The synovial tissue is a primary target of multiple diseases characterized by different pathogenic mechanisms, including infective, deposition, neoplastic, and chronic immune-inflammatory pathologies. Synovial biopsy can have a relevant role in differential diagnosis of specific conditions in clinical practice, although its exploitation remains relatively limited. In particular, no validated synovial-tissue-derived biomarkers are currently available in the clinic to aid in the diagnosis and management in most frequent forms of chronic inflammatory arthropathies, namely rheumatoid arthritis (RA) and the spondyloarthritides (SpA). In this brief review, we will discuss the current spectrum of clinical applications of synovial biopsy in routine rheumatologic care and will provide an analysis of the perspectives for its potential exploitation in patients with chronic inflammatory arthritides. The assessment of the pathologic process at peripheral sites has been proved as a source of clinically relevant information in different human pathologies including cancers and systemic autoimmune diseases. Examples of the latter group are the assessment of the salivary glands in sialo adenitis, the muscle in idiopathic inflammatory myopathies or the kidney in systemic lupus erythematosus, in which the qualitative and/or quantitative analysis of local inflammatory processes can be exploited to corroborate diagnosis/classification, facilitate discrimination among disease entities, evaluate prognosis and guide the choice of appropriate treatments –3. SimilBased on the existence of major unmet needs and on data derived from previous proof-of-concept studies (see next paragraphs), there is now growing attention in expanding the translational applicability of synovial tissue analysis also in this direction . One of In the following paragraphs, we will provide a summary of current applications of synovial biopsy in clinical practice and of the background data that are allowing to conceive their extension into the field of stratified medicine.For the majority of rheumatologic diseases, patients' interview, clinical examination, imaging and serological tests are usually sufficient to establish a diagnosis and monitor treatment response. The analysis of the synovial tissue can be, however, of assistance for diagnostic purposes in course of arthritis of undetermined origin, allowing the identification of specific traits of a restricted, though defined, spectrum of pathologies, including infective, neoplastic and some deposition diseases . Whilst Beyond expanding the analytical substrate, the availability of biopsy specimens may also offer specific information by allowing the integration of microbiological and molecular screenings with the analysis of characteristic histopathologic traits of some infections and rare diseases (see next paragraphs for details).US-guided dry needle synovial tissue aspiration  or synovBoth synovial fluid or synovial tissue analyses can also contribute to the differential diagnosis of other rarer deposition diseases including amyloid arthropathy, through Congo-red stain and the identification of typical apple-green birefringent deposits of immunoglobulin free light chains, as well as ochronotic arthritis, typically associated to local accumulation of homogentisic acid polymers and characteristic yellow-brown cartilage debris –28.in situ through conventional stainings , as well as the evaluation of indirect signs, such as the presence/pathologic aspect of local granulomatous reactions or the degree of perivascular neutrophilic infiltration  stain or CD15 immunohistochemistry  conditions characterized by typical synovial histopathologic features and less traceable changes in the synovial fluid. Examples of these conditions, in which the synovial biopsy may have a primary diagnostic role, include primary synovial malignancies , metastatic tumors and some benign proliferative lesions like pigmented villonodular synovitis and synovial chondromatosis . In all these conditions, the iagnosis , 41–43.The standard histologic analysis of the synovium can be instrumental also in the diagnosis of arthritis in patients affected by some non-Langerhans cell histiocytic disorders, uncommon conditions characterized by multi-system involvement due to dysregulated accumulation of mononuclear phagocytes. In some forms with adult-onset , patients can display severe joint involvement, typically associated to abnormal sub-lining infiltration of CD68-positive (CD1a- and S100-negative) histiocytes and multinucleated giant cells with a lipid-laden or PAS-positive ground-glass cytoplasm –46.Altogether, these data, generated over the last decades, demonstrated the utility of synovial tissue collection for differential diagnosis, but left partially unclear the actual output of the procedure in the real-life setting of a rheumatology clinic, in particular for what concerns most recent approaches, such as US-guided biopsy. This issue has now been addressed by independent groups demonstrating, quite consistently, a success rate of around 82–96% in obtaining samples suitable for analysis with the potential to achieve a diagnosis in between 16 and 20% of the cases , 20, 47,Beyond its possible application for differential diagnosis of unclassified arthritis, synovial tissue examination has been also proved to be a valuable source of surrogate biomarkers of response-to-treatment. Evidence supporting this concept derives from pioneering studies performed in the last two decades demonstrating, through the evaluation of serial arthroscopic biopsies, the sensitivity-to-change and external responsiveness of sub-lining CD68+ macrophages in relationship to variations of clinical composite indices in rheumatoid arthritis (RA). The reduction of CD68+ sub-lining macrophages has been shown to be associated to effective treatment, less influenced by placebo compared to clinical parameters, and to be a valid measure of response to treatments characterized by different mechanisms of action –51. CollIf, on the one hand, the studies presented in the previous sections delineated the conditions that can be diagnosed through a synovial biopsy, on the other, they also shed further emphasis on what synovial tissue sampling cannot currently offer in routine clinical practice. The possibility to identify specific traits for most common forms of systemic chronic inflammatory arthropathies, namely RA and spondyloarthritis (SpA) remains, indeed, impracticable. This issue is relevant both in patho-biologic and clinical terms and has been the object of intense investigation in the past. Indeed, since early diagnosis and treatment in these conditions are linked to improved long-term outcomes , 53, theRA and SpA synovitis  do display measurable differences compared with post-traumatic and degenerative conditions, in terms of gene expression , histopaDespite these data and the observed gross analogies, there is now growing evidence from independent studies that a detailed comparative analysis of specific components of the inflammatory process may actually allow to detect multiple and congruent biological differences among diseases, in particular if patients' characteristics and the overall degree of joint inflammation are appropriately matched. One of the most compelling aspects that has been reproducibly confirmed relates to the characteristics of the vascular system. Synovial vascularity has been shown to display macroscopic and microscopic differences between RA and SpA, with the latter associated to an increased distribution of tortuous blood vessels in the sub-lining both in early and established disease –67. AccoThe existence of peculiar biological traits characteristic of SpA synovial stroma has been confirmed by gene expression analyses. In this context, of particular interest is the work performed by Yeremenko et al.  who, by No differential characteristics, instead, have been reproducibly recognized in the distribution of major lymphocyte populations  and of lining/sub-lining CD68+ macrophages –67, 70, In conclusion, data derived from several independent studies demonstrate that, despite a shared inflammatory background, the inflamed synovium of different forms of chronic inflammatory arthritides can associate to differential cellular and molecular traits. Further research and novel multi-center observational studies , 73 are If differences in the synovial characteristics can be captured between different clinical entities, a cutting-edge question is whether clinically relevant differences can be reliably distinguished also within the same disease, a fundamental premise to conceive the possible integration of synovial biopsy into a precision medicine algorithm.per se, are not sufficient to entirely explain the heterogeneity of the disease and that a finer profiling is required , all reported data were falling within the feasibility range of a routinely applicable procedure. As a complementary observation, the assessment of different characteristics  in one joint has been shown to be representative of the same parameters in other joints in RA  the myeloid phenotype, also characterized by processes associated with TNFα and IL-1β production, TLR and NOD-like receptor signaling, Fcγ-receptor-meditated phagocytosis; (iii) the fibroid phenotype, enriched for genes associated with TGFβ and BMP signaling, together with SMAD binding, but lacking enrichment of any immune system processes; (iv) the low inflammatory phenotype, showing only enrichment for inflammatory and wound response processes. These phenotypes, or similar patterns according to a recently revised classification (A critical issue remains the interpretation of the observed heterogeneity and two main models are currently emerging. In particular, whilst some studies have described the variability of synovial characteristics primarily as a function of the overall degree of inflammation intensity , 94, othfication , have befication , 97.In clinical terms, despite it remains unclear whether the heterogeneity of synovial features does reflect fixed characteristics of specific disease subsets or dynamic phases conditioned by fluctuations of the inflammatory process, we have now proof-of-concept evidence that the assessment of synovial inter-individual differences does actually have the potential to predict clinically-relevant outcomes. Data supporting this idea derive from independent observational studies based on patients' stratification through either histological parameters or molecular signatures. Associations between synovial pathologic traits and clinical response to specific treatments has been obtained in studies focusing on agent targeting different molecular pathways, including anti-TNF , 98–104,Despite current advancements, the development of a valid personalized approach to RA or SpA, based on synovial biopsy and applicable at community level still remains a very ambitious target. It should be indeed emphasized that data derived from available prediction studies, though promising, did not always led to univocal conclusions. Although differences might be obviously related to the limited sample size, differences in the definition of exposure variables and pre-set confounders, we might also consider that both RA and SpA are likely to be determined, as the majority of immune-inflammatory diseases, by a complex series of events controlled by polygenic, environmental and endocrine factors , 111. SoTaken together, the studies discussed in this review highlight the important, though circumstantial role that synovial biopsy can have in current clinical practice. Depending on the clinical context, it may complement and in some cases substitute less invasive procedures, offering the possibility to integrate microbiologic and histopathologic data. The combination of these approaches in certain circumstances can be essential to achieve a definite diagnosis in patients with arthritis of undetermined origin. The spectrum of applicability of synovial biopsy remains, however, relatively limited mostly due to the lack of validated markers for the diagnosis and management of major forms of chronic inflammatory arthritis.The next challenge is thus to define the exploitability of the heterogeneous molecular and cellular patterns that characterize the synovial tissue in RA and SpA for the development of novel diagnostic markers and multi-dimensional precision medicine algorithms. Based on recent data from observational studies and the technological advancements in synovial tissue sampling and analysis this perspective seems now more realistic. Of considerable relevance in this direction is the recent introduction of novel cutting-edge tools allowing transcriptional profiling and single-cell RNA sequencing of infiltrating cells isolated from synovial samples , 119. ThAM, SB, and SR contributed to literature review and preparation of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer AN declared a past co-authorship with one of the authors AM to the handling editor.


Pseudomonas aeruginosa is one of the six bacterial pathogens, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp., which are commonly associated with antimicrobial resistance, and denoted by their acronym ESKAPE. P. aeruginosa is also recognized as an important cause of chronic infections due to its ability to form biofilms, where the bacteria are present in aggregates encased in a self-produced extracellular matrix and are difficult or impossible to eradicate with antibiotic treatment. P. aeruginosa causes chronic infections in the lungs of patients with cystic fibrosis and chronic obstructive lung disease, as well as chronic urinary tract infections in patients with permanent bladder catheter, and ventilator-associated pneumonia in intubated patients, and is also an important pathogen in chronic wounds. Antibiotic treatment cannot eradicate these biofilm infections due to their intrinsic antibiotic tolerance and the development of mutational antibiotic resistance. The tolerance of biofilms to antibiotics is multifactorial involving physical, physiological, and genetic determinants, whereas the antibiotic resistance of bacteria in biofilms is caused by mutations and driven by the repeated exposure of the bacteria to high levels of antibiotics. In this review, both the antimicrobial tolerance and the development of resistance to antibiotics in P. aeruginosa biofilms are discussed. Possible therapeutic approaches based on the understanding of the mechanisms involved in the tolerance and resistances of biofilms to antibiotics are also addressed. Pseudomonas aeruginosa biofilms consists mainly of polysaccharides, proteins, extracellular DNA and lipids, and its composition is strain dependent, and also depends on the growth conditions and the age of the biofilm  is a key regulator of the biofilm lifecycle in many bacteria, including ruginosa . High cemponents . As a prmponents . Quorum ruginosa . The QS  fashion . PQS is  fashion , whereasne cells .One of the most important features of microbial biofilms is that the bacteria are able to survive antibiotic treatment administered at high doses . If the The term antibiotic tolerance can also be used in the context of planktonic bacterial populations, and here it describes bacterial cells that survive treatment with bactericidal antibiotics without having acquired antibiotic resistance determinants. Antibiotic tolerance of planktonic bacteria is mainly caused by an altered physiological state of the cells as a consequence of environmental stress, and is mediated by cellular stress responses and related systems . In biofin vivo by systemic administration without toxicity ppGpp, which regulates the expression of many genes. P. aeruginosa ΔrelAspoT double knockout mutant, and provided evidence that this phenotype was due to impaired anti-oxidant capacity and consequently increased endogenous oxidative stress. However, in the case of colistin, this is in contradiction to reports showing that the bactericidal effect of this antibiotic is independent of ROS (OH.) production . In biofilms, the SOS response can be activated due to several biofilm-specific stresses such as oxidative stress  and more tolerant to colistin  than wild type biofilms  of the antibiotic tolerance can be explained by oxygen limitation under these conditions  showed increased sensitivity to antibiotics. The anti-oxidant systems, such as catalases and superoxide dismutase, are upregulated by the activation of the stringent response in biofilms , which have been shown to be important for the bactericidal effect of antibiotics . Antibioinolones  and Burkycosides . Biofilmbiofilms  improvinAs bacterial metabolism plays an important role for the tolerance of biofilms to antibiotics, several strategies targeting energy production have been proposed to overcome the antibiotic tolerance .rpoS, spoT, relA, dksA, dinG, spuC, algR, pilH, ycgM, and pheA) are involved in persister formation in P. aeruginosa, suggesting that multiple pathways can lead to the development of persister cells (E. coli has suggested that the second messenger ppGpp drives persister formation through the Lon protease and activation of toxin-antitoxin (TA) modules  resulted in an increase in the sensitivity of P. aeruginosa biofilms to tobramycin and gentamicin. In addition to affecting the expression of genes encoding proteins involved in efflux pumps and ABC-multidrug transporters, BrlR also affects the expression of genes encoding modification of LPS and membrane protein composition, as well as metabolism and energy generation. This multitude of different potential targets might explain the involvement of BrlR in the regulation of biofilm tolerance to various antibiotics , while high concentrations of imipenem led to induction of ampC throughout the biofilm. The inactivation of the beta-lactam molecules by the enzymes in the biofilm matrix has pharmacokinetic/pharmacodynamic consequences, as the time-dependent effect of ceftazidime, a cephalosporin that is hydrolyzed by the AmpC beta-lactamase of P. aeruginosa, was changed to a dose-dependent effect  of P. aeruginosa are more similar to the planktonic MICs   .P. aeruginosa biofilms toward aminoglycosides may also play a role in biofilm tolerance to antimicrobial peptides. For example, it may be expected that binding of antimicrobial peptides by the negatively charged biofilm matrix components, such as eDNA, can play a role in tolerance of P. aeruginosa biofilms toward antimicrobial peptides. In addition, the Psl exopolysaccharide has been shown to confer protection of P. aeruginosa biofilms against colistin and polymyxin B (P. aeruginosa biofilms toward colistin (Most antimicrobial peptides are positively charged similar to the aminoglycosides. Therefore, some of the mechanisms that are involved in the tolerance of ymyxin B . Moreovecolistin .pmr and arn genes in P. aeruginosa biofilms may occur through eDNA-mediated cation chelation and acidification (P. aeruginosa biofilms to colistin leads to upregulation of the pmr and arn genes in the metabolically active peripheral subpopulation (arn gene products mediate a reduction of the net negative charge of LPS, which reduces the interaction with and uptake of polymyxins (pmr/arn operon plays a role in colistin tolerance of P. aeruginosa biofilms together with activation of MexAB-OprM efflux pump (mexCD-oprJ and muxABC-opmB genes are expressed in response to colistin exposure (P. aeruginosa biofilms grown under anaerobic conditions (As described previously, upregulation of the fication . Moreovepulation . The arnlymyxins . Upregullux pump . In addiexposure . Colistiexposure . This isnditions .P. aeruginosa biofilms in vitro (in vivo (P. aeruginosa lung infections after the first identification of the bacteria in airway secretions (The combination of colistin targeting the metabolically inactive population with antibiotics targeting the metabolically active population, such as ciprofloxacin and tobramycin, has been shown to be able to eradicate in vitro  and in v(in vivo . In cystcretions .P. aeruginosa imipenem can induce production of alginate (It has been shown that sub-MIC concentrations of the different classes of antibiotics can stimulate biofilm formation both in Gram positive and Gram negative bacteria. The involved mechanisms have been reviewed recently  and inclalginate , ciproflalginate .The described tolerance mechanisms, all contribute to the persistence of biofilms, which provide a fertile ground for the emergence of antibiotic-resistant mutants. In planktonic cultures, it has been reported that tolerance precedes the occurrence of resistance .Antibiotic resistance implies mutations in resistance genetic determinants leading to increased antibiotic minimal inhibitory concentrations for bacterial cells disrupted from biofilm and it is accepted as a side-effect of prolonged maintenance antibiotic therapy.In contrast to the planktonic, fast-dividing cells that are traditionally used to study antibiotic resistance development in shaking cultures, biofilm-grown bacteria encounter gradients of nutrients and oxygen which lead to a heterogeneous bacterial population including slow-growing or non-dividing cells . KnowledA common trait observed in biofilm evolution experiments is the diversification of the bacterial population to a higher degree than encountered in evolution studies using planktonic cultures . Anotherin vitro experimental evolution studies that antibiotic resistance developed by distinct pathways in planktonic cultures and biofilms exposed to sub-inhibitory levels of ciprofloxacin (nfxB mutants overexpressing the MexCD-OprJ efflux pump in biofilms exposed to low doses of ciprofloxacin, with mutants comprising 80–90% of the biofilm population after 4 days treatment, was also demonstrated (It was recently shown by floxacin . While tnstrated . This suPseudomonas aeruginosa, is an important cause of persistent infections due to its ability to form biofilms which display tolerance and resistance to antimicrobial agents. The multiple and various mechanisms employed by P. aeruginosa to survive antibiotic treatment while growing in a biofilm represent an important therapeutic challenge. The molecular mechanisms underlying tolerance of biofilms to antibiotics are targets for therapeutic interventions for potentiating the anti-biofilm effect of antibiotics. To identify the best intervention targets, a clarification of the relative contribution of the mechanisms involved in the tolerance of biofilms to specific classes of antibiotics is needed. In addition, different tolerance mechanisms might be triggered in vivo at the biofilm infection site depending on the specific environmental conditions, such as the access to nutrients and oxygen, which depend on several factors such as tissue vascularization, and degree and type of inflammation (The ESKAPE pathogen ammation . ThorougOC and TT-N contributed equaly to the concept and writing of the review.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.


This review briefly retraces the main discoveries linking the activity of gut microbiota to prevalent brain neurodegenerative disorders, and then provides a deep insight into the state-of-the-art for in vitro modeling of the brain and the blood-brain barrier (BBB), two key players of the MGBA. Several brain and BBB microfluidic devices have already been developed to implement organ-on-a-chip solutions, but some limitations still exist. Future developments of organ-on-a-chip tools to model the MGBA will require an interdisciplinary approach and the synergy with cutting-edge technologies  to achieve multi-organ platforms and support basic research, also for the development of new therapies against neurodegenerative diseases.We are accumulating evidence that intestinal microflora, collectively named gut microbiota, can alter brain pathophysiology, but researchers have just begun to discover the mechanisms of this bidirectional connection . The most noticeable hypothesis for a pathological action of gut microbiota on the brain is based on microbial release of soluble neurotransmitters, hormones, immune molecules and neuroactive metabolites, but this complex scenario requires reliable and controllable tools for its causal demonstration. Thanks to three-dimensional (3D) cultures and microfluidics, engineered in  Research on microbiota is rooted in Antonie van Leeuwenhoek's pioneering work (XVII century) Bardell, , but in In homeostatic conditions, it interacts with the intestinal mucosa and maintains its integrity  activate the immune system (including T-cells) and increase the levels of inflammatory mediators, the permeability of the gut barrier  and specific pathogen-free (SPF) mice treated with antibiotics  is not limited to digestive functions and satiety, but the gut microbiota also influences behavior and cognitive abilities  in susceptible neurons in the form of Lewy bodies and Lewy neurites  were increased in MS patients, while Parabacteroides distasonis (which stimulates anti-inflammatory interleukins in humans and mouse models) was reduced  protect the brain against microbial invasion. These barriers also allow the delivery of nutrients, the removal of metabolites and the protection of the brain from abrupt changes in blood biochemistry  can reach the BBB and then the brain , a non-protein neurotoxic amino acid that can trigger pathological processes  frequently observed in neurodegeneration  and its proteolytic enzymes called gingipains can translocate to the brain  and epithelial cells  perfused cell cultures and the difficulties of managing co-cultures of different cell populations are just examples of this complexity when modeling these two human compartments. Thus, if the main purpose is to study the effects of the secretome produced by the gut microbiota on the BBB and the brain, we have to develop more advanced and reliable systems to overcome the technical weaknesses that hinder the modeling of multi-organ communication.Taking into consideration all the possible communication pathways between the microbiota and the brain that we have described so far, it is quite apparent that the route based on the passage of soluble microbial secretome from the microbiota to brain cells is actually the most reproducible pathway to model the MGBA Organs-on-a-chip offer several advantages  and glial cells.Neurons are composed of a cell body, dendrites (picking up the messages from other nerves), and an axon (transmitting impulses from the cell body to the periphery). They communicate by several connections (synapses) that allow the passage of nerve impulses. The neuronal circuitry is composed of afferent and efferent pathways that send information by chemical or electrical signals. In chemical synapses, the signals propagate by the release of neurotransmitters, while electrical signals are due to action potentials caused by different ions crossing the neuronal membrane. They are important for the transmission of information that exploits currents flowing along the membrane of the axon to the synapses . Oligodendrocytes are essential for neuronal myelination and they contribute to the regulation of the concentration of extracellular ions. However, they are rarely included in Researchers have studied brain structure and functions extensively, but including the different cell populations and considering its surface dimensions when modeling the entire brain is an enormous challenge. They have started with single cell cultures, neglecting the possible interactions between neurons and glial cells. More recently, they have moved to the study of cell-cell interactions and improved the mimicking of natural tissue in both physiological and pathological conditions. For instance, co-cultures have supported the study of physical communication between cells  are powerful 3D biological platforms to investigate neuronal development, drug transport, and the pathogenesis of neuronal diseases. They rely on the capability of small cell aggregates to create in vitro, without any existing pattern or foreign material, polarized floating structures similar to in vivo tissues. They recapitulate more complex cell-cell interactions and have a higher level of functionality  and applied as an Similarly, Wang et al. have developed a microfluidic chip to obtain human-induced pluripotent stem cell (hiPS)-derived brain organoids to study neurodevelopmental disorders at early stages of gestation. They have used conventional soft lithography to fabricate a PDMS-based device with five independent channels. A central perfusion channel  separated two culture channels for the formation and culture of brain organoids . Embryonic bodies were immobilized in Matrigel and infused into the culture chambers, while medium in static conditions filled the two remaining external channels. Their results have confirmed that organs-on-a-chip provide a controllable microenvironment for an efficient development, maturation, and extended growth of brain organoids  into neuronal and astroglial cells, and study cell migration in response to gradients of chemotactic cues. Their device showed three PDMS layers attached to a glass substrate for mechanical support and high-resolution imaging. The first PDMS sheet modeled the lumens of blood vessels (vascular compartment) and exhibited support pillars to avoid membrane collapse, the second substrate had a porous PDMS membrane (pore size: 5 μm) as a scaffold for the BBB and the remaining PDMS substrate modeled the neural tissue . The top and bottom compartments had four perfusion channels  . They have filled the brain chamber with a collagen hydrogel embedding N2a neuroblastoma cells, C8D1A immortalized astrocytes and BV-2 immortalized microglia. After gelation, they have defined the BBB compartment by plating bEnd.3 EC facing the hydrogel. However, in this model a rocker shaker (instead of a micro-pumping system) ensured fluid flow and shear stresses and fluid flow, and the Authors had to refresh the medium every 2 days  onto defined regions of a commercial microelectrode array (MEA), without the need of physical or chemical barriers. Their system is a miniaturized device lacking perfusion, but the Authors have proposed an effective method to separate distinct neuronal populations on microfabricated devices, and it offers a unique approach to develop a complex cell environment for anatomically-relevant brain-on-a-chip devices , while specific transporters  allow the transfer of hydrophilic molecules (such as glucose). Larger hydrophilic molecules translocate by endocytosis, while the TJ between the EC lining the microvessels of the CNS regulate the paracellular pathway for the movement of ions and molecules in-between cells. In particular, the transmembrane proteins of TJ  are anchored inside the EC by another protein complex, including zonula occludens-1  and associated proteins , Lucifer yellow (444 Da), FITC-labeled sucrose, mannitol or dextrans, inulin and bovine serum albumin  from the parenchymal one . According to the complexity of the model, the cell populations are plated on the plastic well and on the top side of the membrane, on the plastic well and on both sides of the membrane or only on both sides of the membrane  separated by a microporous semipermeable membrane, acting as a physical barrier.Dynamic Griep et al. have modeled the BBB to study barrier dysfunctions in neurodegenerative conditions. Their chip was made up of two PDMS layers divided by a Transwell®-like polycarbonate membrane  to culture CMEC/D3 EC. To measure the TEER, the Author haves included Pt electrodes inside furrows in the PDMS sheets after assembling the top and bottom parts of the chip  a PDMS sheet, a glass electrode layer, a PDMS substrate, a porous polycarbonate membrane for cell culture, another PDMS layer, a glass electrode substrate and finally a last PDMS sheet. The two central PDMS layers hosted the perfusion channels . They were perpendicular and their aspect ratios were designed to induce a uniform shear stress distribution on bEnd.3 EC  and minimize the shear stresses on C8D1A astrocytes . The electrodes in the glass substrates opposite to the membrane allowed to monitor the TEER. To reduce the noise due to the wiring resistance, the Authors have used two sets of two thin-film AgCl electrodes forming a four point sensing structure , while the neural channel perfused rat cortical neurons adherent to the vascular network. The Authors have observed a good agreement between the permeability coefficients obtained by 20 and 70 kDa FITC-dextran in their NVU and Similarly to Adriani et al., Campisi et al. have highlighted the importance of a 3D matrix mimicking the ECM, but they have also focused on culturing human brain cells to remove cross-species incompatibility and achieve physiologically relevant results. They have filled the ports of their chip with a fibrin hydrogel embedding the co-culture of hiPS-EC, pericytes and astrocytes from human brains and pipetted hiPSC-EC into the channels. The results have suggested that their model shows physiologically relevant structures and is suitable for several applications, such as the study of the neurovascular function, transport across the BBB and metastatic cancer extravasation to the brain  Cronin, , in vitr2D cultures on plastic surfaces are valuable tools for cell-based studies because of their cost-effectiveness, ease of handling and robustness across different cell types. They allow the diffusion of secreted soluble factors in the medium, but they force cell attachment in a planar direction and prevent cell-cell and cell-ECM interactions, thus affecting proliferation rate and differentiation. In their physiological microenvironment, cells are surrounded by their own 3D matrix; moreover, they are exposed to endocrine signals from distant tissues, paracrine signals from nearby cells and physical stimuli . In 2D cultures, we lack most of this biological, mechanical and topographical complexity Paşca, . Assemblin vitro.Organs-on-a-chip allow cell culturing in single chambers, promoting their communication by microfluidic channels and controlling the spatial and temporal distribution of their microenvironment. They offer the possibility to establish more complex, physiologically-relevant and reliable conditions by creating gradients, setting the medium flow to ensure the exchange of nutrients and metabolites and stimulate cell growth, proliferation and differentiation, applying mechanical forces to mimic the physical microenvironment of living organs , and monitoring the operating parameters   hold potential for supporting research aimed at the comprehension of the molecular mechanisms underlying disorders involving different organs, exploring frontier hypotheses with a multidisciplinary approach and leading to the development of new therapeutic strategies. For instance, Ingberg's research group has provided crucial results for the study of host-microbiome interactions and the development of microbiome-related therapeutics, probiotics and nutraceuticals. In particular, by a microfluidic device ensuring oxygen gradients, Jalili-Firoozinezhad et al.  have co-in vitro tools are required to overcome technical and biological limitations, and develop reliable platforms to study the whole MGBA.However, the path to model the entire MGBA with a single multi-organ platform is still long. However, from the previous discussion, it is apparent that interdisciplinary strategies combining bioengineering, biochemistry and medicine and leading to innovative To this respect, a contribution may come from bioprinting and its applications. This is a technological approach for the simultaneous incorporation of materials, cells and biologically active factors  would add complexity to recreate the microenvironment by mimicking the composition and anatomical properties of the ECM and providing key mechanical, biochemical and topographical cues as gradients or localized hotspots . It aims at applying a bioengineering approach to the MGBA, by developing technological platforms to elucidate the effect of microbiota secretome on brain functions, in a context of AD-related neurodegeneration. The engineered platform will be suitable not only to study the MGBA, but also to examine other pathologies involving several organs.in vitro cell modeling (Tunesi et al., in vitro with a very complex biochemical pathway as the MGBA, paving the way to significant advancements in the field, for example the discovery of new, not invasive therapies for neurodegenerative disorders based on microbiota management by food ingredients or probiotics.The main challenge of the MINERVA project lies in the great complexity arising from the involvement of many biochemical pathways, multi-organ functions and crosstalks. Five optically accessible, microfluidic organs-on-a-chip will compose the final platform. They will model the microbiota, the gut, the immune system, the BBB, and the brain compartment. Each compartment will permit both sampling and cell perfusion. A microporous membrane will divide each chamber into two parts, separating the cells from culture medium flowing to the next device. Therefore, before reaching brain cells, culture medium will be enriched with secretome from the gut microbiota, gut epithelial cells, immune cells and BBB cells . A hydroIR, LI, and MT wrote, reviewed, and edited the original draft. MC reviewed the original draft. DA reviewed and edited the original draft. CG reviewed and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The available data, including experimental studies, clearly indicate an excessive intravascular activation of circulating platelets in multiple sclerosis (MS) and their hyper-responsiveness to a variety of physiological activators. Platelet activation is manifested as an increased adhesion and aggregation and is accompanied by the formation of pro-thrombotic microparticles. Activated blood platelets also show an expression of specific membrane receptors, synthesis many of biomediators, and generation of reactive oxygen species. Epidemiological studies confirm the high risk of stroke or myocardial infarction in MS that are ischemic incidents, strictly associated with incorrect platelet functions and their over pro-thrombotic activity. Chronic inflammation and high activity of pro-oxidative processes in the course of MS are the main factors identified as the cause of excessive platelet activation. The primary biological function of platelets is to support vascular integrity, but the importance of platelets in inflammatory diseases is also well documented. The pro-thrombotic activity of platelets and their inflammatory properties play a part in the pathophysiology of MS. The analysis of platelet function capability in MS could provide useful information for studying the pathogenesis of this disease. Due to the complexity of pathological processes in MS, medication must be multifaceted and blood platelets can probably be identified as new targets for therapy in the future. Multiple sclerosis (MS) is a major human putative autoimmune, demyelinating, and degenerative disease of the central nervous system (CNS) with a variety of pathophysiological mechanisms, such as axonal injury, neuronal damage, inflammation, demyelination, astrogliosis, and remyelination ,2. ProceThe pathophysiological mechanisms of this multi-component disease are difficult to clearly characterize. The nature of the pathophysiology of autoimmunity is very complex and involves the various type of cells. The etiology of MS is still unknown, however, accumulating body of evidence points to a complex interplay of genetic and environmental factors disturbing the functioning of the immune response. MS pathomechanism is associated with many factors, the most important of which include immune system dysfunctions combined with biochemical disturbances and disruption of the blood–brain barrier (BBB) . The cauThe disruption of the permeability, especially for small molecules and blood cells, that enter the bloodstream, contributes to the infiltration of a large number of leukocytes, thus inducing the neurotrophic changes causing demyelination in the CNS. However, there is growing evidence that indicate the co-existence between co-stimulatory pathways and the function performed by inflammatory cells, both in the periphery nervous system (PNS) and in the CNS. Platelet activation significantly affects the interactions between leukocytes and endothelial cells . It is bAs already mentioned, MS is a demyelinating and inflammatory disease of CNS, however, it is closely related to the damage of intracerebral blood vessels, mainly as a result of increased BBB permeability, and/or as a consequence of the closure of the blood vessel . MultiplWell-documented epidemiological reports show an augmented risk of cardiovascular disease in patients with MS, and some studies have found that the risk of death due to ischemic stroke or myocardial infarction in MS was significantly greater (over 30%) than for the age-matched overall population. Moreover, according to statistics, the cause of death in more than 10% of MS patients considered a stroke ,22. AnalDemyelinating lesions within specific areas in the central nervous system may be involved in the pathogenesis of autonomic dysfunction in MS . It has Moreover, MS these patients lead a more sedentary lifestyle, which promotes the occurrence of stroke , and aboIt is well-known that the lack of physical activity is closely related to the emergence of so-called metabolic syndromes—such as obesity, hypertension, dyslipidaemia, elevated glucose concentrations, and higher levels of high-density lipoprotein (HDL) and cholesterol—Which significantly increase the risk of vascular incidents. According to research carried out by Laroni et al. indicate that treatment of MS patients with disease-modifying drugs (DMDs), especially glatiramer acetate, fingolimod, and dimethyl fumarate significantly enhances the risk of cardiovascular diseases . While iAlthough the contribution of platelets to inflammation is well established and the inflammatory response has been recognized to be critical in neuronal diseases such as MS, the participation of blood platelets in neurodegeneration is weakly investigated till now. In the 1950s, there were only a few early studies on the importance of platelets in the CNS demyelination, which presented elevated platelet adhesion in the initial stage of MS (RRMS) . Since tMS affects only human, however, several animal models of this disease have been identified. The most commonly used models include EAE and cuprizone model. The choice of the appropriate model depends on the method of inducing the process of demyelination in the CNS. In the EAE model, induction occurs with myelin antigens or passive transfer of myelin-specific T lymphocytes induces an inflammatory demyelination in the animal CNS. The copper model is based on the toxic induction of demyelination with the participation of copper chelator cuprizone . There are many of research based on the cuprizone animal model. One of them is research conducted by Berghoff et al., which was based on the impact of demyelination on BBB hyperpermeability. This research indicates that proinflammatory molecules such as interleukins (IL-6 and IL-1β), tumor necrosis factor (TNF), and chemokine CCL2 are the most likely candidates to contribute to BBB disorders in the cuprizone animal model . The stuBlood platelets are the main cell effector in hemostasis, coagulation, and pathologic thrombosis. Adhesion of blood platelets at sites of vascular injury is a crucial first step in hemostasis and thrombosis, prompted by the exposure of subendothelial matrix compounds, mainly collagen or other thrombogenic proteins. These receptor-mediated processes involve adhesion surface molecules and lead to signal transduction, platelet aggregation, degranulation, and secretion of pro-thrombotic factors from platelet granules. Further reinforcement of platelet activation and secondary acquisition of extra blood platelets contributes to the creation of the haemostatic plug. Various physiological stimuli activate signalling pathways, consequently causing platelet adhesion, degranulation (secretion of compounds magazine in their granules), excretion of microparticles, and inducing expression of the receptors that finally mediate platelet aggregation. Blood platelets excessively activated by endogenous agonists contribute to various types of pathogenesis . PlatelePlatelet quantity and activation status are critical to physiological and pathological hemostasis. Understanding the molecular mechanisms responsible for the increased platelet activation in MS can have important implications for finding effective therapies. A common feature of myocardial infarction and stroke is vessel occlusion. Pathological activation of blood platelets and formation of thrombotic lesions within the coronary arteries are considered to be the major cause of myocardial infarction . It has Except for the pivotal role in primary hemostasis, platelets are essential for the maintenance of normal function of endothelial barrier. One of the cardinal symptoms of inflammation is the increased vascular permeability caused by changes in the functioning of the barrier . It was Several mentioned studies have shown that activated platelets can modulate dendritic cell maturation and their activation. Dendritic cells represent highly specialized APCs and presence both in the innate and adaptive immune system by stimulating naive, memory, and effector T-cells, promoting B cell activation, and interacting with NK cells . There aIt is well-known that blood platelets are a rich source of biologically active molecules, like inflammatory mediators. In the circulatory system, platelets after activation release various types of components that are stored in the highly organized characteristic cell granules. A plurality of biologically active compounds released from platelet granules, sometimes at high concentrations, can disturb the permeability of BBB. Due to activation of T-lymphocytes which are responsible for the emergence of new inflammatory injuries in MS . It has Platelets after activation produced inflammatory lipid mediators such as eicosanoids, like thromboxane A2 (TXA2), or platelet activating factor (PAF) which transmit outside‑in signals to diverse cell types involved in innate immune defence, furthermore, PAF likewise induces severe endothelial barrier leakiness. PAF is endogenous phospholipid of proinflammatory, hemostatic, and vasoactive properties, is naturally synthesized by a various of cell types, such as platelets, neutrophils, monocytes, endothelial cells, and neurons. This metabolite of arachidonic acid induces, both platelet and neutrophil adhesion and activation. Additionally, provide variations in local cerebral blood flow and blood rheology. PAF as a solid platelet activator, causes platelet synthesis of IL‑1β—The pro-inflammatory cytokine which has been suggested to have a major role in vascular inflammation by activation of the cytokine regulatory network . PAF secSimilarly, platelet-derived microparticles (PMPs), which contain a large amount of the platelet-derived membrane proteins, such as the MMPs that have been discerned as a key player in disruption of BBB in patients with MS . MicropaInteraction between platelets and leukocytes is thought to signify an important trigger for induction of proteolytic MMP activity. Platelet-derived MMPs fundamentally contribute to a variability of pathologies by supporting cell migration, tissue degradation, and finally inflammation. Platelets and exude a diversity of MMPs, such as MMP-1, MMP-2, MMP-3, MMP-9, and MMP-14. Moreover, platelets elevate MMPs concentrations at sites of vascular detriment by leukocyte recruitment on the endothelium cell surface and stimulation both of them to produce the MMPs ,82. PlatBlood platelets probably can also perform a role of direct effector cells during immune processes due to their increased surface expression of IgE receptors, which is an important immune defence mechanism. Binding of IgE by platelet membrane lids to platelet activation results in their aggregation, formation of heterogeneous platelet-lymphocyte conjugates and release of cytotoxic mediators ,87,88.As we demonstrated in this paper platelets have a multi-form participation in development of MS. Therefore, we should take into account that platelets may become a target for new drugs administered in the course of MS mainly to relieve symptoms and prevent inflammation. The reduction of their activity will also have a significant effect on the level of risk of selected cardiovascular diseases in MS. All of the studies cited herein highlight the significance of platelet activation in MS. This leads to the supposition that blood platelets are probably constantly activated in development of MS, and are engaged in the pathogenesis of the disease by promoting inflammatory processes. Nevertheless, as emphasized by the authors of most researches, the role of blood platelets in pathogenesis and development of MS stay unknown. Further studies are still needed to validate the correctness of the information received to date, and to more broadly determine the role of platelet activation in the neuro-pathogenesis of MS. All the more so, as the clear mechanisms of increased platelet activation in MS also remain unknown. Further research on platelets in MS are particularly important in clarifying the reason for increased risk of ischemic events observed in MS. Further analysis of platelet function in MS lesions might elucidate new features of pathology, and could open the opportunities for therapy. The precise definition of the mechanisms of increased platelet activity in MS will determine the new therapeutic goals of this disease. The existing reports indicate that blood platelets could be a possible target for pharmacotherapy in MS, then their over-activation can be implicated in the development of the neuroinflammatory process correlated with neurodegenerative diseases. The summarizing table of proven changes in platelet activity and functions in MS has been presented in The different treatment options of MS are based on different mechanisms of action, influencing their use and effect. MS is a disease of unclear etiology, which makes its treatment difficult and is limited only to symptomatic treatment, which slows down the development of the disease. Perhaps in the future, disease-modifying drugs will be direct against platelet activation and especially to combat their interaction with immune cells for prevent the progression of inflammatory process. Currently, several disease-modifying drug therapies are available for the treatment of MS and are the standard treatment for MS patients. However, it should be remembered that they have various side effects, including they can indirectly affect the function of platelets and blood vessels. More common observed side effects with the drugs are low white blood cell count and slower heart rate. The variety of drugs used in MS is presented in the last meta-analysis, performed by Hamidi et al., including 11 disease-modifying drugs used for treatment of adult patients diagnosed with RRMS, along with their administration form and recommended dose .


Studies conducted in the last years have demonstrated that human milk represents a continuous supply of beneficial bacteria to the infant gut, which contribute to the maturation of the digestive and immune functions in the developing infant. Nevertheless, the origin of bacterial populations in milk is not fully understood yet and they have been proposed to originate from maternal skin, infant’s mouth, and (or) endogenously, from the maternal digestive tract through a mechanism involving immune cells. Understanding the composition, functions and assembly of the human milk microbiota has important implications not only for the infant gut microbiota establishment, but also for the mammary health since dysbiosis in the milk bacteria may lead to mastitis. Besides, host, microbial, medical and environmental factors may affect the composition of the human milk microbiome, with implications for the mother-infant health. Application of both culture-dependent and -independent techniques to assess the milk microbiome faces some practical limitations but, together, have allowed providing novel and complementary views on its origin, composition and functioning as summarized in this minireview. In the next future, the application of the ultimate advances in next-generation sequencing and omics approaches, including culturomics, will allow a detailed and comprehensive understanding of the composition and functions of these microbial communities, including their interactions with other milk components, expanding the opportunities to design novel microbiome-based modulation strategies for this ecosystem. Human milk is the gold standard for infant nutrition as it contains multiple biologically active components, including immune factors, lipids, oligosaccharides, microRNAs and hormones among others , whose cLactobacillus salivarius, Lactobacillus fermentum, Lactobacillus gasseri, Bifidobacterium breve, Bifidobacterium adolescentis, and Bifidobacterium longum subsp. infantis have demonstrated potential to promote mother and infant health, including the prevention or treatment of lactational mastitis, the promotion of a normal gut bacterial colonization in preterm neonates, or the amelioration of diarrhea in IBS patients  the roles that milk bacterial communities play on infant health and development, including their influence on the acquisition of the gut microbiota in early life; (b) the roles that they may play on maternal health, including breast health; (c) the maternal, infant, environmental and medical factors that shape and modulate their compositions, both under physiological conditions and when dysbiosis occurs; and (d) their origin, in order to confirm or refuse the existence of an endogenous oral-entero-mammary route allowing the selective translocation of some bacteria from the maternal digestive tract to the mammary gland.The ever-growing asset of-omic tools is opening new avenues to understand the benefits of breastfeeding from microbiological, metabolic and immunological perspectives, and to establish new microbiota-based strategies to promote maternal-infant health, including the development of procedures for the transfer of the milk microbiota to neonates or infants that are not breastfed or the development of human milk-derived probiotics . This reStaphylococcus aureus) or infant infections  . Other bacterial groups present in milk from healthy lactating mothers are more fastidious to grow under routine laboratory conditions as they require either specific atmospheric incubations  or specific nutrients or ingredients in the cultivation media , and thus may have passed unnoticed in early microbiological studies not specifically designed to recover such species. For instance, lactic acid bacteria  and bifidobacteria have only been isolated from milk following the utilization of specific growth media and longer anaerobic incubations  genotypically, the bacterial isolates, though these approaches fail to obtain information on non-culturable bacteria. Additionally, most microbial studies on milk were performed on samples from women whose infants were suffering from potential milk-borne infections and thus, microbial growth media and conditions have been biased toward detecting pathogens including viral particles , and pathogenic microbial species typically associated with mammary infections  . Overallubations . Globallactarius .Lactobacillus and Bifidobacterium species  from milk have awakened strong interest as potential probiotic bacteria, enjoying the GRAS  and the QPS  status conceded by the Food and Drug Administration and the European Food Safety Authority, respectively.Culture-dependent approaches have been pioneering in demonstrating that healthy human milk harbors a microbial community, representing a source of commensal microorganisms for the neonate. Albeit their limitations to access unculturable bacteria, culturing enables preserving the strains facilitating further studies and exploitation of potential biotechnological applications , 2015. Sin vitro assessment of probiotic properties in human milk isolates has included their antimicrobial activity against pathogenic microorganisms and the compounds that may be responsible for such activity , the ability to survive when exposed to conditions similar to those found in the human gastrointestinal tract, their adherence to human intestinal cells and mucin, their ability to degrade mucin or to produce biogenic amines or their susceptibility to antibiotics  analyses, are essential steps to characterize and evaluate the safety and the probiotic potential of bacterial isolates from human origin, including those isolated from milk. Generally, the ibiotics , 2010. Tl models  and humal models . Finallytococcus , 2013.In the framework of recent research conducted in the field of the gut microbiota, the “culturomics” approach, which is based on the utilization of a wide range of culturing and incubation conditions, is emerging as a promising strategy to aid in the discovery and description of novel microorganisms associated to the human host, including pathogenic and commensal species . Such apSince cultivable microorganisms represent a fraction of the communities inhabiting a specific ecological niche, culture-independent molecular techniques, i.e., quantitative PCR, denaturing (or temperature) gradient gel electrophoresis, and Next generation sequencing (NGS) approaches, from metataxonomics  to metagenomics , have allowed valuable complementary assessment of the human milk microbiota, including information on microorganisms which remain unculturable to date . NonetheBesides, culture-independent techniques cannot differentiate between live and dead microorganisms, as DNA from dead organisms may persist in the environment, affecting the biological significance of the conclusions from sequencing-based analysis of microbial communities. Several methodologies have been implemented to incorporate a viability assessment in DNA-based analysis . Among tStaphylococcus, Streptococcus, Corynebacterium, Propionibacterium, lactic acid bacteria, and Bifidobacterium , which are either non-culturable or hard to culture in the laboratory, and that have not been recovered through milk culturing to date , a DNA binding dye that enters only dead and membrane-compromised cells, thus preventing detection of damaged cells by qPCR or NGS approaches (It is worth highlighting that DNA-based studies provide information on the community’s composition and their metabolic potential but do not demonstrate the activities they perform proaches , althougproaches . In factproaches . Other Nproaches .Finally, little attention has been paid to the associations of milk microbiota with other milk components  althoughLR and JR conceived this review article. All authors wrote and approved the final version of the manuscript.CG-C is now employed by Probisearch SL, but did not at the time of the research being undertaken. The company had no role in the study. 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.


Enterotypes are used for classifying individuals based on the gut microbiome. A number of studies are available to find the Enterotypes inhealthy individuals; however, most of them lack comparisons at the world level. We analyzed the healthy human gut microbiomes of 495datasets available in the European Nucleotide Archive (ENA) database derived from fifteen countries from four continents. Firmicutes andBacteroidetes were the two most abundant phyla in the healthy human gut, worldwide. A high ratio of Proteobacteriato Actinobacteria anda low abundance of Prevotella were identified as the indicators of IBD. Prevotella, Bacteroides, and Bifidobacterium were identified as theEnterotypes in the inter-continental comparisons. At the intra-continental level, two (Bacteroides and Ruminococcaceae), four, and two  Enterotypes were identified in theAmerican, European, and Asian continents, respectively. In addition, a high abundance of the unknown genus of Ruminococcaeae wasobserved in the Colombian human gut microbiome. A substantial impact of the geographical distance was observed on human gutmicrobiome variations, demonstrating a cumulative effect of factors, including dietary habits, genetics, lifestyle, environment, and climate,etc. Healthy human harbors trillions of microbial cells with healthpromoting effects inside its gut, in a ratio of 1:1 with respect to thehuman cells Due to the underlying inter-individual variations, healthyindividuals can be clustered separately based on the differences inthe enrichment of the microbial taxa in their guts An initial study conducted on the healthy individuals from theEuropean countries, Japan, and USA demonstrated an existence ofthree Enterotypes in the healthy human gut microbiome In addition to the characterization of the Enterotypes, the impact ofdifferent factors, including age, geography, birth mode, antibiotics,diet, and genetics, on healthy human gut microbiome has beeninvestigated In the present study, we have conducted a comprehensiveEnterotype analysis by using publicly available 16S rRNA geneamplicon data from fifteen countries belonging to four continents,viz., Asia, Africa, America (North and South), and Europe. We havedetermined the compositional variation and diversity  patterns present within the healthy human gut microbiomeswith respect to their geographical locations followed by theirquantifications.The publicly available raw 16S rRNA gene based metagenomicdatasets from fifteen countries, viz., Burkina Faso, Egypt, India,Malaysia, China, Japan, Taiwan, Thailand, Indonesia, Italy, Sweden,Spain, USA, Argentina, and Colombia were retrieved from theEuropean Nucleotide Archive (ENA) (https://www.ebi.ac.uk/ena).Paired-end sequences generated by the Illumina sequencingmethod were merged using the fastq-join method The alpha diversity of the datasets were calculated using threealpha diversity based methods, including Shannon index, Observedspecies, and Phylogenetic diversity (PD) whole tree. The t-test wasemployed for the determination of the significance among the alphadiversities of the countries. The microbial diversity among thedatasets was estimated by calculating the beta diversity indicesusing weighted and unweighted unifrac distances. Further, aPrincipal Component Analysis (PCA) was carried out using theweighted and unweighted unifrac distances to calculate thesimilarity and dissimilarity among the datasets based on theirmicrobiome structure and diversity. PCA results were visualizedusing the Emperor tool To determine the Enterotype clusters in the human gut microbiome,a widely used enterotyping pipeline (http://enterotyping.embl.de)was used. The optimum number of the clusters was identified usingthe Calinski-Harabasz (CH) index, whereas robustness of theclusters were accessed using the Silhouette index. The Kruskal-Wallis test was implemented to check the significant differences(p < 0.05) of taxa abundance and diversity across Enterotypes.The OTUs were considered as core OTUs of a given continent ifthey were present in at least 80% of the human gut microbiomesamples with a relative abundance of at least 0.001 in any twocountries of a given continent. Further, the shared and uniquegenera among the continents were calculated by only consideringthe presence or absence of the genera in the continents. To identifythe shared genera at the inter-continental level, at first the presenceof the given genus is confirmed in all the countries of a givencontinent. The genera from all the countries which qualified thisstepwere further compared to identify those genera which wereshared by all the continents. Similarly, unique genera wereidentified for the continents which were present uniquely in a givencontinent and was absent in all other continents.The latitude and longitude of the countries analyzed in this studywere retrieved using the ggmap R package We used 597 datasets representing healthy human gut microbiomefrom fifteen countries for this analysis. A total number of31,126,235raw read sequences were obtained from these 597datasets. The quality-filtering step resulted in a total number of29,229,968reads. After removing the chimeric sequences, a totalnumber of 28,427,739 read remained in the processed datasets.Following this, OTUs were predicted per sample and those samplescontaining low number of predicted OTUs (< 250 OTUs) wereremoved from the further analyses. This resulted in 495 datasetsbelonging to fifteen countries. In order to estimate the number ofOTUs per country, all the OTUs predicted in each dataset of acountry were combined together. The total number of predictedOTUs was found to vary from 1,257 to 5,660 .ThesequIn the present study, we have retrieved 24 phyla, 56 classes, 114orders, 202 families, and 427 genera across all the 495 samples.Firmicutes and Bacteroidetes were found as the most abundantphyla , howeverThe phyla Actinobacteria and Proteobacteria were found to be thenext major taxa in the healthy human gut microbiome .The phylum Actinobacteria is an important part of healthy humangut microbiome. Particularly, Bifidobacterium of Actinobacteriais ofhigh relevance as it is one of the commonly used probiotics It is known that in the human gut, Bifidobacterium has highabundance of glycoside hydrolases to degrade starch than the othermicrobes residing in the gut The overall alpha diversity showed differences within the datasetsof the different countries, but without any statistical significance(p < 0.05). Of these, the Shannon based alpha diversity,which represents the richness and evenness of the taxa, revealed asimilar diversity pattern across the European countries. Within theAsian continent, also a similar pattern was observed, except forIndia and Malaysia, which exhibited very low alpha diversities incomparison to the other Asian countries. The African continentshowed a high diversity in Egypt as compared to Burkina Faso.Further, Colombia exhibited a very high alpha diversity ascompared to the other countries in the American continent. Amongall the fifteen countries, Colombia and India exhibited the highestand the lowest diversities, respectively. This indicates Colombia tobe the most and India to be the least diverse in the richness andevenness of the taxa.The PD whole tree based index was used to infer the phylogeneticdiversity of different datasetsto resolve the effect ofphylogenetically related taxa on the overall alpha diversity. A lot ofvariation in the phylogenetic diversity was observed within thedatasets of all the four continents, except for Asia .Although all the European datasets exhibited a similar alphadiversity in terms of the evenness and richness of the taxa,however, differences were observed among their phylogeneticdiversities. Maximum and minimum phylogenetic diversities wereobserved for Swedish and Italian datasets, respectively. However,the datasets of the American and African continents exhibitedsimilar trends of the Shannon and Phylogenetic based alphadiversities. Among the Asian samples, only few minor differenceswere observed between the Shannon and Phylogenetic alphadiversities in different countries. Overall, Sweden exhibited themost phylogenetically diverse gut microbiome, whereas Italy wasthe least diverse in its gut microbiome.In order to estimate the beta diversity, a PCA was performed on allthe datasets using weighted unifrac distance metric .This metric encompasses the effect of the abundance of taxa, whichimpacts robust clustering of the datasets than their separation intodifferent clusters. Only the Asian datasets exhibited a distinctcluster along the PC1 and PC2 axes. A clear separation could not beobtained for all the other continents using this method. Hence, weopted for an unweighted approach as it is known to demonstrate aneffect of only the presence and absence of the taxa on the separationof the gut microbial datasets in three-dimensional (3D) spaces. Thisanalysis resulted in a clear separation of the samples belonging tothe Asian, American, European, and African continents .However, a mixed cluster was observed for a few datasetsprimarily belonging to the South American population followed bysome datasets from the other countries, including, Egypt, India,Italy, Malaysia, and Sweden. It is interesting to note that althoughthe Asian datasets differed from the samples of the othercontinents, however, they exhibited a large variation in the gutmicrobiomes among themselves, which is shown by the spread ofthe datasets along the PC2 axis. Similar findings were observed forthe American datasets. Further, a better separation of the datasetsinto distinct clusters using the unweighted unifrac distance methodindicated significant roles of the less abundant taxa for thedistinction of the datasets.We identified three optimal and robust Enterotype clusters usingthe Principle Coordinate Analysis (PCoA) from the gut microbiomeof the 495 datasets . The genera Bifidobacterium, Prevotella,and Bacteroides were the highly abundant Enterotype taxa in thecluster 1, cluster 2, and cluster 3, respectively ). Amajority of the datasets from the Asian countries, including Japan,Taiwan, China, and Thailand were clustered into the Enterotypecluster 1 (Available with Authors). Thus, this cluster, dominatedby the genus Bifidobacterium, might represent a signature taxon forthe Asian-population. Cluster 1 also contained a few samples fromsome of the non-Asian countries, primarily including Colombia andItaly. The Enterotype cluster 2, which is mainly driven by thegenusPrevotella, demonstrated a mixed cluster of various countriesfrom different continents. A majority of the datasets from BurkinaFaso, India, Indonesia, and Sweden were the members of thiscluster. Further, a majority of the human gut microbiome datasetsfrom Spain, Argentina, USA, Egypt, and Malaysia were clusteredinto the Enterotype cluster 3 with Bacteroides as the major drivinggenus for this cluster. Thus, this cluster might represent signaturetaxa for the Western-population.Previously, Gorvitovskaia et al. had compared the worldwidepopulation using a dataset of 747 healthy human individuals fromthe countries belonging to the four continents, viz., Asia, America,Europe, and Africa The Enterotype clusters identified in the American continent werealso clearly separated into two clusters and exhibited no overlap.This indicated the occurrence of two sub-types of the Americanpopulation dominated by the Enterotypes Bacteroides andRuminococcaceae. The datasets from the USA were distributedalmost equally between these two Enterotype clusters. A fewprevious studies carried out on adults and children of the USA alsodemonstrated Bacteroides as the driving Enterotype of the healthygut microbiome of this country We also investigated the top ten highly abundant genera in eachcountry (Available with Authors)and observed a high abundanceof the members of the unknown genus of Ruminococcaeae in theColombian human gut microbiome. The genus Bifidobacterium wasabsent in the top ten highly abundant genera in the Colombiandatasets, thus confirming its very low abundance. Thus, it is anintriguing question that how a majority of the Colombian datasetsstill got clustered into the Enterotype cluster driven by the genusBifidobacterium in our inter-continental analysis. The difference inthe relative abundances of Bacteroides and Prevotella was found to bevery low in the Colombian datasets as compared to those of theother countries. This might have prohibited the clustering of theColombian datasets in the Enterotype clusters driven by these twogenera and might have forced these samples to be a part of the thirdEnterotype cluster. A large dominance in the number of thesamples from the Asian continent might be responsible for theformation of the third Enterotype cluster, which was primarilydriven by the genus Bifidobacterium.Unlike the Asian and American continents, the European continentwas found to harbor four Enterotype clusters, which weredominated by the genera including, Faecalibacterium, Bacteroides,Prevotella and Clostridiales. Interestingly, the clusters belonging tothe genera Bacteroides and Clostridiales were partly overlapping. Thisindicated that the European population could be further dividedinto three sub-populations based on the gut microbiome. Two ofthese sub-populations were dominated by the generaFaecalibacterium and Prevotella, while the third sub-populationcomprised a mixture of the individuals carrying the generaBacteroides and Clostridiales as the dominant taxa. This also indicatedthat the European population is more diverse as compared to theAsian and American populations in terms of the dominating generain their gut microbiomes.At the continent level, we could not detect any distinct Enterotypeclusters in the African continent. However, during our intercontinentalcomparisons, a majority of the datasets from BurkinaFaso got classified into the Enterotype cluster driven by thegenusPrevotella. The genus Prevotella has been identifiedas one of the driving Enterotype taxon in the adult rural Africanpopulation in an earlier study The members of the microbiome residing in the same niche areknown to cross-talk among themselves, thus, might also affect thegrowth and survival of one another. In order to delineate the effectof the dominant driving taxa on the overall microbial diversity, weinferred the species richness in the Enterotype clusters. Thisanalysis revealed a statistically significant lower microbial diversity only in the datasets belonging to the Enterotypecluster driven by Bacteroides (Available with Authors) which is incorroboration with an earlier study During the last century, IBD was more prevalent in the Westerncountries Taken together, a higher P/A ratio and a lower abundance ofPrevotella in the human gut microbiome might be the factorsassociated with the rising incidences of IBD. For example, theWestern countries exhibited a higher P/A ratio and a lowerabundance of Prevotella and are known to exhibit higher incidencesof IBD. On the contrary, the Asian countries exhibited a lower P/Aratio and a higher abundance of Prevotella thus corroborating withcomparatively lesser incidences of IBD. African countries exhibiteda higher ratio of P/A and also a higher abundance of Prevotella. Thisindicated that these two factors have been counter-balancing eachother and might be eventually promoting healthier phenotype. Ashift in the trends of these factors might act as the indicator for theoccurrence of IBD.Though the individual gut microbiomes differ from each other,there exists a core set of microbial members, which performsessential functions in the healthy human gut. Towards this, weidentified 11, 98, 55, and 15 core OTUs in the African, Asian,European, and American continents, respectively (Available withAuthors). In addition to the core OTUs in different continents, weidentified a total of 115 genera which were present across all thefour continents (Available with Authors). Although the continentsshare these common genera, however with varyingabundances(Available with Authors). A majority of the shared taxa werefound to be belonging to the phyla Firmicutes, Bacteroidetes,Actinobacteria, and Proteobacteria. At the family level, a majority ofthe shared taxa across the continents were found to be belonging toLachnospiraceae, Coriobacteriaceae, Veillonellaceae,Enterobacteriaceae, Ruminococcaceae, and Erysipelotrichaceae.The functional roles of the core taxa were analysedcomprehensively by performing thorough literature survey(Available with Authors).The members of the familyLachnospiraceae are the known producers of short chain fatty acids(SCFAs), including acetate, butyrate, and propionate by thedegradation of the complex polysaccharides. These SCFAs arefurther utilized by the host for their energy needs. The members ofthe family Ruminococcaceae are well known butyrate producers ofbutyrate from carbohydrate. Some members of the familyVeillonellaceae have been known to produce propionate fromlactate. Coriobacteriaceae family is found to be implicated in thebile acid metabolism processes. Further, recent studies indicated theassociation of family Erysipelotrichaceae with the lipid metabolismof host. These results indicate that the healthy human gutmicrobiome consists of the common microbial members which aremainly responsible for fulfilling the metabolism and energyrequirements of the host. In addition to the presence of the commongenera across the continents, we also observed 58, 70, 10, and 25unique genera, which were found to be exclusively present in thecontinents, Asia, Africa, Europe, and America, respectively(Available with Authors).We have investigated the effect of the overall geographical distanceof the fifteen countries on the healthy human gut microbiomevariations based on the weighted and unweighted unifrac distancemetrics. The weighted unifrac distance reflects the effect of theabundance of the microbial taxa. A Mantel correlation value of 0.34(Rw) was observed between the geographical distance and theweighted unifrac distance matrix. This implied that 11% (Rw2) of thehealthy gut microbiome variations based on the abundant taxamight be explained by the geographical distance. A higher Mantelcorrelation value of 0.64 (Ru) was observed between thegeographical distance and the unweighted unifrac distance matrix.It is important to note that the unweighted unifrac distance reflectsthe effect of only the presence and absence of the microbial taxa andis independent of the relative abundance of the taxa. This impliedthat 40% (Ru2) of the gut microbiome variations based on the taxapresent in gut, including rare taxa, might be explained by thegeographical distance. These observations indicate that thegeographical variations might be affecting the diversities of therarer abundant taxa more than these of the highly abundant taxa.The present study has demonstrated significant differences amongthe healthy human gut microbiomes of different countries, in termsof the composition, diversity, and Enterotypes. Three dominantEnterotype clusters driven by the taxa Bifidobacterium,Prevotella,andBacteroideswere identified across the worldwide population. ThegeneraBifidobacteriumand Bacteroides were identified as the majorEnterotype taxa of the Asian and Western (American andEuropean) populations, respectively, whereas, the African datasetswere distributed between Prevotella and Bacteroides. The Enterotypeclusters driven by the genera Bacteroides and Prevotella were morerobust as compared to the third cluster. The composition of thethird cluster might vary with respect to the datasets included in theanalysis. The members of the family Ruminococcaceae wereidentified as the signature taxa of the Colombianpopulation.Besides these distinctions in the gut microbiomes, coretaxa conserved across the continents were identified withpredominant involvements in the process of metabolism andenergy production.The low abundant taxa were identified as theimportant factors for the distinction of the microbiomes fromdifferent geographical regions. Such, low abundant taxa were alsofound to be predominantly affected by the geographical distances.In addition, a higher P/A ratio and a lower abundance of Prevotellamight be an indicator of the rising incidence of IBD, however,comprehensive studies on IBD patients are required to confirm thishypothesis. It is important to note that the possibility of ourobservations being affected due to the differences in the variableregions of the 16S rRNA gene used in the independent studies,cannot be ruled out. This analysis provides a snapshot of the effectof the geographical factor on the healthy human gut microbiomewhich may be impacted by various other factors, including dietaryhabits, genetics, lifestyle, environment, and climate, etc.