Recent advances in the field of meta-omics sciences and related bioinformatics tools have allowed a comprehensive investigation of human-associated microbiota and its contribution to achieving and maintaining the homeostatic balance. Bioactive compounds from the microbial community harboring the human gut are involved in a finely tuned network of interconnections with the host, orchestrating a wide variety of physiological processes. These includes the bi-directional crosstalk between the central nervous system, the enteric nervous system, and the gastrointestinal tract . The increasing accumulation of evidence suggest a pivotal role of the composition and activity of the gut microbiota in neurodegeneration. In the present review we aim to provide an overview of the state-of-the-art of meta-omics sciences including metagenomics for the study of microbial genomes and taxa strains, metatranscriptomics for gene expression, metaproteomics and metabolomics to identify and/or quantify microbial proteins and metabolites, respectively. The potential and limitations of each discipline were highlighted, as well as the advantages of an integrated approach (multi-omics) to predict microbial functions and molecular mechanisms related to human diseases. Particular emphasis is given to the latest results obtained with these approaches in an attempt to elucidate the link between the gut microbiota and the most common neurodegenerative diseases, such as multiple sclerosis (MS), Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS). In the last 15 years, the growing awareness of the sustained association between the intestinal microbiota and human health has led to many efforts to better understand its role and contribution in the pathogenesis of various diseases. 14 cells, approximately 10 times the number of cells of the human body [At birth human intestine is essentially sterile; the onset of the gut microbiota starts as a dynamic ecosystem where the microbial composition increases both its diversity and richness until achievement of the “mature” level in the adult . At thisman body . Since iman body .Firmicutes and Bacteroidetes phyla, and, to a lesser extent, by Actinobacteria, Verrucomicrobia, Proteobacteria, Fusobacteria, and Cyanobacteria phyla [Bacteria is the most represented kingdom in the gut-associated microbial community and, although featured by a high inter-individual variability, a balanced composition of the human gastro-intestinal tract (GIT) microbiota is mainly represented by ia phyla . Bioactiia phyla ,6,7, orcIn this view, accumulating data suggest that alteration in the optimal microbiome composition and activity (a condition named “microbiome dysbiosis”) may contribute to the onset of several pathologic conditions, such as neurological and neurodegenerative disorders ,9. The technological progress witnessed in the last two decades has marked a profound change in the methods employed for the study of the human microbiota and its pivotal role in both physiologic and pathologic processes. To date, studies based on germ-free (GF) animal models, gut microbiota manipulation with antibiotics and fecal microbial transplantation, have been performed to investigate the modulatory effect of the microbiota on gut–brain axis and its implications in neurodegeneration. Omics sciences, such as metagenomics, metatranscriptomics, metaproteomics, and metabolomics, represent the last frontier in elucidating host-microbiota cross-talks, providing invaluable contributions for unravelling this intriguing issue . The intIn this review, mainly taking into account peer-reviewed studies of the last five years, we aim to provide a general description of the state-of-the-art of the investigation methods used in the study of microbiota, with particular emphasis on meta-omics sciences such as metagenomics, metatranscriptomics, metaproteomics, and metabolomics, along with their complementary integration made feasible by the advances in bioinformatics tools. In addition, we focused on the latest achievements of these approaches in elucidating the influence of gut microbiota in the onset and/or progression of the most commonly studied forms of neurodegeneration, such as multiple sclerosis (MS), Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS). To achieve the stated aim, a detailed and in-depth search procedure was carried out. The literature analyzed in this review includes original studies available in qualified databases, such as Medline/Pubmed, Scopus, Web of Sciences, and Google Scholar. The literature searching and evaluation covers the last 20 years. However, it should be noted that the oldest references are related to the early advances in the omics sciences or definitions of diseases. The literature of the last 10 years was mainly considered throughout the manuscript. Instead, for the list of the latest achievements obtained from the application of meta-omics sciences , only thA combination of key words and terms was used: microbiota/omics sciences; microbiota/ neurodegeneration; gut–brain axis/neurodegeneration. Each disease  was combined with all the meta-omics sciences , respectively.The references of all identified articles and recent review articles were cross-checked to ensure a valid and effective search.The technological progress of the last decades has marked enormous changes in the methodology adopted for the investigation of the microbiota and its relationship with the host, moving from the traditional culture-based approach to the omics sciences. Conventional culture-based methods fail to identify all the microorganisms that make up the microbiota and are limited to analyze and elucidate up to only 10%–30% of the cultivated microbial community both in terms of composition and functions ,44. Based on a holistic perspective, omics and meta-omics sciences, including shotgun metagenomics, metatranscriptomics, metaproteomics, and metabolomics, use attractive and powerful tools to characterize the microbial consortia, investigate functions and dynamics, and quantify the biomolecules they produce. In this perspective, the resident microbial genomes (metagenome), transcripts (metatranscriptome), proteins (metaproteome), and metabolites (metabolome) are investigated in the human frame, providing a comprehensive overview of the complex network of interconnections that regulate the functional dynamics of each anatomical district ,45.These techniques provide detailed information on taxa strains of the microbial population, evaluate potential microbial functions and molecular networks, and quantify their protein and metabolic products. In addition, the application of these meta-omics approaches to clinical samples has identified microbial species, protein and metabolic pathways that could be associated with the development and treatment of human diseases. The main challenge of the omics-based microbiome studies relies on the strong computational effort required to deal with the impressive amount of data generated by the constantly improving next generation sequencing (NGS) and mass spectrometry (MS) technologies . On the other hand, the integration of such omics sciences in the so-called multi-omics approach, provides more evidence of biological mechanisms and, ultimately, opens new perspectives for the development of novel therapeutic strategies and personalized medicine.The technical aspects of omics sciences have been widely described elsewhere ,47,48,49The following paragraphs will provide an overview of the elective technologies used in the study of the gut microbiota and its crosstalk with the brain, and how these disciplines have contributed to elucidate the link between the human microbiota and neurodegenerative diseases. Metagenomics is a community-based powerful tool that studies the microbial genomes collected from the ecological niche  where the microorganisms coexist, in order to describe the phylogenetic, physical, and functional features of the microbiota, in a culture-independent manner. Current metagenomics techniques are based on the shotgun approach in order to provide a microbial community census starting from reads from DNA and alignment with reference genomes . In paraHere, targeted sequencing of the 16S rRNA (prokaryotes) or 18S rRNA (eukaryotes) gene is one of the most attractive strategy for a reliable and cost-effective investigation of the overall microbial community diversity . The obtShotgun metagenomics, instead, by sequencing all the microbial genomes, allows for a more complete picture of the functional gut microbiota overcoming the marker-gene limitations .The investigation of the whole metagenome is based on the construction of the metagenomics library following DNA extraction and cloning . The metThus, in general, metagenomic sequencing not only provides accurate information on the microbial composition and family classification, but can also allow functional annotation, and gene de novo prediction ,57. NeveMetagenomics studies should be supported by the constant updating of curated reference databases and calculation sequencing algorithms. The choice of database is a fundamental step.The Human Microbiome Project has provided a database of bacterial genome information useful to predict OUT functions . PreferrTo overcome the metagenomics limits, metatranscriptomics is aimed at investigating the gene expression and activity of the whole microbial community ,71. Despite the wide choice of techniques, metatranscriptomics studies mainly focus on mRNA sequencing to assess which genes are expressed in the analyzed community . The seqSome limitations are related to the difficulties of obtaining sufficient amounts of RNA to be analyzed and, mostly, separate mRNA from the other abundant RNA, such as rRNA. Lastly, metatranscriptomics classification is limited to not enough reference databases. As for metagenomics, bioinformatic data analysis is of fundamental importance for the comprehensive functional characterization of the RNA molecules. In this perspective, efforts are still required to improve and develop bioinformatics tools that integrate the metagenomics sequence with metatranscriptomics data . Simple Annotation of Metatranscriptomes by Sequence Analysis (SAMSA) was the first open-source bioinformatics pipeline designed for metatranscriptomic data. It works with the metagenomics (MG) RAST server, a public resource for handling both metagenomic and metatranscriptomic data . SAMSA’s latest implementation, SAMSA2, based on several tools such as DIAMOND for sequence alignment and local databases, manages end-to-end metatranscriptome analysis for stand-alone use on a computing cluster .Along with MG-RAST, COMAN is a web-based tool to analyze metatrascriptomics data, although both do not support mapping to reference database. MetaTrans is another open-source pipeline as well as Anvi’o and IMP for the integrated metagenomics and metaproteomics analysis .The important role of proteomics for the effective reliable identification of the metabolically active microorganisms has been widely recognized ,77. TherMetaproteomics  is the study of all protein samples recovered directly from environmental sources. Along with metabolomics, metaproteomics provides the most realistic picture of the key effectors that directly mediate the biochemical functions operated by the organisms at the specific moment of sampling ,79.The recent interest in the investigation of more complex samples, such as the gut microbiota, leads to the advent of high-performance MS platforms, which are now the dominant approaches for metaproteomics investigations. The actual MS technologies enable the investigation of different features of the metaproteome, such as differential proteins expression , investigation of sub-proteomes , post-translational modification (PTM) pattern, protein–protein interactions, and absolute protein quantitation ,79. AlthFollowing computational assistance and using protein reference databases, MS data are processed to analyze the peptide sequences and, subsequently, provide protein identifications. A typical MS experiment generates hundreds of thousands of fragmentation spectra and an enormous amount of MS data that cannot be manually elaborated. Efficient bioinformatics software and tools are currently available to perform the computational operations required to translate the myriad of spectra into a meaningful output that is both concise and informative . CommonlMoreover, the bioinformatics data analysis and the availability of reference genome/metagenome sequences is undoubtedly of paramount importance in order to obtain a comprehensive picture of the metaproteome analyzed. Similarly to metatrascriptomics, the availability of enough reference databases can represent one of the technical limitations of this omics approach. In addition, robust, well-standardized protocols should be integrated in the experimental workflow especially for quantitative metaproteomics studies, which are still lacking .Metabolomics provides a snapshot of the metabolites array produced by the microbial community at the moment of sampling, enabling a comprehensive investigation of the microbial population and the interactions between the microbial ecosystem and its host . TogetheThe main steps in metabolome investigation include the pre-resolution of the complex metabolite mixture, following the sample lysis and/or purification of the extracted metabolites from samples of interest. Metabolites separation is generally accomplished through high performance liquid chromatography (HPLC) or gas chromatography (GC), enabling a wide range of metabolites to be analyzed through high sensitivity MS platforms. Similarly to metaproteomics, metabolites identification and their subsequent quantitation is computed on the basis of the mass spectral fingerprint and the MS fragmentation pattern.Nevertheless, nuclear magnetic resonance (NMR) spectroscopy is also widely used in analyses that specifically target a reduced number of metabolites . The metabolomics raw data requires complex processing steps to obtain small molecules and metabolite identification . SeveralAs we previously described, each omics technology sheds light on an important aspect of the multifaceted microbial intestinal community, however, it is limited to a single perspective. Although each single approach has its own limits linked to technical issues, multi-omics integration can generate a comprehensive scenario that includes more evidence while explaining biological mechanisms and phenomena. In this context, insights integrated by combinations of different multi-omic sciences  can provide a more detailed description of microbiota–host interactions, in order to reveal the bilateral flow of information that underlies different diseases, including neurodegeneration.An important task in the field of multi-omics sciences concerns the annotation and integration of the identified molecules  into predicted classes, and clustering them into functional groups. For this purpose, data repositories commonly used are Kyoto Encyclopedia of Genes and Genomes (KEGG) ,99, GeneGut microbiota is in close connection with the central nervous system (CNS) through the GBA, enabling a bidirectional communication from gut to brain and vice versa. Briefly, the GBA includes the following key components: the CNS, the autonomic nervous system (ANS), the enteric nervous system (ENS), the hypothalamic–pituitary–adrenal axis (HPA), the immune system (cytokine and chemokines) ,102. ComBiologically active neurochemical molecules of bacterial origin provide a mechanistic basis on how the microbiota may influence brain homeostasis and physiological processes .Lactobacillus and Bifidobacterium produce microbial metabolites, such as gamma-aminobutyric acid (GABA), which is the main inhibitory neurotransmitter in the brain. Candida, Escherichia, and Enterococcus produce the neurotransmitter serotonin (5-HT), while some species of Bacillus secrete dopamine. In particular, 5-HT has different roles. In the peripheral system, it is involved in the regulation of gastrointestinal secretion, motility , and pain perception, while in the brain 5-HT is involved in regulating mood and cognition. The gut microbiota also plays an important role in the metabolism of tryptophan, precursor of 5-HT production, and in the stimulation of enterochromaffin cells, which are the main producers of 5-HT in the intestinal mucosa (about 95%) [Much research on the brain–gut microbiota axis is based on the use of GF animals. Many of these studies suggest that the intestinal microbiota produces relevant levels of neurotransmitters that are, in part, responsible for many aspects of brain health and disease ,105. Forout 95%) ,106. Bacout 95%) . In addiout 95%) .A strong functional integrity and interplay of the GBA is required for the homeostasis of the nervous systems. Alterations of these connections in the GBA may influence the progress of neurodegeneration or even contribute to its onset ,109.Over recent years, accumulating evidence has suggested that the gut microbiota is involved in neurodegeneration. The alteration of the brain–gut microbiota homeostasis could worsen the etiology, the pathogenesis, and/or the progression of some disorders such as MS, PD, AD, ALS, and others . Neurodegenerative diseases are multifaceted disorders in which a close interaction of genetic and environmental factors seem to initiate the pathological process. Microbial metabolites and molecules trigger and/or amplify inflammatory brain processes, including perturbation of host immune homeostasis, alteration of blood–brain barrier and brain structure .It is well known that oxidative stress (OS) is closely related to mitochondrial dysfunctions and is one of the main factors associated to neurodegenerations . InteresMoreover, the reduced diversity of gut microbiota during aging, also influenced by dietary habits accumulated over the years, could have a role in the development of neurodegeneration. Bacteriodetes and Firmicutes remain dominant, although their relative proportions may change significantly. An increase in pathogenic bacteria (pathobionts) usually occurs along with a concomitant decrease of beneficial bacteria (symbionts) [It is well known that composition of microbial community changes in diversity during aging . The phymbionts) .Omics and multi-omics sciences support ongoing research studies to elucidate these intriguing connections and open new perspectives for therapeutic approaches for various neurodegenerative diseases.The following sections describe the implications of an altered gut microbiota in some of the most relevant neurodegenerative disorders. An overview of some recent and relevant meta-omics studies has been proposed.In MS is an inflammatory disease characterized by immune-mediated axon demyelination leading to distinct signs, including autonomic and cognitive alterations as well as motor, sensory, and visual defects . It is wExperimental evidence suggests that MS is also characterized by alterations of the intestinal permeability and bile acid metabolism which may consequently further contribute to complications in the immune regulation of the nervous system .The role of intestinal dysbiosis in the onset or development of MS is currently not well understood. Lactobacillus, that contribute to an impairment of immune system in fecal samples of EAE mice [Experimental observations support the hypothesis of a cross-correlation between inflammatory demyelination of the CNS and modification of the microbiome. A few experimental evidences have been collected from analysis based on 16S rRNA sequencing on GF mice and experimental autoimmune encephalomyelitis (EAE) mice, a well-established mouse model of MS. . In thisEAE mice .Akkermansia muciniphila, and consequently suppress neuroinflammatory processes [Recently, an integrated studied based on 16S rRNA sequencing and computational metabolomics has revealed that basis of the beneficial effect of the use of cannabidiol for the treatment of muscle spasticity of MS patients. The authors showed in EAE mice that cannabinoids directly prevent microbial dysbiosis, acting by the reduction of mucin degrading bacterial species, such as rocesses .Some few attractive studies have also been performed on human samples.Through 16S rRNA biomarker sequencing, a characterization of gut microbiota composition has been carried out in fecal samples of pediatric MS compared to control children matched for age and sex. Perturbations in the gut microbial community composition have been shown in recent onset pediatric MS  and subsInterestingly, two shotgun metagenomics based studies have been performed on cerebrospinal fluid of MS patients providing information about bacterial and viral composition also in such important biofluid ,14.Faecalibacterium [cClostridial species [In fact, previous research studies, based on marker-16sRNA gene, have highlighted a MS dysbiosis related to bacterial abundance such as acterium  and cClo species  in fecalThe use of antibiotics cocktails or probiotics, which clearly modify the composition of intestinal microflora commensals, improves immune responses as well as can generally attenuate the symptomatology of the disease .These results provide a basis for future studies in which the controlled microbiota modulation could potentially contribute to the treatment of MS.PD is the second common worldwide neurodegenerative disease characterized by the death of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Clinical symptoms generally appear when significant neuronal loss has already occurred. These include characteristic signs, especially related to the motor system, such as tremor, bradykinesia, rigidity, and postural instability. The recognition of these symptoms is the basis for a clinical diagnosis of advanced PD. Pharmacologic interventions are targeted at alleviating symptoms rather than preventing and/or resolving the disease . Indeed,Growing experimental evidence focused on the microbiome demonstrating the potential role of gut microbiota on the disease state, as extensively reviewed in a recent paper by Sampson et al. .As it is well known, Lewy bodies, characterized by toxic alpha-synuclein (αSyn) aggregates, are the main pathological hallmark of PD SNpc neurons . It has Enterobacteriaceae and reduced Prevotellaceae in fecal microbiota of PD patients compared to age-matched controls. Interestingly, the increased abundance of Enterobacteriaceae was correlated to the severity of postural instability and walking difficulties as well as a decrease of Prevotellaceae. Indeed, Prevotella produces SCFA, and thiamine and folate as by-products, that promote a healthy intestinal environment [A large number of metagenomics studies have been focused on the identification of microbial taxa in PD gut microbiota, its taxonomic diversity and abundance. Many of these have been performed especially in fecal samples due to the non-invasiveness of the sample collection. Although some controversies occur in the results, it is well evident the idea of a clear correlation of alteration of gut microbiota in PD in comparison to a healthy status. In particular, 16S rRNA analysis showed higher prevalence of ironment .A shotgun metagenomic approach has been used by Bedarf et al. to investigate also the fecal microbiota of PD patients and healthy participants. The observed results revealed differences in the colonic microbiome composition and ẞ-glucuronate and tryptophan related metabolism at an unprecedented detail that was not achievable through other investigation techniques such as 16S rRNA gene sequencing .Proteobacteria has been detected in PD mucosa [Further metagenomics analysis has been shown that fecal microbiota of PD patients is featured by a decreased abundance of butyrate producing bacteria, known for their anti-inflammatory properties. Moreover, a parallel increase of pro-inflammatory D mucosa .Bacteroidetes and Prevotellaceae and an increase of Enterobacteriaceae have been shown as the key players in PD fecal samples [An integrated metagenomics and metabolomics approach has highlighted that impaired concentrations of SCFAs, among the main metabolic products of gut-associated bacteria fermenters, may contribute to the gut microbiota-mediated alterations. Such alterations could worsen the ENS environment and, consequently, contribute to the gastrointestinal dysmotility of PD patients. Particularly, the reduction in SCFAs levels, along with a decrease of  samples . Studies performed on GF mouse models have been remarkably contributing to elucidating the molecular mechanisms underlying the microbiota–PD connection. As stated above, alterations of gut microbiota could be a trigger for αSyn aggregations ,132. Enterobacteraceae, and in particular Proteus spp. [Using αSyn overexpressing mice as a well-validated model for PD, it has been reported that gut microbiota promotes motor deficits, microglia activation and neuroinflammation, and ultimately αSyn accumulation . Remarkaeus spp.  which aleus spp. . Following an innovative approach, Flores Saiffe Farías et al. performed an in-silico framework to associate metaproteins with the brain proteins expression through ontological labels. Out of the metaproteome-derived data, PD was found to be associated with selected bacterial taxa, and functional classes related with neuronal communication, DNA/RNA metabolism, and alterations in the Major Histocompatibility Complex-I . In general, it must be taken into account that PD is a multifaceted disease due to a complex interaction among genetic and environmental factors. These studies underline a pivotal role of the microbiome dysbiosis in PD, and further investigations are desirable to determine how it could trigger and/or amplify the neuronal damage.AD is a chronic neurodegenerative disease characterized by synapses loss and neuronal death leading to a progressive decline in cognitive function, loss of memory and, lately, to dementia. Deposition of amyloid beta peptide (Aβ) in plaques and accumulation of hyperphosphorylated tau in the so-called neurofibrillary tangle (NFT) are the main hallmarks of AD . The mechanisms underlying these toxic depositions, which lead to an increased neuro-inflammation, are not fully understood, but it has been supposed an important role of the gut microbiota in this context .Eubacterium deficiency (known for the peculiar anti-inflammatory function) and an overabundance of Escherichia/Shigella, when compared to healthy patients. [Cattaneo et al. highlighted a close connection between brain amyloidosis and pro-inflammatory gut bacterial taxa. Following a deep metagenomics approach, patients with cognitive amyloidosis damage showed higher levels of pro-inflammatory cytokines, a rectal atients. . In GF Aβ precursor protein (APP) transgenic mouse model, a remarkable reduction of cerebral Aβ amyloid deposition has been shown when compared to control mice with intestinal microbiota. The absence of intestinal microbiota caused a significantly decreased cerebral Aβ amyloid pathology. Based on 16rRNA sequencing, the colonization experiments showed the importance of the nature of the donor (diseased transgenic versus wild-type model) for the promotion of AD. Thus, persistent gut microbial dysbiosis can regulate host innate immunity mechanisms that affects Aβ amyloidosis, and, consequently, microbiota modulation can induce positive effects on neuronal pathways, slowing down the progression of AD .A recent study of Haran and colleagues has applied a metagenomics approach to comparatively evaluate the gut microbiome of AD elders versus the stool microbiome of non-AD elders. The study highlighted numerous microbial taxa and functional genes to be considered as predictors of AD dementia. Specifically, AD-microbiome is characterized by a lower proportion and prevalence of butyrate-producing bacteria along with a higher abundance of bacterial taxa commonly known to cause proinflammatory states .ALS is a neurodegenerative disease characterized by the death of lower and upper motor neurons in the spinal cord, brain stem, and motor cortex, leading to progressive paralysis and weakness. Although many factors are known to be implicated in the neuronal damage, such as microglia activated inflammation, neurotoxicity, redox unbalance, and a severe mitochondrial dysfunction, the deep and complex pathological mechanism affecting motor neuron is still not fully understood . As widely reviewed by McCombe et al., theoretical reasons support the hypothesis of the involvement of gut microbiota in the pathogenesis of ALS. They include the important connections with the impaired metabolism, host immunity, and production of toxins that induce brain damage . G93A mouse model. Most of the experimental evidence of gut dysbiosis comes from studies on mouse models for ALS, such as the SOD1G93A mice, compared to wild-type mice, by Wu et al. Alterations in tight junction structure at the intestinal level and a reduced expression of the related protein (ZO-1), have been correlated to an increased intestinal permeability enabling the passage of toxins from intestinal lumen into blood circulation [A dysfunction in the intestinal tract has been highlighted in transgenic SOD1culation . Parabaceroides distasonis, Lactobacillus gasseri, Prevotella melaninogenica, Ruminococcus torques, Akkermansia muciniphila (AM), and others, is related to disease condition. Interestingly, AM has been shown to decrease in a time dependent manner with the disease progression [In ALS mouse altered microbiome composition and function have recently been highlighted, even before the onset of symptoms due to motor neuron dysfunction . In partgression . In general, a few studies have been performed on human ALS, and with conflicting results.Ruminococcaceae. The authors concluded that ALS cannot undoubtedly be associated with a significant gut microbiota impairment [Brenner et al. have compared an ALS patient group, strictly selected without symptoms of dysphagia, gastrostomy, non-invasive ventilation, or low body mass index to an age- and gender-matched healthy control group. The metagenomics analysis of fecal microbiota has revealed that ALS patients do not show substantial alterations in gut microbiota composition, rather in the total number of microbial species and in the abundance of uncultured pairment .Ruminococcus abundance, and low Firmicutes/Bacteroidetes (F/R) ratio, as an indicator of dysbiosis. In contrast, most patients showed inflammatory markers, such as fecal secretory IgA, eosinophilic protein X, and calprotectin. The authors therefore assumed that gut-mediated inflammation is likely to be involved in ALS onset or progression [Similarly, Rowin et al. showed a low diversity of intestinal microbial composition in fecal samples of ALS compared to controls, inferring that there is no direct and significant gut microbiota–disease correlation. Only a few ALS patients showed low gression . Firmicutes/Bacteroides ratio has also been shown in ALS patients along with an increase of the abundance of genus Dorea and a decrease of the abundance of genus Oscillibacter, Anaerostripes Lachnospiraceae [In a previous metagenomics study, a reduced piraceae . Escherichia coli and Enterobacteria, and a decrease of Clostridium and yeast [Conversely, Mazzini et al. highlighted an altered ALS gut microbiota by quantitative PCR analysis. Specifically, the authors showed a cluster distinction between bacterial profiles of ALS patients compared to controls, especially related to an increase of nd yeast .Fecal microbiota transplantation (FMT) has emerged as a promising strategy to restore gut microbiota dysbiosis involved in complex pathologies including neurodegenerative diseases . To this regard, a very recent paper has proposed a multicenter randomized double-blind clinical trial employing FMT as a therapeutic intervention for ALS patients at an early stage, opening new avenues for the treatment of neurodegenerative diseases by acting on the microbiota modulation . It is nowadays well established that the gut microbiota is able to influence human health by acting on several physiological processes. In recent years, huge efforts have been made to shed light on the myriad of established cross-communications between the gut microbiota and distant organs, such as the brain. There is still an open debate about whether dysbiosis is a factor that determines neurodegeneration or rather an epiphenomenon resulting from it. The mutual correlation has undoubtedly been confirmed by many animal and human studies.However, this field is still in its infancy and further complementary studies are needed to address the outstanding issues. In this context, the integrated approach based on the potential and experimental strength of the omics and meta-omics sciences currently opens new perspectives and provides powerful tools to support ongoing research and clinical studies. The valuable contribution meta-omics sciences have already made in the investigation of taxonomic characterization and functional dynamics of gut microbiota should be acknowledged. These sciences make it possible to obtain functional data and results not achievable with the other investigation methods available so far.We are confident that the continuous technological progress and the development of innovative omics-based investigation methods will allow, in the near future, even more in-depth studies on a wide range of microbiota related disorders, including but not limited to neurodegenerative diseases.


Research on emotion suggests that individuals regulate their emotions to attain hedonic or instrumental goals. However, little is known of emotion regulation under low emotional clarity. The theory of motivated cue integration (MCI) suggests that emotion regulation under low emotional clarity should be understood as dissociation between a high-level individual hierarchical system of goals and low level interoceptive and exteroceptive embodied cues. MCI conceptualizes low emotional clarity as the product of low access to signals of emotion that result in prediction error associated with mismatch between the current bodily state and the predicted state. This deficit in emotional processing could be understood as a problem of means substitution, suggesting that use of alternative multisensory data may facilitate situational evaluation. Based on this reasoning, a new perspective on emotion regulation under low emotional clarity is presented, according to which interchangeable attention to multisensory data associated with words, associations, and images may help in cue integration, enabling the creation of a link between concrete bodily cues, abstract mental representation, and a more accurate prediction. Based on the idea that emotional episodes are conceptualized as special types of goal-directed action episodes, this process will lead to the creation of broader integrative meaning, results in the development of emotion goal. Emotion regulation is an essential aspect of mental health and refers to processes that amplify, attenuate, or maintain the strength of emotional reactions  such thaEmotional clarity is defined as the extent to which people unambiguously identify, label, and characterize their own emotions. A majority of theories consider awareness and clarity of emotions as the building blocks of emotion regulation e.g., . For exaMotivated cue integration (MCI) theory , 2019 prThe theory of MCI , 2019 atAccording to MCI, this process occurs both in the bottom-up and the top-down directions through selective attention to multisensory information and affective signals, which, in turn, are integrated into meaning and result in action generation. Awareness of the association between high-level processes and low-level processes has been demonstrated in various studies. Based on the rationale that many voluntary actions aimed at ensuring homeostatic properties, The distinction between high-level and low-level processes has been illustrated in earlier emotion theories . For exaThis variability in findings and the challenge of replication  help expMCI suggests that the monitoring of the flow of interoceptive and exeroceptive cues, associated with words, mental images, accessible memories along with the individual’s goals, will contribute to the formation of integrative meaning . Goals aIndividual differences are conveyed by the unique associations that individuals make between goals and means of attainment , the repThere is evidence that both emotional and non-emotional action tendencies are determined by high-level goal-directed processes, which differ only in the degree of control precedence that they have . AccordiAccording to the predictive coding model e.g., , emotionMCI suggests that dissociation between high-level personal goals and low-level multisensory data results in a prediction error. Under this condition, automatic emotion regulation of preferences does not contribute to relief of psychological distress. While automatic prediction may be efficient in different contexts, low emotional clarity requires increased awareness.via a prior means was unrecognized or thwarted, alternative means should be used. Following this view, alternative types of encoding  may increase access to the dissociated aspects of the psychological experience and clarify emotion. Supportive evidence exists for the possible substitutability of emotion and alternative types of encoding, indicating the association between multisensory data and mental images as alternative types of encoding , enabling emergence of new details which may reconstruct the emotional experience. Clinicians and neuropsychologists can train individuals to use awareness-of-sensation techniques to increase emotional clarity by integrating peripheral multisensory data and creating associations between perceptual cues, symbols, and the individual’s personal needs to create an emotion regulation goal. Integrating peripheral cues in the moment-by-moment creation of meaning may bridge the gap between research on higher level emotion regulation  and variThe author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This systematic review of randomized clinical trials aimed to evaluate the available evidence regarding the efficacy of propolis-based mouthwash on dental plaque and gingival inflammation.PubMed, Scopus, and Web of Science databases were searched up to November 2019. Clinical trials that evaluated the efficacy of propolis mouthwashes compared with chlorhexidine (CHX) were included. The primary outcomes comprised dental plaque and/or gingival inflammation. Two authors assessed the risk of bias using the Cochrane tool. Due to marked heterogeneity of the available data, studies were assessed qualitatively, and no metaanalysis was performed.Nine clinical trials, comprising 333 subjects, fulfilled the eligibility criteria. Most of the included studies showed high risk of bias. Overall, propolis mouthwashes showed good efficacy on plaque and gingivitis in all of the included studies. Out of the eight studies that reported on plaque index, 5 studies found equal efficacy of propolis and CHX in reducing plaque, two studies found superior efficacy in favor of CHX, while one study found superior efficacy in favor of propolis. Six studies assessed gingival inflammation outcome, four of which reported better results with propolis, while two studies reported comparable results.The results suggest that propolis-based mouthwashes have potential benefits in reducing plaque and gingival inflammation. However, methodological limitations along with small sample sizes in some of the included studies weaken the strength of the evidence. Therefore, further well-designed clinical trials with large sample sizes and adequate follow-up period are recommended to discern the efficacy of propolis mouthwash on plaque and gingivitis. Dental plaque is the main etiological factor of periodontal diseases, mainly plaque-induced gingivitis , 2. The Improper oral hygiene is the main risk factor for periodontal diseases, irrespective of the age , 2. TherOwing to their ease of use and availability over the counter, the public highly appreciate mouthwashes. Chlorhexidine mouthwash (CHX) is considered as the gold standard antiplaque agent . CHX  guidelines and PICO principles . The proThe following eligibility criteria were applied: 1) Study design: Randomized controlled trials (RCT); 2) Participants: Systemically healthy adults aged ≥18 years; 3) Intervention: propolis-based mouthwash of any concentration; 4) Comparison group: CHX mouthwash of any concentration; 5) Outcomes: gingival inflammation and/or plaque index; 6) Follow-up period: any time with no restriction on the follow-up time; and 7) Language: articles published in English.A comprehensive literature search of PubMed/Medline, Scopus, and Web of Science (ISI) databases was conducted in December 2019 for all relevant studies from the date of inception up to and including November 2019. A combination of the following keywords and Medical Subject Heading (MeSH) terms were used: (“propolis” OR “honey” OR “honeybee” OR “bee glue”) AND . The titles and abstracts of the retrieved studies were screened independently by two reviewing groups composed of two reviewers each  and , and irrelevant studies were excluded. Disagreements, if any, were resolved by discussion or by a reviewer not involved in the screening. Full-text of the potentially eligible studies were obtained and evaluated independently by the two reviewing groups for inclusion. Respective authors were contacted whenever deemed necessary. Additionally, the references of the retrieved studies were hand-searched for any additional studies. One of the included studies lacked some important data, and hence the respective authors were contacted via email; thankfully, the authors responded and provided all missing information.Risk of bias of the included studies was carried out independently by two reviewers (AS and AA) according to the Cochrane risk of bias assessment tool . The folThe following data were extracted and tabulated by two authors (AS and AM): authors and year of the study, the study design, country, number of participants, mean ages, gender, concentration of propolis and CHX, frequency of use per day, follow-up period, side effects, clinical parameters; and main outcomes.A total of 849 articles were retrieved, 248 of which were duplicates and thus were removed. The titles and abstracts of the remaining 601 articles were screened independently by the two-reviewing groups, and 559 were excluded because they did not fulfill the eligibility criteria. The full-text of the remaining 42 articles were obtained and evaluated for inclusion. Of these, 33 articles were excluded for various reasons (list of the excluded studies and the reason of exclusion are presented in supplementary Table General characteristics of the included studies are presented in Table All included studies compared propolis-based mouthwashes with CHX , 17–22 (Dental plaque was assessed in eight included studies , 18–22. Out of the eight studies , 18–22 tOut of the six studies that reported on gingival inflammation outcome, four studies , 20, 22 Eight studies , 18–22 rThe risk of bias assessment results are summarized in Table To the best of our knowledge, this is the first systematic review that assessed the available evidence regarding the efficacy of propolis-based mouthwashe in comparison to CHX in reducing plaque accumulation and gingival inflammation. Overall, the findings of the qualitative analysis suggest that propolis-based mouthwashes are comparable to CHX in reducing plaque, but more efficacious in reducing gingival inflammation. Additionally, the results showed that propolis-based mouthwashes are safe with no or minimal reported side effects. Nevertheless, these findings should be interpreted with caution, given some methodological weaknesses discussed in the subsequent sections.One of the main outcomes assessed in the present review was the efficacy of propolis-based mouthwashea in reducing dental plaque. The results showed positive antiplaque efficacy of propolis comparable to CHX, the gold standard mouthwash for plaque. Propolis has been shown to have antibacterial properties against different strains of aerobic and anerobic oral pathogens, probably due to the flavonoid content of propolis , 15, 18.Undoubtedly, CHX is the most widely used mouthwash for plaque and gingivitis . HoweverDefinitive conclusions can only be drawn from high quality studies. For this purpose, we critically analyzed the included studies using the Cochrane Collaboration’s tool for assessing risk of bias. The results showed that most of the included studies were of low quality as reflected by the high risk of bias in these studies, making it difficult to draw a robust conclusion. Another concerning issue is the obvious methodological discrepancies in reporting and measuring the outcomes across the included studies. This in fact precluded us from pooling the data and performing meta-analysis.Adequate follow-up period is central for evaluating the clinical efficacy of any mouthwash . In the Certainly, the results of the present systematic review support the clinical efficacy of propolis-based mouthwashes for controlling plaque and gingivitis. However, there are many limitations that should be acknowledged. The main limitation is the small sample sizes along with low quality of some of the included studies. Another important limitation is the substantial heterogeneity among the included studies with respect to outcomes measurements, duration of evaluation, dose and formulations of the propolis-based mouthwashes, and gender and age of the participants. Furthermore, the present systematic review included only studies published in English and searching only three databases for the relevant studies and thus we may have missed some potential studies.The present study suggests that propolis-based mouthwashes may have good clinical efficacy in reducing plaque and gingival inflammation. However, due to the marked methodological variability across the included studies as well as the high risk of bias in some of the included studies, further well-designed clinical trials with adequate follow-up period and standardized methodologies are highly recommended to discern the clinical efficacy of propolis-based mouthwashes before advising to use them in clinical practice.Additional file 1: Table 1. Excluded studies and reasons for exclusion.Additional file 2: Table 2. Means and standard deviations of PI and GI of the included studies.


Combining entransy theory with constructal theory, this mini-review paper summarizes the constructal optimization work of heat conduction, convective heat transfer, and mass transfer problems during the authors’ working time in the Naval University of Engineering. The entransy dissipation extremum principle (EDEP) is applied in constructal optimizations, and this paper is divided into three parts. The first part is constructal entransy dissipation rate minimizations of heat conduction and finned cooling problems. It includes constructal optimization for a “volume-to-point” heat-conduction assembly with a tapered element, constructal optimizations for “disc-to-point” heat-conduction assemblies with the premise of an optimized last-order construct and without this premise, and constructal optimizations for four kinds of fin assemblies: T-, Y-, umbrella-, and tree-shaped fins. The second part is constructal entransy dissipation rate minimizations of cooling channel and steam generator problems. It includes constructal optimizations for heat generating volumes with tree-shaped and parallel channels, constructal optimization for heat generating volume cooled by forced convection, and constructal optimization for a steam generator. The third part is constructal entransy dissipation rate minimizations of mass transfer problems. It includes constructal optimizations for “volume-to-point” rectangular assemblies with constant and tapered channels, and constructal optimizations for “disc-to-point” assemblies with the premise of an optimized last-order construct and without this premise. The results of the three parts show that the mean heat transfer temperature differences of the heat conduction assemblies are not always decreased when their internal complexity increases. The average heat transfer rate of the steam generator obtained by entransy dissipation rate maximization is increased by 58.7% compared with that obtained by heat transfer rate maximization. Compared with the rectangular mass transfer assembly with a constant high permeability pathway (HPP), the maximum pressure drops of the element and first-order assembly with tapered HPPs are decreased by 6% and 11%, respectively. The global transfer performances of the transfer bodies are improved after optimizations, and new design guidelines derived by EDEP, which are different from the conventional optimization objectives, are provided. Heat and mass transfers exist widely in nature and engineering. Some heat and mass transfer enhancement problems exist, such as how to effectively dissipate heat flow that is generated in an electronic device, strengthen convective heat transfer in a confined space ,3,4,5,6,Bejan  found thT is the temperature of the object. It was called the heat transport potential capacity and phenomenologically corresponded to energy in the electrical capacitor.Various performance indexes have been taken as optimization objectives in heat and mass transfer optimizations. To reflect the essence of the heat transfer process, based on the analogy between heat conduction and electrical conduction processes, Guo et al. ,17 propoHeat quantity as a kind of energy is conserved in the heat transfer process but the heat transfer ability of the object is dissipated. That is, the entransy or heat transport potential capacity should be dissipated. In the heat transfer process, the dissipation rate of entransy per unit time and volume is named as the entransy dissipation function Using the concept of entransy, EDEP, which can guide heat transfer optimization, is deduced as follows ,17: “ForThe optimizations based on the maximum potential difference minimization can only reduce the maximum potential of a body and reflect its local transfer performance. To improve the global transfer performance of transfer bodies, the entransy theory ,19,20,21Based on the constructal theory and entransy theory, heat conduction and finned cooling problems, cooling channel and steam generator problems, and porous medium mass transfer problems have been investigated. Comparisons of the optimal results obtained by the extremes of entransy dissipation and other optimization objectives have been conducted.Bejan  introducFins with convective heat transfer area useful way to dissipate heat from a heat generating body . Bejan aA tapered assembly with tree-shaped HCCs is shown in problems  with theT-shaped , Y-shapeT-shaped , umbrellT-shaped  and treeT-shaped  fins) weThe results showed that the optimal constructs of the tapered assembly, disc-shaped assembly, and the four kinds of fins discussed, based on the minimizations of EDR and MTD, are different. The optimal constructs based on EDR minimization decrease the mean heat transfer temperature differences (MHTTDs) and improve their HCPs. For the tapered assembly, the MHTTD is not always decreased when the internal complexity of the assembly is increased , but an For the disc-shaped assembly, the critical radii based on the minimizations of EDR and MTD, which determine whether the radial-patterned disc or tree-shaped disc is adopted, are different . In Figu complex . In FiguAnother effective heat dissipation method is the convective heat transfer in the cooling channel. Bejan and Errera  insertedOpposite to the heat dissipation processes with HCCs or cooling channels, the heat should be absorbed by the steam generator as much as possible. Kim et al. ,71 maximConstant cross-sectional channels in a rectangular heat generating body are shown in ing body  75] was wasD2, Dmization  76]. In. InD2, DThe results showed that the optimal constructs of cooling channels in rectangular and cuboid bodies based on the minimizations of EDR and MTD, as well as those of a steam generator based on maximizations of EDR and HTR, are different. For the cooling channels in the rectangular body, the flow resistance is evidently reduced when EDR minimization of the rectangular element is conducted, and it can be further reduced by adopting variable cross-sectional channels . In Figumization , the ETRmization  and theyThe mass transfer problem has many similarities with the heat conduction problem, and some scholars have conducted mass transfer constructal optimizations ,78 by anx and y directions, HPP width, and width, length, and area of the rectangular element, respectively. The mass flow uniformly generates in the assembly and flows out from the left side of the HPP. The total areas of the assembly and HPP are fixed. The minimizations of MPD and mass EDR were taken as optimization objectives, respectively. Constructal optimizations of the assemblies with the premise of an optimized last-order construct and without this premise were implemented by varying their shapes. Comparisons of the constructal results with constant and tapered HPPs and the results based on the two optimization objectives were also conducted. Similar to the work of the rectangular assembly, the “disc-to-point” mass transfer problem with HPP  is lower. Compared with the rectangular mass transfer assembly with constant HPP, the MPDs of the element and first-order assembly with tapered HPPs are decreased by 6% and 11%, respectively, and the mass transfer performances (MTPs) of the assemblies are improved. When the premise of an optimized last-order construct is adopted, the optimal constructs of disc-shaped mass transfer assembly obtained based on the minimizations of MPD and mass EDR are different . In Figuonstruct . In Figu(1)For the heat conduction and finned cooling problems, the MHTTD of the tapered assembly is not always decreased when the internal complexity of the assembly increases, but there exists an optimal order of the assembly that leads to the minimum MHTTD. The critical radii of the disc-shaped assembly based on the minimizations of EDR and MTD, which determine whether the radial-patterned disc or tree-shaped disc is adopted, are different. The HCP of the disc-shaped assembly can be further improved by releasing the premise of an optimized last-order construct. The global HTPs of Y-shaped and tree-shaped fins are better than that of a T-shaped fin. The global HTP of a tree-shaped fin is not always better when the internal structure becomes more complex.(2)For the cooling channel and steam generator problems, the optimal construct of cooling channels in rectangular and cuboid bodies based on the minimizations of EDR and MTD, as well as those of a steam generator based on the maximizations of EDR and HTR, are different. The flow resistance of the cooling channels in the rectangular body is evidently reduced when EDR minimization of the rectangular element is conducted, and it can be further reduced by adopting variable cross-sectional channels. There exists an optimal volume fraction of cooling channels in the cuboid when EDR minimization is conducted, but it does not exist in the MTD minimization. Compared with the optimal results obtained by MTD minimization, the ETR of the cuboid body obtained by EDR minimization is reduced by 23.12% and its global HTP is improved. The average HTR of a steam generator obtained by EDR maximization is increased by 58.7% compared with that by HTR maximization, which illustrates an evident improvement of the global HTP.(3)For the porous medium mass transfer problems, the optimal constructs of rectangular mass transfer assembly with tapered HPP obtained by the minimizations of MPD and mass EDR are different. Compared with the rectangular mass transfer assembly with constant HPP, the MPDs of the element and first-order assembly with tapered HPPs are decreased by 6% and 11%, respectively, and the MTPs of the assemblies are improved. When the premise of an optimized last-order construct is adopted, the optimal constructs of a disc-shaped mass transfer assembly obtained based on the minimizations of MPD and mass EDR are different. The latter construct reduces the APD of the disc-shaped assembly evidently, which reflects the essential requirement of MTP optimization. Moreover, the APD can be further decreased by releasing the premise of an optimized last-order construct.Heat and mass transfer enhancements exist widely in engineering problems and the constructal theory has become a useful tool to solve these problems. The optimizations based on maximum potential difference minimization can only reflect the local transfer performances of transfer bodies. To improve the global transfer performances of transfer bodies, a series of constructal optimizations were conducted by combing the entransy theory with the constructal theory. This paper reviews constructal optimizations, performed at the Naval University of Engineering, for heat and mass transfers based on EDEP. Optimal constructs of the heat conduction and finned cooling bodies, cooling channel bodies, steam generators, and porous medium mass transfer bodies were obtained by optimizing their external and internal structures. Comparisons of the optimal results obtained by different optimization objectives and assembly methods were conducted. The results show the following: The constructal optimizations for heat and mass transfers based on EDEP make the global HTPs and MTPs of the transfer systems effectively improve. These optimizations show the merit of the entransy theory and illustrate the significance of entransy optimization. The corresponding results reviewed in this paper can provide new guidelines for the designs of heat and mass transfer systems.


The protein glycosylation is a post-translational modification of crucial importance for its involvement in molecular recognition, protein trafficking, regulation, and inflammation. Indeed, abnormalities in protein glycosylation are correlated with several disease states such as cancer, inflammatory diseases, and congenial disorders. The understanding of cellular mechanisms through the elucidation of glycan composition encourages researchers to find analytical solutions for their detection. Actually, the multiplicity and diversity of glycan structures bond to the proteins, the variations in polarity of the individual saccharide residues, and the poor ionization efficiencies make their detection much trickier than other kinds of biopolymers. An overview of the most prominent techniques based on mass spectrometry (MS) for protein glycosylation (glycoproteomics) studies is here presented. The tricks and pre-treatments of samples are discussed as a crucial step prodromal to the MS analysis to improve the glycan ionization efficiency. Therefore, the different instrumental MS mode is also explored for the qualitative and quantitative analysis of glycopeptides and the glycans structural composition, thus contributing to the elucidation of biological mechanisms. N-acetylgalactosamine , N-acetylglucosamine (GlcNAc), glucuronic acid (GLCA), iduronic acid (IdoA), mannose (Man), sialic acid (SA), and xylose (Xyl)   ,16. The lcNAc-R] . This suthaliana ,19, it hal. 2003 ,22. WithStudies on the glycoforms structures have been conducted in plants by Taylor et al. 2012; Tryfona et al. 2012; highlighting that a residue of Gal can be transferred to Ser residues on specific proteins and arabinose chains, and that structurally complex arabinogalactans are present on the hydroxyproline residues of the proteins belonging to the cell wall ,24,25.O-mannosylation characterized the O-glycosylated proteins displaying a higher variable in sugar components and the linkage type of glycans determining the multiple functions  and sialyl Lewis x (SLex) ) is observed in advanced stages of cancer +, m/z 204 [HexNAc + H]+, m/z 292 [NeuAc + H]+, and m/z 366 [Hex-HexNAc + H]+  glycosylation on Asn52 site of L.VQKN52VTSESTCCVAKSY.N peptide) was reported in A glycosylation profile of standard (Sigma) pituitary human follicle stimulating hormone (hFSH) was performed by LC-MS/MS analysis using Q-Exactive plus spectrometer by Chiara Guerrera and coworkers (paper under revision). An example of high resolution MSThe fragmentation pattern of high-energy collision dissociation (HCD) is an alternative type of fragmentation method characterized by higher activation energy and shorter activation time comparing to the traditional ion trap CID . HCD forThe MS/MS spectra offered a valid support for the choice of specific transitions to be used during the development of the quantitative MRM method. Actually, once established, the glycosylation pattern of the glycopeptide of interest, the most intense signals were selected to set up the MRM method. The extracted ion currents (EIC) associated to the different transitions for the same glycopeptide were recorded at the same retention time. This finding ensured the unequivocal identification above to contribute glycoprotein quantification. In Therefore, transitions are representative of intense fragments ions that are unique for the peptide to be quantified, thus ensuring high sensitivity and reduced interference from other peptides ,140.Major complications can also arise in developing a glycopeptide quantification method because of the absence of exogenous glycopeptide standards and incomplete proteolytic digestion caused by steric hindrance due to the glycan chains ,141,142.6 has been used for the labeling of glycans, resulting in an Δm of 6 Da [13C and 15N of proteotypic peptides (Δm 8 and 10 Da). Anyway, in order to conduct a quantitative analysis of the glycoforms present in a mixture, it is essential to quantified non-glycosylated peptides in order to normalize the glycosylation profile to the total protein content [As reported by Lebrilla et al., the application of MRM in the field of glycomics can be divided into three main areas: quantification of glycoproteins, glycopeptides, and oligosaccharides, showing each of them intrinsic troubles. In order to quantify glycoproteins, the crucial step is the selection of glycopeptides to be monitored . Sample  of 6 Da  or lysin content .Quantitative protein assays by using targeted MRM strategies have been developed to investigate protein concentrations in various biological fluids ,153,154 Some examples of glycoproteins quantification based on mass spectrometry in MRM mode are annotated below to support the high potential and versatility of approach even in rapid routine clinical screening. Recently, a method for the quantification of total glycosylated and sialylated prostate-specific antigen (PSA) was recently developed. Periodate-oxidized PSA tryptic glycopeptides are captured using immobilized hydrazide, released by PNGase F, and quantified by MRM using a triple quadrupole LC-MS . In a reThe extensive literature on protein glycosylation reveals numerous examples in which these post-translational modifications play essential roles in the events of biological recognition, signaling and cell–cell communication. In fact, oligosaccharide structures perform crucial functions throughout the cell: in the cytosol, on the cell surface, in the secretory compartments, and in the extracellular space. Among the many roles that glycans play on the cell surface, the importance of specific glycosylated forms of the protein domain to facilitate or modulate biological recognition events has been highlighted. Therefore, the knowledge of the diversity of the structures of glycans becomes a further level of information content in the understanding of biological systems, laying the foundations for the greatest challenge of the near future, that is, identifying the critical contexts in which the functions of the glycans contribute to the biological regulation of the inside the surprisingly large array of heterogeneous glycan structures.As widely described in this review, recent advances in qualitative and quantitative analytical strategies based on the use of mass spectrometry provide the necessary breadth, depth, and sensitivity of the analysis to define the entire spectrum of the complexity of glycan in various biological contexts. The continuous high-performance adaptations of these methodologies enable the collection of essential structural datasets to reveal the mechanisms in the biosynthetic pathway regulation, to define unique glycan signatures for pathological states and to provide correlations between structure and biological functions.In this way, by increasing the ability to decode the numerous functions of glycoprotein or glycans in complex biological systems, the development of new therapeutic approaches, such as vaccines or targeted pharmacotherapies, is encouraged. Although technological progress in the field of targeted mass spectrometry has made great strides in the recognition of specific glycan structures, future studies are necessary for the realization of standardized methodologies that can be used in common clinical and diagnostic practice.With the improvement and evolution of MS technology and sample preparation techniques, these types of test will play a more important role in the quantification of glycoproteins. In a futuristic scenario, to build high-performance platforms for the verification of cancer-exclusive glycoforms, these MRM-MS assays could be associated with methods to enrich robotic immunoaffinity ,163.


Since 2019, China has been the second largest medical device market in the world. At present, high-end radiotherapy equipment such as particle therapy system has a huge market potential due to the grim situation of cancer prevention and control and the growth of people's wealth in China. However, China's MedTech industry, especially the particle therapy equipment field, still faces the influence of policy, fluctuation of market demand, strengthening of industry supervision, and even geopolitical realities. This paper reviews the market prospect of particle therapy medical devices from the perspective of China's medical device policy and demand information analysis, which is conducive to the research on the industrial layout of particle therapy medical physics, and also helps high-performance medical device manufacturers to expand their business visions. MedTech manufacturers should actively adjust their business strategy and implement scientific and technological innovation on the basis of compliance with industry regulatory requirements in order to seize opportunities from challenges and gain profits growth. The Chinese medical device market is fostered by the burgeoning demand from the increasingly affluent and a rapidly aging population and the rising prevalence of chronic diseases, such as diabetes, heart disease, and cancer.Today, the market for medical devices in China is rapidly developing, far exceeding the country's economic growth rate. Currently it is increasing in value by around 20% a year, and it was worth an estimated value of $96.3 billion in 2019, making it the second largest medical device market in the world after the United StatesParticle therapy equipment in particular is in great need in China.From 2015 to 2020, a total of 77 proton and heavy ion center projects have been reported in China, including two heavy ion (Wuwei and Shanghai) and four proton therapy projects in operation, 26 proton projects under construction, and 60 proposed items. Many hospitals in China have signed purchase agreements for proton heavy ion radiotherapy equipment. Enthusiasm for investment presents the potentially attractive and capacious market future of particle therapy equipment.However, profit growth in China's MedTech industry still faces the influence of policy, shifting of market demand, strengthening of industry supervision, and even geopolitics. Specifically, the shifting of domestic demand, regional share, and strict supervision are the challenges faced by the high-end medical equipment industry, especially the particle therapy equipment field.China's “made in China 2025” initiative calls on China's top hospitals to increase the use of domestically produced equipment by 50% by 2020, focusing on the development of high-performance equipment, such as diagnostic imaging equipment, robotic surgical instruments, etc. . The treThe state pushes forward the implementation of demand localization strategy through centralized bidding policy and governmental procurement redirection. Those technology roadmap and business strategy of medical device products with access to the Chinese market need to adapt to the shifting demand .The state-owned public and private hospitals respond quickly for the localization strategy in China. In 2019, six quotas of proton therapy equipment were obtained by large-scale regional public hospitals, at least half of which are to be locally made. Although Chinese medical device manufacturers are the leaders in the domestic market, hospitals and healthcare providers nationwide prefer imported devices, especially when purchasing innovative and high-tech devices. Significant scale of demand for foreign-made, high-end devices is also fueled by continuous growth of China's aging population, and driving forces to improve the quality of care by the Chinese government. Obviously, the strategy of using legal commercial means to transform foreign brand products into local-made ones helps to cope with the changes in market demand trend.Due to the worry that hospitals introduce large-scale medical equipment blindly, resulting in waste of resources, the state has strengthened the market-oriented demand management of this equipment . With reFor those medical equipment companies that really want to maximize profits by utilizing market demand, it is time to adjust their product, supply chain, and commercial settings flexibly. In this way, medical equipment manufacturers have the chance to seek out high profits in the value chain by providing unique high-quality equipment that meets both the market demand and market regulatory requirements.The concept of China's medical device regulation also helps to standardize the medical device market. In recent years, the supervision notion has gradually integrated with the international mainstream regulatory mechanism, laying stress on ensuring the safety and effectiveness of public use of medical devices through strengthening the awareness of subject responsibility , 8. SincThe market supervision principle of Chinese government requires that MedTech manufacturers must comply with obscure requirements. In view of this industry and market trend, the medical device industry tends to adopt a localization strategy, which helps to win purchase orders on the one hand and has the additional benefits of reducing costs and enhancing market familiarity on the other hand. The foreign medical device enterprise, which occupy the high position of the value chain, also needs to be adjusted to maintain brand advantage and earn high profits. MedTech companies from the developed world should find other ways to enter China such as finding OEM partners, licensing technology, establishing factories, etc.Despite many challenges, both foreign and domestic medical device manufacturers still have many opportunities to make profits under the innovation strategy. In order to achieve its goal of promoting China's economy up the value chain, creating “national champions” and lowering dependence on foreign imports, China has made the development of the country's biomedical and high-end medical device manufacturing sector a key priority , 12. IncMedTech innovation was encouraged with a series of policies intensively promulgated throughout the development process of China's medical equipment industry. In 2013, the National Health Commission of China launched the application development and promotion plan of domestic large-scale equipment; in 2015, China grouped medical devices into the key realm of the medical industry, and was determined to accelerate the localization of medical device manufacturing during the 13th Five Year Plan period  regulations has listed medical device innovation as a priority, which speeds up the market access of innovative products. By the end of 2019, NMPA has approved the market access of 73 innovative medical device products, including the carbon ion therapy system independently developed by China .In addition, domestic medical device manufacturers are encouraged to increase investment in technology and technological innovation to enhance long-term competitiveness and move up the value chain. Some local manufacturers are growing stronger and more competitive with foreign suppliers, such as Lanzhou Kejin Taiji, Shanghai Lianying, Beijing Guoke ion, Shenzhen Aowo, Xinhua medical and Neusoft Medical Co etc., which have been competitive in CT, gamma knife, linac, carbon ion therapy system, and other products in recent years.At present, China's medical device market is full of uncertainty. In addition to being associated with the outbreak of a new coronavirus disease , the medical device industry market is expected to be alert to the impact and consequences of the trade war between the United States and China.However, opportunities outweigh challenges in China's high-end medical device market nowadays accordingly.Specifically, the influence of policy, fluctuation of market demanding, industry supervision enhancement, and even geopolitical realities are the challenges faced by the high-end medical equipment industry, especially the particle therapy equipment field. The opportunity comes from the flexible adaptation of policy and market demand fluctuation through technological innovation and product improvement.In the next national planning cycle, China's 14th Five year plan (2021–2025), the national intellectual property system will be further strengthened, and the more mature supervision mode of the medical technology market, such as two vote system and GMP (Good Manufacturing Practice) / GSP (Good Supply Practice) management evaluation system, will be improved. With the rapid development of a highly digital medical device ecosystem, the market access path of innovative products is expected to be shortened.In view of this, the opportunities for localized innovative medical devices are greater, compared with the fierce market competitive pattern of China's medical device market, especially in high-end equipment.ZD: data collection and drafting papers. YM: make important revisions to the paper. QL: approval of final papers to be published. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In the last decades, the therapeutic decision-making approach to metastatic renal cell cancer (mRCC) has dramatically changed thanks to the introduction in the treatment scenario of, first, anti-angiogenic agents and, afterward, immune-checkpoint inhibitors (ICIs). Immunotherapy is now the standard of care in pretreated mRCC patients and has recently entered even the first line setting. Nevertheless, in mRCC as well as in other tumor settings, a durable and clinically meaningful benefit from treatment with ICIs is not obtained for all patients treated. Therefore, the necessity to identify and validate predictive biomarkers of response to immunotherapy has emerged, in order to design the optimal treatment strategy for mRCC patients.In this review, we present and discuss the most promising predictive biomarkers of response to ICIs in mRCC with the recent data available. In details, the first marker that was investigated is the immunohistochemical expression of programmed death receptor ligand 1 (PD-L1), showing a negative prognostic role in mRCC, but the debate about its potential predictive value is still open. Additionally, the high heterogeneity in PD-L1 determination methods adds complexity to this issue. Second, the tumor mutational or neoantigen burden is an emerging biomarker of increased response to immunotherapy, hypothesizing that the higher the TMB, the higher is the production of neoantigens, and thus the stimulation of anti-tumor immune response, even though controversial results have been obtained. Third, the tumor microenvironment, namely the different populations of the immune infiltrate, plays a key role in tumor progression and in the response to immunotherapy. Finally, several studies have collected evidence on the potential association of the occurrence of immune-related adverse events (irAEs) with the benefit from ICIs, first in non-small cell lung cancer (NSCLC) and melanoma, and recently even in mRCC.Several promising biomarkers of response to immunotherapy with ICIs have been identified, though without conclusive results upon their potential predictive value in mRCC. Therefore, the results of the exploratory analyses of the recently presented first-line trials and hopefully of future prospective, biomarker-driven studies could provide useful tools to be applied in the everyday clinical practice. Renal cell cancer (RCC) represents about 3% of all solid tumors worldwide, with an estimated incidence of approximately 330,000 new cases per year . RCC is In the past decades, thanks to a deeper understanding of the biological and molecular disease scenario, the clinical approach to mRCC has undergone a dramatic change with the introduction of novel drugs . First, In details, the targeting of the immune system deeply changed the disease course and the prognosis of patients affected by several tumors, among which non-small cell lung cancer (NSCLC), melanoma and RCC. The introduction of the immune-checkpoint inhibitors (ICIs) targeting first the Cytotoxic T-Lymphocyte Antigen 4 (CTLA4) and subsequently the programmed death receptor 1 (PD1) and its ligand (PD-L1) provided a new therapeutic standard and a changing paradigm in different tumor settings .For what concerns mRCC, immunotherapy entered the clinical scenario in patients who had received a prior line with anti-angiogenic agents, in light of the results of the CheckMate 025 trial , 9. The Subsequently, in order to optimize the potential benefit of immunotherapy, ICIs have been investigated in the first-line setting, either alone  or associated with anti-vascular agents , thanks to several clinical trials that recently presented their first results . Altogetad hoc studies should be designed and conducted in order to potentially validate this therapeutic approach in mRCC patients  was initially identified in the first clinical study investigating the anti-PD1 agent nivolumab and, afterward, it has been widely studied in several settings .The reported rate of PD-L1 expression in the different tumors is endowed with a high variability across studies, and, considering specifically the neoplastic diseases with enhanced response to ICIs, namely NSCLC, melanoma and RCC, it ranges between 14 and 100% . NeverthConsidering the association of the expression of PD-L1 with prognosis, several studies collected evidence on a potential negative prognostic value of PD-L1 overexpression in gastric, hepatocellular, esophageal, pancreatic, ovarian, and bladder cancer, whereas a positive value in breast cancer and merkel cell carcinoma and controversial results in lung cancer, colorectal cancer, and melanoma . For exapost hoc analyses of the COMPARZ study and the METEOR and CABOSUN trials, respectively, PD-L1 expression on tumor cells was associated with shorter survival in mRCC patients, irrespectively of the type of targeted therapy received  is a recently defined emerging biomarker of increased response to immunotherapy. The definition of TMB is the total number of mutations per coding area of tumor genome, measured as mutations per megabase (mutations/Mb). The genomic alterations occurring in tumor cells display the capacity to generate tumor-specific antigens (neoantigens), potentially processed and presented on the surface of cancer cells and therefore recognized by the T cells, eliciting the anti-tumor response . The potIn the setting of mRCC, initial evidence has been collected on this topic in recent studies. TMB scores display a deep variability through the different histological subtypes of RCC, ranging from a very low level in chromophobe type to a comparable value of TMB in clear cell and papillary tumors and, additionally, TMB is not associated with the clinically defined prognostic groups according to IMDC and MSKCC . Interesad hoc prospective trials are awaited, with the aim to unravel the potential predictive role of TMB in the setting of mRCC. The ongoing NIVES study (NCT03469713), investigating the combination of nivolumab with stereotactic radiotherapy in pretreated mRCC patients, recently presented the first data, and its exploratory analyses could provide evidence on this topic in the next future , it was reported that the presence of tumor-infiltrating lymphocytes is associated with an enhanced response to immunotherapy . MoreoveAnother emerging topic in the setting of the identification of potential predictors of response to immunotherapy is the occurrence of immune-related adverse events (irAEs). In details, ICIs determine a peculiar spectrum of treatment-related toxicities, arising from an excessively augmented activity of the immune system and potentially involving any organ with a multifaceted variety of presentations , 67. TheNevertheless, the specific criteria for the definition of irAEs have not been established so far and whether any toxicity arising from an immune-mediated mechanism or specific classes of irAEs display a meaningful correlation with response to immunotherapy remains to be elucidated yet, thus warranting further investigation in this setting.Immunotherapy revolutionized the therapeutic strategy and the clinical outcome of patients affected by several different tumors, among which RCC. Nevertheless, not all patients derive a significant and durable benefit from immunotherapy with ICI, indeed, this underlines the unmet clinical need of identifying reliable predictive markers of response to immune agents. The development of biomarkers endowed with high sensitivity, specificity and accuracy able to identify which patients may truly benefit from the treatment with ICIs would allow to refine the therapeutic selection and to better tailor the treatment strategy. Furthermore, recent studies provided evidence that maybe even the prognostic stratification of mRCC patients should be refined in those treated with ICI, including the role of systemic inflammation besides the canonical criteria .ad hoc clinical trials should be collected in order to change the clinical decision-making process in the everyday real-life.The amount of data collected so far in the setting of mRCC, encompassing the expression of PD-L1, the TMB and the widespread role of immunity, has not yet established a predictive biomarker applicable in the clinical practice. The first remark is that probably the huge complexity of the scenario of response to immunotherapy could not be captured by a single biomarker, thus potentially suggesting the need for more comprehensive and integrated approaches. Another point to underline is that the high heterogeneity of the studies conducted, as well as of the techniques and methodologies used and the variety of markers investigated, adds further complexity and limits the validation of these biomarkers with the aim to realize a widely exploitable tool in the clinical setting. Finally, although extremely promising results have been obtained for several biomarkers so far, solid evidence-based data deriving from large, prospective AR and GP conceptualized and designed the manuscript. AR drafted the manuscript. PS, EZ, AM, MS, MC, VG, EV, FB, and GP critically reviewed the manuscript. All the authors approved the final version of the manuscript submitted.GP declares receiving honoraria for advisory board from Bayer, Bristol Myers Squibb, Ipsen, Merck, Novartis, and Pfizer. EV declares receiving honoraria for advisory board from Pfizer, Bristol Myers Squibb, Ipsen, and EUSA pharm. FB reported receiving honoraria for speaker activities and participation in advisory boards from Amgen, Inc., Roche, and Novartis International AG. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.