This Special Issue enqueues a series of publications dealing with arteriogenesis, which is the growth of a natural bypass from pre-existing arteriolar connections, as defined by Wolfgang Schaper, Werner Risau and Ramon Munoz-Chapuli in the late nineties of the last century. In times of increasing numbers of patients with cardiovascular occlusive diseases not only in highly industrialized but in almost all countries of the world, it is of major importance to understand the molecular mechanisms of this tissue- and life-saving process, which was given to us by mother nature to compensate for the function of a stenosed coronary or peripheral artery non-invasively. Since our first investigations on collateral artery growth more than 20 years ago, a lot of progress has been made, which we try to give access to in this issue on arteriogenesis. International Journal of Molecular Sciences, Zeen Aref and Margreet de Vries, from the group around Paul Quax, give an overview of the currently available variations of murine hind limb models for the study of angiogenesis and arteriogenesis, and highlight their advantages and disadvantages [In the current special issue, entitled “Arteriogenesis – Molecular Regulation, Pathophysiology and Therapeutics II“ in the vantages . Huan Zhvantages .The trigger for arteriogenesis is increased fluid shear stress, which is exerted on the endothelial cells of pre-existing collateral arteries by blood flow being redirected around stenosed vessels. For a long time, it was completely unknown how this increased mechanical stress results in local leukocyte recruitment promoting collateral artery growth. Co-workers of the group of Elisabeth Deindl describe the functional role of extracellular RNA in that process and highlight the role of this nucleic acid during ongoing arteriogenesis . MoreoveArterial occlusion results in reduced perfusion, and hence ischemia, in distally located tissue. As a consequence of the thereto related hypoxia, angiogenesis is induced. In contrast to other ischemic conditions, where capillaries are required to supply tissue locally with oxygen and nutrients, capillaries in regions distal to occluded arteries are necessary for the removal of cell debris. Yvonn Heun and the group around Hanna Mannel show that the tyrosine phosphatase SHP-2 inhibits 26S proteasome and thereby activates hypoxia-induced HIF-1α (hypoxia inducible factor 1α). This mechanism, which was identified in wounds, seems to be generally applicable, and results in pro-angiogenic gene expression under hypoxic conditions in ischemic tissue [The process of collateral artery growth is often severely compromised in patients with diabetes mellitus. Srinivasan Vedantham and Elisabeth Deindl investigated the role of insulin for arteriogenesis in diabetic mice. They were able to attribute to this peptide hormone a function in the expression of Egr-1 (early growth response-1), a transcription factor relevant for collateral outward remodeling . CathrinThomas-Schmitz-Rixen, Kerstin Troidl and co-workers have developed an exercise training protocol in mice allowing the investigation of the effect of this physical force on collateral artery growth in mice , and, toLast but not least, the interventional cardiologists and specialists in the field of arteriogenesis, Bigler Marius Reto and Christian Seiler, provide an overview of human coronary collateral circulation and introduce the different clinical therapeutic approaches for the promotion of arteriogenesis in patients. They explain the problematic nature of biochemical concepts involving, for example, G-CSF (granulocyte-colony stimulating factor) and GM-CSF (granulocyte-macrophage colony stimulating factor), review the feasibility of biophysical concepts such as physical exercise and ECP , and focus on the promising approach of permanent occlusion of the internal mammary arteries in promoting natural bypass growth in patients . With all our investigations and efforts, we think that we have come much closer to the goal of all of us to understand the mechanisms of collateral artery growth, finally enabling clinicians to promote arteriogenesis effectively in patients with vascular occlusive diseases.


During the current corona pandemic, new therapeutic options against this viral disease are urgently desired. Due to the rapid spread and immense number of affected individuals worldwide, cost-effective, globally available, and safe options with minimal side effects and simple application are extremely warranted. This review will therefore discuss the potential of zinc as preventive and therapeutic agent alone or in combination with other strategies, as zinc meets all the above described criteria. While a variety of data on the association of the individual zinc status with viral and respiratory tract infections are available, study evidence regarding COVID-19 is so far missing but can be assumed as was indicated by others and is detailed in this perspective, focusing on re-balancing of the immune response by zinc supplementation. Especially, the role of zinc in viral-induced vascular complications has barely been discussed, so far. Interestingly, most of the risk groups described for COVID-19 are at the same time groups that were associated with zinc deficiency. As zinc is essential to preserve natural tissue barriers such as the respiratory epithelium, preventing pathogen entry, for a balanced function of the immune system and the redox system, zinc deficiency can probably be added to the factors predisposing individuals to infection and detrimental progression of COVID-19. Finally, due to its direct antiviral properties, it can be assumed that zinc administration is beneficial for most of the population, especially those with suboptimal zinc status. The importance of the trace element zinc for the development and function of the immune system across all kinds of species has been proven in numerous studies –3. As ziThe most common symptoms of COVID-19 are impaired smell and taste, fever, cough, sore throat, general weakness, pain as aching limbs, runny nose, and in some cases diarrhea . In the The entry of infectious agents into the human body is prevented by tissue barriers equipped with cilia and mucus, anti-microbial peptides like lysozymes and interferons. Regarding SARS-CoV2, the angiotensin-converting enzyme 2 (ACE2) and the cellular protease TMPRSS2 are the major mechanism for entering the cells .Mucociliar clearance of viruses is affected by zincInfections with coronaviruses go along with damage of the ciliated epithelium and ciliary dyskinesia consecutively impairing the mucociliar clearance . It was Zinc is essential for preserving tissue barriersex-vivo model of the chronic obstructive pulmonary disease (COPD) showed that decreasing zinc levels raised the leakage of the epithelium of the respiratory tract -4 can potentially recognize outer components of SARS-CoV2's like the viral spikes balancing power regarding immune cell numbers and functions might be highly beneficial in regard to therapy of COVID-19.Our suggested benefits of zinc supplementation to prevent and treat COVID-19 are supported by a row of successful supplementation studies focusing on respiratory tract infection, of which we listed some selected publications in As illustrated in Several studies indicate that there is an association between chemosensory dysfunctions and COVID-19 –133. Smestreptococcus pneumoniae (About 50% of patients that died of COVID-19 had bacterial or fungal co-infections , underlieumoniae . As menteumoniae . During eumoniae . In combeumoniae , underliVascular complications resulting from atherosclerosis development, microangiopathic organ failure, and venous thromboembolism were found as a major cause of death in COVID-19 patients –149, sugIn this perspective, we reviewed the most important literature on the role of zinc homeostasis during viral infections, focusing on the potential benefits of zinc supplementation to prevent and treat SARS-CoV2 infections. Although data specifically on SARS-CoV2 are unfortunately still pending and randomized controlled studies have not been conducted, the enumerated evidence from the literature strongly suggests great benefits of zinc supplementation. Zinc supplementation improves the mucociliary clearance, strengthens the integrity of the epithelium, decreases viral replication, preserves antiviral immunity, attenuates the risk of hyper-inflammation, supports anti-oxidative effects and thus reduces lung damage and minimized secondary infections. Especially older subjects, patients with chronic diseases and most of the remaining COVID-19 risk groups would most likely benefit. Although studies are needed testing the effect of zinc as therapeutic option for established disease, preventive supplementation of subjects from risk groups should begin now, as zinc is a cost-efficient, globally available and simple to use option with little to no side effects. The first clinical trials on zinc supplementation alone and in combination with other drugs such as chloroquine have been registered , 160–162The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.LR is a member of ZINC Net.IW, BR, and LR drafted and corrected the text, table, and figure. 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.


The diffusion and adoption process of a learning management system (LMS) at higher education institutes faces several obstacles; some of which are unique to contexts while others are shared experiences. This diffusion case study compares the adoption process of the LMS Blackboard at two universities, Texas A&M University and Monash University in Australia, investigating the factors which impacted the adoption of the innovation at each context as well as the extent to which social capital influenced the diffusion process. The study specifically examined the different adopters involved, the objections raised, the barriers encountered, and the significant factors either resulting in the success or the failure of the innovation, employing a social capital-infused theoretical framework of diffusion within organizations outlined by Frank, Zhao, and Borman . Primary and secondary data were analyzed and examined from three peer-reviewed, empirical articles for comparison within the study.Findings highlighted strong alignment with Rogers’  diffusion of innovations theory as well as the importance of social capital maintained by Frank and colleagues . Though describing different adoption processes and factors, each context supported the universal idea behind diffusion theory that members of a social system communicate an innovation through social channels over time and that innovators and early adopters play a vital role in this process . As higher education institutes advance further into the twenty-first century and adopt more innovations within their learning frameworks and systems, this diffusion case study stresses the importance of understanding diffusion theory, having an in-depth knowledge of the stakeholders involved in the adoption process, and creating and implementing a meticulous diffusion plan to ensure a successful diffusion and adoption process. This study investigates the diffusion of learning management systems (LMSs) in educational institutions through the review, analysis, documentation, and evaluation of the innovation adoption process in the select contexts. Four significant factors impact diffusion and the adoption of an innovation, which include the actual change, the innovation’s communication process, the period for the innovation process, and the social system of the innovation’s integration Rogers, . The focRQ1: What factors impacted the adoption of LMSs in the studied contexts?RQ2: To what extent did social capital influence the adoption of LMSs in the studied contexts?LMSs are defined as software applications that support the administration, documentation, internal review and tracking, reporting, and delivery of educational and training course-related materials, allowing communication between students and faculty in virtual spaces Sanga, . StandarThese platforms changed the manner in which higher education institutions, as well as businesses, provided educational opportunities to their students and employees, offering easily accessible online options of learning interaction to substitute for face-to-face components. While LMSs have been widely adopted throughout different educational institutions due to their accessibility, cost-effectiveness, and quality, they have encountered challenges and failure, and therefore, the adoption process of LMSs requires further investigation , the research identified two candidates with related case studies on which to base the investigation: Texas A&M University and Monash University.n = 3949), staff (n = 5997), and students  during the spring 2011 semester and its potential replacements Moodle, Sakai, and Blackboard Learn. The primary data source is a peer-reviewed, empirical study by Walker and Lindner  as well as inductive coding related to grounded theory to identify emerging categories and subcategories to add to the code system , (b) compatibility with existing values (n = 7), (c) ease of use (n = 3), (d) trialability (n = 4), and (e) observability (n = 4). Additional key reasons affecting the motivation to adopt included top-down authority innovation directions, demand by students at the university, economic and political necessity, communication opportunities, and supportive social systems (Samarawickrema & Stacey, Similar to Benson and Palaska , SamarawAt Texas A&M, the Blackboard Vista LMS was first employed in 1998 as a top-down, authority-innovation decision associated with a teaching with technology initiative implemented to improve the teaching and learning process for faculty and students (Walker & Lindner, Similarly, in the Monash University context, Benson and Palaskas  describeWalker and Lindner  did not Whereas sources on the Texas A&M context remained somewhat vague on adopter categories, Benson and Palaskas  detailedThe success of the adoption process at Monash University was reflected in 2017 when Monash College, a pre-university feeder institution for the university, was awarded the 2017 Blackboard Catalyst Award, highlighting an institution which fully integrated the LMS delivery model as well as supported and enhanced faculty and staff with high-quality professional development (Bertsche, From the Texas A&M University context, the long-term engagement with the Blackboard LMS, particularly the ease of use analysis, described a high level of self-efficacy amongst faculty and student users and therefore a potential easy ability to adopt a new LMS, such as Moodle, Sakai, or Blackboard Learn (Walker & Lindner, Walker and Lindner  also desSeveral studies have described the adoption process of various LMSs within different contexts Arpaci, , identifAntecedents to the adoption of new information technology system practices, such as LMSs, include, but are not limited to, perceived advantages and user characteristics Arpaci, , social Each context also highlighted the importance of social pressure, technical compatibility, and management as having significantly more influence on the adoption by early adopters than late adopters (Benson & Palaskas, In a quantitative study focused on the critical success factors for web-based learning management systems, Lwoga  maintainRucker and Downey  pinpointIt is critical to understand the factors that influence the adoption and usage of LMSs. Through this understanding of the adoption process, administrators and LMS providers can offer more beneficial design and implementation aspects as well as more ongoing professional development support for instructors and learners (Rhode et al., The two contexts examined in this diffusion study showcased a substantial alignment with ideas related to Rogers’  diffusioDespite the different social and cultural backgrounds, the contexts and related resources point toward the universality of diffusion and the importance of understanding the adoption of innovations within higher education to improve the process for all stakeholders significantly.It is critical to engage key champions, opinion leaders, and early adopters in the adoption process to ensure a smooth and efficient diffusion procedure throughout higher education institutes while using various strategies to mitigate the potential pitfalls. Through a meticulously executed diffusion plan, a university can ensure a successful innovation adoption process and the future goals of the institution toward development and improvement.The case studies examined support a social capital infused theoretical model of diffusion within higher education institutions while also highlighting other key adoption factors to consider when facilitating an effective adoption process. From the standpoint of contribution to the field, this comparative study has several important implications. First, although the study by Frank et al.  pinpoint


There are many risk factors associated with breast cancer (BC) such as the familial history of BC, using hormone replacement therapy, obesity, personal habits, and other clinical factors; however, not all BC cases are attributed to these risk factors. Recent researches show a correlation between patient microbiome and BC suggested as a new risk factor. The present review article aimed at evaluating the role of the microbiome as a risk factor in the occurrence of BC, investigating the proposed mechanisms of interaction between the microbiome and human genes involved in BC, and assessing the impact of the altered composition of breast, gut, and milk microbiome in the physiological status of normal breast as well as cancerous or non-cancerous breast lesions. The study also evaluated the growing evidence that these altered populations may hinder chemotherapeutic treatment. The role of microbiome in the development and maintenance of inflammation, estrogen metabolism, and epigenetic alterations was properly investigated. Finally, clinical and therapeutic applications of the microbiome- e.g., probiotics, microbiome genome modulation, and engineered microbiome enzymes in the management of BC were reviewed. Breast cancer (BC) is the most common cancer among women worldwide. It includes Luminal A, Luminal B, Her2-enriched, and triple-negative subtypes based on the expression of estrogen, progesterone and Her2 receptors, and Ki67 protein . GeneticBased on recent studies, the microbiome is a risk factor for BC and an explanation for different responses to therapy . The queAlthough it is not proved yet that dysbiosis can cause BC, the comparison of breast tissue samples show differences in the composition and abundance of some specific bacterial taxa between patients and healthy individuals . TherefoThere are fewer studies on microbiome and BC than other cancers. Particular composition of gut, breast tissue, and milk microbiome and the critical evidence of interaction with the inflammatory system, estrogen metabolism, and genetic and epigenetic alterations were also discussed in the current review. Moreover, the effects and mechanisms of microbiome on different therapeutic approaches such as hormone, chemo-, radio-, and immuno-therapy of BC were scrutinized. Although there are some reviews on microbiome and breast cancer –18, littIt was believed that breast tissue is sterile, but now it is known that breast tissue has its specific microbiome, which is different from those of other tissue such as gut . Since bNext-generation sequencing services paved the way for finding the microbiome composition of the breast . The breBacillus and Staphylococcus spp. in patients with BC. Interestingly, Escherichia coli (Enterobacteriaceae family) and Staphylococcus epidermidis induce double-stranded DNA break in BC cells  was dominant in the normal adjacent tissue samples , bactericidal/permeability-increasing protein (BPI), and myeloperoxidase (MPO) were expressed minor in the tumor in comparison with healthy tissue.Quantification of bacterial DNA in BC showed that  samples . AdditioThe difference in breast tissue microbiome profile between healthy individuals and patients with BC was also confirmed in another study . This diThis evidence suggests that bacteria may maintain the healthy status of breast tissue by stimulating host inflammatory responses. Reduction of bacterial load in a healthy individual may exacerbate the risk of BC. These findings demonstrated an unknown link between dysbiosis and BC, and the potential diagnostic and therapeutic implications of these discoveries should be investigated in further investigations. Although many studies were performed on the body-wide impacts of microbiota, there is still no clear pattern about the direct effect of these microorganisms on the risk of BC.Findings of the bacterial biodiversity in human breast milk and its changes over time are limited. However, culture-independent molecular techniques such as qPCR and NGS approaches allow valuable complementary assessments of the human milk microbiota.Firmicutes, Actinobacteria, and Bacteroidetes , Firmicutes (34%), and the genera of Pseudomonas (61.1%), Aureus (33.4%), and Streptococcus (0.5%). Milk has less biodiversity in comparison with infants' and mothers' stools at the level of phylum. Researches show that enriched immune-modulatory DNA motifs of Lactobacillus contribute to immune development by modulating the immune responses  in digestive system. Shifting to a plant-based diet would increase Firmicutes population  produced by Streptococcus mitis, and extracellular death factor (EDF) produced by E. coli, cause in vitro angiogenesis and BC cells invasion  for 2–4 weeks, three times a day. Another trial (NCT03760653) determined the effect of probiotics supplementation  and physical exercise on the bacterial balance of gut, immune system, and the quality of life in BC survivors.In the (NCT03358511) clinical trial, the role of probiotics on the number of CD8in vitro and in vivo studies, using them as a promised treatment in the clinical practice is not established yet. Although not confirmed, it seems that the tissue-specific microbiome cannot be changed even with long-term applications of probiotics; hence, the aforementioned positive effects might be obtained by modulating the gut microbiome and subsequently preventing tumorigenesis in the breast.Although using probiotics inhibited tumor growth, induced apoptosis, and also enhanced the immune system in S. typhimurium flagellin in BC cells mediates the pro-inflammatory responses to obtain an effective anti-tumor activity, which may serve as a novel therapeutic target for BC therapy , and not affect the toxicity of the rest 15 drugs. Thus, the response to therapy in BC tumors may be improved by microbiome modulation  recruited 80 participants to explore more information about the intestinal microbiome of patients with BC undergoing chemotherapy. These studies can prove that gut microbiome analysis holds the potential to predict patient response to chemotherapy before treatment and personalize medicine. However, further studies are required to understand the biochemical interactions between therapeutic agents and bacteria. In addition, new combinations of chemotherapy with probiotic treatments should be taken into consideration.Microbiome engineering may open new horizons in prevention, diagnosis, and treatment of cancer. As mentioned above, alterations in gut bacterial populations may increase the risk of cancer. Therefore, designing antibiotics that target a particular spectrum of the microbiome might help regulate the gastrointestinal microbiome as a possible way to reduce the BC risk . Since cConsequently, engineered probiotics might be useful in targeting these signaling pathways. The combination of microbiome engineering and recombinant DNA technology can be utilized to modify the genome of vital microflora compartments and reprogram microbial mechanisms . The futMicrobiome diversity across different people caused by pathological, physiological, and environmental differences is a crucial challenge when trying to define beneficial or pathological microbial signatures. The complex diversity of these microorganisms makes it challenging to identify specific cancer signatures that are stable over time.The microbiota plays a crucial role in preserving the health status of the human body, and their impairment causes pathobiological changes, including BC. Although the evidence of the correlation of microbiome with BC is undeniable, there are essential questions to unlock the exact role of the microbiome in the development and treatment of BC. Strains, dosage, and regimen of probiotics based on the clinical feature of BC and probiotics interaction with the conventional treatment are not determined entirely yet. Further studies are needed to find the exact relationship between microbiome and cancer. In other words, it needs to be clarified whether microbiome alteration leads to cancer or cancer occurrence leads to microbiome alteration, whether dysbiosis is carcinogenic or if there is a way to regulate dysbiosis. To answer these questions, large-scale studies including animal models, retrospective and prospective ones, as well as clinical trials should be designed. Engineered bacteria such as probiotic products might be new modalities to develop a therapeutic approach on clinical scale.This manuscript was originally submitted to the Cancer Metabolism section of Frontiers in Oncology.RE and KM-A: conceptualization. RE, ZE-S, SH, and FB: drafting of the manuscript. RE, SH, and ZE-S: designing figures and tables. RE, SH, ZE-S, and KM-A: review and editing of the manuscript. All authors read and approved the final 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.


Despite continuous advancements in systematic treatment of chronic pain there is still a subset of clinical conditions where the standard medical and surgical approaches are not uniformly effective. Moreover, in addition to these relatively rare but remarkably treatment-refractory diagnoses , there are many other, much more prevalent, conditions , painful peripheral neuropathy, complex regional pain syndromes, etc.) where even a small percentage of treatment-refractory patients constitutes a large group of chronic pain sufferers.The treatment algorithms for these patients include gradual escalation of various modalities, usually chosen based on efficacy and safety, with treatment invasiveness being interpreted as direct measure of surgical risks and complications. The latter argument (on significant treatment-related risks) has been a deterrent for widespread acceptance of certain surgical interventions and a major source of reluctance for many pain-treating specialists and patients alike. This is mainly concerned the surgical neuromodulation of cerebral targets—the deep brain stimulation (DBS) and motor cortex stimulation (MCS). However, despite all obstacles , the worldwide experience with cerebral continues to grow.The article by the neurosurgical team from Oxford  providesMuch has been written on DBS for pain; the fascinating history behind this remarkable procedure is, in essence, a reflection of the entire modern functional neurosurgery: the innovation driven by need and purpose, careful observation and continuous advancement based on countless trials and errors, and gradual expansion of both targets and indications based on multiple enthusiastic centers of excellence.DBS for pain remains an underused modality. While widely used for movement disorders ,3 and unIn times of serious societal concern about opioid epidemics, there is now more than ever a need in critical re-evaluation of surgery for pain, particularly when it comes to non-destructive interventions with decades of clinical experience. As the matter of fact, we have recently suggested a revision of the World Health Organization ladder approach to the pain management in favor of considering surgery earlier in the treatment algorithm , and the


Cerebral stroke, which is one of the most frequent causes of mortality and leading cause of disability in developed countries, often leads to devastating and irreversible brain damage. Neurological and neuroradiological diagnosis of stroke, especially in its acute phase, is frequently uncertain or inconclusive. This results in difficulties in identification of patients with poor prognosis or being at high risk for complications. It also makes difficult identification of these stroke patients who could benefit from more aggressive therapies. In contrary to the cardiovascular disease, no single biomarker is available for the ischemic stroke, addressing the abovementioned issues. This justifies the need for identifying of effective diagnostic measures characterized by high specificity and sensitivity. One of the promising avenues in this area is studies on the panels of biomarkers characteristic for processes which occur in different types and phases of ischemic stroke and represent all morphological constituents of the brains’ neurovascular unit (NVU). In this review, we present the current state of knowledge concerning already-used or potentially applicable biomarkers of the ischemic stroke. We also discuss the perspectives for identification of biomarkers representative for different types and phases of the ischemic stroke, as well as for different constituents of NVU, which concentration levels correlate with extent of brain damage and patients’ neurological status. Finally, a critical analysis of perspectives on further improvement of the ischemic stroke diagnosis is presented. Despite constantly increasing understanding of ischemic stroke pathophysiology, resulting among others from laboratory and clinical studies, improvement of neuroradiological imaging technics, better understanding of the significance of risk factors and application to clinical practice the results of large population studies, diagnosis of acute stroke is frequently very difficult. An improvement of the organization of stroke centers and emergency transport has resulted in a significant reduction of door-to-needle time. However, despite the efforts made, this does not concern all countries and all stroke centers. In fact, a significant proportion of stroke patients still arrive to the hospital and are diagnosed late or even too late to start thrombolytic therapy . The pro2+ ion concentration, and oxidative stress, accompanied by free radical production . The difference in the dynamics of its expression in the early stages (between 2 and 6 h) of the course of both forms of stroke can, however, shed light on the etiology (hemorrhagic or ischemic) and thus, in combination with the data from the patient’s history and the results of the neurological examination and radiological tests (if available), can facilitate differentiating diagnosis. Despite the increasing availability of neuroimaging tests, there is still a fairly large group of patients, in whom these examinations were not performed in the early stages of stroke or clinical evaluation in pre-hospital settings was undertaken by a non-specialist. In such cases, an assessment of GFAP level and dynamics of its changes may be useful in diagnostic process and considering most appropriate therapy. The characteristic difference in dynamics of its concentration changes may provide some help in differentiating diagnosis in the absence of precise data on the time point of stroke onset. In addition, there are premises indicating GFAP value in determining outcome prognosis and neurological status of stroke patients. In summary, GFAP cannot be considered a characteristic marker of only one type of stroke and is also activated in the course of traumatic brain injury. However, different dynamics of its concentration changes in the blood serum, association with the size of tissue damage, and patients’ outcome indicate the diagnostic value. Determining possibilities of its clinical application requires further studies.The S100β protein, a representative of the S100-calmodulin-troponin cytoplasmic proteins family, is present in astrocytes, oligodendrocytes, and Schwann cells. The S100β protein reveals neuroprotective or neurotoxic function, depending on its concentration level , 41. In In summary, the unique significance of S100β protein as a potential biomarker of ischemic stroke results from the fact that it undergoes early upregulation in the course of brain tissue damage  and is a good predictor of dynamically occurring destructive processes. Depending on the extent of cerebral blood flow reduction, gradual transition of penumbra into the ischemic core can occur very dynamically, within the first 24 h from ischemia onset. In a significant proportion of cases, the ischemic “at risk zone” is not visible on CT imaging, even up to 24–48 h from its onset . In thesAQP4 is a member of the water-channel proteins family, expressed in the end-foot of astrocytes, which surround capillary vessels, ventricular walls or subarachnoid space . AQP4 reBadaut et al.  showed iThe abovementioned data are of important practical value for elaboration of future diagnostic protocols, based on the distribution of ischemic biomarkers and results of functional neuroradiological imaging studies. This approach, based on evaluation of biomarkers’ concentration and distribution, together with the results of neuroradiological studies, could also enable assessment of status of physiological brain barriers  in the course of ischemic stroke.Fatty acid–binding proteins (FABPs) represent a family of cytoplasmic proteins, responsible for maintaining an appropriate serum concentration of the long-chain fatty acids and expression of genes regulating cellular growth and differentiation , 146. TwThe advantage of using both FABPs as potential stroke biomarkers is related with early increase of their serum concentrations during stroke, which occurs already within 2 to 3 h after ischemia and remains elevated up to 120 h. The disadvantage is concerned with their relatively low specificity, whereas increase in H-FABP concentration has also been observed in the hemorrhagic stroke . The serReassuming, perspectives of diagnostic application of FABPs as ischemic stroke biomarkers are promising, although require further investigation. Their advantage is in principle concerned with rapid increase of serum concentration and multifunctional value, enabling indirect assessment of the volume of ischemic lesion, severity of neurological deficits, and differentiation among types of ischemic stroke.The results of numerous studies indicate changes in concentration of both excitatory and inhibitory amino acid neurotransmitters, such as glutamate (Glu) and serine (Ser) or γ-aminobutyric acid (GABA) and glycine (Gly), respectively, in the course of ischemic stroke. These changes may be a good indicator of the depth of neuronal and neuroglial dysfunction. They may also correlate with the severity of neurological deficits, of both focal character  and general nature , depending on localization of the lesioned area. However, dynamic changes in concentration of neurotransmitters, in the course of various types of ischemic stroke, are poorly understood. Further studies on these processes will provide important information concerning dysfunction of particular brain structures affected by ischemia. Another group of potential biomarkers, deriving from the NVU, is represented by enzymes responsible for synthetizing of neurotransmitters and metabolites significant for diagnosis of the ischemic stroke. Among these, one could encounter glutamine synthetase (GS), serine racemase (SR), and neuron-specific enolase (NSE). Their potential role as stroke biomarkers is presented in the following paragraphs.In ischemic stroke, an increase in Glu concentration in both blood plasma and CSF was observed. Due to the easy passage through the cerebral functional barriers, these changes were positively correlated, which implies diagnostic value of Glu concentration measurement in both physiological fluids , 55. A pThere is well-documented evidence showing characteristic difference in dynamics of Glu level changes between patients with stable and progressive ischemic stroke , 56. WhiIt has been shown that Gly concentration in blood plasma and CSF increases in stroke patients, correlating with progression of neurological deficits , 55. As Concentration of this inhibitory neurotransmitter also reveals characteristic changes in the course of different types of ischemic stroke. Considering the widespread distribution of GABA in cerebral structures, in particular, in areas related to motor function, such as frontal cortex or basal ganglia , it can be expected that dysregulation of GABA-ergic neurotransmission, resulting from ischemia, may be responsible for enhancing of disorders, such as paresis, muscular tone dysregulation, or involuntary movements . It can In summary, decrease in GABA concentration in the blood plasma could be a predictor of the worse outcome in patients with progressive stroke. Demonstration of the relationship between GABA and Glu concentration changes, localization of the ischemic area, and severity of functional disorders in the course of ischemia can provide interesting data supplementing diagnosis and prediction of complications in the course of ischemic stroke.d-Ser is converted from l-Ser in neurons and astrocytes [d-Ser relays on binding to Gly site and co-activating NMDA receptors, whereas overstimulation can result in glutamatergic excitotoxicity. After transient ischemia, d- and l-Ser blood concentrations transiently increase, reaching their maximal levels at 20 h, which suggests their role as biomarkers of the acute phase of ischemia [d-Ser binding to the NMDA receptor, which has been described in pathological processes related to reduced synthesis of Ser [This amino acid co-neurotransmitter and gliotransmitter is widely distributed in the CNS in two isoforms, of which trocytes , 161. Phischemia . Some aus of Ser . This reThis glutamine (Gln) synthetizing astrocytic enzyme is responsible for maintaining of Glu/Gln balance in physiological conditions . It is sl-Ser into d-Ser [Function of SR is concerned with isomerizing of to d-Ser . Recent to d-Ser . In mousto d-Ser . The exato d-Ser . HoweverNSE is an isoform of glycolytic enzyme, which expression changes in acute phase of ischemic stroke . In thisAnother group of potential ischemic stroke markers consists of pro- and anti-inflammatory mediators. Majority of these biomarkers is not characteristic for one cellular subpopulation but rather for all constituents of NVU , 169. ThIL-6 is a proinflammatory cytokine secreted by microglia or astrocytes, depending on the acute or subacute phase of ischemic stroke, respectively , 171. ThMMP-9 is an inducible enzyme, representing a large family of over 20 proteinases, involved in degradation of the extracellular matrix and secreted by different components of NVU. In the course of acute ischemic stroke, MMP-9 function is related to disintegration of the components of extracellular matrix and disruption of BBB, through degradation of the basal lamina, in particular laminin, type IV collagen, and fibronectin , 179. CoTNFα is expressed by macrophages and neurons in the infarcted area that enhances astrocytic and microglial inflammatory response in the course of ischemia-reperfusion injury . Its funvWF is a hemostatic agent secreted by the endothelial cells and megakaryocytes. It is involved in platelet adhesion, aggregation, and activation of complement system . The vWFThis anti-inflammatory cytokine contributes to reduction of the acute ischemic damage and infarct volume, through the M2 microglia phenotype stimulation , 110. ILIL-10 is a cytokine of anti-inflammatory and neuroprotective function, released by astrocytes and microglia. It inhibits the production and secretion of IL-1β , TGFβ 1, and TNFThe fourth group of potential stroke biomarkers is represented by the brain-derived neurotrophic factor (BDNF), glial cell line–derived neurotrophic factor (GDNF), and nerve growth factor (NGF). Neurotrophic factors could be considered an interesting supplement among the ischemic stroke diagnostic indicators, which results from their predictive value in assessment of neurological status and threatening complications. However, currently available results concerning dynamics of changes in expression of these markers in various types of stroke are fragmentary and frequently inconclusive.r = − 0.363; P < 0.001) in patients with acute ischemic stroke [BDNF is expressed by several constituents of NVU. Apart from neurons, it is also detected in astrocytes and microglial and ependymal cells, as well as in the endothelial cells of cerebral vessels, which reflects its important regulatory function , 205. Inc stroke . Recent c stroke , 207. Thc stroke . The resc stroke . The BDNc stroke . Clinicac stroke . The resc stroke . Some auc stroke .GDNF, being a representative of the transforming growth factor-beta family, is secreted by reactive astrocytes . The resNGF is expressed by astrocytes and pericytes after stimulation by inflammatory mediators and proinflammatory cytokines , 212. NeAlthough the results of experimental studies on numerous candidate stroke biomarkers and meta-analyses have been presented and discussed in many publications, so far, none of potential biomarkers have met clinical expectations , 217. AmAn ischemic stroke is a heterogeneous process which cannot be adequately signalized by activation of a single biomarker. Therefore, it is critically important to evaluate a broad range of molecules involved in pathophysiology of stroke, to identify and choose a set of potentially the best indicators of this process . Recent In this review, we have concentrated on a selection of substances, which are representative for the components of NVU and released from the brain into blood or CSF after stroke. According to this approach, the new diagnostic panels should contain the representatives of glial proteins , pro- and anti-inflammatory cytokines , factors representing hemostatic system (vWF), markers of NVU metabolism  and neurotransmitter synthesis , representatives of amino acid neurotransmitters , and, finally, neurotrophic and growth factors . Representation of different biochemical and functional categories by the abovementioned substances will enable monitoring of main pathophysiological processes, which occur during NVU damage. However, in several cases of the presented potential candidates for stroke biomarkers, available data related with their function are based only on experimental studies and not supported with sufficient clinical evaluation, in particular, concerning their role in various types of stroke. This justifies the need for further studies aimed at verification of their diagnostic utility.Apart from the abovementioned functional division, another one based on the time point of expression in relation to the onset of stroke symptoms may be of practical value . AssessmProposed in this review approach, based on the NVU concept, for selection of potential ischemic stroke biomarkers takes into account the complexity of brain tissue and functional relationships between its cellular components. For example, the ischemic lesion affecting perivascular axonal terminals, together with the astrocytic end-foots and functional BBB, can be manifested by a changed concentration of specific biomarkers  signaling the extent and dynamics of brain damage. Similarly, disintegration of structural and functional relationships between neurons and astrocytes results in decreased level of energetic metabolism, dysfunction of tripartite synapse and impaired gliotransmission, which is signalized by release of several biomarkers . Furthermore, the NVU involvement in inflammatory response initiated by ischemic process can be inferred from increased secretion of characteristic mediators  and their transfer into the vascular system. Thus, the NVU concept proves to be useful not only for selection of potential biomarkers, representing different components of this unit, but also for determination of those of them, which are representative for pathophysiological processes occurring at different phases of stroke. In our view, another possibility for application of the NVU concept for improvement of cerebral stroke diagnosis could be related with development of methods, which combine the results of morphological studies on distribution and concentration of selected markers  with the results of neuroradiological studies, assessing the depth of cerebral blood flow disturbances, evolution of brain edema and penumbra.Ischemic stroke is a dynamic process which consequences result from the coexistence of several phenomena, such as oxidative stress, immunological response, reactive gliosis, and dysfunction of hemostatic system. In spite of continuous development of new diagnostic methods, recognition of the cerebral ischemic infarct, especially in its acute phase, is often very difficult or even impossible. Despite activation of numerous markers, their diagnostic value is not satisfactory and requires further investigation, especially in different types of stroke. This justifies the approach relying on selection of several potential markers, characterized by acceptable sensitivity and specificity, associated with acute or later phases of stroke and representative for various pathophysiological processes. Taking into account still unsatisfactory results of studies on ischemic stroke biomarkers, it may be assumed that further efforts in this area will concentrate on searching for new components of diagnostic panels representing various elements of NVU. New complex diagnostic protocols will include analysis of biomarkers’ content, neuroradiological studies, and patient’s genetic profile, including the gene polymorphism studies associated with an increased risk of stroke and its complications.


Ex vivo cell/tissue-based models are an essential step in the workflow of pathophysiology studies, assay development, disease modeling, drug discovery, and development of personalized therapeutic strategies. For these purposes, both scientific and pharmaceutical research have adopted ex vivo stem cell models because of their better predictive power. As matter of a fact, the advancing in isolation and in vitro expansion protocols for culturing autologous human stem cells, and the standardization of methods for generating patient-derived induced pluripotent stem cells has made feasible to generate and investigate human cellular disease models with even greater speed and efficiency. Furthermore, the potential of stem cells on generating more complex systems, such as scaffold-cell models, organoids, or organ-on-a-chip, allowed to overcome the limitations of the two-dimensional culture systems as well as to better mimic tissues structures and functions. Finally, the advent of genome-editing/gene therapy technologies had a great impact on the generation of more proficient stem cell-disease models and on establishing an effective therapeutic treatment. In this review, we discuss important breakthroughs of stem cell-based models highlighting current directions, advantages, and limitations and point out the need to combine experimental biology with computational tools able to describe complex biological systems and deliver results or predictions in the context of personalized medicine. Over the past decades, much of our understanding of human physiology and pathologies has been derived from studies on animal models. However, during the years, emerging limitations have raised doubts about the reliability of this investigational approach . The maiHere, we review the progress made in establishing the most proficient cell/tissue modeling, from basic cell culture systems to more innovative organoid platforms.Since their establishment, cell cultures have been proven to be the first and most powerful tool for the in vitro investigation of cell biology, from basic research to more complex translational approaches . ClassicAll of the above-mentioned techniques have been developed to respond to the compelling need to find better preclinical models for the study and modeling of disease pathogenesis.Currently, stem cells (SCs) disease models represent the most powerful tool for having a better understanding of human disease pathogenesis and for developing new therapies . As a maAmong stem cell types, the most used for disease modeling purposes are adult stem cells (multipotent)  and induThe advent of iPSCs opened a wide range of new stem cell applications. It is well-known that iPSCs can be derived from differentiated somatic cells, such as fibroblasts, by introducing defined transcriptional factors according to the Yamanaka and Takahashi protocol  and, likNevertheless, when somatic cells are reprogrammed to generate iPSCs, they acquire a series of new intrinsic properties that might generate hurdles in their applications. In fact, in addition to teratoma formation, karyotypic abnormalities and immature phenotype, they are characterized by unpredictable genetic background variations, thus making less reliable the corresponding disease model . In thisThe selective modification of cells’ genetic information was the groundbreaking discovery that revolutionized the concept of cell models. Innovative biotechnologies, together with other previously established, allowed the generation of pathological cells by introducing a defined mutation responsible for the onset of the disease (genome editing) as well as the restoration of a missing gene function in defective cells (gene therapy) . In the Briefly, gene therapy can be achieved through the use of advanced viral vectors  in the double helix then resolved via non-homologous end joining (NHEJ) or homologous direct recombination (HDR). These molecular mechanisms are the bases of all the principal genome editing methods that, listed from the first used to the last discovered, are: Zinc finger nucleases , transcription activator-like effector nucleases  and the Clustered Regularly Interspaced Short Palindromic Repeat/Cas9 associated system  ,100. WhiTissue engineering applies the concepts of engineering and biology to develop scaffold-based systems with the aim of reproducing the structure and the physiological functions of healthy tissues. The use of stem cells and biomaterials allows the generation of ad hoc bio-hybrid systems that represent 3D-models of tissues either for disease modeling  or, afteThese scaffold-based platforms rely on the mechanobiology concept by which biomaterials provide the 3D-structure of native tissues and through their chemical/physical properties are able to specifically steer stem cells toward tissue-specific phenotypes . As a maAmong the many scaffold fabrication methods, 3D-bioprinting has recently received a great deal of attention as one of the best tool to obtain specific precise architecture and biochemical composition of biocompatible materials and supporting components . With thAccordingly, 3D-bioprinting was used for generating in vitro disease models of tumor microenvironments , ovarianIn recent years, progress in 3D-stem cell cultures led to the production of new systems with great potential as cell model platforms . These mA more recent advance in stem cell biology and 3D-tissue engineering is the innovative application of microfluidic techniques for the development of organ-on-a-chip platforms (OOC) . The ratIn spite of the many advantages that OOCs have, they rely on the ability of the scientist to artificially reconstruct the correct microenvironment using the correct cell types and arranging them as they would be in the physiological state. Therefore, future perspective of 3D cell modeling will involve the synergic combination of OOCs and organoids, for the possibility of precisely control microenvironmental conditions and exploit organoids self-organization, with the aim of obtaining the best ex vivo cell modeling technology .In this review, we have documented the significant improvements in disease modeling using human adult stem cells and iPSCs, highlighting how their contribution in generating more elaborated 3D-models  have greatly facilitated our understanding and ability to treat human disease. Noteworthy, we have also pointed out that the tremendous advancement of the field has highlighted the need for these models to be accurate, efficient and safe, in order to accomplish valid differentiation protocols and development of effective therapies. In this regard, computational tools able to predict complex biological systems, together with genome editing and gene therapy technologies support researchers in the development of more proficient disease models. Collectively, the issues discussed emphasized ex vivo-stem cell models as an alternative or complementary system to animal disease models.


Obesity is one of the major social and health problems globally and often associated with various other pathological conditions. In addition to unregulated eating behaviour, circulating peptide-mediated hormonal secretion and signaling pathways play a critical role in food intake induced obesity. Amongst the many peptides involved in the regulation of food-seeking behaviour, somatostatin (SST) is the one which plays a determinant role in the complex process of appetite. SST is involved in the regulation of release and secretion of other peptides, neuronal integrity, and hormonal regulation. Based on past and recent studies, SST might serve as a bridge between central and peripheral tissues with a significant impact on obesity-associated with food intake behaviour and energy expenditure. Here, we present a comprehensive review describing the role of SST in the modulation of multiple central and peripheral signaling molecules. In addition, we highlight recent progress and contribution of SST and its receptors in food-seeking behaviour, obesity (orexigenic), and satiety (anorexigenic) associated pathways and mechanism. Somatostatin (SST), also known as somatotropin release-inhibiting factor, is a growth hormone inhibitory peptide that was first discovered in the hypothalamus in 1973 ,3. SST aSST mediated regulation of obesity and satiety is most controversial and varies in a species- and dose-specific manner. Impaired energy regulation, in general, is believed to be the leading cause of obesity. The concept that SST might involve in obesity emerged from the regulation of insulin release ,16. HypeSST and its analogues block the insulin secretion from pancreatic β-cell and seem, by all accounts, to be a promising agent to treat obesity ,24. HoweObesity is an imminent global pervasive with the increasing prevalence and repercussions such as multiple aetiologies, which subsequently leads to pernicious effects on normal physiological functioning . In the At present, the pharmacological interventions available for the regulation of food intake and obesity are inadequate and unable to collectively address the clinical diversity of various side effects and contraindications. The number of drugs that have been approved for weight reduction are very few . It is iContrary to obesity, satiety is a sense of fullness due to ephemeral lack of interest in further ingestion of food, which eventually leads to the suppression of appetite. Fullness is supposed to have a vital role in weight management because it will impact the frequency and amount of food and drink consumed later . The feeThe role of CNS, specifically the hypothalamus in food-seeking behaviour is well established . PerturbThe distribution of neuronal cells in the different hypothalamic regions with selective and preferential expression of hormonal peptides with orexigenic and anorexigenic stimuli attested the hypothalamus as a key centre in the regulation of appetite and energy homeostasis . PreviouMost neuroendocrine signals and functions displayed by central and peripheral stimuli in regulation and maintenance of appetite are confined in different regions of the hypothalamus. Amongst them ARC is the hypothalamic nuclei that maintains the balance between food intake and energy requirement . ARC remIn addition to POMC, the presence of leptin receptors in the hypothalamus contributes significantly to the regulation of food intake and energy balance. The perturbed leptin signaling is often seen in obese person and ob/ob and db/db mouse model devoid of leptin and leptin receptor gene ,86. It iThe SST positive neurons in the hypothalamus are mostly confined to the PeVN and account for the major portion of SST immunoreactivity in hypothalamus . In addiFood-seeking behaviour does not always depend on CNS stimuli; the peripheral system including GI and adipose tissue also plays a prominent role. Endogenous gut hormones such as CCK, glucagon-like peptide 1 (GLP-1), oxyntomodulin, peptide tyrosine, and pancreatic polypeptide have anorexigenic effect in humans which decrease food intake. Conversely, ghrelin, an appetite modulator, has an orexigenic effect in humans and leading to increased food intake . On the Physiochemical properties of the ingested food stimulate the secretion of various hormones from the lining of the GIT. GI hormones are also expressed in the CNS, which relay metabolic information between the GIT and the brain, influencing meal initiation and termination. The majority of the gut hormones that act on various sites such as the hypothalamus, brainstem, and vagus afferent decrease the food intake . GhrelinIn contrast to the well-accepted role of the hypothalamus, food intake is also regulated by the peripheral hormonal system either through the vagus nerve or by crossing the BBB . The dorLeptin, a satiety hormone secreted from adipocytes, is linked to appetite, energy homeostasis, and glucose metabolism and stimulates POMC and CART expressing neurons directly in the hypothalamus . Body faSST is widely distributed in most parts of the body. The CNS, GIT, and endocrine glands are the major sites of SST production and action . SST is Soon after the discovery of SST-14 and SST-28 and widespread distribution in different body parts, five different receptor subtypes, namely SST receptors 1–5, were identified, which are encoded by five different genes and exhibit 70% structural similarity between human and rodents. SSTR subtypes belong to a family of GPCRs and all five SSTR subtypes have been pharmacologically characterized . In addiThe divergent roles of SST in the target tissues are either direct or indirect. Direct biological actions of SST are mediated through five different receptor subtypes, namely SST receptors 1–5 (SSTR1–5). SSTR subtypes couple to inhibitory G-protein and exert inhibitory role on adenylyl cyclase and are consequently involved in inhibition of the second messenger cAMP. SSTR subtypes play a crucial role in receptor and tissue-specific manner . SSTR1 tThe role of SST in the regulation of food intake and energy store and expenditure is most controversial. Previous studies support the pro- and anti-satiety effect of SST that depends on species, doses, and mode of administration ,163,168.The presence of SST in the brain regions which are involved in the regulation of food intake displays the role of SST in appetite. SST and its analogues have been proven to induce satiety effect in children and adults . SST proSST-14 and SST analogue OCT injection directly into the brain or by icv route in the hippocampus and cortex of the rat prompted food intake ,188,189.The hypothalamus regulates glucose metabolism through parasympathetic activity. Hypothalamic damage, mainly due to brain tumour, may result in upregulation of parasympathetic activity resulting in hyperinsulinemia . Often, The hedonic eating behaviour of palatable food that is regulated by several cortical brain regions is a crucial contributor in obesity and satiety in a region-specific and neuronal phenotype manner. Whether SST plays any role in this direction is not well understood. Recently, Zhu et al. demonstrated that basal forebrain regions rich with GABAergic neurons play a key role in food intake . This stSSTR2 gene has been associated with obesity and food intake in the Mediterranean population [Like SST five different SSTR subtypes also display widespread distribution in different parts of the brain, including the hypothalamus, and serve as a key central regulator of feeding behaviour in rodents and humans. The distributional pattern of SSTR subtypes is selective in a species-specific manner. We demonstrated significant variation in SSTR subtypes in hypothalamus. SSTR5 is absent in the hypothalamus of the rat but strongly expressed in the hypothalamus of mice ,167. Mospulation . Thus, cSST expression and physiological functions in central and peripheral target are modulated by several factors, among them BDNF, a neurotrophic factor, affects its regulation ,224. HowThe molecular mechanisms for the role of BDNF in food-seeking behaviour and its distribution in different region of the hypothalamus with high expression in VMN and interaction with orexigenic and anorexigenic are more complex than it seems, as reviewed by Rosas-Vargas et al. . The exoIn addition to the regulation of anorexigenic and orexigenic response in the hypothalamus, BDNF plays an essential role in neuronal development, neuronal survival, differentiation, synaptic neurotransmission, and synaptic plasticity as well as many neuropsychological disorders. These divergent effects of BDNF are associated with modulation of key neurotransmitters in the brain and indicate the role of BDNF in the regulation of food intake by interrupting neurotransmission that not is only limited to hypothalamus but is also found in some other brain regions including the cortex, hippocampus, and reward area. In this context, Pelleymounter et al. described that infusion of BDNF in rats resulted in an increased ratio of 5 hydroxyindole acetic acid and serotonin (5HIAA/5HT) in the hypothalamus that leads to suppressed appetite and body weight . FurtherThe role of BDNF in food intake, appetite, energy homeostasis, and obesity is undisputed; however, the mechanism and mode of action of BDNF in such a complex process is not well understood. Most importantly, how SST might be involved in the regulation of BDNF mediated function in obesity is not known yet. In this perspective, the functional role of SST via activation of different SSTR subtypes in the modulation of BDNF and regulation of obesity is well supported by the regulation of hypothalamic–pituitary–adrenal axis (HPA) in presence of BDNF, CRH, and glucocorticoids (GCs). There are several possible mechanistic explanations by which BDNF and SST may affect obesity and energy homeostasis. Previous studies have shown decreased SST expression at molecular levels in BDNF KO mice or in mice with disrupted BDNF transcript ,248,249.Pre- and post-episode of stress and anxiety contribute to food intake and suppressed desire to eat ,252,253.The activation of brain SSTR2 interferes in the prevention of CRH mediated anorexic effect ,263. As The expression of SSTR2 in the brain regions involved in anxiolytic effect in the stress model of anxiety support its role in anxiety. Furthermore, evidence also exists from behavioural studies showing that SSTR2 specific agonist but no other subtypes suppressed anxiety ,263. SSTGCs are prominent hormones and a crucial player in the regulation of metabolic homeostasis. In addition to the well-established role of GCs in the regulation of GH synthesis and release as well as in inflammatory and cardiac function, they are also linked to several behavioural and pathological conditions including anxiety, depression, and post-traumatic stress disorder ,266,267.Similar to CRH, stress-associated changes in GCs affect ACTH levels that participate in appetite via stimulation of NPY/AgRP . Most apSSTR subtypes are well expressed in different nuclei of the hypothalamus and colocalized with SST and tyrosine hydroxylase as well as cannabinoid receptor 1 in receptor and region-specific manner ,166,270.Previous studies have shown the role of insulin and leptin (two prominent members of adiposity negative-feedback model of energy homeostasis) towards stabilization of fat stores by well-organized information to the brain through circulating signals ,64. The SST gene expression [Leptin is primarily confined and released from adipocyte and exerts a crucial role in the regulation of body weight, appetite, and energy homeostasis, especially in anorexigenic behaviour . Furtherpression ,293. Stepression ,142,164.pression ,289. Thepression . In vivopression . The prepression ,296,297.POMC and AgRP exert negative and positive effects on food intake, respectively ,299,300.The activation of AgRP positive neurons in ARC prompt response in food-seeking behaviour is contrary to the lack of food desire in absence or inhibition of AgRP neurons ,212,298.Ghrelin, an orexigenic hormone, is prominently expressed in gastric mucosa . The firSeveral overlapping physiological functions including appetite and comparable distributional pattern of SST, nNOS, and CB1R in the hypothalamus are compelling evidence to debate the possible interaction between these prominent orexigenic players in rodents and human. In agreement with previous studies and our recent observations with colocalization of CB1R and SST in different hypothalamic regions involved in food intake, we speculate a collaborative function between SST and CB1R in food intake behaviour ,320,321.Mounting evidence supports the role of many receptor proteins including the receptors for SST, endocannabinoid, serotonin, epinephrine, DA, GABA, glutamate, and opioid in the regulation of appetite and energy homeostasis. These receptors have shown selective and preferential co-expression as prominent players of anabolic and catabolic function including NPY/AgRP, leptin responsive neurons, POMC, MC3R, and MC4R in different nuclei of hypothalamus including ARC, PVN, VMN, and LHA. However, the distribution of these receptors is not limited to the hypothalamus but also exists in other brain regions including the hippocampus, cortex and most importantly reward pathway, i.e., VTA-NAc, the brain region associated with food intake reward behaviour. Despite the profound impact on appetite associated hormonal secretion and regulation, currently nothing is known on whether these receptors functionally interact with each other and function in concert in food control in areas such as the hypothalamus, which needs to be addressed. Furthermore, the requirement of the N-methyl-D-aspartate receptor in the activation of AgRP neurons during fasting is important but uncertain physiologically in food-seeking process specifically in fasting . MountinThe expression of SST in hypothalamic regions including ARC, VMN, and LHA and D cells of GIT demonstrating its role in food-seeking behaviour and in the regulation of hormonal secretion as well as gut motility are compelling pieces of evidence to support the role of SST in satiety and obesity . There iIn the regulation of satiety, various contradictory mechanisms are involved and are still not fully understood. However, the involvement of various receptors, satiety factors, and mechanisms of various pathological conditions linked to the regulation of satiety are tempting to explore further in details. Recently, the development and synthesis of new receptor agonists and antagonists provide a broader spectrum for the role of SST in several pathological conditions including appetite and satiety. SST induces satiety by delaying GI motility and emptying. SST mediated inhibitory action on satiety associated factors such as CCK may decrease the effect on satiety. On the other hand, the central action of SST on SSTR2 in the brain stimulates food and water intake. SST analogues, with their broad array of actions, are better candidates for the treatment of obesity and future studies might uncover the new role and mechanism in certain diseases, which lead to the loss of appetite. There is a scope for the development of receptor-specific agonists and antagonists for selective therapeutic intervention in food-seeking behaviour. In several pathological conditions, including tumours of different origin, HIV, etc., the loss of appetite is the major problem while SST and its analogues provide a potential therapeutic option. In addition to hypothalamus, elucidating the role of other efferent and afferent projections of the brain stem, cortex, and reward system as well as the exploration of multiple downstream signaling pathways involving SSTR subtypes in regulation of anabolic and catabolic hormone will shed new light on food-seeking behaviour.


The nature of memory and the search for its localization have been a subject ofinterest since Antiquity. After millennia of theoretical concepts, shifting fromthe heart to the brain, then from the ventricles to solid parts, the corememory-related structures finally began to be identified through modernscientifically-based methods at the diencephalic and cortical  levels, mostly in the late Modern period, culminating in thecurrent state of knowledge on the subject. At this point, the faculties were understood to bein the solid parts of the cerebrum, as proposed by Johannes Jakob Wepfer (1658), and ashort time later by Thomas Willis (1664).,The nature of memory (function and failure), as well as the search for its localization,have been an object of interest since Antiquity. Wepfer 158, and aThe long preceding period of these initial localization theories gave way to a phase ofmodern, more concrete and scientifically-based facts, in a bid to relate memory failure(amnesia) to solid brain structures, which took place mainly in the XIX century,extending into the XX century (late Modern period). At this time. numerous anatomicalbrain structures were found to be related to this dysfunction (and function), furtheringunderstanding on the matter and culminating in the present state of knowledge.The memory-related brain structures known today can be divided into diencephalic andcortical , and involve the core components of thedeclarative memory circuits. The nuclei with modulatory functions, such as thecholinergic Meynert’s basal nucleus, will not be addressed. The development of thisknowledge is outlined in the rd and4th ventricles, and in the acqueductal region. For this condition, heproposed the diagnosis of ‘Superior Hemorrhagic Poliencefalitis’ (1881).,The modern studies of memory were triggered by Carl Wernicke 1848-1904) and SergeiSergeievich Korsakow (1854-1900). First, Wernicke described three acute cases , without memory impairment, which evolved to death. Theautopsy revealed punctiform hemorrhages in the floor of the 348-1904 aTheir studies were not able to objectively identify the underlying anatomicstructures. Wernicke’s pathological findings would only be later understood,retrospectively.,,The close relationship between both syndromes was not recognized by their authors.However, Adolf Elzholz and Karl Ludwig Bonhoeffer identified this relationship,recognizing the strong relation between the two syndromes, regarding them as part ofthe same pathological process.rd ventricle, superior brainstem, and 4thventricle, and also lesion of the anterior tubercle of the thalamus, and markedatrophy of the mammillary bodies, while the fornix and Vicq d’Azyr bundle showingabnormal changes in one of the cases.,Two publications that followed Korsakow’s are worthy of note, as they furtheredunderstanding on the subject, namely, the studies of Hans Gudden 1866-1940) and ofEduard Gamper (1887-1938), which provided entirely novel anatomical information.Gudden presented clinical and pathological data on five patients with severe chronicalcoholism who died in the acute or chronic phase of the condition (1896). Theypresented a clinical picture resembling that of Korsakow’s and pathologicalexamination showed lesions compatible with Hemorrhagic encephalitis, affecting thewalls of the 366-1940 a,At a later date 1928), Gamper held a conference about chronic alcoholics who died inthe acute, subacute or chronic phase with the clinical manifestation of Korsakow’spsychosis, with or without Polioencephalitis haemorrhagica (Wernicke’s).Neuropathological examination showed lesions of the brainstem, mammillary bodies,and thalamic nuclei . He held that the mammillary bodies constituted animportant nodal point of the vegetative mechanism underlying the psychic processesof the amnesic syndrome, considering their strong connections, on one side with themidbrain and the thalamus (and through this certainly to the cingulate gyrus) and onthe other side with the Ammon’s formation by way of the fornix.28, GampeApparently Gudden was the first author to relate the amnesic symptoms of his caseswith anatomic structures, such as the mammillary bodies and related tracts (fornixand Vicq d’Azyr bundle), and the anterior tubercle of the thalamus. Gamper endorsedand extended Gudden’s finding, and proposed a circuit formed by the connections ofthe mammillary bodies, thalamus, and cortical structures (hippocampus and cingulategyrus), related to memory failure.The findings of Gudden and those of Gamper, with the suggestion of theabove-mentioned circuit, can be viewed as possible precursors of the circuitdescribed later by James Papez (1937), initially proposed as a mechanism ofemotion.The cited studies, particularly those of Gudden and Gamper, have revealed thecritical role of diencephalic structures in memory function, prompting furthersearch for distinct brain structures and neural circuits underpinning the memoryprocess.,Vladimir Michailovich Bechterew (1857-1927) presented a case with severe memoryweakness [amnesia], falsification of memories (pseudo-memories) [confabulation], andapathy (1900). The autopsy revealed softening of the cerebral cortex in the regionof the anterior (gyrus uncinatus) [uncus], and the internal part of both temporallobes (gyrus cornu Ammonis) [hippocampus proper – fascia dentata – subiculum] and adjacent parts. He commented that the casepresented the characteristic symptom complex of Polyneuritic psychosis [Korsakow’s]that could also occasionally manifest in organic lesions of the cerebralcortex.Bechterew’s report was the first relating memory impairment with lesion of thehippocampal region.About half a century later, this feature was confirmed by William Beecher Scoville(1906-1984). He performed bilateral medial temporal resection for refractoryepileptic seizures and other conditions. One of these patients, later known as theHM case, had surgical removal of the medial surface of the temporal lobes, anterior⅔ of the hippocampus and the [para]hippocampal gyrus bilaterally, as well as theuncus and the amygdala. The patient was assessed by Brenda Milner who detectedsevere loss of anterograde memory and partial loss of retrograde memory, whereasearly memories appeared normal and technical skills also remained intact. Thesefindings indicated the importance of the hippocampal region [hippocampus and relatedstructures] for the normal functioning of anterograde memory.,The first to suggest the involvement of the cerebral cortex with memory handling wasWillis (1664), albeit in a conjectural manner.,The HM case, as seen earlier, in which long term-memory (‘early memories’) waspreserved after the surgery,,The first to demonstrate experimentally that the human cerebral cortex was related tomemory was Wilder Graves Penfield (1891-1976). He stimulated the cortex of awakepatients who underwent surgery for epilepsy treatment and stated (1959): “There isan area of the surface of the human brain where local electrical stimulation cancall back a sequence of past experiences”.,Today, it is known that the frontal and temporo-parieto-occipital associativeneocortical areas constitute the sites where long-term memories are presumed to bestored.The nature of memory function, as well as the search for its localization, has been asubject of interest since Ancient times. After millennia of theoretical concepts,the core memory-related structures began to be identified, mostly in the late Modernperiod (XIX and XX centuries), through modern scientifically-based methods. Firstthe diencephalic, then the hippocampal structures were found, and finally theneocortical structures, culminating in the current state of knowledge.


Ramsay Hunt syndrome (RHS) is caused by a reactivation of varicella-zoster virus (VZV) infection, and it is characterized by the symptoms of facial paralysis, otalgia, auricular rash, and/or an oral lesion. Elderly patients or immunocompromised patients, deep pain at the initial visit and no prompt treatment are significant predictors of postherpetic neuralgia (PHN). When PHN occurs, especially involved cranial polyneuropathy, multiple modalities should be administered for patients with the intractable PHN. The use of thermography in the follow-up of PHN secondary to RHS with multicranial nerve involvement has not yet been described yet in the literature.The patient was a 78-year-old man with the chief complaint of a 3-month history of PHN secondary to RHS with polycranial nerve  involvement. Multimodality therapy with oral gabapentin, pulsed radiofrequency (PRF) application to the Gasserian ganglion for pain in the trigeminal nerve region, linear-polarized near-infrared light irradiation for pain in the facial nerve region, and 2% lidocaine spray for pain in the glossopharyngeal nerve region was used to the treat patient, and follow-up evaluations included thermography. This comprehensive treatment obviously improved the quality of life, resulting in considerable pain relief, as indicated by a decrease in the numerical rating scale (NRS) score from 9 to 3 and a decrease in thermal imaging temperature from higher to average temperature on the ipsilateral side compared with the contralateral side. Lidocaine spray on the tonsillar branches of the glossopharyngeal nerve resulted in an improvement in odynophagia, and the NRS score decreased from 9 to 0 for glossopharyngeal neuralgia after three applications.Although the use of thermography in the follow-up of RHS with multiple cranial nerve  involvement is very rare, in this patient, thermal imaging showed the efficacy of combination therapy  and that is a good option for treatment. Ramsay Hunt syndrome (RHS) is a multicranial neuropathy caused by the varicella-zoster virus (VZV) infection, and it is characterized by the symptoms of facial paralysis, otalgia, auricular rash, and/or an oral lesion , 2. CorrTo monitor the response to treatment in patients with PHN, infrared thermography  is used to study the physiology of thermoregulation and reveals a correlation of thermal imaging with the intensity of pain. Infrared thermography has been shown to quickly detect someone’s surface skin temperature without being physically close to the person being evaluated for the duration of the public health emergency related to coronavirus disease 2019 (COVID-19) . PreviouA 78-year-old man with the a chief complaint of a 3-month history of gradually increasing pain and numbness in the right ear, submandibular region, temporal region, and the right side of the tongue root and right-sided facial weakness was seen Fig. . ParoxysThe delivery of pulsed radiofrequency (PRF) to the Gasserian ganglion was recommended to alleviate the pain of the mandibular nerve division of the trigeminal nerve. A radiofrequency generator  was inserted through the foramen ovale into Meckel’s cave under common fluoroscopic control guided by bony landmarks Fig. e, whereaPeripheral facial paralysis was treated with linear-polarized near-infrared light irradiation   caused by VZV infection and its subsequent activation may cause herpes zoster cephalicus, facial paralysis, otalgia, an auricular rash and/or an oral lesion . In 1915When PHN occurs, resulting in severe physical, occupational, and social impairments due to unceasing pain, combination therapy should be administered to patients with intractable PHN. First, the application of lidocaine spray to the tonsillar branches of the glossopharyngeal nerve resulted in complete resolution of the lateral glossopharyngeal neuralgia due to Eagle’s syndrome . TopicalInfrared thermography was used to study the physiology of thermoregulation and revealed a correlation of thermal images with the intensity of pain . Warm spThis comprehensive treatment improved the quality of life, provided considerable pain relief, and showed a correlation of thermal images with the intensity of pain. Further prospective investigations might be needed to estimate the effectiveness of comprehensive treatment for polycranial nerve PHN secondary to RHS with multicranial nerve  involvement.


Personalised dietary modulation of the gut microbiota may be key to disease management. Current investigations provide a broad understanding of the impact of diet on the composition and activity of the gut microbiota, yet detailed knowledge in applying diet as an actionable tool remains limited. Further to the relative novelty of the field, approaches are yet to be standardised and extremely heterogeneous research outcomes have ensued. This may be related to confounders associated with complexities in capturing an accurate representation of both diet and the gut microbiota. This review discusses the intricacies and current methodologies of diet-microbial relations, the implications and limitations of these investigative approaches, and future considerations that may assist in accelerating applications. New investigations should consider improved collection of dietary data, further characterisation of mechanistic interactions, and an increased focus on -omic technologies such as metabolomics to describe the bacterial and metabolic activity of food degradation, together with its crosstalk with the host. Furthermore, clinical evidence with health outcomes is required before therapeutic dietary strategies for microbial amelioration can be made. The potential to reach detailed understanding of diet-microbiota relations may depend on re-evaluation, progression, and unification of research methodologies, which consider the complexities of these interactions. Faecalibacterium prausnitzii , 4948, 4Finally, the typical faecal sample represents the final point of a developing and maturing ecosystem through the gastrointestinal tract . With vaMicrobiome data processing pipeline, collection, storage, and analysis of samples are particularly vulnerable to significant error , 58 thatFaecalibacterium tend to be overrepresented by these methods [Traditional culture-based technologies that have been used to investigate the microbial ecosystem have recently regained attention with the development of new methods enabling the culturing of an extended number of bacteria from the human gut. Though bacterial taxa including Ruminococcaceae and  methods . Besides methods , thereby methods , consequ methods .Development of new methodologies should assist in addressing sample processing bias. For example, whole shotgun metagenomic sequencing has allowed investigators to bypass the PCR amplification used within 16s rRNA sequencing related to an overestimation of certain taxa . OutsideImproved accuracy and throughput of DNA sequencing techniques, together with multi-omic analysis and mechanistic experiments in animal models, increased our understanding on the structure and function of the microbiome in health and disease . SeveralThe complexity of the human gut microbiome is largely mirrored by that of diet . While wWhile a shift towards bigger datasets is undoubtedly required, this cannot be considered a ‘catch-all’ solution. With dietary bioactive compounds acting synergistically, and present within a multitude of food sources, almost all nutritional variables correlate between each other and health outcomes, particularly evident in large datasets . Other fAdvancing nutritional research techniques has not progressed at the same pace as the rapid development of microbial investigations in the last decade. To improve understanding of diet-microbial relations, drastic progress is required to further our ability to characterise diet beyond the established macro and micronutrients. Emphasis on the importance of accurate dietary data methodologies and application of techniques typically applied within other scientific research fields, such as machine learning, may assist. While the nutritional community includes outstanding scientists, a large number of dietary research are undertaken by investigators in other fields, without the input of dietitians, nutritionists, or scientists trained in nutritional epidemiology. This may limit the quality of dietary data collection, processing, analysis, and reporting. For diet-microbial investigations, the involvement of a trained nutrition research professional should undoubtedly improve research outcomes and aid in the elucidation of some of the intricacies within these relations.In designing study protocols for diet-microbiome investigations, collaborations between dietitians and/or nutritionists and microbiologists, epidemiologists, and biostatisticians are essential in order to capture a broad spectrum of accurate data. Establishing the cause and effect of diet has been acknowledged as challenging . NutritiInvestigators of diet-microbiome relations often rely on food frequency questionnaires and self-reported food diaries . Yet, muBesides collection methodology, the majority of nutrition research investigates diet as a limited series of macro and micronutrients  covered Although epidemiological research has succeeded in identifying a link between the gut microbiome and nutrients derived from food composition databases , the assWhile many further limitations and biases are worthy of discussion, the final note on this subject should highlight the importance of rigorous reporting to allow for scientific reproducibility. Standardised reporting guidelines of all future research efforts should be developed following a consultative process . ExtensiThe pliable nature of the gut microbiota composition facilitates its modulation via environmental factors, the most important of which is diet . Yet, toF. prausnitzii, S. variabile, and Roseburia, has been shown to be auxotrophic for B vitamins, and therefore rely on exogenous sources [Diet influences not only the microbial composition, but also regulates the activity of the ecosystem (and its effects on the host) without noticeable compositional alterations  Fig. 3)3). Furth sources , with li sources . By incl sources , 119.FiReconceptualization of the current approaches towards big data technical methodologies and improved study design may assist in further characterisation of the diet-microbial landscape . TechnolLongitudinal large multi-centre studies are required which employ standardised protocols for the collection of validated biomarkers of health for phenotyping, subject demographics, dietary information, biological samples, laboratory processing, genetic analyses, and data analysis and manipulation . GeneratLarge databanks typically include limited phenotypes to limit researcher burden and cost, whereas typically small finite samples facilitate more in-depth phenotyping . MaximisTo advance our understanding of the role of diet-microbiota interactions on human health and disease, it is crucial to step back and re-evaluate current approaches. Standardisation and optimisation of methodologies may assist in capturing the complex spectrum of these relations. To inform dietary strategies for the prevention and amelioration of chronic metabolic disease states, we first need to ensure intrinsic data is sufficient and relevant, in a manner that considers the deeply individual aspects of both diet and microbiome. Characterisation of the natural intricacies of the ecosystem and the interactions existing between its multiple members at various levels of complexity remains critical. Both micro- and macro-scale influences that drive microbial variation should be considered, from spatial organisation to transmission amongst hosts and between the host and the environment. A comprehensive, multidisciplinary research agenda is required to accurately describe the gut microbial composition and function. Individual and combined complexities of both microbial research and nutrition research demand reconsideration of standard approaches, with a push towards gold standard protocols, further emphasis on the use of randomised control trials, and mechanistic studies, and analysis techniques that include big data, multi-omics, and machine learning approaches. Without multifactorial approaches towards investigations of the diverse aspects of the microbiome, diet, and diet-microbiome relations, we will be limited in our progression towards therapeutic interventions on a personalised or population level. While the path ahead may be unclear in how we may reach these targeted strategies to improve host health, the approaches outlined within this review may assist in a collaborative move forward.


The increasing number of data studies on the biological impact of anthropogenic chemicals in the marine environment, together with the great development of invertebrate immunology, has identified marine bivalves as a key invertebrate group for studies on immunological responses to pollutant exposure. Available data on the effects of contaminants on bivalve immunity, evaluated with different functional and molecular endpoints, underline that individual functional parameters  and the expression of selected immune-related genes can distinctly react to different chemicals depending on the conditions of exposure. Therefore, the measurement of a suite of immune biomarkers in hemocytes and hemolymph is needed for the correct evaluation of the overall impact of contaminant exposure on the organism's immunocompetence. Recent advances in -omics technologies are revealing the complexity of the molecular players in the immune response of different bivalve species. Although different -omics represent extremely powerful tools in understanding the impact of pollutants on a key physiological function such as immune defense, the -omics approach has only been utilized in this area of investigation in the last few years. In this work, available information obtained from the application of -omics to evaluate the effects of pollutants on bivalve immunity is summarized. The data shows that the overall knowledge on this subject is still quite limited and that to understand the environmental relevance of any change in immune homeostasis induced by exposure to contaminants, a combination of both functional assays and cutting-edge technology  is required. In addition, the utilization of metagenomics may explain how the complex interplay between the immune system of bivalves and its associated bacterial communities can be modulated by pollutants, and how this may in turn affect homeostatic processes of the host, host–pathogen interactions, and the increased susceptibility to disease. Integrating different approaches will contribute to knowledge on the mechanism responsible for immune dysfunction induced by pollutants in ecologically and economically relevant bivalve species and further explain their sensitivity to multiple stressors, thus resulting in health or disease. Bivalves, the second largest group in Mollusca, show a large diversity in anatomical structure and size, physiology and behavior, and adaptation to different environments. Moreover, several bivalve species represent an important food source for humans and are of considerable economic value in aquaculture in estuarine and coastal areas . CoastalThe bivalve's affords efficient protection against different potential invaders, based on the activity of circulating immunocytes, the hemocytes, which can recognize potential pathogens and foreign particles through phagocytosis and different cytotoxic reactions, secreting soluble factors in the hemolymph fluid –14. BivaThe first overview of the immune responses of marine invertebrates, including bivalves, to environmental perturbation, including contaminant exposure, was provided by Ellis et al. . In thisThe first review article focusing on pollutants and immunity in marine bivalves was from Renault , where dMytilus galloprovincialis, on immune responses evaluated after exposure (in vitro and in vivo) to a number of estrogenic compounds, alone and in mixture  supported these observations , but also the number and type of immune biomarkers measured, and the type of responses observed to be increasing the concentration of pollutants. With regard to this latter point, an example was first provided by data obtained in the model bivalve, the mussel  mixture . Classicrvations –27. In trvations . Thanks rvations , 29–34, Overall, available data generally show that whatever individual parameter is measured , each can change in response to contaminants depending on the conditions of exposure . In this light, in analogy with the general biomarker approach, where integrated multiple biomarker responses are utilized to identify the response to environmental stress , 36, onlin vivo effects of contaminants can often be reversible, suggesting the establishment of compensatory mechanisms . In particular, the gills, which through their filter-feeding activity represent the first contact with water-borne pathogens, are also the first site of uptake and accumulation of water-soluble pollutants . In this light, the possibility to evaluate the immune response also at the tissue level, not only as hemocytic infiltration as a sign of inflammation  . AlthougIn the last decade, the application of -omics approaches in ecotoxicology has been proposed and applied in order to identify toxicity pathways, to more precisely quantify classical biomarkers, to identify novel ones, and to draw adverse outcome pathways (AOPs) –53. In pHowever, the -omics approach has only been utilized in the last few years to investigate the effects of contaminants on bivalve immunity. Although a PubMed search for “Bivalve Immunity & Pollutants” gave 29 hits for “transcriptomics,” 14 for “proteomics,” and seven for “metabolomics,” respectively, only a few of these publications focused on immune responses. In this mini-review, we made a first attempt to highlight the main advancements in knowledge, the limitations of available data, and future developments of the application of the -omics approaches to evaluate the impact of contaminants on bivalve immunity in health and disease.As mentioned above, the transcriptomic approach has been largely applied to the study of disease processes in marine bivalves [reviewed in Nguyen and Alfaro ]. AlthouTranscriptomics has also been increasingly utilized in evaluating the effects of exposure of several inorganic and organic contaminants in different bivalve species. A number of studies identified a significant impact of a wide range of contaminants , on different pathways, such as response to stress, redox balance, metabolism, biotransformation of xenobiotics, apoptotic processes, and immunity , 54–61. Mytilus edulis, the effects of cadmium (Cd) were evaluated in an in vitro model of mussel hemocytes by transcriptomics  and the determination of functional parameters, namely phagocytosis and hemocyte viability  and challenge with V. splendidus, alone and in combination , on immune responses were investigated under two salinity regimes, evaluating both functional immune parameters and changes in proteome  on the unionid mussel Lampsilis fasciola were evaluated by traditional behavioral and reproductive endpoints and a metabolomic approach  and arsenate induced oxidative stress in the digestive gland. A metabolomic analysis demonstrated that both chemical forms of As induced immune stress, as shown by the increase in branched-chain amino acids  and osmotic stress  effluent  exposed to copper (Cu)  . The obsper (Cu) . Althougper (Cu) , the posHigh-throughput sequencing technologies also provide the ability to fully characterize the diversity of complex microbial populations  in environmental matrices, both at the taxonomic  and functional  levels . It is nThe rich and complex microbiome of bivalves has been shown to be influenced by host genetics, environmental conditions, stress, and infection , 95–100.M. galloprovincialis, in vivo exposure to nanoparticles induced significant shifts in the composition of microbial communities of the hemolymph, at the same time inducing changes in immune parameters, with distinct effects observed with different NPs (2) induced an overall stimulation of immune responses, which was associated with an almost complete disappearance of Vibrio and Psychrobium from mussel hemolymph. These data indicate that nTiO2-induced activation of immune defenses may contribute to creating an unfriendly medium for the most sensitive bacterial communities present in hemolymph  in bivalve hemocytes and hemolymph aquaculture research have been recently reviewed . The autHowever, when we look at the state of the art on the application of -omics technologies to evaluate the effects of pollutants on immune responses of bivalves, only a few studies demonstrate a clear relationship between contaminant exposure and alterations of immune responses . This unAlthough the integration of different -omics approaches would greatly expand our knowledge on the bivalve immune system, and its responses to contaminant exposure, the need for complex methodologies and the analysis of large-scale datasets from different -omics platforms, currently makes this approach unfeasible in environmental monitoring. However, the differently expressed molecules that have been identified through -omics studies can be used as candidate immune biomarkers with applications in biomonitoring of environmental pollutants as well as in aquaculture for early disease diagnosis. At present, integrating data on functional immune responses and susceptibility to pathogen challenge with the application of individual -omics platforms at cellular and tissue level appears to be the most suitable approach to better define the mechanisms underlying the immune response to pollutants and their significance for the health status of bivalves . UnderstLC conceived and designed the review. CC collected and analyzed available data. MA and TB designed the artwork. LC, MA, and TB critically wrote the manuscript. All authors contributed to manuscript revision, read, 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.