The synaptic cleft has been vastly investigated in the last decades, leading to a novel and fascinating model of the functional and structural modifications linked to synaptic transmission and brain processing. The classic neurocentric model encompassing the neuronal pre- and post-synaptic terminals partly explains the fine-tuned plastic modifications under both pathological and physiological circumstances. Recent experimental evidence has incontrovertibly added oligodendrocytes, astrocytes, and microglia as pivotal elements for synapse formation and remodeling (tripartite synapse) in both the developing and adult brain. Moreover, synaptic plasticity and its pathological counterpart  have shown a deep connection with other molecular elements of the extracellular matrix (ECM), once considered as a mere extracellular structural scaffold altogether with the cellular glue . The ECM adds another level of complexity to the modern model of the synapse, particularly, for the long-term plasticity and circuit maintenance. This model, called tetrapartite synapse, can be further implemented by including the neurovascular unit (NVU) and the immune system. Although they were considered so far as tightly separated from the central nervous system (CNS) plasticity, at least in physiological conditions, recent evidence endorsed these elements as structural and paramount actors in synaptic plasticity. This scenario is, as far as speculations and evidence have shown, a consistent model for both adaptive and maladaptive plasticity. However, a comprehensive understanding of brain processes and circuitry complexity is still lacking. Here we propose that a better interpretation of the CNS complexity can be granted by a systems biology approach through the construction of predictive molecular models that enable to enlighten the regulatory logic of the complex molecular networks underlying brain function in health and disease, thus opening the way to more effective treatments. Neuronal synapses are, at a biochemical level, stations of electrochemical signaling between the dynamic circuits underlying the complex and deeply interconnected processes of motor and learning functions. Intricate as brain processing may seem, the synapse and its plasticity represent the anatomic and functional unit that can explain it. Hundreds of proteins form the synaptic elements, and their correct expression, structural organization, turnover, and reshaping capability are pivotal for the proper function of the central nervous system (CNS) ,2.The pivotal characteristic of the brain is the continuous and strategic ability to modify itself in an experience-based fashion. The subsequent behavior could rely on the strength of circuit transmission and the reinforcing of active synapses or the pruning of new ones. Although development and adulthood show different patterns of synaptic plasticity, this function is fundamental for brain homeostasis ,4. SinglFinally, the extracellular matrix (ECM), acting as a functional scaffold, represents almost one-fifth of the brain volume. The complex network constituted by proteoglycans, glycoproteins, and glycosaminoglycan sustains neuronal function and provides, together with structural support, a reservoir of trophic factors, signaling molecules, biochemical pathways and long-distance gradient-like communication between cellular components of the CNS ,12.exempla of integration among cellular compartments  and the ECM, that can selectively permit the transmembrane active transport, the diffusion of molecules through tight junctions, and the selective loosening and remodeling of the BBB . Al. Al74]. One single astrocyte can enwrap about 120,000 synapses in rodents. A human astrocyte might unsheathe from 270,000 to 2 million synapses ,80 that Astrocytes are interconnected with gap junctions formed by connexins (Cx), Cx43 and Cx30 being the most expressed ,94. The Astrocytes are highly sensitive to plasticity processes and may act with structural changes involving their cytoskeleton and different expression of the glial fibrillary acidic protein (GFAP). These cells have a high motility rate of morpho-functional re-shaping in a timescale of minutes . SustainAll the discussed regulatory functions of astrocytes in synaptic plasticity make puzzling and intriguing the fact that these cells do not cover all synapses ,103. TheMicroglia can rapidly sense differences and homeostatic perturbations by scanning other cells (astrocytes and neurons) and the brain ECM  108], a, a108], Their scavenger role has practically led to the strong parallelism between microglia and the macrophage system in other tissues. However, microglia is an ontogenically distinct population of the phagocyte system, with a different embryonic origin, compared to resident macrophages of other tissues . For insThese sensors have been shown to contribute to synaptic plasticity, neurogenesis, myelination, and blood vessel formation . ConcernAnother paramount role during CNS development is the microglia-dependent synaptic elimination, essential for the correct wiring of the system, particularly studied in the visual cortex . The intOverall, these data show an interaction between the microglial system and multiple levels of synaptic plasticity. The mentioned pathways are strongly associated with the penta-partite model and pivotal in the development of systems biology approach to the adaptive/maladaptive tuning. The activation of both astrocytes and microglia in maladaptive plasticity and various neurological diseases, known as reactive gliosis ,53,137, Vβ3 associated with PDGF receptor) to exert its mitogenic activity, with the relevant contribution of Tenascin-C and NG2 (Oligodendrocytes are the myelin-forming elements of the CNS. They develop from the oligodendrocytes precursors cells (OPC). OPCs are important in CNS development but, unlike other progenitors, remain abundant in the adulthood and maintain the ability to modify the state of the white matter both in physiological  and pathological conditions  . OPCs ex and NG2  139]. T. TVβ3 asThe oligodendrocyte lineage proliferation, differentiation and survival, however, can be influenced also by neuregulins, NT-3 and NGF, further validating their role in synaptic plasticity ,141. ApaOligodendrocytes, although maintaining the ability to regenerate, are long-lived cells, their lifespan apparently is independent from the brain area or the grade of axon myelinization  Figure . New oliSynaptic plasticity processes require structural and functional modifications, including shape, density, formation, and elimination of synapses . The funThe molecular composition of ECM in the CNS has been deeply investigated to elucidate the intricate structure of this functional scaffold ,12. The Morpho-structural modifications of the ECM during CNS injury, aging and reactive astrocytosis have been studied for their implication in neuroinflammation, neurodegeneration and the maladaptive synaptic plasticity.Elegant experiments were made to discriminate the astrocytic and neuronal ECM contribution using co-cultures of neurons and astrocytes obtained from quadruple knockout mice for Tenascin-C/R, brevican and neurocan. These co-cultures showed reduced production and organization of the ECM, with lowered neuronal activity. The integration of wild-type astrocytes did not rescue the neuronal phenotype  and co-cFurthermore, MMPs and ADAMTS  proteases are secreted by both glia and neurons and can reshape the ECM in response to external or internal perturbations of the homeostasis . These sThe PNNs, a specialized form of ECM, seem to be key elements in synaptic stability, creating a functionally permeable barrier that allows or limits the formation of new connections between neurons . FurtherConsidering their role in synaptic stability, PNNs were shown as fundamental elements for memory maintenance . CSPGs, The increased expression of CSPGs, Tenascin-C, Tenascin-R, and HA can regulate axon elongation mediated by reactive astrocytes ,168. CSPThe maturation of NTs  can occur as abovementioned in the ECM. In this case, proNTs are processed by serine proteases like plasmin and MMPs, particularly MMP-7 and MMP-9 ,172. PlaCell adhesion molecules (CAM) are pivotal for the interaction between cellular elements and ECM. In particular synaptic CAM (SynCAM), neuroligins and hevin (involving both neurons and astrocytes) have demonstrated a role in the developmental synaptic plasticity, and more recently, also a member of the leucine-rich repeat transmembrane (LRRTM) proteins ,175. LRRFinally, we need to consider the complexity of the NVU. This component allows the CNS homeostasis, metabolic supply, and immunological privilege, being regulated by a fine tuning mode between the BBB elements , neurons, glia and interstitial ECM ,182.The immune privilege of the CNS applies to both brain and spinal cord   and can Thrombin is one of the main activators of neuro-immune responses and is strictly linked to the NVU. This protease can exert both adaptive and maladaptive plasticity modifications on neurons and glia ,187 depePAR-1 canonical activation seems to be neurotoxic and is achieved with pathologically high concentrations of proteases  activatiSerpins and thrombomodulin are also considered NIRegs and could reduce the toxicity of thrombin on the CNS by preventing the canonical activation of PAR-1 . LPS admThe role of pericytes, on the other hand, was largely ignored, although it was reported that a mutant mouse model with pericyte deficiency showed an significant increase of BBB permeability to both water and solutes of low and high molecular mass . This inlocus coeruleus. The increased ECM space alters the synaptic transmission and contributes with the arterial pump to the CSF influx and interstitial solute exchange during wakefulness-sleep rhythms [The CSF dynamic exchange of solutes with a directional flow from the arterial perivascular space into the brain parenchyma and the consequent venous drainage was recently added to the regulating functions of the NVU. In vivo studies demonstrated an active flow from the cisterna magna to the subendothelial space, regulated by astrocytic end-feet of the so-called glial-limiting membrane ,92. This rhythms  and poss rhythms ,200.Recent data suggest that a paradigm-shift in CNS studies is mandatory. To understand CNS complexity, novel experiments should focus on the functional cooperation between several cellular, sub-cellular and molecular components in synaptic plasticity. This can be achieved by a systems biology approach. Neurons, although paramount for synapse functioning, are not able to develop, reshape and reinforce the circuitry of the brain on their own.The support of the glia is essential for trophic factors and neurotransmitters modulation, axon myelinization, and synapse re-localization and elimination. On the other hand, the structural scaffold of the ECM consistently regulated by all resident cells acting as a functional unit can be pivotal in both developmental and several physiological changes. Moreover, the ECM is modified by pathophysiological processes of CNS diseases due to its competence in NTs storage, axon guidance, circuitry protection and intercellular communication. This synaptic model also accounts for the role exerted by the NVU in the metabolic supply, BBB maintenance, glymphatic system, coagulation and immune-system intervention in both adaptive and maladaptive plasticity. NVU and its peculiar structure support selective and controlled exchange between CNS and the blood flow, with precise rules and the possibility to modulate synaptic transmission.Here we have enlightened the state-of-the-art evidence of the main pathways that should be considered if we want to develop a comprehensive view of synaptic function under physiological and pathological modifications. The increasing amount of omics data  gained us with an extremely complex picture of the molecular events underlying perturbed conditions linked to neurological and neurodegenerative disorders. On the other hand, we need to organize these big-data into a dynamic, integrative model that takes into account not only the molecular networks but also their relative distribution between cellular and sub-cellular elements. Indeed, modular systems biology is a strategy that allows structuring the information about complex biological processes to obtain modular and mathematical/computational models that may favor the identification of the key steps of the process, as well as the prediction of how the molecular events of the network will respond to specific perturbations of the system. The perspective is to be able to comprehend the regulatory logic of the complex molecular network, which belongs to different cellular and non-cellular domains . A clear understanding of these mechanisms, through an iterative process of computational and experimental validation, could lead to the design of new drugs and innovative effective treatments for neurological diseases.


Arthropods comprise a predominant and well-succeeded phylum of the animal kingdom that evolved and diversified in millions of species grouped in four subphyla, namely, Chelicerata (arachnids), Crustacea, Myriapoda (centipedes), and Hexapoda (insects). It is agreed that the success of the arthropods’ flourishment and evolutionary story are in great part due to the diversification of venom apparatus and venom usage ,2. ThousCentruroides limpidus [Dinoponera quadriceps [Odontomachus monticola [O. monticola venom sac, besides the crude venom. Apart of numerous structural and functional classes of polypeptides found in a given venom proteome and peptidome, short membrane active peptides with or without definitive characterized antimicrobial activity have also been found in the venom of these species of ant and scorpion, like in other arthropods. The structural and molecular characterization of antimicrobial peptides are the focus of four articles: the antimicrobial and antibiofilm effects of peptides agelaia-MPI, polybia-MPII, polydim-I from the venom of social wasps, and the peptides Con10 and NDBP5.8 from scorpion venom against multidrug-resistant Acinetobacter baumannii, investigated and reported by das Neves and colleagues [A. lycosa erithrognata venom toxin, LyeTxI-b, that is effective in treating bacterial keratitis caused by drug-resistant Staphylococcus aureus, reported by Nunes da Silva et al. [Solenopsis invicta was evaluated by de Carvalho and colleagues [Berberomeloe majalis (Coleoptera: Meloidae), was demonstrated by Whitman and coworkers to display an important effect against distinct class of parasites [Considering the natural history of the evolutionary success of arthropods based on the molecular arsenal contained in their venom, a study reported here by Justin Schmidt explores and correlates the pain and lethality induced by hundreds of insect stings, pointing the direction to screen pharmacologically active venom components of pharmaceutical interest . To disslimpidus , the preadriceps , and theonticola . In a lalleagues ; a detaia et al. ; the artlleagues . Canthararasites .Tenebrio molitor [Aethina tumida were identified by Doupnik [One of the most studied animal venoms, bee venom, still has many interesting aspects to be discovered and explored. Crude venom and isolated components were reexamined in a review dealing with the potential therapeutic applications of bee venom to treat skin diseases , and in  molitor , while C molitor . In anot Doupnik  as molecLoxoceles) to be developed into therapeutics [Sphex argentatus, Sa12b and Sh5b, on ASIC currents in rat dorsal root ganglion neuron, contributing with the first discovery of a wasp peptide toxin that acts on such a kind of ion channel [Loxoceles as toxin moiety, a chimeric hybrid was produced by Calabria and colleagues to raise protective antibodies in Loxoceles antivenom therapy [Buthacus leptochelys [Neural receptors on excitable tissues, particularly ion channels, are a sort of preferential targets for arthropod venom components, notably from spider and wasps. Dongol and coworkers reviewed the structural determinants of diverse spider knottins (inhibitor cystine knot toxins) that influence voltage-gated sodium (Nav) channel activity on neuronal signaling, their role in the modulation of pain, and as a platform to develop analgesics . In the apeutics . The purapeutics . Acid-se channel . The pre therapy . Last butochelys . These nOverall, the compilation of such special articles highlights the huge potential of the discovery of arthropod venom. The diversity of peptide scaffolds and structures found in the numerous species of arthropods are amenable to be developed into specific and selective ligands and biotools. These, apart from being useful in basic research, are usable for precise intervention and modulation of the physio-pathological processes of diseases such as neurological disorders, or even for pest control, such as in the preparation and use of environmentally friendly biopesticides. So far, the future is bright for the usage of selective arthropod peptides.


Chronification of inflammation is the process that lies at the basis of several human diseases that make up to 80% of morbidity and mortality worldwide. It can also explain a great deal of processes related to aging. Atherosclerosis is an example of the most important chronic inflammatory pathology in terms of public health impact. Atherogenesis is based on the inflammatory response of the innate immunity arising locally or focally. The main trigger for this response appears to be modified low-density lipoprotein (LDL), although other factors may also play a role. With the quick resolution of inflammation, atherosclerotic changes in the arterial wall do not occur. However, a violation of the innate immunity response can lead to chronification of local inflammation and, as a result, to atherosclerotic lesion formation. In this review, we discuss possible mechanisms of the impaired immune response with a special focus on mitochondrial dysfunction. Some mitochondrial dysfunctions may be due to mutations in mitochondrial DNA. Several mitochondrial DNA mutations leading to defective mitophagy have been identified. The regulatory role of mitophagy in the immune response has been shown in recent studies. We suggest that defective mitophagy promoted by mutations in mitochondrial DNA can cause innate immunity disorders leading to chronification of inflammation. Chronification of inflammation is relevant to many human disorders and a subject of active current research that aims to answer the numerous questions remaining to date. If we could explain why the immune response can be prolonged in time, creating sites of chronic inflammation, it would help to clarify the mechanisms of diseases that account for up to 80% of current morbidity and mortality, as well as the mechanisms involved in aging. In this review, we discuss the role of the immune response disturbance at the level of mitochondrial dysfunction, in particular, defective mitophagy. Being a specialized form of autophagy, mitophagy clears the cell from dysfunctional mitochondria that can also be dangerous for the cells. It is known that mitophagy defects may be caused by some mitochondrial DNA (mtDNA) mutations. We describe the possible input of mtDNA mutations in the chronification of inflammation that can cause defects in mitophagy and, as a consequence, cause a massive release of pro-inflammatory factors and an unresolving inflammatory reaction.Atherosclerosis is a convenient model to study chronification of inflammation. Atherosclerotic lesions develop in the arterial wall locally, and even focally. Early stages of the disease development are characterized by prominent local differences between the adjacent areas of the arterial wall in terms of inflammatory activation. Comparison of such areas may reveal the cells, molecules and signaling pathways that are responsible for inflammation chronification.Despite the common understanding that atherosclerosis is an inflammatory disease, use of general anti-inflammatory drugs, such as non-steroidal anti-inflammatory drugs (NSAIDs) could not be proven beneficial for treatment of the disease. Some of the suggested approaches were doomed to failure, since they impacted targets not related to atherosclerosis ,2,3. It In this review, we discuss cellular mechanisms of atherosclerosis development. During the last decade, studies of atherosclerosis pathogenesis have been conducted mostly on transgenic animal models. However, the cellular composition and architecture of the arterial wall in animals and humans differs significantly, as does the structure of atherosclerotic lesions ,6. One oAtherosclerosis is currently regarded as an inflammatory disease, which is dependent on the chronic inflammation at all stages of its development. The disease can affect any artery, but the most dangerous lesions evolve in the aorta, coronary artery, arteries supplying brain and kidneys, and lower limb vessels. Atherosclerotic lesions develop locally, and even focally, progressively increasing in size and obstructing the blood vessel lumen, sometimes leading to a complete obstruction of the blood flow.Local inflammatory processes accompanying atherosclerotic lesion development are quite distinct by their mechanisms from the classical symptoms of inflammation, such as pain and erythema. Histological analysis of atherosclerotic lesions reveals local infiltration and activation of monocytes/macrophages and cytokine release followed by tissue reparation and remodeling. Such local inflammation driven by activated immune cells also result in the local healing response. Resolved inflammation is generally believed to result in diffuse intimal thickening which is normal for an adult artery. Thanks to modern imaging techniques, not only plaque morphology, but also disease activity can be observed in patients’ vessels affected by atherosclerosis . TherefoHistological features of inflammation include three consecutive stages: infiltration, reparation, and scar formation, which can also be observed during atherosclerotic lesion formation . Early sAt the organism level, increased levels of inflammation markers have long been known to be associated with atherosclerosis development ,12 and hThe use of statins for reducing cardiovascular risk have taken a firm place in current clinical practice. It was shown that beneficial effects of statins are not limited to lipid metabolism normalization, but are actually pleiotropic. Importantly, statins possess beneficial effects on vascular inflammation, protecting against atherosclerotic plaque development . BlockinAtherosclerotic lesion development takes place in the arterial wall intima, the innermost layer of the arterial wall . The intArterial wall intima is separated from the blood vessel lumen by a monolayer of endothelial cells (ECs) that are located on the basal membrane. The endothelial layer plays an important function in the arterial wall maintenance, regulating the penetration of cellular and chemical components of circulating blood into the arterial wall . The endBeneath the basal membrane, there is a thick proteoglycan-rich layer, also called elastic-hyperplastic layer, which is populated by various cell types . In the Atherosclerotic lesion development is accompanied by profound changes of the cellular contents of the intimal layer of the arterial wall . First, Atherogenic modified LDL is the primary source of lipids accumulating in foam cells . LipoproNative LDL interacts with a specialized LDL receptor (LDLR), and is normally internalized via receptor-mediated endocytosis, being destined for lysosomal degradation . This prIntracellular lipid accumulation is likely to be responsible for the loss of intercellular contacts and the destruction of the network of pericyte-like cells in the subendothelial space . This prThe innate immunity response is evolutionarily the oldest system of organism defense against pathogen invasion. It can be triggered by microbial infection and intrusion of other foreign matters, and is represented by cellular immune response, primarily executed by tissue macrophages . The maiOf special interest is the link between inflammasome activation and mitochondrial dysfunction, which appears to be especially important in atherosclerosis ,59. SeveMitochondria are semi-autonomous organelles derived from ancient endosymbiotic prokaryotic organisms that retain some important features of their bacterial ancestors . ImportaMitochondria realize fission and fusion independently of cell division, and can increase or reduce their numbers within the cell . The innMitochondrial homeostasis may be impaired due to the infection that leads to mtDNA release and excessive reactive oxygen species (ROS) production. These processes are aimed at destroying the invading pathogens, but can be dangerous to the host cells and tissues as well. Mitochondrial damage triggers NLRP3, which promotes the activation of caspase-1 and generation of the pro-inflammatory IL-1β and IL-18 ,74. ThesMitochondrial quality control is executed by several mechanisms that help monitor mitochondrial damage, reduce mitochondrial stress, and ensure selective removal of defective mitochondrial proteins or entire damaged organelles ,78. An eBeing a central player in maintaining cellular energy homeostasis and regulators of apoptosis and cell survival, mitochondria are naturally implicated in numerous human disorders. However, their role in chronification of inflammation appears to be especially important for chronic human disorders that are known to be associated with inflammatory processes. Interesting parallels can be drawn between human diseases that involve chronic inflammation and presence of mitochondrial dysfunction and mtDNA mutations. The existence of such parallels also makes mitochondrial dysfunction an attractive point of therapeutic intervention.The link between mitochondrial dysfunction and atherosclerosis has been the subject of extensive research . BesidesChronic obstructive pulmonary disease (COPD) is associated with aberrant inflammatory reactions in the lungs, which also involves mitochondrial dysfunction. In particular, mitochondrial genes MAVS and NLRX1 were shown to play a role in COPD pathogenesis ,92,93. IAmong the many processes involved in aging, impaired mitochondrial function and alteration of mitochondrial count have been identified as prominent players . RecentlHis m.12201T>C, Gong et al. found that overexpression of the human mitochondrial histidyl-tRNA synthetase gene corrected the mitochondrial dysfunction caused by this mutation [Age-related accumulation of mtDNA mutations has been observed in many studies, and the possible role of mtDNA mutations in aging was widely considered ,67,68,69mutation . These rA recent study conducted by our group has reported a relationship between mitochondrial functions and atherosclerosis-associated mtDNA mutations investigated on cybrid cell lines carrying various variants of the mitochondrial genome obtained from atherosclerotic patients . EarlierMutations of the mtDNA associated with conditions that increase the risk of atherosclerosis development, such as hypertension, type 2 diabetes and obesity have been described ,116,117.Functional activity of cells involved in the immune-inflammatory response is highly dependent on correct functioning of the mitochondria. Impairment of mitochondrial respiration may result in both normal aging of blood vessels and in various cardiovascular diseases, including atherosclerosis, heart failure, and the formation of aneurysm . MitochoSeveral heteroplasmic mtDNA mutations positively or negatively correlating with the extent of atherosclerotic lesions in human arteries have been identified in circulating cells, atherosclerotic arteries, and other tissues. These mutations are located in the mitochondrial genes encoding rRNA12S, tRNA-Leu (UUR recognition codon), tRNA-Leu (CUN recognition codon), subunits of 1, 2, 5, and 6 NADH-dehydrogenase, and cytochrome B ,132. HetThe focal nature of the atherosclerotic lesion distribution within the arterial wall is clearly seen in The observed mosaicism in mtDNA mutation distribution may have different explanations. First, it may reflect the focal nature of atherosclerotic lesion initiation, which preferentially occurs in the sites of local disturbance of the endothelial function and increased permeability. Second, due to the fact that even non-involved loci are significantly differed from each other by the content and composition of atherosclerosis-associated mutations , the mosA study conducted by our group has shown that mtDNA mutations are related to the degree of monocyte activation . In thisThree cybrid lines carrying mitochondrial mutations associated with atherosclerosis (see above) were used to evaluate the response to inflammatory stimulation with lipopolysaccharides (LPS). Cybrid lines were created from macrophage-like cells of the THP-1 line, ρ-0 cells, that do not have functional mtDNA, fused with platelets isolated from different patients. Thus, each cybrid cell line contained a nuclear genome of THP-1 cells and the patient’s mitochondrial genome. Each cybrid line had the mitochondrial genotype of a single patient A. The leThe right panel of Thus, cells differing in the mitochondrial genome and carrying atherogenic mutations were dramatically different in their immune response from intact THP-1 cells. Two cybrid lines did not respond to inflammatory stimulation, but the third line not only responded to the first but also to the second stimulation, i.e., this cybrid line lacked tolerance of innate immunity. Presence of such cells in the focus of inflammation will complicate the resolution of the inflammatory response and will contribute to the chronification of inflammation.MtDNA mutations may also be involved in mitophagy impairment, and, as a consequence, insufficient clearance of dysfunctional mitochondria. To ensure mitophagy, several molecular mechanisms that preserve cellular and tissue homeostasis are activated and coordinated . ViolatiSeveral independent groups have investigated the relationship between mitochondrial mutations and mitophagy. The role of m.12338T>C mutation in decreased mitophagy was demonstrated by reduced levels of autophagy protein light chain 3 and accumulation of autophagic substrate p62 in the in mutant cybrids as compared with control lines. These data demonstrated the direct link between mitochondrial dysfunction caused by complex I mutation and apoptosis or mitophagy . In Lebe2+ uptake, which caused stimulation of the autophagic mechanism through the signal axis of AMP-activated protein kinase [Another study showed that mtDNA mutations that disrupted mitochondrial function stimulated mitophagy and reduced the regenerative capacities of cardiac progenitor cells . Human fn kinase . A studyn kinase .The relationship between mitochondrial functions and atherosclerosis-associated mitochondrial mutations was investigated on cybrid cell lines carrying various variants of the mtDNA obtained from atherosclerotic patients. The identified mtDNA variants associated with increased mitophagy (as assessed by LAMP gene expression) were: m.3336T>C, m.3256C>T, m.5178C>A, m.3336T>C, m.3256C>T, and m.5178C>A .The relationship between mitophagy and innate immunity is discussed in recent review articles ,146,147.The effect of mitophagy modulators on the ability of human monocytes to respond to pro-inflammatory stimulation was studied. Four mitophagy modulators were used . None ofThus, modulation of mitophagy can affect the inflammatory response of innate immunity. Further research should establish a link between defective mitophagy and chronification of inflammation.A model that could link the mtDNA mutation-induced mitochondrial dysfunction and deficient autophagy may help understanding of the importance of these events in chronification of inflammation and identifying potential points of therapeutic intervention. Based on the available data discussed above, we propose a plausible mechanism, according to which, phagocytosis stimulation by circulating large associates of modified LDL activate the pro-inflammatory response of the innate immunity system . AccordiThe proposed concept allows speculation that atherogenesis is due to two errors made by the cell of the arterial wall. The first one is that the cell perceives the associates of modified LDL as a pathogen that is taken up by phagocytosis, which causes an inflammatory response and the accumulation of intracellular lipids, which in turn is a trigger of atherogenesis at the cellular level. The second is that due to mutations, the mitochondria becomes dysfunctional and due to a defect in mitophagy, the cell cannot free itself from this mitochondrion and perceives it as a bacterium-like pathogen. This triggers an ongoing inflammatory signaling that may lead to inflammasome activation. Mitochondrial dysfunction may be linked to TLR and NLRP3 signaling, with mitochondria therefore playing a key role in the sterile inflammation. Together, these processes result in chronification of inflammation. Such a development may be characteristic not only of atherosclerosis, but of any pathology associated with chronification of inflammation. The specificity of atherosclerosis is that the main triggers of the immune response are not PAMPs or DAMPs but rather, modified LDL. Future studies should investigate the mechanisms of sterile inflammation in atherosclerosis in more detail, paying special attention to mitochondrial dysfunction.The important role of mitochondrial mtDNA mutations in the development of various chronic human diseases is currently beyond any doubt. Moreover, mtDNA mutations may serve as important disease-modifying factors. However, outlining the direct links between certain mtDNA variants and pathophysiological features is challenging due to genetic heterogeneity of the mitochondria, the pleiotropic nature of mitochondrial dysfunction input in the overall pathology, and the lack of knowledge about relevant mtDNA variants, although the list of different mtDNA mutations associated with human diseases is constantly growing. Nevertheless, the results available to date position mitochondria as likely therapeutic targets for treatment of such diseases as cancer, chronic inflammatory states, and atherosclerosis. Selective mitochondria-targeting drugs, such as mitochondrial antioxidants, are being tested in preclinical and clinical trials ,152. MorClarification of the causes of the transition of acute to chronic inflammation (chronification of inflammation) can reveal the mechanisms of diseases that make up to 80% of morbidity and mortality, as well as explain the causes of aging. Atherosclerosis is one of the most important inflammatory pathologies. The cellular mechanisms of atherogenesis are based on the inflammatory response of innate immunity arising locally or focally. The trigger for this response appears to be modified LDL. With the quick resolution of inflammation, atherosclerotic changes in the arterial wall do not occur. However, a violation of the response of innate immunity can lead to chronification of local inflammation and, as a result, to the formation of an atherosclerotic lesion. Mitochondria dysfunctions are the cause of innate immunity disorders in the case of atherosclerosis and many other pathologies. Some mitochondrial dysfunctions may be due to mutations in mitochondrial DNA. Mitochondrial mutations have been found that can lead to defective mitophagy. The regulatory role of mitophagy in the immune response has been shown in recent studies. We suggest that it is defective mitophagy as a result of mutations in mitochondrial DNA that can cause innate immunity disorders leading to chronification of inflammation.


The head represents the most complex part of the body and a distinctive feature of the vertebrate body plan. This intricate structure is assembled during embryonic development in the four-dimensional process of morphogenesis. The head integrates components of the central and peripheral nervous system, sensory organs, muscles, joints, glands, and other specialized tissues in the framework of a complexly shaped skull. The anterior part of the head is referred to as the face, and a broad spectrum of facial shapes across vertebrate species enables different feeding strategies, communication styles, and diverse specialized functions. The face formation starts early during embryonic development and is an enormously complex, multi-step process regulated on a genomic, molecular, and cellular level. In this review, we will discuss recent discoveries that revealed new aspects of facial morphogenesis from the time of the neural crest cell emergence till the formation of the chondrocranium, the primary design of the individual facial shape. We will focus on molecular mechanisms of cell fate specification, the role of individual and collective cell migration, the importance of dynamic and continuous cellular interactions, responses of cells and tissues to generated physical forces, and their morphogenetic outcomes. In the end, we will examine the spatiotemporal activity of signaling centers tightly regulating the release of signals inducing the formation of craniofacial skeletal elements. The existence of these centers and their regulation by enhancers represent one of the core morphogenetic mechanisms and might lay the foundations for intra- and inter-species facial variability. The shape of the face possesses species-specific features that give each vertebrate group its unique facial appearance. In some species such as humans, the otherwise conserved shaping mechanism is flexible enough to allow for shape fine-tuning, resulting in an impressive facial variability . The shaThe chondrocranium formation is an intricate, multi-step process utilizing cells derived from all three germ layers and driven by, for instance, complex cell and tissue interactions, specific cellular behavior, or by a multitude of spatiotemporally active morphogens. In this review, we will summarize the advancements in our understanding of its developmental complexity. In the first part, we will focus on mechanisms driving the specification, delamination, and migration of cranial neural crest cells to their respective destinations, and their interactions with surrounding cells that result in sensory placode formation. Next, we will pinpoint to mutual interactions between different cell and tissue types, the cellular response to changing physical forces, and specific clonal behavior such as oriented cell division. In the end, we will expose the importance of the spatiotemporally defined activity of signaling centers driving the early craniofacial morphogenesis and discuss the possible genetic basis of facial variability.A milestone in the process of head formation is the emergence and migration of the neural crest cells (NCCs). The NCCs are transient multipotent progenitors arising from the borders of the closing neural tube . DependiInterestingly, cranial and trunk NCCs utilize distinct migration strategies . Trunk NThe NCCs give rise to a broad spectrum of cell types such as neurons and glial cells of the peripheral nervous system, smooth muscles, endocrine cells, melanocytes, chondrocytes, odontoblasts, connective tissue, and bone . In mousEpigenetic regulation is one of the further mechanisms controlling the cranial NCC plasticity, especially concerning their axial regional identity. The cranial NCCs maintain broad facial patterning competence and can contribute to different structures in the forming head. Cranial NCCs show similar chromatin accessibility patterns even though their transcriptional profile differs. The chromatin pattern is maintained until post-migratory stages when the cells commit to a specific fate in response to local cues . In XenoThe combination of the single-cell transcriptomics and bioinformatical analysis of branching trajectories of murine NCCs, revealed new aspects of NCC multipotency, EMT, and fate specification . Recent Migration of single cells and large-scale collective cell movements represent important aspects of early facial morphogenesis. Such movements enable both mechanical and molecular interactions between cells and tissues, and their environment. One of the examples of such coordinated movement are the reciprocal interactions between the cranial NCCs and cranial placode cells. Cranial placode precursors arise at the anterior part of the neural plate border, and form a common pre-placodal region (PPR) characterized by the expression of the transcription factor Six1 . The PPRThe cranial NCCs then follow stereotypical migratory streams and proliferate on their way to finally settle in various compartments of the head and produce ectomesenchyme . While aFunctional experiments targeting different components of the Wnt/PCP signaling highlighted the importance of the ectomesenchyme polarity for the proper facial outgrowth in several developmental time-points. Overall, the rate of cell divisions in Wnt/PCP mutants does not appear to be affected, however, the impaired direction of daughter cell allocation during cell division results in a variety of pathologies, such as shorter and wider facial proportions, abnormal mesenchymal patterning during early palatogenesis or misoriented and misshaped chondrocytes affecting the facial morphometry . OrienteXenopus  that alter the levels of gene expression. Deletion of enhancers or changes in their sequence can result in a wide range of facial phenotypes . The seqThe development and availability of new techniques in the last decade have significantly advanced our understanding of the complexity of craniofacial development and opened new perspectives for future research. Single-cell omics analysis can be applied to uncover the gene regulatory networks driving the transitions from mesenchyme to cartilage or to reveal the enhancer-driven control of gene expression levels in different cellular populations. This approach, in combination with genetic tracing and live imaging, can further dissect the interactions between various cell or tissue types in detail and resolve, among others, the still enigmatic processes of placode specification and individualization. The precise spatiotemporal mapping of the signaling centers in the developing embryos of various model organisms and exploring their genomic regulation will advance our understanding of both the intra- and inter-species facial variability and the genetic basis of facial features heritability. Additionally, such investigation will allow us to better comprehend the nature of rare craniofacial pathologies. Further research of the conserved developmental link between the formation of the nervous system and the skull may identify new therapeutic targets to reduce the impact of severe congenital syndromes manifested with craniofacial deformities or offer new strategies to enhance cartilage regeneration in adulthood.AM-R and MK wrote the manuscript and designed the figure. Both 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.


The human gastrointestinal tract harbors most of the microbial cells inhabiting the body, collectively known as the microbiota. These microbes have several implications for the maintenance of structural integrity of the gastrointestinal mucosal barrier, immunomodulation, metabolism of nutrients, and protection against pathogens. Dysfunctions in these mechanisms are linked to a range of conditions in the gastrointestinal tract, including functional gastrointestinal disorders, ranging from irritable bowel syndrome, to functional constipation and functional diarrhea. Irritable bowel syndrome is characterized by chronic abdominal pain with changes in bowel habit in the absence of morphological changes. Despite the high prevalence of irritable bowel syndrome in the global population, the mechanisms responsible for this condition are poorly understood. Although alterations in the gastrointestinal microbiota, low-grade inflammation and immune activation have been implicated in the pathophysiology of functional gastrointestinal disorders, there is inconsistency between studies and a lack of consensus on what the exact role of the microbiota is, and how changes to it relate to these conditions. The complex interplay between host factors, such as microbial dysbiosis, immune activation, impaired epithelial barrier function and motility, and environmental factors, including diet, will be considered in this narrative review of the pathophysiology of functional gastrointestinal disorders. In the human body there are about 39 trillion microbial cells   and quantitative  comes first in IBS etiology. The usefulness of describing the microbiota at higher taxonomic levels may be limited, since this may not provide meaningful information. New metagenomic tools allow an integrated analysis of taxonomic and predictive functional dynamics of the microbiota, providing improvements in genus-species analyses, more detailed insights into the effect of microbial metabolic pathways on crucial aspects of IBS pathogenesis, as well as of the potential host-microbiota interactions in health and disease. In addition, current techniques relying for example on 16S rRNA gene analysis, may also overlook potential pathogens, such as colonic spirochetes, which may be linked to symptoms of IBS, due to the incompatibility of standard primers , regulatory proteins acting on the epigenome through chromatin-remodeling changes  signaling leads to Bacteroidetes overgrowth  proteins in both normal and pathological conditions  in inducing shifts in the GI microbiota, influencing host metabolic and immune markers  or non-immunoglobulin E-mediated (non-atopic) reactions. Acute-phase immunoglobulin E-mediated hypersensitivity results in the activation of mast cells, eosinophils, and other immune cells and the release of molecules  involved in GI symptom generation , or related organic or inorganic acids, have the potential to trigger a non-specific antigen-induced pseudo-allergic hypersensitivity reaction, causing the release of cysteinyl leukotrienes , which are short-chain, soluble, highly fermentable carbohydrates. Their fermentative properties make FODMAPs closely linked to symptoms generation in IBS , increasA diet low in FODMAPs is very restrictive and although long-term restrictive diets seem to still allow for an adequate nutrients intake  (Clavel et al., Erysipelotrichaceae seem to be linked to diets high in fats and to play a role in host lipid metabolism (Harris et al., Erysipelotrichaceae may play a role in the development of colonic inflammation or if their relative abundance is reflecting more the dietary and/or the lipid and cholesterol status of the host.On the other hand, the consumption of diets rich in saturated fats of animal origin has been associated with low-grade inflammation in the GI tract, through the activation of TLR-dependent signaling by microbial factors (Kim et al., Clostridium spp. have long been considered as major producers of ammonia from protein fermentation (Vince and Burridge, A high intake of dietary protein, specifically animal-based proteins, has been implicated in the pathogenesis of IBS through multiple mechanisms (Kakodkar and Mutlu, Fusobacterium spp., which is known to generate cysteine through the cysteine desulfydrase activity, has been associated with impaired colonic function in IBS or inflammatory bowel diseases (Strauss et al., Hydrogen sulfide, another end-product of protein fermentation, is produced by the microbiota mostly through the degradation of the sulfur-containing amino acid cysteine. Understanding the mechanisms underlying host-microbe interactions and symptoms pathophysiology will likely improve the current knowledge of pathways involved and the predictive value of IBS biomarkers. Biomarkers can be measured in blood, fecal, urine or breath samples, to potentially discriminate IBS from other GI disorders or from health, and more importantly within the IBS subtypes and to characterize improvements in well-being and quality of life of IBS subjects.General observations in IBS vs. health include differences in microbial composition, immune profile, GI motor and sensory function, pain perception, serotonin metabolism, and the expression of genes involved in immune activation (Camilleri et al., Some of the parameters that have been studied include biomarkers of GI and immune function and biomarkers of GI microbiota (Bischoff, Bacteroides (Sundin et al., Recent studies have highlighted the role of immune dysregulation and microbial dysbiosis in IBS and some molecules of the immune system measured in blood or in GI luminal contents could be putative biomarkers. Fecal CgA plays a role in pain regulation and antimicrobial activity, and their fecal levels have been negatively correlated with colonic transit time in individuals with IBS (Öhman et al., Calprotectin, a protein released by neutrophils during GI inflammation, can be easily measured in stool samples, as it is resistant to degradation in the colon, and can therefore be considered as a non-invasive marker of low-grade inflammation. Although calprotectin is primarily used to distinguish IBS from IBD (Chang et al., Serine proteases, such as tryptases, which are released by colonic mast cells and bacteria, have been also reported to be elevated in IBS-D (Róka et al., −/− mice showed a decreased amplitude and frequency of the contractions in the proximal colon (Forcén et al., TLRs are a family of receptors present on both epithelial and immune cells in those tissues exposed to the external environment, such as the lungs and GI tract (Zarember and Godowski, Consistent with these observations, chronic low-grade inflammation and differences in pro- and anti-inflammatory cytokine concentrations in the colonic mucosa or systemically have been also associated with IBS (Sundin et al., +, CD4+, and CD8+ T cells and activated macrophages has been observed also in subjects with a diarrhea-predominant phenotype persisting after an episode of infectious gastroenteritis (Spiller et al., An increased count of lamina propria CD3FGIDs present a highly variable clinical phenotype associated with early childhood events, somatisation, different diets, psychological, hereditary and environmental factors. To date, specific immune cell populations, cytokine concentrations and bioactive metabolites have been investigated in an independent manner, resulting in contrasting findings on the exact role of immune activation in the development of FGIDs (Lazaridis and Germanidis, Several studies have provided new insights into bacterial mechanisms influencing the immune system in the context of inflammatory bowel diseases (Gonçalves et al., The evaluation of the consequences of dysbiosis in FGIDs has some limitations. Firstly, there is still a lack of integration between taxonomic and functional data for the identification of specific microbes and to better understand their contribution to the optimal function of the GI tract and associated organs, for example the brain via the gut-brain axis. Indeed, the interactions between microbial community and host could not be gathered from single analyses as most metabolic pathways in nature take place within communities, rather than pure cultures. High-throughput DNA sequencing technology has enabled a shift from descriptive analysis of different taxa of the microbial community to an investigation of the predictive functional contribution of the microbiota to health and disease (Rooks and Garrett, Secondly, IBS clinical symptoms are heterogenous and fluctuating and there are no confirmed molecular or organic biomarkers to diagnose this condition. Finally, the identification of a microbial signature in IBS is confounded by the individual complexity, instability and variability of the microbiota, which can be influenced by the psychological status, medications and diet. In this regard, diet can affect microbiota composition and function as well as colonic motility, sensitivity and epithelial barrier function. However, further research is needed to elucidate the role of specific macronutrients and micronutrients in IBS.Finally, discrepancies between studies may also reflect differences in DNA extraction methods, analytic techniques, number of subjects and the sample collection method. Indeed, fecal samples do not precisely represent the microbiota composition or function in the proximal colon, and colonic biopsies do not physiologically reflect the microbiota, because of the extensive sample preparation.A possible microbial pathogenesis in IBS has also therapeutic implications. In this regard, probiotic, prebiotic, synbiotic and antibiotic treatments have been largely investigated although with contrasting results, and the manipulation of GI microbiota represents a promising strategy in the treatment of FGIDs.In this review, the recent evidence proposing FGIDs as systemic conditions has been discussed. This involves not only individual systems, such as the GI microbiota, the digestive, immune and enteric nervous systems, but also their intricate interplay. The mechanisms involved in FGID pathophysiology can be investigated at the cellular and molecular level, including the analysis of the genome, trascriptome, proteome, metabolome and brain connectome. Therefore, we suggest an integrative system biology approach as the most appropriate to investigate the complex interactions underlying FGIDs, considering the broad range of different and interacting elements, which are responsible for the highly variable clinical phenotype.CC prepared the first draft and WY, RG, NT, WM, and NR edited and approved the final manuscript. 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.


Cardiomyopathies (CMPs) represent a diverse group of heart muscle diseases, grouped into specific morphological and functional phenotypes. CMPs are associated with mutations in sarcomeric and non-sarcomeric genes, with several suspected epigenetic and environmental mechanisms involved in determining penetrance and expressivity. The understanding of the underlying molecular mechanisms of myocardial diseases is fundamental to achieving a proper management and treatment of these disorders. Among these, inflammation seems to play an important role in the pathogenesis of CMPs. The aim of the present study is to review the current knowledge on the role of inflammation and the immune system activation in the pathogenesis of CMPs and to identify potential molecular targets for a tailored anti-inflammatory treatment. Cardiomyopathies (CMPs) represent a diverse group of heart muscle diseases, defined as myocardial disorders in which the heart muscle is structurally and functionally abnormal in the absence of hypertension, valvular heart disease, congenital heart disease and coronary artery disease sufficient to explain the observed myocardial abnormality. According to the recent classification of the working group on myocardial and pericardial diseases of the European Society of Cardiology (ESC) , they arThe development and progression of a cardiomyopathic phenotype depends on a complex interaction between several cellular signaling pathways, environmental stressors and individual genotypes. Our understating regarding the development and the progression of each cardiomyopathy has improved over the last decades. Among the several mechanisms implicated, inflammation seems to have an important role in the pathogenesis of CMPs ,4,5.Myocardial inflammation has recently emerged as a pathophysiologic process that contributes to cardiac hypertrophy, fibrosis and dysfunction in the context of heart disease . ElevateMoreover, the evidence that CMPs, such as heart failure, can be associated with elevated circulating levels of proinflammatory cytokines has provided a new research area that has revealed a potential role of inflammation and immune system activation in the pathogenesis of these disorders.The aim of the present review is to summarize the current knowledge on the role of inflammation and immune system activation in the pathogenesis of CMPs and to identify potential molecular targets for a tailored anti-inflammatory treatment.To explain their role in the pathogenesis of CMPs, we briefly describe the most important cellular and molecular mechanisms that contribute to cardiac inflammation and fibrosis. Inflammation and fibrosis occur in nearly all forms of myocardial injury and seem to play an important role in the pathogenesis of different myocardial diseases . MyocardSeveral cell types play an important role in cardiac inflammation. First, neutrophils and monocytes migrate to the site of cardiac injury and release harmful mediators such as reactive oxygen species (ROS) and several proteases, in order to eliminate the factors that caused the cardiac injury. However, their response is nonspecific and could result in additional cardiac insult . Thus, mSeveral inflammatory pathways are involved in cardiac inflammation, such as the TNF-NF-κβ pathway (implicated in cardiac infection and injury)  or caspaTLRs are activated by molecular motifs from pathogens  or from endogenous material released by stressed or damaged cells  . The engThe persistent activation of cardiac inflammatory pathways may lead to significant fibrotic changes, which represent a substrate for pathological myocardial remodeling. Myocardial fibrosis results in electromechanical disturbances and reduces the nutrient supply toward the myocardium, perpetuating a vicious cycle of fibrosis, myocyte death and inflammation .Arrhythmogenic cardiomyopathy (ACM) is a heart muscle disease characterized by a fibro-fatty replacement of the right ventricular and/or left ventricular myocardium, which leads to anatomical, contractility and electrical rhythm abnormalities with life-threatening complications ,19. The A tailored pharmacological treatment that directly targets the underlying disease mechanism in the various forms of ARVC is under investigation. Recently, an inhibitor of GSK3𝛽, a major regulator of Wnt/𝛽-catenin signaling identified in a zebrafish model of ARVC , showed On the other hand, dominant-left ACM (ALVC) is principally associated with nondesmosomal arrhythmia-associated variants  ,27,28 anIn ACM, the replacement of the myocardium by fibro-fatty tissue has been related to three principal mechanisms : apoptosHowever, the pathophysiology of ACM is still partially unknown, and in recent years a relationship between myocardial inflammation and ACM has been described; in particular, the role of infectious agents has been proposed because of the common findings of patchy cell death with inflammatory infiltration in the myocardium , suggestMyocardial inflammation in ARVC can be analyzed though invasive procedures such as endomyocardial biopsies or through postmortem autopsy studies, and it was noted in the form of lymphocyte infiltrates in nearly two thirds of ARVC cases .67Ga uptake [Moreover, myocardial inflammation in ARVC can also be identified noninvasively with the combined analysis of the plasma levels of inflammatory cytokines and cardiac scintigraphy . In parta uptake . There ia uptake . These ca uptake . In parta uptake . Finallya uptake .Similarly, inflammatory cell infiltrates have been described in patients with ALVC . The subSmith et al.  evaluateIn conclusion, these findings suggest that desmosomal dysfunction may stimulate the inflammatory response, thus favoring the recruitment of the inflammatory cells and further weakening the junctional stability. However, it is unclear if myocardial inflammation represents a primary insult to disease mutations or a secondary response to cardiomyocyte death.Hypertrophic cardiomyopathy (HCM) is a heart muscle disease characterized by left ventricular hypertrophy that is not solely explained by abnormal loading conditions  ,41. In nDifferent mechanisms are implicated in the pathogenesis of HCM . The priThe initial defect causes a cascade of secondary molecular events, including the activation of the calcium-sensitive and stress-responsive molecular pathways that collectively mediate the programming of cardiac hypertrophy and induce the morphological and histological phenotypes that are recognized as HCM, principally represented by myocardial hypertrophy, myocyte disarray and myocardial fibrosis. The variability of the cardiac phenotype suggests that several epigenetic and environmental factors are involved in the pathogenesis of this disorder, but the mechanism by which the genetic mutation translates into the phenotype remains poorly understood . Since tSeveral studies documented the leucocytes’ infiltration in the myocardium and the elevation of inflammatory cytokines in HCM patients ,49,50, aKuusisto et al.  evaluateSubsequently, Fang et al.  showed tThe trigger for early inflammation in HCM may be linked to cardiomyocyte disarray and sarcomere injury, mitochondrial oxidative stress, and microvascular disease with tissue injury; however, to date this trigger has not been clearly determined.A recent study by Becker et al. explained the key role of neutrophil extracellular traps (NETs) in the pathogenesis of hypertrophic cardiomyopathy . In respIn brief, the stages of the pathogenesis of HCM can include a genetic predisposition, represented by a sarcomeric gene mutation in about 40–60% of patients, an early phase characterized by tissue level inflammation and NETs, an intermediate phase with platelet activation, microvascular thrombosis and transforming growth factor (TGF) β1 signaling, and a late phase of myocardial fibrosis, ventricular disarray and hypertrophy, finally responsible for the clinical features and adverse events. The animal HCM models provide an opportunity to determine if tissue level inflammation occurs, the underlying mechanisms that may involve ETs, and whether they participate in the phenotypic expression of HCM and in the transition from a hypertrophic state, characterized by myocyte hypertrophy to a dilated (hypokinetic end-stage) state with diffuse myocardial fibrosis and an impaired systolic performance.The term dilated cardiomyopathy (DCM) is used to refer to a spectrum of heterogeneous myocardial disorders that are characterized by ventricular dilation and depressed myocardial performance in the absence of hypertension, valvular heart disease, congenital heart disease and ischemic heart disease . This coNext to the genetic cause, various acquired causes  are responsible for the remaining portion of DCM patients ,59.The term dilated inflammatory cardiomyopathy (DCMi) refers to a broad group of disorders presenting with a dilated phenotype in which inflammation of the heart is the cause of myocardial dysfunction and can be viewed in contradistinction to a secondary immune response to other mechanisms of injury  . NeverthInfectious myocarditis is the leading cause of DCMi worldwide. The diagnosis of viral DCMi has been facilitated by the introduction of new-generation protein chain reaction (PCR) techniques in endomyocardial biopsy (EMB). The prevalence of specific pathogens involved in infectious myocarditis varies according to the geographical location, different molecular methods used and epidemiologic shift . NoninfeA pathophysiological model of DMCi development has been validated for some forms of infectious myocarditis and noninfectious pathogens. In this model, a myocardial injury occurs and triggers a chronological sequence comprised of three pathogenic phases  62,65,6,665,66:(i) the myocardial injury is determined by a specific pathogen, directly and by nonspecific immunity. Then, the innate immune response, through recognition receptors such as TLR, determines a proinflammatory cascade of cytokines  and interferons when it detects pathogen-associated (infectious triggers) or damage-associated (noninfectious triggers) molecular patterns.(ii) In the second phase, one to four weeks after the onset of the myocardial injury, there is a specific immune response with autoimmune or hypersensitivity features, based on the cross-reactivity of viral epitopes and some cardiac structures (molecular mimicry phenomenon), as well as the exposure of intracellular structures to the immune system, consequent T and B lymphocytes activation, and antibodies production against myocyte structures .(iii) Finally, after several weeks to months, the complex interplay between the infectious or noninfectious myocardial damage and immune response may lead to ventricular dilatation and dysfunction.Immunosuppressive therapy has been considered to be a promising approach to DCMi. However, large trials showed mixed results ,68. It hThe growing evidence for the major role of inflammatory mechanisms in CMPs has prompted the development of new targeted therapies in this setting. Although potential targets in HCM and in ACM have not yet been identified, novel targeted inflammatory treatments seem to be feasible in DCM.Since the inflammatory cascade in infectious DCMi is triggered by a viral internalization by the common cellular receptor and by consequent intracellular signaling, its prevention could alter viral infection. Tyrosin kinase p56lck seems a logical target for pharmaceutical agents . After tAdditionally, the IL-1β blockade seems to be a promising novel target therapy. Recently, the anakinra (an IL-1β blockade) showed a beneficial effect on inflammation, myocardial performance and clinical status in acute decompensated heart failure (HF) patients . Its safThe immunoadsorption and subsequent administration of polyclonal IgG has been proposed for chronic inflammatory autoimmunity with promising results . In DCM Inflammation plays a central role in the disease pathogenesis and progression of myocardial disease. To date, there is no evidence to support the use of anti-inflammatory treatment in these patients, and the modulation of the inflammation remains a promising target for the treatment of CMPs. Additional studies are needed to further delineate the mechanisms and to identify novel target molecules.


The original technologies for their preparation preserve the valuable microbial content and improve their nutritional and probiotic qualities. This review emphasises the features of LAB starters and the autochthonous microflora, the biochemistry of dairy food production, and the approaches for achieving the fortification of the foods with prebiotics, bioactive peptides , immunomodulatory exopolysaccharides, and other metabolites  with reported beneficial effects on human health. The link between the microbial content of dairy foods and the healthy human microbiome is highlighted.The reported health effects of fermented dairy foods, which are traditionally manufactured in Bulgaria, are connected with their microbial biodiversity. The screening and development of probiotic starters for dairy products with unique properties are based exclusively on the isolation and characterization of lactic acid bacterial (LAB) strains. This study aims to systematically describe the LAB microbial content of artisanal products such as Bulgarian-type yoghurt, white brined cheese,  Dairy products are indispensable, consumed daily and one of the most desired foods by a large part of the world population. They are prepared at home , or produced industrially in tonnage quantities everywhere in the world. To obtain them, fermentation technologies are used with the participation of various lactic acid bacteria (LAB). Lactic acid fermentation improves the taste, enhances the digestibility of the milk, and offers the manufactures a wide variety of valuable products. Fermented milk is very useful for many reasons. The related products have a prolonged shelf life; they are safe because LAB act as preservatives and inhibit the development of pathogenic microflora, they are extremely suitable for the absorption of nutrients from milk, and they are beneficial due to the health impact of lactic acid bacteria on various body functions .kefir consumption was found in China in a Bronze Age tomb 1−6Gal(β1–4)Glc). They are usually mixed in yoghurt, with some branched structures composed of multiple galactose moieties linked to glucose at the reducing end. MO are even more heterogeneous and are composed of five monosaccharides: D-glucose, D-galactose, ic acid) . A brandic acid) . Both GOiniphila ,107,108.rebiotic ,110. A. muciniphila, which was revealed by developing animal models of obesity [Other compounds found in dairy products, which have recently attracted attention due to their potential prebiotic properties, are flavonoids and short-chain fatty acids (SCFA) ,112,113. obesity . Flavono obesity .Traditional Bulgarian dairy products contain unique bacterial microflora that has evolved under specific climatic conditions over the centuries. In addition to the excellent technological qualities leading to wonderful taste, aromas, organoleptic properties and durability, LAB strains, both starter and autochthonous, contribute to the functionality of dairy foods. The beneficial effects of dairy food consumption may be attributed to the biologically active compounds in these products; to the modification of milk components by starter cultures; and, importantly, to the direct consumption of LAB strains as preventive and therapeutic agents. The beneficial effect may be direct, through the interaction of the human body with consumed microorganisms, or indirect, as a result of microbial metabolites generated during the fermentation process. The development of new dairy products should be based on current knowledge of the food-gut axis, as well as new well-thought-out and thorough experiments to reveal the mechanisms by which their metabolites can affect health problems. Research on the probiotic potential of dairy products and the main mechanisms by which LAB contribute health benefits will continue with new findings, both in healthy and disabled people. This approach would turn the food into medicine and the diet into treatment.


Clinical management of colo-rectal liver metastasis would benefit from a refined stratification of patients in prognostic groups, in order to identify the best therapeutic option. Efforts are ongoing in the definition of parameters associated with clinical behaviors, which could help classifying patients in clinically relevant groups. Here we aimed at discussing the recent advances in this field, and we introduced current and new promising candidates, such as morphological tumor features and immune components, which have been showing significant association with survival. Some of these parameters are slowly reaching the clinic and further efforts are ongoing in the attempt to combine them in multiparametric scores.Prognostic studies are increasingly providing new tools to stratify colo-rectal liver metastasis patients into clinical subgroups, with remarkable implications in terms of clinical management and therapeutic choice. Here, the strengths and hurdles of current prognostic tools in colo-rectal liver metastasis are discussed. Alongside more classic histopathological parameters, which capture features related to the tumor component, such as tumor invasion, tumor growth pattern and regression score, we will discuss immune mediators, which are starting to be considered important features. Their objective quantification has shown significant results in prognostication studies, with most of the work focused on adaptive immune cells, namely T cells. As for macrophages, they are only starting to be appreciated and we will present recent advances in evaluation of macrophage morphological features. Deeper knowledge acquired by multiparametric analyses is rapidly uncovering the variety of immune players that should be assessed. The future projection is to implement deep-learning histopathological tools and to integrate histopathological and immune metrics in multiparametric scores, with the ultimate objective to achieve a deeper resolution of the tumor features and their relevance for colo-rectal liver metastasis. Based on the last update of the World Health Organization (WHO), colorectal cancer (CRC) represents the third most common cancer worldwide as well as the second cause of cancer mortality . The livIn recent years the prognostication of CLM has changed, introducing new classifiers that could perform better in terms of patient stratification. Much inspiration has been taken from the parameters used in the primary CRC setting, however, while for primary CRC pathologists routinely report these parameters, a narrower description of tumor features is provided for CLM. Here we will review current histopathological parameters as well as immune mediators that are emerging as novel classifiers in CLM. Many of the studies done in this clinical setting reflect the progress made in the prognostication of colo-rectal cancer, in which the consensus immunoscore, i.e., the quantitative assessment of number of T and cytotoxic T cells in tumor regions, has proven a strong prognostic relevance ,4,5. FurThe aim of this review is to present the available data that are propelling the introduction of new factors in human CLM prognostication. Among the variety of biomarkers currently evaluated, we will focus our attention on those parameters that can be assessed on histological sections  and that have shown prognostic impact in available studies. This choice is explained by the possibility to integrate histopathological parameters, primarily related to tumor features, with immune parameters, which consider the number, type and localization of immune cells in cancer tissues . ProvideRAS and BRAF mutations, as well as histopathological variables, particularly the metastatic tumor invasion, the tumor histopathological growth pattern (HGP), the tumor regression grading (TRG) and the chemotherapy-associated liver injury (CALI). Discussion is still lively on which histopathological characteristic should be evaluated and included in the pathological report.Over the years, a fruitful integration among surgeons and pathologists has contributed to identify some critical parameters that can be reproducibly and quantitatively assessed in resected metastasis specimens and that have shown significant prognostic relevance ,8,9. TheThe spreading of tumor cells occurs via different routes, including via vascular and lymphatic vessels, bile ducts and along the nerves. The presence of cancer cells within or in close proximity to these areas can therefore be considered a potential prognostic histopathological feature. Colorectal cancer cells can potentially spread within or outside the organ using the same pathways and originate liver metastases ,10.Intrahepatic vascular invasion refers to the occurrence of tumor invasion in either hepatic or portal veins. It is common  to evaluate the potential of invasion of the portal vein or hepatic vein as two distinct elements. To date, the clinical relevance of intrahepatic vascular invasion in CLM patients is still controversial. Some studies have reported hepatic and portal vein invasion to have poor prognostic significance, either as independent histopathological feature or in combination with others, such as lymphatic invasion ,12,13,14A similar argument can be formulated with lymphatic invasion, i.e., the presence of neoplastic cells within the luminal structure lined by endothelial cells in the portal area. The correlation between this model of tumor spreading and prognosis, initially studied in intrahepatic cholangiocarcinoma, was first investigated in CLM patients by Sasaki et al., using standard H&E . In thisThe invasion of biliary ducts is another variable that has been considered as a possible means of stratifying patients according to their outcomes. When this parameter is investigated by immunohistochemistry, biliary duct epithelium is highlighted by targeting cytokeratin-7. Moreover, the visualization of tumor cells from the colon mucosa (positive for CDX2 and CK20) may help to differentiate between metastatic colon cancer, showing intrabiliary growth, and other cancers, such as primitive biliary neoplasias  or hepatocellular carcinoma  with bile duct invasion ,18,19. BPerineural invasion is an active phenomenon though which tumor cells migrate along axons, supported by the favorable microenvironment orchestrated by neoplastic cells, nerves and the immune system . This feOverall, histopathological features of CLM represent important characteristics with reasonable impact on the course of disease, however, to date, there is a remarkable lack of consensus on their prognostic value and further analyses are needed to clarify this matter. Recently, de Oliveira et al. performed a systematic review of clinical observational studies and a meta-analysis aimed at identifying the impact of histopathological factors on the prognosis of resected CLM patients . The anaColo-rectal metastases show different manners to growth in the liver and to interact with the organ, as appears from the morphological variations that characterize the interface between the tumor and the surrounding normal tissue. The identification of distinct histopathological growth patterns (HGPs) has given the possibility to test this parameter as possible prognostic factor, revealing promising results in several studies.Two systems are primarily used to classify the HGPs. To date, the most accepted system, according to the international consensus guidelines , identifThe existence of multiple metastatic growth patterns reflects different interactions that the tumor can establish with the host, and vice versa. This concept partially explains the distinct outcomes observed in CLM patients according to their metastatic HGPs. Thus, the desmoplastic and pushing tumor borders are characterized by a large number of immature blood vessels and proliferating endothelial cells, reflecting the intense angiogenesis occurring in the metastases for the construction of its supporting stroma. Moreover, elevated lymphocyte infiltration in the tumor–liver interface is observed in this type of cancers. On the contrary, tumors with replacement growth pattern co-opt the blood vessels and the connective tissue of the surrounding healthy liver for their expansion and have a general non-inflamed status .Partial hepatectomy to resect liver metastases represents a curative therapeutic approach in 20% of patients with CLM , but unfMacroscopical evaluation of tumor response to neoadjuvant treatments before surgery, using computed tomography or magnetic resonance, is thus crucial for the optimization of CLM management; however, microscopical evaluation of resected metastases after partial hepatectomy is also essential. Indeed, pathological response has been shown to be predictive of favorable prognosis ,36. DiffIncrease of T cell infiltration in resected CLM previously treated with chemotherapy has been reported ,41. A rePreoperative chemotherapy plays a critical role in the improvement of the management of CLM patients. Nonetheless, it presents some shortcomings, the most important being the development of chemotherapy-associated liver injuries (CALI), which include sinusoidal obstruction syndrome (SOS), nodular regenerative hyperplasia (NRH) and steatohepatitis. SOS can be considered a consequence of endothelial damage related to preoperative treatment. NRH would be the advanced stage, whereby nodular modification of the liver parenchyma is recorded ,44. In aThe density and types of immune cells infiltrating cancer tissues, commonly referred to as the immune contexture ,45, has The integration of quantitative information related to the density, type and localization of T cells in colo-rectal cancer has been the strength of the Immunoscore. The universe of adaptive immune cells, namely T cells, and their activation states has been compressed into three markers, which, quantitatively evaluated, hold a strong prognostic value in stage I-III CRC patients. Fewer studies are available on the metastatic setting, namely CLM, although much inspiration is taken from the evidence collected for primary CRC. One important question is whether primary CRC lesions and their hepatic metastases display comparable immune landscapes. If this was the case, one could capture relevant information on the immune environment of the metastatic nodule by analyzing the primary tumor . AnotherThe attention reserved to macrophages as critical orchestrators of pro-tumor circuits in liver diseases is being translated into efforts aimed at testing these phagocytes as prognostic factors in colo-rectal liver metastasis ,56. MacrAlmost every tumor tissue is populated by macrophages, which can be either the result of local differentiation and maturation processes from circulating monocytic precursors, or they can represent the fraction of macrophages resident in the tissue. This ontogeny-related difference has important implications in terms of prognostic studies. For instance, except for a few situations in which a specific marker is available allowing a clear discrimination of recruited versus resident macrophages , question is whether it is correct to evaluate both the components. Tissue-resident macrophages of the liver, commonly referred to as Kupffer cells, are traditionally considered macrophages with peculiar tissue tasks , thus exPaying more attention to localization in the tissue context and the interaction with various types of endothelial cells could be the way forward. Macrophages occupy very specific regions in the liver as well as in tumor tissues, such as the sinusoidal space, the peritumor region, the tumor core, or the invasive margin . KupfferThe dramatic advancement of image-based profiling is revolutionizing our approach to prognostication. High-dimensional cell profiling allows capturing multiple features of a cell population, or even of single cells, and finds applicability in high-throughput screenings, drug discovery systems, immuno-monitoring studies . SeveralWith the development of new surgical techniques and more effective therapeutic protocols, eligibility of CLM patients for surgical resection has progressively increased over years. While drastically lowering morbidity and mortality rates, this improvement in the management of CLM patients imposes identification of prognostic classifiers, to help clinical oncologists as well surgeons in selecting the best therapeutic options and follow patients’ response. Recent successful studies describing the prognostic and predictive value of immune mediators in primary colo-rectal cancer have fueled the interest in the development of immune-based tools to aid in the critical prognostication process in colo-rectal liver metastasis. Further efforts are needed to understand how much of the complexity of immune cells in the spotlight can be exploited to identify nonredundant markers of disease progression. The value of immune mediators is being evaluated alongside more common histopathological parameters, whose impact on survival of CLM patients is still controversial. Provided that quantifiable parameters are generated, these could be integrated into prognostic scores, potentially complementing the ones available.


Overall, the current evidence shows that exposure to green space may potentially increase prosocial behaviour among children and adolescents, with some contingencies . However, the volume and quality of this evidence is not yet sufficient to draw conclusions on causality. Further, heterogeneity in the indicators of green space exposure could lead to mixed findings. In addition, none of the included studies investigated potential mediators. Nevertheless, this review provides preliminary evidence and a basis for further investigation with rigorous study methodology capable of drawing causal inferences and testing potential effect modifiers, linking pathways, and relevant green space measures.The plausible role of nearby green space in influencing prosocial behaviour among children and adolescents has been studied recently. However, no review has been conducted of the evidence testing the association between green space and prosocial behaviour. This systematic review addresses this gap among children and adolescents. Within this review, we propose a conceptual framework describing potential pathways linking green space to prosocial behaviour, discuss the direction, magnitude, moderators, and mediators of the association, and develop a narrative synthesis of future study directions. Out of 63 extracted associations from 15 studies, 44 were in the positive or expected direction, of which 18 were reported to be statistically significant ( Prosocial behaviour is increasingly recognised as an important part of child development  and adolescents (13–18 years). These age ranges were selected based on a previous systematic review on prosocial behaviour among adolescents  harm mitigation , (ii) restoring capacities , and (iii) building capacities  and PM2.5 (particulate matter < 2.5 μm in diameter) during the full gestation period were reported to be associated with increased odds of abnormal range of prosocial behaviour after controlling for child-related factors, maternal factors, and socio-economic status. Meanwhile, past work suggested that air-related pollution can be reduced by the presence of green space , natural environments are best suited for humans as places where we initially evolved and humankind's survival was reliant on nature before the agricultural revolution. Emotional responses upon natural environments are viewed as part of feeling connected to nature and as being “central to the psychological components of stress and restoration”  were peer-reviewed research articles, (2) had quantitative observational or experimental design; (3) investigated association between green space as an exposure that includes objective and/or subjective measures  and prosocial behaviour as either an outcome or as a mediator of a health outcome; (4) were published in English; and (5) included participants ≤ 18 years of age. No restriction on publication date was applied. Published articles that only contained an abstract  were excluded.Prosocial behaviour among children and adolescents was the outcome of interest. In this review, prosociality was defined as a range of positive behaviours that include offering help, sharing, cooperating, and comforting. The outcome focuses on the behavioural aspect rather than cognitive or affective responses . Meanwhile, green space refers to naturally-created areas or built environments that bear natural vegetation. Green space exposure in this review considered all characteristics of green space in accordance with the keywords provided  independently assessed the title and abstract of the published articles using the same inclusion criteria, followed by the full-text assessment. Further, any discrepancies between the two reviewers were discussed and consulted with a third reviewer (TA). Information about publication details, study design, sample size, participant characteristics, exposure concept and measurement, measure instrument of prosocial behaviour, and the results were extracted into Quality and risk of bias of the articles were assessed using the quality assessment tools developed by the National Institutes of Health  for obseSample size and age of participants differed by included study. Small sample sizes (< 100) were reported in two experimental studies  improving the appearance of frequently accessed green space by children and adolescents  and (2) spending time in green space or participating in activities involving contacts with natural vegetation . Improvements in the quality of schoolyards by increasing the presence of natural components and other facilities was evaluated in studies in the US . However, measurements based on teacher-reports , combined parent- and teacher-report , and self-report  were also observed. In addition, prosociality was assessed through in-person observations in four experimental studies (27%). The Strengths and Difficulties Questionnaire  and does not take into account the quality of green space including abandoned or unsafe areas  and did not account for time-variant measures of green space and prosocial behaviour. This prevents the examination of possible variations in prosocial behaviour as a response to changes in green space exposure over time. According to the conceptual framework, the accumulation of exposure to green space might elevate the benefits for prosocial behaviour development and greater impact may be observed during the late childhood as the sensitive period. Therefore, testing this hypothesis in longitudinal studies will provide new insights that will be beneficial for policy recommendations. In addition, mediation analyses are needed to test mechanistic pathways that may underlie the documented associations between green space and prosocial behaviour.To our knowledge, this is the first systematic review evaluating the relationship between green space and prosocial behaviour. The findings are presented and discussed by different measures of green space exposure with additional explanations on potential moderators. The use of nine databases with keywords adopted from current published systematic reviews, no restriction on publication date, and screening of references of included studies allowed a comprehensive search. The process of developing and reporting this review following the PRISMA guidelines lends credibility to the findings.There are some limitations of the evidence reviewed and review method. Firstly, there was only a limited number of longitudinal studies which preclude drawing causal inferences. The findings from experimental studies without control groups are also prone to low internal validity. Secondly, area-level measures of green space varied by study and resulted in mixed-findings, making it difficult to define absolute amount of green space needed in the neighbourhood for positive development of prosocial behaviour. Thirdly, all studies were from high-income countries. Thus, findings can be applicable to these countries, including high-income countries with hot climates and rapidly growing populations where the presence of green space is substantial for mitigating harmful environmental stressors  and bridging people to the community . However, findings may not be widely applicable to middle- and low-income countries. A limitation of the review method is that some articles that were not published in English may not have been retrieved.This review provides preliminary evidence of positive associations between green space exposure and prosociality. However, experimental studies are just as limited as observational studies, the exposure to green space can be randomly assigned, but individual compliance in reality is agentic. Therefore, it might lead to the question of what aspects or characteristics of green space might further influence the use of green space. It is conceivable that individuals might not use green space if it is not well-maintained, physically attractive, or generally of poor quality. Therefore, the quality of green space might be an important aspect that should be considered in understanding the potential benefits of green space on human health.Green space quality has been associated with health outcomes independently of the green space quantity  are required to edge closer to causal inferences and evidence-based policy recommendations. Based on a conceptual model described above, using a longitudinal approach may also help to understand to what extent the accumulation of green space exposure affects the levels of prosocial behaviour in different stages of development, particularly during critical and sensitive periods of the green space-prosociality association. Assessment of potential mediators could help to test plausible pathways linking green space with prosocial behaviour. Moreover, measuring green space exposure as perceived quality is needed due to a sensitive measurement in relation to child health and behaviour outcomes. Lastly, given reported effect modifiers from previous studies, analysis of green space and prosocial behaviour should be tested across strata of other variables .The current evidence shows that exposure to higher levels of green space may be associated with greater prosocial behaviour. Different measurements of green space exposure led to mixed findings. Area-level green space measures were less consistent in demonstrating statistically significant associations between green space and prosocial behaviour, whereas associations were more consistent when green space was measured using in-person observation. The number of studies was too few to draw conclusions on subjective green space measurements. Further investigation on the association between green space and prosociality is warranted, especially with studies employing longitudinal designs to confirm temporality and sensitive period, as well as, capable of testing potential effect modifiers, mediators, and measures of green space quality.IP, TA-B, and XF conceptualised the review. IP conducted the systematic search, study quality assessment, summarised the findings, wrote, and revised the manuscript. EJ peer-reviewed the systematic search, performed full-paper assessment of the eligible articles, and reviewed the manuscript draft. TA-B, DC, SV, and XF provided critical inputs throughout the process and edited the manuscript. All authors approved the final version 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.