inhibit intracellular aggregation of PrPSc replication in a persistently infected neuroblastoma cell line, prolonging the survival of mice infected with intracerebral inoculation of RML scrapie prion (Karapetyan et al., Astemizole, an H1 histamine receptor antagonist, has the capacity to inhibit mTOR signaling and is able to prevent PrPSc in a neuroblastoma cell line persistently with permanent infection (Heiseke et al., Metformin is a first-choice drug for type II diabetes that is currently repurposed for independent clinical indications (Wurth et al., 3 production (Sarkar and Rubinsztein, Sc, trehalose reduced the amount of protease-resistant PrPSc accumulated within the cells, protecting them from oxidative damage (Beranger et al., Mood stabilizers, as lithium and valproic acid, have been recently demonstrated to possess the ability to stimulate neuronal autophagy and increase neuronal resistance to neurotoxic proteins, inhibiting Intracellular accumulation and toxicity of Aβ peptides, mutant huntingtin, and α-synuclein. The most exhaustive characterization of the pharmacodynamical basis of their neuroprotective activity is obtained with lithium that stimulates the formation of autophagosomes through the blockade of phosphoinositol turnover and IPC misfolding by preventing its interaction with PrPSc templates. On the basis of encouraging results, obtained in silico or in experimentally infected rodents, a number of drugs, whose chronic administration was considered ethical, were tested as inhibitors of the PrPC-PrPSc transition. One of the first strategies, which has been pursued to prevent PrPC-PrPSc conversion or stimulate PrPSc removal, employed natural and synthetic polymers endowed with PrPSc and misfolded PrPC binding ability, acting as chaperone-like agents (Teruya and Doh-Ura, C-PrPSc interface, by virtue of its planar three-dimensional, resulted in neuroprotection in cellular and animal models of TSEs (Forloni et al., in vitro (Forloni et al., C, administered by intravenous administration (Mead et al., Sc occurs. Thus, diagnosis and treatments start when the prion burden in the brain and neuronal death are already beyond sensitivity to any therapy (Forloni et al., Four decades have passed since the first formulation of the prion hypothesis, and an apparent scientific heresy has become a major milestone for the biology of living beings and the pathology of some sporadic, familial, and infectious neurodegenerative disorders of humans and animals (Prusiner, All authors contributed to the design, draft preparation, reviewing, and editing of the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by IRCCS Ospedale Policlinico San Martino Ricerca Corrente 2022 grant to TF.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.


Food production, along with the constant demand for higher yields, is an imperative of contemporary agricultural production. In order to accomplish this goal, it is necessary to control pests, weeds, and the causative agents of plant diseases, which implies the application of plant protection products. However, the intensive and/or unsuitable application of synthetic pesticides has led to a series of consequences for agricultural production itself and for the environment, non-target organisms, and for human health as well. The presence of resistant populations, side effects on pollinators and other beneficial organisms, and pesticide residues in food, water, and soil are just some of the consequences. The fact that in recent years the number of active substances approved for use in agricultural production is rapidly decreasing, all of the above requires continuous and comprehensive studies of the response of the living organism  to various new agrochemicals and to products of biotechnology, followed by the mandatory risk assessment of the effect on humans and animals as well as the environment. In order to minimize the use of chemical pesticides, the Special Issue “Advances in Alternative Measures in Plant Protection” intends to offer an insight into the alternative measures of plant protection in contemporary agriculture. Special Issue contains twenty-two scientific articles: sixteen original research articles and six original reviews. With these research and reviews, authors contribute to the development of new, biological ways of controlling pests in agricultural production.Gracilariopsis persica has drawn attention for its biologically active compounds which show antifungal activities against Botrytis cinerea, Aspergillus niger, Penicillium expansum, and Pyricularia oryzae on the mycelial growth. The antifungal activity is most likely associated with its phenolic compounds, such as rosmarinic, oleic, and palmitic acid and quercetin, whose antifungal activity has already been reported [Ganoderma boninense in inoculated oil palm woodblocks [Rubia tinctorum root also shows antifungal activity, alone or in combination with chitosan oligomers or with stevioside, against three Botryosphaeriaceae taxa by the inhibition of their mycelial growth. Considering the complexity of the extracts' chemical composition, the activity most likely should be ascribed to the combination of a group of them [As an alternative to synthetic pesticides for the control of plant diseases, especially for the management of plant pathogenic fungi, bioactive metabolites derived from algae/plant extracts, essential oils, and other materials can be used. The extract of seaweed reported . The antodblocks . The hyd of them . In addi of them . Phytophthora infestans (Mont.) de Bary [An important part of sustainable pest management is natural enemies. Root fungal endophytes isolated from solanaceous plant species expressed activity in the control of a late blight of potato incited by  de Bary . When it de Bary . Pseudomonas have a high potential in the control of plant pathogens. They can be used as an alcoholic extract derived from bacterial isolate, which manifests exceptional activity against some of the most common pre- and post-harvest fungal diseases [For the management of plant pathogens, as an environmentally friendly method of plant disease control, microorganisms can be used usually because of their capability of counteracting the growth. Species belonging to the bacterial genus diseases . Bacillus spp. or B. subtilis and the foliar application of Reynoutria sachalinensis, Melaleuca alternifolia, harpin αβ proteins, and bee honey. The use of these biorational products in the control of these diseases in greenhouse production has the potential to be incorporated into an integrated program for the management of the examined diseases in tomatoes [Sclerotium rolfsii, a destructive disease for many plants, including tomatoes, could be successfully undertaken using some antiseptic and disinfectant agents [Indoor agricultural production is particularly challenging due to conditions affordable for pests. As an alternative to chemical products, the control of causal agents of the major tomato diseases in greenhouse production was undertaken by tomatoes . Moreovet agents .Thymus vulgaris hydrolate showed an inhibitory effect on the weed species, including Amaranthus retroflexus (L.), Chenopodium album (L.), Portulaca oleracea (L.), Echinochloa crus-galli (L.) P. Beauv., Sorghum halepense (L.) Pers., and Solanum nigrum L., through the inhibition of the seed germination and seedling growth. T. vulgaris hydrolate had the least negative phytotoxic effect on the germination of soybean, sunflower, and maize [Plant extracts can indicate an herbicide effect as well. nd maize . Ambrosia trifida L. have an allelopathic influence on the oxidative stress parameters and phenolic compounds in maize, soybean, and sunflower. The sunflower was the most sensitive crop to A. trifida allelochemicals, maize showed a mild sensitivity, while the soybean did not demonstrate sensitivity [Metarhizium robertsii (2693) causes the death of 73% Tenebrio molitor larvae, but also significantly increases the maize root length [Understanding the plant–plant interaction is also one of the Advances in Alternative Measures in Plant Protection. The phytotoxic substances released by sitivity . The physitivity . Furthert length . Fusarium proliferatum, the addition of low concentrations of Se could improve conventional fungicides and decrease their side effects [Erysiphe alphitoides (Griffon and Maubl.). Evaluating the influence of the irrigation levels (overhead sprinklers) on the damage caused by powdery mildew to Quercus petraea in a nursery setting, it was showed that controlling the irrigation rate can become an effective component of integrated protection strategies against this pathogen [Tetranychus urticae Koch 1836 could be successfully controlled by eco-friendly approaches, such as black soap and detergents, without having a negative impact on predators [Aside from plants, microorganisms, insects, and nematodes, micronutrients have to be considered as a source of bioactive compounds. Recently, some micronutrients are recognized as an alternative to synthetic fungicides. Due to the influence of Selenium on the inhibition of the development of a  effects . One of pathogen . The negredators . Photorhabdus spp. as biocontrol agents against a broad range of insect pests [Furthermore, the authors review the latest knowledge regarding the importance of plant-parasitic nematodes in agricultural production  and genect pests . As an ect pests .Finally, the utility of biocontrol agents composed of microorganisms, plant-based compounds, as well RNAi-based technology has to be emphasized , not onlAs Guest Editors, we are grateful to all the authors for choosing this Special Issue to publish their research. We hope that together we have contributed to the understanding of the importance of alternative measures in plant protection and opened up new possibilities for the improvement of this field.