Editorial on the Research TopicRecent advances in our understanding of NEC pathogenesis, diagnosis, and treatmentNecrotizing enterocolitis is a leading cause of death among premature infants, and despite research spanning over six decades, the pathogenesis is still not completely understood. The onset of NEC can be rapid and unpredictable, with clinical signs such as abdominal distension and bloody stools, progressing to fulminant bowel necrosis and death within hours. Even though the clinical and pathological descriptions of NEC were first described many decades ago, the management options have not progressed significantly and continue to be supportive care, such as cessation of feedings, intravenous fluids, antibiotic administration, and, in some cases, surgical bowel resection. Although treatment options for NEC remain limited, one effective preventative strategy is the administration of human milk. Recent advances in identifying the precise nutrients in human milk shed light on its bioactive components and their impact on the intestine. In recent years, several studies have highlighted the benefit of using prebiotics and probiotics as additional preventative options for NEC. Clinical studies focused on diagnostic tools such as using serum biomarkers or big data and artificial intelligence may pave the way for earlier detection to minimize disease progression, avoid the negative impact on other organ systems, and improve the poor neurodevelopmental outcomes associated with NEC. The primary objectives for this topic were to focus on recent advances in our understanding of NEC pathogenesis, new diagnostic strategies such as biomarkers and artificial intelligence, and explore new therapeutic options for treating this devastating disease.This editorial highlights recent developments in the underlying pathogenesis of NEC, including the use of animal models, bench-to-bedside approaches, and machine-learning approaches for diagnostic purposes. This series of publications comprises state-of-the-art reviews, meta-analyses, and original research.Singh et al. discuss bench-to-bedside approaches to understanding NEC pathogenesis, including a summary of the immunological status of infants with NEC and several defense mechanisms that become impaired in prematurity and NEC. The article by Klinke et al. describes the function of neutrophil extracellular traps (NETs) in necrotizing enterocolitis. NETs are released by neutrophils after contact with pathogens, and studies have shown that NET release is seen in mice and infants with NEC. This review discusses the various roles that NETs play in NEC, and specifically, that excessive NET formation can lead to hyperinflammation, contributing to disease pathogenesis. The manuscript by Bautista et al. is a state-of-the-art review describing the in vivo models of NEC. This review focuses on the descriptions of the different animal models, the phenotypic considerations, the strengths and weaknesses of each model, and how they recapitulate the human disease in vivo.Sami et al. describe the role of human milk nutrients in preventing NEC. Preterm infants represent the most fragile population susceptible to developing NEC. Shortly after birth, their intestines face a series of challenges, including ongoing maturation, dietary demands driven by high nutritional needs, and the establishment of their gut microbial communities. Human milk is instrumental in shaping the gut microbiome, and this article summarizes the components of human milk, including lactoferrin, human milk oligosaccharides, dietary amino acids, vitamins, trace elements, and the interactions of these nutrients on the gut microbiota in NEC.Sajankila et al. and a meta-analysis evaluating probiotics to prevent NEC in premature infants by Zhou et al. are helpful updates on this important aspect of preventing NEC. The review article discusses the various probiotic formulations, including single-strain formulations vs. multiple-strain formulations. In the meta-analysis, which included 27 randomized controlled trials with several different treatment interventions, they found that Lactobacillus rhamnosus GG and bovine lactoferrin can significantly reduce NEC incidence in preterm infants. While some questions are yet to be answered in terms of optimal probiotic combination and dosage, and there are concerns about sepsis related to non-medical grade probiotic use in premature infants, Sajankila et al. provided hope for the future with a discussion about the next generation of “designer probiotics,” which will need detailed study and evaluation.A review article of the current probiotic therapies for NEC by Bethell et al. focus on recent advances surrounding NEC diagnosis, imaging modalities, and a discussion on the surgical approach to NEC. The state-of-the-art review on machine learning and artificial intelligence in NEC by McElroy and Lueschow explores using machine learning methods as a biomarker for NEC diagnosis, including using stool microbiome data and clinical demographics to predict infants at the highest risk for NEC. The limitations and pitfalls of our current use of machine learning and artificial intelligence should not dissuade us from utilizing these powerful tools for earlier diagnosis of NEC and improving outcomes.Manohar et al. review the impact of the gut-brain axis on the long-term complications of NEC. NEC is associated with impaired long-term neurodevelopmental outcomes, including a higher incidence of cerebral palsy and cognitive deficits. The authors discuss the ways in which neurodevelopmental impairment is assessed, including cognitive developmental tests, as well as magnetic resonance imaging, and regions of the brain affected by NEC. This review discusses how the gut-brain axis plays a role in the neurodevelopmental impairment seen in NEC and how the microbiome, the innate immune system, and various neurotransmitters play a role in the pathogenesis of NEC-related neurodevelopmental impairment.Cuna et al. discuss the use of early antibiotics and the risk of NEC in premature infants and mouse models of the disease, highlighting the mechanistic work evaluating the effects of early and prolonged antibiotic exposure in neonatal mice and piglets on the gut microbiome and intestinal immunity.Early randomized controlled trials in the 1970s–1990s demonstrated that prophylactic antibiotics decreased the risk of NEC. However, more recent retrospective studies suggest prolonged antibiotic exposure is associated with increased risk for NEC. Mackay et al. report a pilot study using an untargeted aptamer-based proteomics assay as a biomarker discovery for NEC. They found ten serum proteins that could differentiate infants with NEC compared to controls with high sensitivity on a small sample volume. We look forward to further detailed study in this area.Finally, research topic has inspired significant discussions in the field of NEC research. Although more studies are desperately needed in this field, it is exciting that new developments are on the horizon.This


Patients suffering from schizophrenia have a higher risk of premature death. An unhealthy lifestyle contributes to increased risk of cardiovascular diseases, metabolic syndromes, suicides among them. In addition to the use of selected therapy with a restriction metabolic risk has become important to influence non-pharmacological factors such as proper diet, introducing the principles of a healthy lifestyle. A diet rich in fiber, the DASH diet, the Mediterranean diet may become beneficial in terms of lowering parameters metabolic, cardiovascular and immune related to premature mortality in schizophrenia.Patients suffering from schizophrenia have a higher risk of premature death. An unhealthy lifestyle contributes to increased risk of cardiovascular diseases, metabolic syndromes, suicides among them. In addition to the use of selected therapy with a restriction metabolic risk has become important to influence non-pharmacological factors such as proper diet, introducing the principles of a healthy lifestyle. A diet rich in fiber, the DASH diet, the Mediterranean diet may become beneficial in terms of lowering parameters metabolic, cardiovascular and immune related to premature mortality in schizophrenia.The objective of this study was to evaluate the influence of the lifestyle on the metabolic parameters in schizophreniaIn our study, we assessed the influence of diet, nutritional knowledge and lifestyle on parameters of metabolic syndrome  in patients with schizophrenia.In the results we have found positive co-relations between unhealthy diet and lifestyle and lack of knowledge on proper nutrition and increased parameters of metabolic syndrome.Dietary intervention may become one of the therapeutic goals in schizophrenia.None Declared


Recent publications on gall formation induced on the leaves of dicotyledonous flowering plants by eriophyoid mites (Eriophyoidea) and representatives of four insect orders  are analyzed. Cellular and molecular level data on the stimuli that induce and sustain the development of both mite and insect galls, the expression of host plant genes during gallogenesis, and the effects of these galling arthropods on photosynthesis are considered. A hypothesis is proposed for the relationship between the size of galls and the volume of secretions injected by a parasite. Multistep, varying patterns of plant gene expression and accompanying histo-morphological changes in the transformed gall tissues are apparent. The main obstacle to better elucidating the nature of the induction of gallogenesis is the impossibility of collecting a sufficient amount of saliva for analysis, which is especially important in the case of microscopic eriophyoids. The use of modern omics technologies at the organismal level has revealed a spectrum of genetic mechanisms of gall formation at the molecular level but has not yet answered the questions regarding the nature of gall-inducing agents and the features of events occurring in plant cells at the very beginning of gall growth. Among the herbivorous arthropods, there are a considerable number of species from six orders of insects , and acariform mites , that induce the growth of galls or cecidia—specialized structures that develop on various organs of flowering plants, especially leaves. Galls develop from the tissues of the host plant and provide the phytophagous parasites inside them with nutrition and protection from predators and adverse external conditions. The estimated total number of galling insect species ranges from 13,000 to 211,000 . The abiThe aim of this review is to discuss the published data on the molecular aspects of gall formation in flowering plants initiated by the eriophyoid mites , as well as representatives of four insect orders . Information on the other three groups of galling arthropods listed above  could not be included because the literature lacks molecular genetic data on gallogenesis induced by these taxa. The present paper differs from a recent review on the molecular aspects of insect galling  in severIn this review, after a brief survey of the functional diversity of leaf galls, the stimuli that are primarily responsible for the induction of gall development, the multistep nature of gallogenesis, links to plant gene expression, and finally the relationship between photosynthesis and leaf gallogenesis are considered in sequence. Herein, we do not discuss the symbiosis of galling arthropods and plants but focus mainly on genetic, biochemical, and histo-morphological differences between several relatively completely investigated model gall systems. The galling arthropods essentially act as “ecosystem engineers” . They syLeaf galls on flowering plants are highly diverse in appearance, shape and color . Many atBiorhiza pallida  (Cynipidae) on the leaves of the oak Quercus robur  (Fagaceae). Later, a phenomenon was discovered that is seemingly an alternative to endopolyploidy or polyteny in gall cells. The typical nutritive tissue of some galls induced on the leaves of the tree Copaifera langsdorffii  (Fabaceae) by a Neotropical gall midge fly  contained anucleated cells [Tibouchina pulchra  (Melastomataceae), during whose development the cell nuclei of the nutritive tissue are lost [There is an important similarity in gallogenesis in the eriophyoid mites , flies of the family Cecidomyiidae (Diptera), wasps of the family Cynipidae (Hymenoptera) and many butterflies (Lepidoptera). During the galling process, stimulation due to parasite feeding causes the formation of typical nutritive tissue rich in carbohydrates, proteins and/or lipids. It lines the gall chambers and serves as a direct food source for the insect and mite parasites inside ,25. Hessed cells . Also, aare lost . In the The functional diversity of galls induced by representatives of various families of the order Hemiptera deserves special attention. Typical nutritive tissue is not formed in galls induced by aphids (Aphididae) and jumping plant lice (Psyllidae) ,31,32. IThe typical nutritive tissue, which appears in the early stages of gall formation induced by eriophyoid mites and insects, arises from the transdifferentiation (metaplasia) of leaf epidermal cells and/or the leaf parenchyma of the host plant ,37,38. TThe first interactions of gall-forming parasites with their host plants occur in different ways, depending on the arthropod group . In caseThe minute eriophyoid mite (body length: 100–300 μm) attacks a single epidermal cell of the host plant by piercing it with stylets and injecting saliva. The damaged cell dies, but the gall-forming effect then spreads to the adjacent leaf area, probably through plasmodesmata and the conducting system ,37,39. AIn the case of gall wasps of the family Cynipidae (Hymenoptera), the primary gall-inducing signal is not the feeding of sexually mature individuals, but the laying of eggs in the leaf tissue . In the Caloptilia cecidophora  , which infests the leaves of the tree, Glochidion obovatum  (Phyllanthaceae), the first and second instars are leaf-miners. Their feeding produces galleries within the leaf lamina, and they do not have any gall-inducing properties. Gall induction is initiated by the third instar, which releases a cecidogenic substance that has not yet been analyzed [Caloptilia, are exclusively leaf-miners and they do not induce galls. Guiguet et al. [Unusual cases are known when insect larvae acquire the ability to initiate galls only after a few molts. For example, in the micromoth, analyzed . This let et al.  reasonabThe salivary glands of a large number of species from all orders of gall-forming insects were demonstrated to contain the phytohormones, auxin and cytokinins, that control normal plant development ,43,44,45B. pallida. In addition, in the genome of the mite, Fragariocoptes setiger  (Phytoptidae), which causes galls on strawberry leaves, phytohormone genes and any other genes that could have entered the mite genome from the plant as a result of horizontal transfer were not found [B. pallida or the mite F. setiger, in which gallogenesis is initiated by a different but yet unexplained mechanism. The leading role in the process of gallogenesis caused by arthropods is now attributed to endogenous cytokinins . On the ot found . TherefoSince many species of eriophyoid mites and insects have symbiotic relationships with various bacteria, there is reason to suggest that along with their saliva, the host plants also receive bacteria (or their metabolic products) that cause the formation of galls ,21,49. IDiplolepis rosae  and B. pallida, which induce galls on the rose, Rosa canina , and common oak, Quercus robur, respectively, has been performed [Important work directed towards the understanding of the molecular mechanisms involved in the initial steps in cynipid and aphid gall induction has been published recently. The transcriptome analysis of the venom glands and ovaries of the cynipid wasps erformed . Some maerformed . Hormaphis cornu  (Aphididae) and witch hazel Hamamelis virginiana  (Hamamelidaceae) revealed a novel aphid secretory protein that was named ‘BICYCLE’. Bicycle genes are strongly expressed in the aphid’s salivary glands [bicycle genes are strongly expressed not only in the salivary glands of a second galling aphid, Tetraneura nigriabdominalis , but also in the salivary glands of a non-gall forming aphid, Acyrthosiphon pisum , as well as in the non-gall forming generation of Hormaphis cornu [Recent molecular research on the interactions between y glands . The samis cornu . Those ris cornu : (p. 1).Eriophyes padi , and bird cherry, Prunus padus  (Rosaceae) [The most comprehensive experimental data on the multistep character of arthropod gall formation were obtained for the combination of the eriophyoid mite, osaceae) . The autosaceae) . The depA multistep, cumulative effect of gall-inducing agents can also be hypothesized in the case of the development of leaf galls induced by flies of the family Cecidomyiidae ,25. By rThus, the primary inducing stimulus produced by the arthropod parasite, which we discussed in There is a reasonable body of published data on gene expression in developing gall tissues induced by the representatives of several groups of herbivorous arthropods on the leaves of various flowering, dicotyledonous plants. Fragariocoptes setiger induces galls on the leaves of the green strawberry, Fragaria viridis  (Rosaceae). During the initiation and growth of young galls, the increase in the expression of the CYCD3 and CYCB1 cell cycle genes in their tissues is associated with active cell proliferation [KNOX and WOX. These two genes are the universal regulators of normal plant development [FviYAB2  and FviREV  [Dactulosphaira vitifoliae  (Phylloxeridae) [The eriophyoid mite feration . By the elopment . Finallygulator) . At the xeridae) . In thisAmphibolips michoacaensis Nieves-Aldrey et Maldonado, 2012, B. pallida, Dryocosmus kuriphilus Yasumatsu, 1951) when they deposit eggs in the meristematic tissues of the leaves of oaks (genus Quercus) and some other trees of the beech family (Fagaceae) has been studied [The development of leaf galls induced by several wasp species of the family Cynipidae  and were later found in many other plants [Particularly noteworthy is a study in which gene expression was analyzed during the development of both participants in a “parasite–host dialogue” involving larval atterns” : (p. 19)r plants . Nodulinr plants ,64,65. Tr plants . B. pallida, the expression of PCWDE genes, which code for plant cell wall degrading enzymes, including six pectin/pectate lyases, four cellulases and four rhamnogalacturonan lyases, occurs [In the young larvae of , occurs . Enzymes, occurs  (p. 1). , occurs ,68; the , occurs ,70,71. IDryocosmus quercuspalustris  on the leaves of the red oak, Quercus rubra . The analysis was carried out during a single stage of gall formation, specifically a young actively growing gall with a feeding larva in the internal gall chamber (approximately 5–6 days after oviposition). For the first time, not only were significant differences (28%) revealed between the transcriptomes of the whole gall and the adjacent leaf tissue, but also between the outer gall tissue, which performs a predominantly protective function, and the internal tissue of the gall, on which the parasite feeds. In general, the transcriptome of the outer tissue of the gall was more similar to the transcriptomes of the tissues of leaf buds, twigs and reproductive structures of oak than to the transcriptome of normal leaf tissue. In this study, as well as in the work on galls initiated by B. pallida [ENOD genes was demonstrated in both the inner and outer tissues of young galls. A very recent study ,54 revea pallida , the actSchlechtendalia chinensis  (Aphididae), and sumac, Rhus javanica  (Anacardiaceae), in the early stages of gall formation, increased expression of the KNOX genes occurs [Dactulosphaira vitifoliae, and coastal grape, Vitis riparia  (Vitaceae), the genes associated with the development of reproductive structures, flowers and fruits, including LFY, AG, SEP, SHP, CAL and FUL, were activated [Leaf galling is also induced by some hemipterans. In the case of a parasite–plant host pair, the aphid, s occurs . In anotctivated . Rhopalomyia yomogicola  —Artemisia montana  (Asteraceae), Caloptilia cecidophora —Glochidion obovatum (Phyllanthaceae) and Borboryctis euryae  —Eurya japonica  (Pentaphylacaceae). Their molecular data were discussed in combination with the molecular data obtained by the same authors for the pair Schlechtendalia chinensis—Rhus javanica [CLE44, BAM3 and WOX [B. euryae on E. japonica showed no activation of genes  associated with the development of reproductive structures, whereas the galls formed by the other three parasite–host pairs showed the activation of genes involved in floral organ development. Takeda et al. [An attempt to identify the genes involved in leaf gallogenesis involving the simultaneous use of several parasite–host pairs was recently undertaken by Takeda et al. ,72. Tranjavanica , which w and WOX . In all Overall, there is relatively little information on the analysis of gene expression changes during leaf galling induced by arthropods. Nevertheless, due to gene expression, galls cease to be typical leaf structures and acquire similarities to other plant organs, but each time in a different way, depending on the gall-forming plant–arthropod combination. Thus, each gall seems to be a unique structural mosaic that includes morphological elements characteristic of the normal development of vegetative and generative organs.Dryocosmus quercuspalustris on the leaves of the oak, Quercus rubra [In the previous section, it was noted that, during gall development induced by representatives of the orders Diptera, Hemiptera, Hymenoptera and Lepidoptera, leaf gall tissues of host plants usually show a significant downregulation of the genes associated with photosynthesis ,59,72. Tus rubra . The intLeaf galls induced by the eriophyoid mites have not yet been studied with respect to the expression of their photosynthesis-related genes. Nevertheless, in the case of mite gallogenesis, there are morphological and physiological data that indicate significant destruction of the photosynthetic apparatus , and inhibition of the process of photosynthesis ,75. At tSmicronyx smreczynskii  or Smicronyx madaranus  , and field dodder, Cuscuta campestris  (Convolvulaceae). Field dodder is an obligate parasitic plant with a very low chlorophyll content and very weak photosynthetic activity. The weevil larvae significantly enhanced the photosynthetic activity in the spherical galls induced by them on the dodder shoots and they therefore acquired nutrient-rich shelters [In general, gall-forming insects and eriophyoid mites have a similar suppressive effect on photosynthesis in the leaf galls induced by them on plants. A notable exception is represented by two unusual galling insect–host plant pairs consisting of the gall-forming beetle weevils, shelters ,78. ThisThe molecular mechanisms involved in the suppression or enhancement of photosynthesis by gall-forming insects and eriophyoid mites currently remain largely unknown. These phytoparasitic arthropods inhabit the leaves of plants and it is difficult to maintain such parasite–tree systems in the laboratory to study the mechanisms involved in the suppression or enhancement of photosynthesis in gall tissues . Among tThe gall-inducing ability of representatives of different phylogenetic lineages of arthropods in representatives of different phylogenetic lineages of plants has arisen repeatedly and independently. The primary inducing stimulus for leaf gallogenesis in most cases is oviposition by galling wasps (Hymenoptera), the larval feeding of flies (Diptera) and micromoths (Lepidoptera), and the feeding of the adult females of Hemiptera , eriophyoid mites (Eriophyoidea) and tenuipalpids . The molecular mechanisms underlying these processes are still unclear. The latest advances in this field involved the detection of the active expression of several secreted proteins  in the salivary glands of gall-forming aphids ,52, as wChanges in the pattern of expression of many genes in gall cells in the course of their development, as well as differences in gene expression between the tissues of galls and normal leaves, have been demonstrated in all the parasite–host systems studied. The use of modern omics technologies at the organism level suggests the involvement of numerous molecular genetic mechanisms in gall formation, but so far has not answered the question of the nature of gall-inducing agents and the features of events occurring in plant cells at the very beginning of gall growth. Breakthroughs in this area can be expected from the use of single cell (SC) technologies. Comparative SC proteomics, transcriptomics and metabolomics could reveal the elements of the functioning of the individual cells in the tissues of gall-forming arthropods , as well as in plant cells directly exposed to these factors. Such studies would contribute to the revelation of the entire sequence of stages of gall development. A general feature of arthropod gall development on plant leaves is the suppression of photosynthesis. It has also been shown that during leaf galling induced by the representatives of several orders of insects, genes associated with the development of reproductive structures are activated. Such data are not yet available for mite gallogenesis. The formation of arthropod galls appears to be a multistep process, all stages of which are still poorly understood. It appears that there is no universal scheme of gallogenesis, and in some cases, some steps may be skipped, e.g., the absence of the formation of typical nutritive tissue in galls induced by insects of the hemipteran families, Aphididae and Psyllidae. In the future, it will be important to conduct molecular analyses of both the initial and advanced stages of gall development induced by representatives of various groups of arthropods, including eriophyoid mites, paying special attention to the very early stages of gall growth, as well as to the mechanisms of nutritive tissue differentiation. This research would potentially yield huge economic benefits because many widely grown tree crops, such as lychees and walnuts, are currently affected by galling.In conclusion, it should be noted that recently generated data indicate a high diversity of gall formation modes across various groups of gall-forming insects. At the same time, the data on insects are incomparably more complete than for mites; in particular, data on the molecular aspects of galling are available only for one mite–plant system ,48. The Brevipalpus, Capedulia, Dolichotetranychus, Larvacarus, Obdulia, Obuloides, Phytoptipalpus) out of about 40 tenuipalpid genera live in bark and leaf galls [Eriophyoid mites have as long a history of association with plants as insects. This superfamily is an ancient lineage of chelicerates related to worm-like, soil nematalycide mites, which adopted phytophagy even before the appearance of gymnosperms and flowering plants. Some gall-like structures on the fossils of extinct plants, attributed to the activity of these mites, date back to the Late Paleozoic ,84,85,86af galls ,89,90,91


Stroke is among the most prevalent causes of disability and is the second leading cause of death worldwide in Western countries . It is aHowever, most of the existing animal models of cerebral ischemia or intracerebral hemorrhage are only imperfect representations of human stroke. Most stroke models are induced in healthy adult rodents instead of in elderly individuals with comorbidities, and the complex organization of the white matter is not comparable in rodent and human brains. Moreover, in most cases, best-practice guidelines for experimental models of stroke are not fully established, reflecting the failure of several neuroprotective and cellular therapies. IJMS, we encouraged the submission of review and original articles focusing on the most advanced and intriguing pathophysiological mechanisms of stroke and on pre-clinical experimental models that are highly relevant for the implementation of clinical studies.For this Special Issue of Regarding possible pathophysiological mechanisms, with the advent of MT, occlusive clots have been made available for histological analysis, providing insights into the composition, structural organization and embolic origin . In the present Special Issue, Essig et al., by analyzing 37 human thromboemboli obtained from acute ischemic stroke patients during MT, find that neutrophils are the main cellular component of cerebral thromboemboli. Neutrophils accumulate in the border region of fibrin-rich structures, thus indicating their possible interaction with distinct structural systems. They also find web-like NETs in 35 out of 37 thromboemboli, in varying amounts within fibrin-rich areas. They conclude that stroke etiology, age, and present oral anticoagulation are associated with morphological patterns and the number of neutrophils, providing insights into the knowledge of clot structural stability and thrombolytic resistance .To explore the pathophysiology of cerebral amyloid angiopathy (CAA), which is a major cause of spontaneous intracerebral hemorrhage and an important contributor to cognitive decline in elderly patients, Gatti et al. present a review on the key pathophysiological mechanisms of disease and the experimental models that facilitate an understanding of disease drivers and potential therapeutic targets . SeveralRegarding possible therapeutic targets and neuroprotective strategies for stroke, Bhattacharya et al. present a review on soluble receptors, which are known to be intriguing concentration-dependent factors with a role in cytoprotection and neuroinflammation . The autIn their review, Chung Yang Yeh et al. review the available results of recent clinical trials for acute ischemic stroke treatment with both a thrombolytic agent and MT, and evaluate trials on neuroprotective agents, with particular attention paid to the role of voltage-gated potassium channel Kv2.1 for neuroprotection . The autWan Leung et al. investigate the neuroprotective effects of Emodin, a traditional Chinese medicinal herb with antioxidative and protective properties in ischemia/reperfusion injury in MCAO rats and PC12 cells exposed to oxygen-glucose deprivation . Emodin In another experimental model, distal middle cerebral occlusion (dMCAO) type 2 diabetes mellitus (T2DM) stroke mice and cultured human brain microvascular endothelial cells (HBMECs) subjected to hyperglycemic and inflammatory injury are studied by Jiang et al. . This stMelia-Sorolla et al. present a review paper on the advantages and limitations of the utilization of pigs, which are large mammals with interesting brain characteristics and wide social acceptance, as a possible alternative animal model to rodents in the study of the pathophysiology of stroke . Pigs, iLastly, Hatekayama present a review of cell therapy clinical trials and an update on the use of polarized cell therapies based on protective cell phenotypes, which are currently employed in pre-clinical studies, to facilitate functional recovery after post-reperfusion treatment in patients with ischemic stroke . In partIn conclusion, this Special Issue, entitled “Pathophysiology and Treatment of Stroke: Present Status and Future Perspective”, highlights a number of research directions with regard to understanding pathophysiological mechanisms and identifying therapeutic targets in order to reduce the significant burden of stroke.


After a period of unprecedented progress against malaria in the 2000s, halving the global disease burden by 2015, gains overall in sub-Saharan Africa have slowed and even reversed in some places, beginning well before the COVID-19 pandemic. The highly effective drugs, treated nets, and diagnostics that fueled the initial progress all face some threats to their effectiveness, and global funding to maintain and increase their use over the long term is not guaranteed. Malaria vaccines are among the most promising new interventions that could accelerate the elimination of malaria. Vaccines are still in early stages of rollout in children, the age group  that has been the focus of malaria strategies for a century. At the same time, over the past decade, a case has been made, based largely on evidence from verbal autopsies in at least a few high-transmission areas, that the malaria death rate among adults has been greatly underestimated. Could vaccinating adults help to bring down the adult malaria mortality rate, contribute to reduced transmission, or both? A randomized trial of a malaria vaccine is proposed in Sierra Leone, a highly endemic setting, to shed light on this proposition. Malaria maintains an intractable hold on much of sub-Saharan Africa. The World Health Organization’s (WHO) 2022 World Malaria Report  tells a During this period, and historically, the emphasis on malaria control has been on young children and pregnant women, considered the most vulnerable populations in high-transmission areas. Those who survive childhood and continue to be exposed to infectious mosquito bites develop a partial immunity that provides some protection against severe illness and death, but not necessarily against reinfection. Such immunity has historically been assumed to persist throughout life as long as one resides in a malaria-endemic region. However, a growing body of evidence suggests, at least in some highly endemic areas, a rising death rate beginning in later middle age, resulting in substantial malaria mortality among adults , 3.This perspective explores the question of whether it is worth considering, and testing, the use of malaria vaccines in adults to materially reduce adult morbidity and mortality and contribute to reducing malaria transmission.The 2000s marked the beginning of a promising new era in malaria control: artemisinins replaced failing chloroquine, and the concept of co-formulated anti-malarials—mainly artemisinin-based combination therapy (ACT)—was accepted globally ; chemoprThe cost of providing the full suite of interventions—treatment, prevention, and vector control—would be high, but establishment of The Global Fund to Fight AIDS, Tuberculosis and Malaria in 2002 and The US President’s Malaria Initiative in 2005 began to deliver greater funding than had been available ever before. The Bill & Melinda Gates Foundation entered the picture in 2000, supporting operational research and capacity development . Global coordination was provided by the Roll Back Malaria Partnership, launched in 1998 by UN agencies and bringing together a wide range of public and private sector actors. The improved tools, substantial financing, and global resolve fueled optimism that the world was on the verge of controlling one of the great scourges of humankind, based on a comprehensive strategy of physical and chemical prevention, testing, and treatment, continuously until elimination and ultimately, eradication.The result of the expanded and improved efforts was impressive: the estimated worldwide age-standardized malaria mortality rate (driven mostly by childhood deaths) was halved between 2000 and 2015, from 30/100,000 population at risk to 15/100,000. The incidence rate dropped from about 80/1000 at risk to about 60/1000, with the gains concentrated among children . ThroughThe reasons for lack of continued progress in Africa are likely due to a combination of lower-than-expected effectiveness of the interventions, suboptimal implementation, and insufficient funding . With grIn addition, the effectiveness of interventions in current use are threatened by:The rise and spread of artemisinin resistance mainly in Asia, but now seen in African settings .Mosquitoes resistant to pyrethroids, the mainstay insecticide used on ITNs .Anopheles stephensi, a mosquito vector that more efficiently transmits malaria in urban environments, is resistant to most insecticides, is an efficient malaria transmitter, and has a high degree of behavioural adaptability [The introduction and spread of tability .False negative rapid diagnostic tests due to histidine-rich protein 2 (HRP2) deletions .Substandard and counterfeit drugs and insecticides and inadequate systems of product surveillance.In addition to these specific threats, the wide and varied current and future effects of climate change loom over malaria control prospects.Research and development continue for new anti-malarial drugs, insecticides, and strategies, but the only wholly new class of intervention that has become widely available since 2000 are vaccines. The first two approved vaccines, both targeting sporozoites, have become available only recently and are recommended for use in children under 5 years old . Should The American Journal of Tropical Medicine and Hygiene [There is no argument that malaria kills more children than any other age group, but the presumption that malaria mortality remains uniformly low throughout the rest of the lifespan has come increasingly into question. A 2020 perspective piece in  Hygiene  summariz Hygiene  was seenNationally representative cause-of-death reporting for Sierra Leone for the years 2018–2020 was published in 2021 [In summary, the existing evidence suggests that there may be a substantial risk of adult morbidity and mortality from malaria in high-transmission settings like Sierra Leone, but the evidence can be challenged. With the recent availability of two approved vaccines and others in late-stage trials, a case can be made for field trials in adults with the intention of reducing mortality, and the possible added benefit of reducing transmission to some degree. In addition to the immediate benefits of an effective malaria vaccine, a vaccine probe trial could provide high-quality evidence of the burden of malaria mortality in African adults and inform the accuracy of VA-attributed malaria deaths.Mass malaria vaccination, rather than vaccinating only children below age 5, has the potential to reduce morbidity and mortality among those vaccinated, both young and old. Under the right conditions, it also may: (1) materially reduce community transmission by reducing the prevalence of circulating gametocytes throughout the population, and (2) reduce the evolution and spread of drug resistance to artemisinins by reducing their use and thus exposure of malaria parasites to them. If vaccination reduces the numbers of infections, the infectious reservoir could be greatly reduced. Individuals with asymptomatic, low-parasitaemic infections, including most infected adults, far outnumber those with symptomatic higher parasitaemias, and as they are unlikely to be treated, their infections may persist for years. Even though they may carry fewer gametocytes per person, they make up a large proportion of the infectious reservoir . AnotherThe gains of the 2000s in malaria control have plateaued in sub-Saharan Africa, in particular, and are in danger of being eroded because of threats to the effectiveness of the core interventions. At the same time, malaria mortality among adults may be underestimated, especially in high-transmission areas. The recent availability of approved malaria vaccines presents an opportunity to better understand adult malaria mortality and to find out whether use of vaccines beyond children could have a material effect on reducing transmission, with the potential to re-energize malarial control efforts. A large-scale trial of malaria vaccines in adults living in high-burden areas is a first step toward generating this knowledge.


Editorial on the Research TopicReviews in reproductive epidemiology: 2022Frontiers in Reproductive Health under the Research Topic Reviews in Reproductive Epidemiology aimed to bring together the most recent evidence synthesis on pressing topics of reproductive epidemiology, to highlight current advances or gaps in knowledge and to propose new associations and/or effective interventions. Summarizing all available evidence in a topic highlights the methodological differences between studies that answer the same question and incorporate quality considerations in the interpretation of the totality of evidence.Under the current landscape of the exponential increase in primary studies of all study designs in reproductive epidemiology on a wide range of reproductive health issues as well as the changing access to reproductive health services, summarizing and interpreting current evidence is of utmost importance for clinicians, policy makers, public health officials and individuals. This special collection of Krokidi et al. summarized available evidence on an important issue related to the effectiveness of health education interventions about uptake, acceptance and awareness on Human Papilloma Virus (HPV) vaccination among people 9–29 years old in India, in light of the new indigenous HPV vaccine . In their systematic review, after searching three databases up to July 2022, seven studies conducted in India were included. Authors concluded that health education and promotion interventions such as audiovisual presentations and workshops proved to be effective in increasing uptake, awareness, and acceptance of the HPV vaccine, while the barriers included, among others, the cost of the vaccine, the lack of awareness, and cultural issues. Another noteworthy conclusion was that males and marginalized populations were underrepresented whereas the involvement of various stakeholders was proven beneficial, and it is highly recommended.This Research Topic consists of four reviews covering topics such as HPV vaccination, attributes of personhood, bacterial vaginosis treatments and male infertility. Hughes and Hughes proposed a framework, based on the biological discrete events and processes spanning pre-fertilization and prenatal development, which implies that personhood should be incrementally attributed, and societal protections should be graduated and applied progressively across the pre-birth timespan (Hughes and Hughes). They provide a novel perspective for considering the biological and ethical aspects of attribution of personhood and individualization of potential humans.In a special and very interesting assessment, Abbe and Mitchell systematically appraised ongoing and potential treatment and prevention strategies for bacterial vaginosis (BV) (Abbe and Mitchell). BV, which is a common cause of vaginitis worldwide, is associated with serious adverse reproductive outcomes including preterm birth, sexually transmitted infections and pelvic inflammatory disease. Given that antibiotics (such as metronidazole and clindamycin) are the only FDA approved treatment and that 50%–80% of women experience recurrence within a year of completing the antibiotic treatment, implementation of new strategies for treatment and prevention is crucial. Thus, the review presents current options of BV management such as smoking cessation, condom use and hormonal contraception, additional strategies considered by many people such as dietary modifications, non-medical vaginally applied products, and treatments from medical practices outside of allopathic medicine. Also, other less studied but promising treatments are presented including probiotics, vaginal microbiome transplantation, pH modulation, and biofilm disruption.In a comprehensive assessment, Kaltsas et al. tried to answer the question whether varicoceles in men with non-obstructive azoospermia (NOA) should be operated or not . The prevalence of this condition in the general population is 1% while it can be as high as 15% in the population of infertile men. Among non-obstructed azoospermic men, 4%–14% have varicocele. Several studies have been performed to assess whether varicocele repair may contribute to the reappearance of spermatozoa in semen. This hypothesis is also contradicted by the small number of spontaneous pregnancies after such interventions. The findings of this review conclude that varicocelectomy in NOA-men may have a beneficial effect on spermatogenesis and the reappearance of motile spermatozoa in the ejaculate. In addition, varicocelectomy increases sperm retrieval rate in men who remain azoospermic after the surgery. However, there is a possibility that NOA-men who are positive for spermatozoa in the semen samples post-varicocele repair will relapse into azoospermia. As a result, NOA-men should be advised to freeze spermatozoa appearing the semen post-varicocelectomy. Finally, this review highlighted that the performance of varicocelectomy in NOA-men and subsequent ICSI procedures using testicular spermatozoa may increase pregnancy and live birth rates in couples without female infertility factors.Finally, Overall, the four contributions published in this Research Topic provide valuable information on the timely topics of HPV vaccination, origins of personhood, sexually transmitted diseases and male infertility. Given the representation of broad reproductive epidemiology research areas, this Research Topic highlights recent advances of the distinct characteristics of each topic, whilst emphasizing important directions and new possibilities for future inquiries. The Research Topic also highlights the limitations that the authors come across when aiming to combine studies with different designs and potential biases and how these considerations were incorporated in the critical appraisal of evidence synthesis.


Providing an accurate localization of the brain functions before proceeding with neurosurgery is of vital importance to spare the removal of crucial brain areas. In this review paper, we investigated how two techniques, namely functional magnetic resonance imaging (fMRI) and magnetoencephalography (MEG), can serve this purpose, with specific reference to pre-operative assessment of sensorimotor and language functions in patients with brain tumors. First, we explored the methodological strengths and issues of each technique from data acquisition, to data processing and result display, also providing a temporal overview of these technical aspects and potential strategies developed to overcome the main issues. Then, we identified 16 studies that made pre-operative assessments by both techniques and accurately scrutinized them. Overall, despite the potential limitations associated with one technique or the other, the majority of these studies underlined the reliable and complementary use of fMRI and MEG. For these reasons, with the aim of the most reliable pre-operative assessment as possible, we recommend the combined use of both techniques, favored by the recent technological advances making MEG use more feasible.Pre-operative mapping of brain functions is crucial to plan neurosurgery and investigate potential plasticity processes. Due to its availability, functional magnetic resonance imaging (fMRI) is widely used for this purpose; on the other hand, the demanding cost and maintenance limit the use of magnetoencephalography (MEG), despite several studies reporting its accuracy in localizing brain functions of interest in patient populations. In this review paper, we discuss the strengths and weaknesses of both techniques, from a methodological perspective first; then, we scrutinized and commented on the findings from 16 studies, identified by a database search, that made pre-operative assessments using both techniques in patients with brain tumors. We commented on the results by accounting for study limitations associated with small sample sizes and variability in the used tasks. Overall, we found that, although some studies reported the superiority for MEG, the majority of them underlined the complementary use of these techniques and suggested assessment using both. Indeed, both fMRI and MEG present some disadvantages, although the development of novel devices and processing procedures has enabled ever more accurate assessments. In particular, the development of new, more feasible MEG devices will allow widespread availability of this technique and its routinely combined use with fMRI. The accurate identification of the brain areas subserving a given brain function is a crucial process in neurosurgery planning. In recent decades, studies have adopted different non-invasive techniques for this purpose to primarily identify potential lesion proximity to eloquent areas of the language and sensorimotor systems. Additionally, due to extensive availability of magnetic resonance imaging (MRI) instrumentation for diagnostic purposes, functional MRI (fMRI) probably represents the most widely used technique. Besides fMRI, electroencephalography (EEG) and magnetoencephalography (MEG) have been employed too, although traditionally deputed to epileptic activity monitoring ,2.The principle of fMRI resides in the blood flow change associated with the hemodynamic response in brain areas involved in a given brain function; this generates a detectable signal, which is measured in terms of a blood-oxygen-level dependent (BOLD) contrast. On the other hand, EEG and MEG measure the brain activity directly, by recording, respectively, the electrical and magnetic fields generated by thousands of neurons that activate in synchrony.−15 T) from external interferences, especially the much stronger Earth’s magnetic field (10−5 T). Despite the need for a shielded room, super-conducting quantum interference detectors (SQUIDs) are also required and need a continuous cooling process by liquid helium (−270 °C). These factors make traditional MEG systems very demanding, being very bulky  and very expensive, not only for the initial set up, but also for their maintenance.EEG and MEG provide similar but not completely equivalent information about brain processes  and eachNevertheless, overall MEG is considered superior to EEG, because it normally has higher spatial accuracy related to the fact that magnetic versus electric fields are less distorted by the skull, scalp, and cerebrospinal fluid and because it better detects activities located in the gyri . FurtherIn light of these technical considerations, in the current review paper we aimed to compare fMRI and MEG in terms of brain activity localization, from the perspective of surgical planning for brain tumor resection . Indeed,Several studies have independently addressed the accuracy of fMRI and MEG in the localization of cognitive and sensorimotor functions in patient populations, for instance comparing the results from those obtained by intra-operative mapping. Localizing the functional activations associated with a given function is crucial, because tumor development can cause brain remodeling through neuroplasticity processes and activations can be displaced with respect to the healthy brain see ,8). Iden. Iden8])Evidence of reliability in the use of fMRI or MEG alone has been widely reported. A recent meta-analysis  reportedIn the current review, we will first provide an overview of the methodological strengths and weaknesses of each technique, to then compare them more directly by commenting the results reported in studies that adopted both of them for pre-operative brain activity assessment. The final aim was to shed light on the advantages and disadvantages in applying one methodology versus the other and the related recommendation about their use in clinical practice.Overall, agreement exists in relation to the pros and cons associated with each technique, regardless of the specific findings observed in the pertaining studies. Apart from the factors representing limitations for the acquisition procedure, for instance the presence of metallic implants in patients (which may prevent fMRI acquisition and cause severe artifacts in the case of MEG), the two techniques present characteristics that influence the quality of the measured brain activity that can either represent a drawback or a strong point for that technique.Executing a given task activates the related brain network, with all the recruited areas simultaneously displayed by fMRI. On the other hand, MEG records the temporal development of the whole brain process, hence its spatiotemporal fingerprints; it detects the earliest component of the evoked response arising from primary eloquent areas, which can be separated from later components mainly arising from non-primary areas . This isConcerning resolution, the two techniques can be considered as complementary. The advantage of fMRI relies in the high spatial resolution of a few millimeters (depending on the voxel size), although a drawback is represented by its poor temporal resolution; indeed, BOLD responses are detected after several seconds following the activation of a given brain region and also lasts several seconds after deactivation.On the other hand, traditional MEG is characterized by a higher temporal resolution (in the order of milliseconds) but a poorer spatial resolution, especially when the source of the detected signal is deep . Furthermore, classical MEG systems could not detect subcortical activity; in fact, MEG mainly detects relatively superficial currents in the fissural cortex, being sensitive to sources tangential to the skull; nevertheless, more recent whole-head systems with hundreds of sensor can cover the whole head and can detect activity even in subcortical regions . HoweverIn the case of presurgical mapping, having high spatial resolution is fundamental, making fMRI more appropriate for this purpose, at least in general terms. In the following paragraphs, we will compare and discuss the in-depth suitability of both techniques from the perspective of neurosurgery.One of the major drawbacks associated with fMRI is represented by the inflow effect. In reality, BOLD signal may be altered by the signal coming from the blood flow in the draining veins, for instance in those supplying the central sulcus; the result is an alteration in neurovascular coupling. This was especially observed when using the conventional 1.5 T system with a gradient-echo sequence ,23. FurtNevertheless, these issues are scaled down by scans performed at high magnetic-field strength (3 T or greater) and the use of a spin-echo or gradient-echo EPI sequence instead of the traditional gradient-echo sequence. These solutions, indeed, decrease the effect of the inflow effect and then increase the BOLD contrast associated with the measured brain process ,23,27,28Acquisition devices also influence signal detection by MEG. Sensors for magnetic signal detection are typically represented by gradiometers , although these are rather insensitive to deep brain sources. The latter can be, instead, collected by magnetometers, but these are also more sensitive to ambient noise. Modern devices include both gradiometers and magnetometers and their combination significantly increases the signal-to-noise ratio , which captures the activity of tens of thousands of cortical neurons that activate in synchrony. A single ECD source can represent a valid solution in the case of strong dipolar patterns with a high signal-to-noise ratio, as is the case of the early components of somatosensory-evoked fields , but is Alternative approaches have been developed to provide a more reliable resolution to the inverse problem; these include spatial filtering and appear even more useful when analyzing widespread activation patterns such as those related to language functions . Some auNevertheless, even signal detection by fMRI has to be evaluated, and this can vary depending on the software used for processing and the selected options. For instance, different basis functions can be used to model the hemodynamic response , potentially giving different results.Finally, another relevant issue pertains to the spatial co-registration procedure to the anatomical MR image. This is likely to be more challenging and less accurate for MEG than fMRI , even thAll these concerns have to be weighted in light of clinical requirements and timing. Indeed, although very promising, these approaches are still time-consuming and it is sometimes difficult to integrate them into the clinical routine, considering also that patients can become tired more easily following intensive pre-operative assessments. For this reason, the best practical solution should be, generally, the one that provides the most reliable assessment within a reasonable time.Following this methodological comparison between the two approaches, we now comment on the findings from studies assessing pre-operative functional activation in patients with brain tumors by means of both fMRI and MEG. In order to identify peer-reviewed papers on this topic, we ran a paper search on the PubMed, Web of Science, and Scholar databases using the following keywords:  AND  AND (glioma OR tumor OR lesion) AND (surgery OR preoperative).We identified 16 studies, reporting findings from a highly variable number of patients (in the range of one to 90 patients). Sample sizes were generally small, with a median of 12 subjects A. The stThe reliability of the findings from each technique was most frequently derived from the comparison with intra-operative mapping ,46,48,49The second variable was the domain assessed. There was a dissociation between the frequency of motor and sensory tasks: a higher number of fMRI studies, compared to MEG studies, used motor localizers; vice versa, a higher number of MEG studies, compared to fMRI studies, used sensory localizers B. In conNevertheless, several cases of discrepancy were reported. Excluding partially different localizations attributable to the use of different tasks for the fMRI and MEG assessments , either fMRI or MEG can be more accurate overall and that is necessary to find a compromise between sensitivity and specificity. Second, in light of these considerations, a combination of the two techniques appears as the best solution to assure reliable findings.p value when needed) when analyzing the data. However, this could be still insufficient to ensure a reliable pre-operative assessment [The gold standard in brain tumors surgery is represented by the awake procedure, during which an intra-operative mapping of the function of interest is performed. Nevertheless, for those patients for whom this procedure is not feasible or refused by the patients themselves, it is fundamental to achieve the most accurate function localization in order to prevent permanent post-operative sequelae. Adopting recent devices and processing procedures and algorithms is essential to increase the pre-operative mapping reliability. Additional cautions should be used. For instance, in order to reliably detect residual activity, especially in the case of fMRI, it is useful to adopt individual thresholds  devices seem promising, although recording is still constrained to a limited signal bandwidth . Importa


The potential of therapeutically loaded nanoparticles (NPs) has been successfully demonstrated during the last decade, with NP-mediated nonviral gene delivery gathering significant attention as highlighted by the broad clinical acceptance of mRNA-based COVID-19 vaccines. A significant barrier to progress in this emerging area is the wild variability of approaches reported in published literature regarding nanoparticle characterizations. Here, we provide a brief overview of the current status and outline important concerns regarding the need for standardized protocols to evaluate NP uptake, NP transfection efficacy, drug dose determination, and variability of nonviral gene delivery systems. Based on these concerns, we propose wide adherence to multimodal, multiparameter, and multistudy analysis of NP systems. Adoption of these proposed approaches will ensure improved transparency, provide a better basis for interlaboratory comparisons, and will simplify judging the significance of new findings in a broader context, all critical requirements for advancing the field of nonviral gene delivery. Recent efforts to develop and translate therapeutically loaded nanoparticles (NPs) have resulted in several advances in the treatment and prevention of disease. Key areas where NP materials have had an impact include their use as vaccines, cancer therapies, and in the treatment of rare genetic disorders –3. StillStandardization of NP characterization is a widely recognized issue that is critically entwined with scientific and technical considerations –8. In faRecommendations from the community to address these issues exist and include encouraging the use of multiple characterization techniques, clarifying focused research questions , and perIn nanomedicine, these challenges are further compounded by the fact that the study of NP interactions with biological systems is a multidisciplinary field that ranges from understanding fundamental biological interactions  to enginFollowing the spirit of MIRIBEL, the current perspective was written with two specific aims in mind. First is to provide an overview of current concerns regarding aspects of NP uptake, transfection efficiencies, payload retention, and interparticle variability in the context of nucleic acid-based nanotherapeutics. In order to illustrate these issues and concerns, we analyzed the materials and methods sections of 50 papers published within the last five years on the topic of NP-mediated delivery of plasmid DNA after reasonable search and selection. Secondly, our objective is to emphasize the importance of standardized practices for the investigation of NP dosing, uptake, and transfection to improve the reusability, quantification, practicality and quality in the field of nonviral gene delivery research. Of note, this perspective is not intended to provide a comprehensive overview of the best practice , but raThe methods chosen to investigate NP uptake and transfection can be biased towards particular properties and may provide limited insights into the efficiency of NP internalization and efficacy. Typically, cellular uptake and transfection efficiency are assessed with the use of fluorescent-labeling carriers and the expression of fluorescent proteins . Both of which are typically assessed by widefield fluorescent microscopy/confocal microscopy (referred to as “imaging”) and/or flow cytometry .Laser scanning or spinning disc confocal microscopy, typically used for qualitative determination of NP uptake, can provide valuable insights into relative distribution and aggregation of NPs, which has been widely used in the field of nonviral gene delivery  rapid action in blocking the uptake route, (ii) equal inhibition of the overall cell population, and (iii) time- and labor-efficient processing ,35. HoweIn order to overcome the poor specificity of pharmacological inhibitors, genetic approaches can be implemented to change the expression of specific proteins . HoweverIn addition to (or in tandem with) inhibition and genetic strategies, imaging-based approaches should be performed. These imaging studies aid in the identification of the internalization mechanisms via fluorescence co-localization analysis of endocytic biological markers and NPs . While cThe combination of multiple methods among those available (each presenting advantages and limits) is probably the best approach to try to fully characterize and investigate the pathways involved and answer essential questions. An idealized scenario may be one in which inhibition results are coupled to both knockdown and imaging data to provide a full understanding of the mode of action in NP uptake studies.There are a variety of approaches for the evaluation of dose in the context of small-molecule therapeutics encapsulated in NP delivery systems –54. HoweConventional colorimetric or fluorescence assays are insufficient for the assessment of NAT-NP systems. These assays often rely on small to mid-size molecular probes that bind and associate nonspecifically  or through explicit interactions with specific NAT residues . UltimatSeveral alternate options exist that can provide reasonable assessment of NAT in NP systems. Firstly, NAT-labeling strategies beyond fluorescent labels, such as radiolabeling and elemental labeling have been explored . HoweverRegardless of the approach, it is worth noting that NAT concentration alone cannot address the extent of loading  or therapeutic efficacy . Nanoparticle tracking analysis (NTA)  can provThese considerations, when taken together, highlight the need for advanced analytical tools that are reproducible, accurate, and non-destructive in the assessment of NP-encapsulated NAT payloads .In the previous discussion, an assumption of particle dose scaling with particle mass is made. However, in the case of large molecules (such as plasmids) in smaller particles, significant deviations from this trend can be observed. To better understand the per-particle variations in payload, high-resolution microscopy, such as total internal reflection fluorescence spectroscopy, near-field scanning optical microscopy, or super-resolution microscopy  can be used to establish relative emission ratios for a labeled payload relative to a labeled carrier.Given the number of possible techniques for the assessment of the size distributions of NP systems, we offer some standards on the most commonly found techniques with an intent to provide a starting point for the development of repeatable protocols.For particles that are readily measured in the solid state, we recommend the ISO 19749:221 standard for SEM analysis, ASTM E2859-11 for AFM analysis, and ISO 21363:2020 for substrate-supported TEM analysis. When at all possible, obtaining the distribution of sizes from these methods with extension to obtain geometric properties  will allow for the comparison within different batches, formulations, particle types, and standards. For particle systems that do not exist in a solid state, cryo-TEM or submersion AFM can offer alternative routes. These measurements will provide high-resolution particle distributions for in-solvent particles, though they may not be as definitive as the solid-state particle distributions.In most instances, it will be valuable to assess the hydrodynamic size of the NPs. First, a quantification of the central tendency of the diameter or radius  should be reported. Additionally, a presentation of population variance  will be of use, specifically for dose/particle calculations. Lastly, a volume or mass distribution should be presented. Dynamic light scattering (DLS) is often be used for the inspection of aqueous suspensions with results bedeviled by the normal constraints of DLS and ensemble detection . Both diCommon techniques used to determine uptake and transfection possess limitations. Image-based techniques, such as widefield fluorescence microscopy and confocal microscopy, are limited by relatively low throughput and can be ambiguous for properly quantifying NP internalization by cells. Flow cytometry allows for high-throughput analysis of thousands of cells but is challenged by inadequate distinction between internalization and association. Imaging flow cytometry is high throughput and possesses the spatial resolution of microscopy, offering a promising route towards routine use in nanomedicine. However, the resolution of the images obtained from imaging flow cytometry is still lower than that of confocal microscopy, somewhat limiting the ability to conclusively distinguish internalized material from external material. Use of approaches with both high throughput and high spatial resolution  should be considered as best practice for the purposes of accuracy, statistical relevance, and comparisons between studies.An in-depth mechanistic understanding of NP uptake will be required to improve the design of NP-based delivery platforms. The sole use of chemical compounds to inhibit or probe NP uptake pathways remains challenging because of issues such as poor specificity, cytotoxicity, cross-active chemical modalities, and varied efficacy. With proper controls, pharmacological inhibitors still provide value for simple screening of uptake pathways. However, genetic knockouts or dominant-negative proteins should be considered as complementary approaches to study uptake mechanisms. Combined chemical and genetic tools will provide data sets that more accurately represent uptake phenomena, while also allowing for closer comparison between studies. Regardless of the uptake pathway, the current understanding of how NPs induce endosomal escape lags behind and is limited by the current techniques used to detect it. Thus, new and accurate endosomal escape assays are required to understand the relationship between NP composition and endosomal escape.NAT are difficult to quantify if encapsulated into NP delivery carriers. The most common forms of nucleic acid measurement  provide limited insights for the characterization of NP-encapsulated NATs. Labeling and destructive methods both have drawbacks, ranging from destruction of valuable media to fundamental changes to the payload. While several multimethod approaches may be capable of providing the ratio between mass of payload and mass of substrate, the mass of payload per unit volume, and the number of payload molecules per particle, there is no single commercial solution available at present. In the interim, with no “silver bullet” method available, there is likely great benefit in a quantification regime that may allow the key dosing values to be obtained through a combination of complementary methods .In summary, when considering NP therapeutic uptake, transfection efficiencies, and NAT dose, there is clear value in utilizing a combination of multiple methods to comprehensively assess features of NP systems. Importantly, our analysis of the literature indicates that the nanomedicine research community as a whole would benefit from multiparameter and multistudy reporting, which would facilitate comparison and assessment of results across studies and research groups.Standardization of high-quality NP-mediated nonviral gene delivery studies based on multimethod, multiparameter, and multistudy reporting will enable interexperimental and interlaboratory comparisons of uptake and transfection results, ideally allowing for placing individual results obtained by one laboratory to apply in a broader context. If such a consensus can be reached, the identification of structure–function and material–biological relations of NPs could be dramatically accelerated.File 1Details about experimental approach and scope.


In recent decades, an exponential surge in technological advancements has significantly transformed various aspects of daily life. The proliferation of indispensable objects such as smartphones and computers underscores the pervasive influence of technology. This trend extends to the domains of the healthcare, automotive, and industrial sectors, with the emergence of remote-operating capabilities and self-learning models. Notably, the automotive industry has integrated numerous remote access points like Wi-Fi, USB, Bluetooth, 4G/5G, and OBD-II interfaces into vehicles, amplifying the exposure of the Controller Area Network (CAN) bus to external threats. With a recognition of the susceptibility of the CAN bus to external attacks, there is an urgent need to develop robust security systems that are capable of detecting potential intrusions and malfunctions. This study aims to leverage fingerprinting techniques and neural networks on cost-effective embedded systems to construct an anomaly detection system for identifying abnormal behavior in the CAN bus. The research is structured into three parts, encompassing the application of fingerprinting techniques for data acquisition and neural network training, the design of an anomaly detection algorithm based on neural network results, and the simulation of typical CAN attack scenarios. Additionally, a thermal test was conducted to evaluate the algorithm’s resilience under varying temperatures. Rapid technological progress has made it possible for a wide range of industries, such as robotics, mechatronics, automation, and the automotive sector, to operate in a highly networked global environment. Although efficiency has increased significantly, these domains are now more vulnerable to growing cybersecurity threats ,2,3,4,5.In modern vehicles, there are numerous electronic control units (ECUs) used for automation and comfort features, both of the driver and passengers . IncludeAlthough data encryption techniques have been proposed in the literature, their application to the CAN bus protocol has shown them to be unsuccessful . MoreoveOur approach makes use of real-time analysis on a simple platform, and thermal testing is used to validate the results. The first part will address data acquisition, explaining the methods and approaches used to collect and organize data. Because of the remarkable multi-class classifier performance of Artificial Neural Networks (ANNs) and their simplicity in embedded system integration, we have decided to use them. The results of experimental testing, which include the evaluation of several attack scenarios put out in the literature to verify the Intrusion Detection System (IDS) algorithm, will then be presented. In the end, the robustness of our proposed method with respect to temperature variation is shown, and this is well known to affect the circuit aspects, and consequently, the physical layer associated with the CAN protocol.(1)Two-Step Algorithm: This algorithm uses a mixed approach of temporal-spatial analysis to detect cyber-attacks over the CAN bus. The algorithm first detects the abnormal behavior of the CAN bus and then identifies the source of the attack ,27,28,29(2)Intrusion Detection System (IDS): IDS is a popular security solution that uses crypto- graphic-based software to address CAN network security issues. The IDS ensures that the exchanged CAN data frame between the two end nodes is authorized. Researchers have proposed various IDS algorithms, such as a lightweight algorithm based on the observance of CAN packets frequencies, an anomaly-based detection method based on the time interval feature of the consecutive CAN packets, and a graph-based feature method that uses machine learning algorithms ,40,41,42(3)CAN-ADF: The Controller Area Network Attack Detection Framework (CAN-ADF) is a framework that uses field classification, modeling, and anomaly detection to detect cyber-attacks on unknown CAN bus networks. The framework uses a holistic approach to detect cyber-attacks and provides a comprehensive solution to the cybersecurity issues related to the CAN protocol ,44.(4)Deep Learning Techniques: Intrusion Detection Systems (IDSs) using deep learning techniques are also proposed in the literature. These IDSs identify cyber-attacks when given a sample of network traffic collected from real-world computer networks. The IDSs using deep learning techniques are powerful and can detect cyber-attacks with high accuracy ,47,48,49In summary, various detection algorithms have been proposed in the literature to address the cybersecurity issues related to the CAN protocol. These algorithms use different approaches, such as temporal-spatial analysis, cryptographic-based software, anomaly-based detection, graph-based feature methods, and deep learning techniques. The selection of the detection algorithm depends on the specific requirements of the application and the level of security needed.There are several detection algorithms proposed in the literature to address the cybersecurity issues related to the Controller Area Network (CAN) protocol. Here is an overview of some of the state-of-the-art detection algorithms.(1)Clock-based IDS (CIDS): CIDS is an anomaly-based intrusion detection system that measures and exploits the intervals of periodic in-vehicle messages for fingerprinting ECUs. The fingerprints are then used for constructing a baseline of the ECUs’ clock behaviors with the Recursive Least Squares (RLS) algorithm. Based on this baseline, CIDS uses Cumulative Sum (CUSUM) to detect any abnormal shifts in the identification of errors, which is a clear sign of intrusion ,54,55,56(2)Physical-Fingerprinting of Electronic Control Unit (ECU) Based on Machine Learning Algorithm: This algorithm uses machine learning algorithms to identify the physical fingerprints of ECUs based on the time and frequency domain features of the consecutive CAN packets. The algorithm classifies the ECUs based on their physical fingerprints and detects any abnormal behavior ,62,63,64(3)ECU Fingerprinting through Parametric Signal Modeling and Artificial Neural Networks: This algorithm uses parametric signal modeling and Artificial Neural Networks to identify the physical fingerprints of ECUs. The algorithm extracts the features of the CAN packets and uses them to train the Artificial Neural Network. The trained network is then used to classify the ECUs and to detect any abnormal behavior ,68,69,70(4)Two-Point Voltage Fingerprinting: This algorithm uses voltage measurements to identify the physical fingerprints of ECUs. The algorithm measures the voltage at two points in the CAN bus and uses the difference between the two measurements to identify the ECU. The algorithm can detect any masquerading attacks on the CAN bus ,74,75,76In summary, ECU fingerprinting algorithms are proposed to detect and to prevent cyber-attacks on the CAN bus. These algorithms use different approaches such as clock-based IDS, machine learning algorithms, parametric signal modeling, and Artificial Neural Networks. The selection of the ECU fingerprinting algorithm depends on the specific requirements of the application and the level of security needed.Electronic Control Units (ECUs) are an essential component of the Controller Area Network (CAN) protocol used in the automotive industry. ECUs communicate with each other over the CAN bus protocol, which ensures high communication rates. However, the CAN protocol is prone to various cybersecurity attacks, and ECUs are vulnerable to these attacks. To address this issue, researchers have proposed ECU fingerprinting algorithms to detect and prevent cyber-attacks on the CAN bus. Here is an overview of some of the state-of-the-art ECU fingerprinting algorithms:Simplicity of Wiring: The CAN bus operates on a message-oriented approach, rather than an address-oriented one. This design allows for the straightforward addition or removal of peripherals (nodes), simplifying the wiring process.Rigid Response Times: CAN bus technology enables the creation of systems with highly predictable and rigid response times. This is achieved through specific techniques that are designed to minimize time-related delays.High Immunity to Interference: The ISO 11898 standard mandates that the CAN protocol must maintain operability, even in scenarios where one of the two wires is severed, or if a bus line to the power supply experiences a short-circuit.Error Confinement: Each peripheral device connected to the CAN bus possesses the capability to self-diagnose hardware issues. In the event of a malfunction, a peripheral can voluntarily remove itself from the bus, allowing other peripherals to continue using it.Multi-Master Protocol: Within the CAN protocol, every node has the capacity to compete for control of the bus. This means that each node can assume the role of a master, taking control of the bus and initiating transmissions.To manage traffic on the bus effectively, the CAN protocol employs the CSMA/BA (Carrier Sense Multiple Access/Bit-wise Arbitration) method. In situations where two or more nodes attempt to transmit simultaneously, an arbitration mechanism based on priority is applied.The Controller Area Network, commonly referred to as CAN bus, is a serial standard for field buses that is primarily employed in the automotive industry. It was introduced in the 1980s by Robert Bosch as a means to connect various electronic control units (ECUs). Notably, the CAN protocol offers a range of key advantages:It is brought to attention that the CAN bus, an essential communication protocol in various automotive systems, is deficient in fundamental security measures, rendering the wired units susceptible to potential breaches orchestrated by malevolent entities. According to the CIA  security model, a comprehensive examination reveals the existence of six critical vulnerabilities within the CAN bus framework. These vulnerabilities emerge from two distinct sources: the vulnerabilities concerning the traffic transmission through the CAN bus and those intrinsic to the protocol’s unique characteristics ,78,79,80The lack of encryption allows potential adversaries to decipher the historical data transmitted via the CAN bus, thereby comprehending the intricate functionalities of the target Electronic Control Units (ECUs) with relative ease ,82.The absence of an authentication mechanism in the CAN frame implies that any transmitter can surreptitiously send deceptive CAN frames to any of the interconnected ECUs, potentially gaining unauthorized control over the target ECUs ,84,85.The absence of integrity checking exacerbates the security concerns, as the receivers might unknowingly accept manipulated data, leading to potential system malfunctions or even complete breaches by malevolent entities ,87.The broadcast transmission characteristic of the CAN bus, where the frames are disseminated to all interconnected ECUs, acts as a double-edged sword, facilitating system-wide communication, but also enabling unauthorized eavesdropping, which jeopardizes the confidentiality of the communication.The priority-based arbitration, which allows frames with higher priority to dominate the communication channel, poses a significant security risk, as it enables an aggressive Electronic Control Unit (ECU) to manipulate the communication channel, potentially disrupting the entire network’s functioning ,89,90.The limited bandwidth and payload capacity of the CAN bus results in the insufficiency of robust access control mechanisms, creating a vulnerability that could be exploited by adversaries attempting to compromise the security of the system.Among the pressing concerns, the absence of encryption, authentication, and integrity checking in the data transmission via the CAN bus represents a severe violation of the fundamental principles of data security, particularly confidentiality and integrity. Furthermore, the characteristics inherent in the CAN bus protocol, such as broadcast transmission, priority-based arbitration, and limited bandwidth, contribute to the system’s susceptibility to various security threats. The combination of these factors contributes to the heightened risk of a Denial-of-Service (DoS) attack, thus compromising the system’s availability. The specific vulnerabilities identified within the CAN bus context can be discerned as follows:(1)Unauthorized access: Since the network is centralized, nodes trust each other, and a malicious node that is attached to the network can have access to all the data flowing and can disrupt the data flow ,95,96,97(2)Replay attacks: An attacker intercepts and records a message, and then replays it later to achieve a malicious goal ,101,102.(3)Denial of Service (DoS) attacks: An attacker can flood the network with messages, causing the network to become unresponsive ,106,107.(4)Spoofing attacks: An attacker can send messages with a fake source address, making it difficult to identify the source of the attack ,111,112.(5)Physical layer attacks: An attacker can manipulate the physical layer of the CAN bus to cause malfunctions in CAN nodes ,117,118.To address these vulnerabilities, various solutions have been proposed, such as intrusion detection systems, encryption, and authentication mechanisms. However, there is no optimal solution, and the problem is mitigated with network segmentation and intrusion detection systems. It is essential to establish a strong security system for automotive networks to maintain the advances in safe technologies and to advance the state of the art in automotive cybersecurity . The primary objective here is to flood the bus, thereby preempting all arbitration phases and effectively monopolizing the communication medium. Consequently, this impedes any legitimate interaction among the other units. A comprehensive depiction of the results derived from this testing is presented in These simulated attacks serve as valuable test cases for evaluating the robustness and effectiveness of the Anomaly Detection algorithm under various security threats, enabling the system to proactively respond to potential vulnerabilities. The results obtained with the simulated attacks are perfectly in line with the accuracy obtained during the validation phase of the classifier model shown in the previous section.Electronic control units are commonly put in situations that are characterized by volatile temperature variations, often varying by large margins. These systems rely significantly on the CAN bus for communication. When these control units are used in automotive applications, their temperatures might vary depending on a number of circumstances, such as how close they are to the engine, how long they run for, and how exposed they are to outside elements like direct sunlight. Because MOSFETs and resistances are intrinsically sensitive to temperature changes, even small changes in temperature can have a significant impact on the complex integrated circuits present in these devices.There is a significant degree of danger associated with subjecting these control units to high temperatures, since the heat generated by these conditions can significantly distort the voltage signals, jeopardizing the accuracy of previously recorded data. As such, careful thermal testing is necessary in order to fully evaluate the possible effects of these temperature-induced changes, particularly with regard to the effectiveness of cybersecurity systems that depend on voltage fingerprinting methods. A deeper comprehension of the true implications of the suggested classification approach may be obtained by attentively analyzing the Softmax outputs in connection to temperature changes. This will allow for the development of more intelligent and reliable security mechanisms. To reduce the measured noise effect, values were collected every 200 temperature values and their mean was calculated. The algorithm is shown in Using the identical configuration as the Second test, the remaining units connected to the bus were set to silent/listen mode. Only Unit C was active in sending messages during the testing process, while the control unit diligently recorded the corresponding data  and a Traffic Analyzer. A pivotal improvement over the existing methods is the adoption of a sophisticated voltage sampling technique that is far superior to the Software Trigger mechanism. The selection of neural network characteristics has been meticulously determined through empirical methodologies. A comprehensive statistical analysis of the features extracted from the data has provided profound insights, guiding the preference for Min-Max normalization over Standardization.Incorporating TensorFlow Lite onto the NXP S32K144 board has enabled the harnessing of cutting-edge tools in the realm of artificial intelligence, effectively unlocking its real-time classification capabilities. To validate the methodology in more complex environments and in the face of potential attacks, a typical CAN network or sub-network scenario was faithfully recreated. To address the challenge of classifying unknown units using the neural network, a novel solution involving the introduction of a Fictitious dataset was proposed. Furthermore, a series of well-documented simulated attacks, inspired by prominent attack methodologies described in the literature, was executed. The efficacy of these attacks was systematically thwarted by the anomaly detection algorithm, thereby affirming its robust functionality. To extend the practical applications of this technology within the automotive sector, the proposed method was rigorously assessed under the conditions of temperature variation. The results underscore the resilience of the methodology to temperature fluctuations, at least within the range of 25 Looking forward, the promising results obtained from this research open doors to a host of potential future developments and extensions. Porting from Laboratory Tests to Real Vehicle Implementation: The next phase following laboratory implementation involves the deployment of this technology in a genuine automotive environment to facilitate functional testing. This real-world application could significantly contribute to the field of automotive cybersecurity. Integration with Other Fingerprinting Techniques: The proposed methodology is amenable to integration with other fingerprinting techniques, including time-based fingerprinting methods and various other fingerprinting approaches. Combining multiple fingerprinting techniques could enhance the overall security of automotive networks. Application to Other Protocols: While this research has been focused on CAN environments, the method’s efficacy encourages its application to diverse network types within the automotive and industrial sectors. This broadening of scope could address security concerns in various communication protocols. Comparison with Deterministic Algorithms: In the realm of classification, an avenue for future exploration lies in the comparison of neural networks with deterministic algorithms, such as Decision Trees, to gauge their relative performances. This could provide valuable insights into the strengths and weaknesses of different approaches. Further Resilience Testing: Extending investigations into the resilience of the system to a broader range of temperature variations can provide valuable information about its practicality under various environmental conditions. Additionally, exploring other potential environmental factors such as humidity and electromagnetic interference can further enhance the system’s robustness. Real-Time Anomaly Detection: Efforts can be directed towards achieving real-time anomaly detection capabilities, potentially reducing response times and increasing the system’s effectiveness in mitigating threats.In conclusion, this research not only validates the effectiveness of the proposed methodology, but also outlines a promising path for future research, development, and practical implementation in the domains of automotive and industrial cybersecurity. The potential for enhancing network security, particularly in the context of the growing significance of connected vehicles and industrial IoT, makes this work a valuable contribution to the field.