In this work, we want to investigate the impact of different substrates and different environmental condition on the biofilm communities growing on plaster, marble, and mortar substrates inside the Herculaneum Suburban Baths. To do so, we measured environmental conditions and sampled biofilm communities along the walls of the baths and used culture-dependent and -independent molecular techniques (DGGE) to identify the species at each sampling sites. We used the species pool to infer structure and richness of communities within each site in each substrate, and confocal light scanning microscopy to assess the three-dimensional structure of the sampled biofilms. To gather further insights, we built a meta-community network and used its local realizations to analyze co-occurrence patterns of species. We found that light is a limiting factor in the baths environment, that moving along sites equals moving along an irradiation gradient, and that such gradient shapes the community structure, de facto separating a dark community, rich in Bacteria, Fungi and cyanobacteria, from two dim communities, rich in Chlorophyta. Almost all sites are dominated by photoautotrophs, with Fungi and Bacteria relegated to the role of rare species., and structural properties of biofilms are not consistent within the same substrate. We conclude that the Herculaneum suburban baths are an environment-shaped community, where one dark community (plaster) and one dim community (mortar) provides species to a “midway” community (marble). The Roman city of Herculaneum was destroyed in AD 79 when the Vesuvius erupted and inundated the town with hot volcanic material, submerging houses and streets . Due to Biological colonization is a complex dynamic, depending on both substrates and environmental factors ,5. UsualVestibulum (on which are still present frescoes traces), marble from the Tepidarium, and mortar from the swimming pool. After collection, we identified the species present in each environmental sample by coupling culture-dependent and -independent techniques and used this information to assemble a co-occurrence matrix and proceeded to analyze the structure and the richness of the communities at various sites. Then we used confocal laser Microscopy (CLSM), the election tool for non-destructive analyses of biofilm on monuments  for the preliminary preparation of images, according to [We used the open source image processing package Fiji ; and alsrding to  and the rding to  plugin trding to ,27. FinaThe Z-stacks in which the algal masses showed little and variable autofluorescence were deconvolved and displayed as MIPs (Maximum Intensity Projection). We manually identified and selected cell outlines to measure cell area and mean fluorescence. The Corrected Total Cell Fluorescence (CTCF) was calculated according to .Scytonema julianum filaments emitting weak self-fluorescent signals, probably due to senescence or death. To reduce the effect of dead filaments on measurements the autofluorescence of single S. julianum cells was equalized using auto-fluorescence values of Leptolyngbya sp. cells, this latter being widely present and with a uniform emission of autofluorescence  to produce an ordination of the communities found at the nine sampling points. Pairwise distance among communities was based on differences among functional groups’ relative richness . All the environmental and structural variables measured were projected on the ordination as nonlinear surfaces to indirectly relate them to the ordination axes.i.e. species) distance as inversely proportional to the number of neighbors a node has . We then split the meta-community network and sampled communities were plot as single graphs using the calculated FR layout. Finally, we subsetted the bath meta-community based on the three substrates, and displayed every subset as a linear graph, i.e. nodes were all put on a single line and sorted first by taxon and then by alphabetical order.The co-occurrence table that defines the sampled communities was used as a starting point for the assembly of an unweighted, undirected meta-community network that links species based on their contemporary co-occurrence in a site. A layout display was calculated using the Fruchterman-Reingold (FR) force-directed algorithm , that deThe sampling campaign was focused on the three rooms of Suburban Thermae not interested by recent restoration works, where the presence of biofilms was evident at a first glance. The general distribution of biofilms appeared patchy to the naked eye. This is a common feature of microbial lithic communities, depending on variation in substrata characteristics correlated to centimetre scale variations .Plotting the environmental features of samples  shows thThe distribution of bacteria, Cyanobacteria, Chlorophyta and Fungi species on each sample from the three selected substrates is shown in Bacillus genus was common to all sites, with an unidentified Bacillus being the most diffused in the sampled areas. The remaining genera, Paenibacillus and Streptomyces, were found on plaster and marble but not on the mortar substrate.Heterotrophic bacterial profiles were different among substrates, and almost equally distributed. The occurrence of members of Leptolyngbya and Phormidium.The highest number of Cyanobacteria bands was found on plaster, with eight phylotypes, whereas the other two substrates hosted limited Cyanobacterial biodiversity. The only two species common to all three substrates were those of filamentous genera Chlorella and Chlorella-like group. The only genera common to the three substrates were Stichococcus, Pseudochlorella, and Microthamnion.We identified thirteen different DGGE bands belonging to Chlorophyta on the three substrates, with dominance from marble and mortar . The genera most represented belong to Aspergillus and Penicillium, but no species appeared in all substrates.Finally, we identified DGGE bands belonging to Fungi on the three substrates, with the same dominance pattern of Cyanobacteria. The genera more represented were Scytonema julianum) and two microfungi (Curvularia geniculata and Lecanicillium sp.) in the environmental sample.DGGE failed to spot one cyanobacterium  and mortar (for Cyanobacteria and Chlorophyta). It is worth noting that the Fungi found on the Mortar substrate are unique to that substrate.After preparation, we analyzed the biofilm samples using a Confocal Light scanning Microscope . The anaThe low intensity of auto-fluorescence with a high Standard Deviation (169.75±96.32) seems to indicate an advanced stage of senescence and limited cellular vitality .Plotting the structural attributes of sampled communities  shows thThe nMDS ordination performed on the nine sites highlight within-substrate similarities .-2s-1 isoline), opposed to the high light condition of the marble ones. Mortar community sits in between. It is worth noting that the Cyanobacteria (and Fungi) to Chlorophyta (and heterotrophic bacteria) gradient is overlapping with the irradiance gradient. The projection of all other variables  on the ordination does not highlight any particular trend, and is such reported as supplemental image  and blue-green algae (Cyanophyta). All four groups are distributed among the walls in the three colonized sites, with a low contribution of heterotrophic bacterial species to richness, and a considerable, yet heterogeneous, pool of species scattered among substrates.Lecanicillium can occur on different substrates of subterranean environments [Actinobacteria belonging to Genus Streptomyces ,46 and fronments ,48. The ronments , and itsronments .Aspergillus, Penicillium and Fusarium, have a cosmopolitan distribution and are common in many different habitats, including caves, where they respectively represent the first, second and fourth most frequently found taxa [The fungal genera und taxa .Vestibulum) to being rich in low-light adapted autotrophs , with the Tepidarium community sharing its dark-adapted species with the Vestibulum and its dim-adapted species with the swimming pool. It is possible to appreciate how the heterotroph/autotroph rates move from the dark vestibule extreme, where fungal richness is maximum, and cyanobacteria steadily compete with green algae, to the dim light swimming pool community, where only E. tuberiformis and Lecanicillium sp. remain as a fungus, and Cyanobacteria are relegated to a small portion of the species pool.The community composition is seemingly influenced by the irradiation gradient, and goes from being a mix of heterotrophic and dark-adapted autotrophic species , sharing almost all of the species with the other two.Our analysis suggests that on the one hand, the meta-communities associated with the three substrates tend to be mostly composed of isolated communities. On the other hand, there is a single meta-community (the Vestibulum) and dim light-adapted  communities, the substrate clustering emerging from the nMDS could hint at a contribution of substrate chemistry in driving community assembly.The results gathered here support the hypothesis of the Herculaneum baths walls as communities where the low irradiance represents one of the limiting factors, influencing community composition. Indeed, while irradiance gradient influences the three meta-communities, driving the assembly of dark- Click here for additional data file.S1 FigPlaster Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigEnvironmental (green) and structural (red) measured variables projected on the ordination as nonlinear surfaces for indirect relation to the ordination axes.(TIF)Click here for additional data file.


Human monocytes/macrophages play a central role in the immune response and defense of the host from influenza virus infection. They classically act as antigen-presenting cells for lymphocytes in the context of an immune cell cluster. In that setting, however, monocytes/macrophages exhibit additional, unexpected, roles. They are required for influenza virus infection of the lymphocytes in the cluster, and they are responsible for lymphocyte apoptosis via their synthesis and expression of the viral neuraminidase. Surprisingly, human alveolar macrophages, expected to be among the first cells to encounter the virus, are not susceptible to direct infection by a human influenza virus but can be infected when the virus is complexed with an antibody. Such monocyte/macrophage responses to influenza virus challenge should be considered part of a very complex but quite effective defense, since the common outcome is recovery of the host with development of immunity to the challenging strain of virus.  Monocytes and macrophages are central to the development as well as expression of the human immune response and its defense against influenza A virus (IAV) infection. Such cells are resident in the respiratory tract ,2,3,4, aMonocytes/macrophages participate in both the expression of innate immunity and in the development of the adaptive immune response that is responsible for recovery from IAV infection. In the former role, they can be formidable producers of interferon ,11 and oEarly studies of human peripheral blood mononuclear cells  and lymphocytes) documented that IAV infection resulted in a depression ,15 or acIt is reasonable to ask why PBM accessory cell function is reduced for responses to non-IAV stimulation. One possible host defense-related reason may be that proliferating cells show greater replication of IAV proteins , and it Notably, the initiation of an immune response to IAV challenge involves the development of immune cell clusters between PBM and lymphocytes ,24. ThisExposure of human PBMC to IAV resulted in the expression of viral antigens by both monocytes/macrophages and lymphocytes , resultiFurther studies demonstrated that the immune cell cluster plays an important role in IAV infection of human lymphocytes, even as that structure also serves to support development of the adaptive immune response . LymphocAfter exposure to IAV in the presence of monocytes/macrophages, both infected and uninfected lymphocytes in the culture developed apoptosis . Fas-FasBoth depletion of monocytes/macrophages and depletion of cells expressing the IAV neuraminidase reduced the extent of lymphocyte apoptosis in the cultures . ApoptosMonocytes/macrophages can serve as required accessory cells for antigen-stimulated and mitogen-stimulated proliferative responses, and they also play a central role in the regulation of immune responses . AlveolaAM and PBM differed in their ability to enhance mitogen- and antigen-stimulated proliferative responses of purified autologous lymphocytes . PBM supMixing experiments were performed in which variable numbers of the two macrophages, PBM and AM, were co-cultured and assayed for support of SK-SD-stimulated proliferation of purified autologous lymphocytes . Again, IAV infection in vitro depresses accessory cell function of human PBM for lymphocyte proliferative responses, as described in Differences between human autologous PBM and AM in the response to IAV, described above, warranted further studies regarding the susceptibility of the AM to IAV infection. PBM, as expected, bound and internalized IAV and synthesized viral RNA and proteins. In contrast, AM showed no evidence of IAV infection despite equivalent exposure . The AM All of the studies discussed above examined human monocyte/macrophage  interactions with seasonal human strains of IAV. The studies are consistent with the concept that alveolar macrophages are immunosuppressive in contrast to the more pro-inflammatory nature of monocytes/macrophages recruited to the lung in response to an infectious challenge . In thisAlthough a human seasonal H1N1 virus caused an abortive infection in human alveolar macrophages, a highly pathogenic H5N1 virus from the 1997 Hong Kong outbreak replicated productively in the alveolar macrophages  and hypeOverall, current evidence indicates that highly pathogenic strains of IAV, such as the avian H5N1 and the 1918 pandemic virus (which was an avian virus), can replicate in human monocytes/macrophages, including alveolar macrophages, but most other IAV infections of monocytes/macrophages are abortive, or nonproductive, and do not result in the release of infectious progeny virions. It is not evident that any published studies of pandemic or avian IAV infections have examined the interactions of human monocytes/macrophages and lymphocytes that are described in Substantial insight regarding IAV infection pathogenesis has been derived from animal models of the infection. Animal models have allowed depletion of monocytes/macrophages, including alveolar macrophages, in order to demonstrate the importance of the cells during IAV infection of the host mouse ,39 or feMouse models have facilitated the examination of the effects of pregnancy on IAV-challenged alveolar macrophages  and on tSeveral caveats apply to murine studies as reflective of human infections. First, murine infections with human seasonal IAV strains, but not highly pathogenic avian strains such as H5N1 viruses or the 1918 human pandemic strain (an avian virus) or the H1N1 2009 human pandemic strain, require prior adaption to the species that are not naturally susceptible ,48. SecoHuman monocytes/macrophages exhibit several unexpected characteristics relating to IAV infection, as described above: (a) an immunofocusing of monocyte/macrophage function, whereby non-IAV-directed immune responses are depressed or suppressed concomitant with the development of active IAV-specific responses; (b) responsibility of the monocytes/macrophages, in the setting of the immune cell cluster of the developing antiviral response, not only for lymphocyte activation, but also for lymphocyte infection by IAV, as well as lymphocyte apoptosis arising from monocyte/macrophage expression of IAV neuraminidase; and (c) a surprising resistance of AM to direct infection by a human IAV.A recent report of sequential experimental H1N1pdm09 infection of volunteers indicated the possibility of re-infection with the same strain of IAV . In gene


Sarcopenia is a physiopathological process associated with aging, caused by reduction of muscle strength, muscle quality and physical performance, and associated with an increased risk of falls, physical disability and premature death. There is no effective treatment for sarcopenia, but physical exercise seems to be highly effective at counteracting the decline in muscle mass and strength associated with aging. Recently, sarcopenia has been recognized as an emerging issue in people living with HIV (PLWH). Despite adequate treatment with combination antiretroviral therapy (cART), PLWH may exhibit an early occurrence of some aging-related conditions, including sarcopenia, frailty and falls, and this is likely resulting from high rates of comorbidities, high-risk behaviours, chronic immune activation and cART-specific factors. In this review, we discuss the potential mechanisms and the clinical relevance of sarcopenia in PLWH, and present data from longitudinal studies of physical activity in this population. Despite none of these studies having specifically addressed the benefits of physical exercise on sarcopenia, there is evidence that exercise is effective to increase aerobic capacity and muscle strength, and to improve body composition and inflammatory outcomes in PLWH. Therefore, the expected benefits of physical exercise are likely to translate into a successful and specific intervention for prevention and treatment of sarcopenia in this population. Sarcopenia is defined by the progressive reduction of muscle mass, muscle strength and function occurring in the elderly and in people with chronic conditions, such as metabolic syndrome, cardiovascular disease or cancer . StartinSarcopenia is clinically relevant because it is associated with an increased frequency of adverse health outcomes, such as frailty, functional disabilities, falls and, in general, quality-of-life impairment ,6,7. TheSarcopenia is an emerging health issue in people living with HIV (PLWH). After more than three decades since the beginning of the AIDS epidemic, and following the introduction of combination antiretroviral therapy (cART), HIV infection has now become a chronic and manageable condition . ConsequThe aim of this review is to discuss the mechanisms and the clinical relevance of sarcopenia in PLWH, to describe the efficacy of physical activity interventions on muscle mass and function, and to provide evidence towards an effective treatment approach for sarcopenia in this population.A number of factors play a role in the development of sarcopenia in the general population, including lack of physical exercise, hormonal changes, nutritional deficiencies and low protein intake, metabolic alterations and chronic inflammation . Among tSarcopenia is characterized by a progressive loss of muscle fibres that are replaced by adipose tissue, increasing fibrosis and changes in muscle metabolism . SeveralAnother consequence of increased immune activation is the accumulation of macrophages and other immune cells in the adipose tissue, with an increased release of pro-inflammatory cytokines and adipokines . These iSome traditional risk factors may also specifically promote sarcopenia progression in PLWH, including a low vitamin D level  and factAccording to the original 2010 consensus of the European Working Group on Sarcopenia in Older People (EWGSOP) on the definition and diagnosis of sarcopenia  and the However, only few studies have reported the prevalence of sarcopenia as a syndrome resulting from the combination of the above measures in treated PLWH ,32,33. T+ T-cell counts at the start of cART and long-time exposure to certain nucleoside reverse transcriptase inhibitors (NRTIs), i.e., zidovudine, stavudine or didanosine [Some of these studies also identified specific risk factors for sarcopenia in PLWH, in addition to traditional risk factors such as female gender, increasing age and high body mass index. These included low education level and employment status, long duration of HIV infection, low CD4danosine ,33,34. Fdanosine .Sedentary lifestyle is one of the principal causes for loss of muscle mass and strength, which, in turn, determines further reduction of activity levels with further muscle weakness . In contIndeed, physical activity represents the most effective strategy in the management of sarcopenia in the general population and in specific patient groups . However2max) is the gold standard for measuring the integrated cardiopulmonary-muscle oxidative function, and studies using the 2max as fitness outcome have shown that and adequate physical exercise intervention invariably improves CRF in PLWH  dynamic strength: measure of the maximum weight that can be lifted once ; (ii) static strength: measure of the maximum force that one can apply to an unmoving object ; and (iii) muscular endurance: measured through multiple lifting repetitions using weights that are below one’s maximum capacity. Muscular strength and endurance improve when muscle fibres grow stronger and new muscles form, and when the supply of oxygen and energy to the muscles becomes more efficient. In PLWH, several studies have proven that adequate resistance training exercises is successful in improving muscular strength, as assessed by using all of the above approaches .In longitudinal studies of physical activity in PLWH, the combination of improved cardiovascular and muscular fitness was associated with other health outcomes with relevance on sarcopenia, including improvements in body composition and inflammatory markers. In particular, a number of studies reported a reduction in fat mass and an increase in fat-free mass, using either dual-energy X ray absorptiometry, computed tomography or bio-impedentiometry. These results were observed irrespectively of type of exercise , its duration and frequency (from 20 to 60 min for 2/3 times a week) and the duration of the study (from 6 to 48 weeks). A few recent studies have also investigated the effects of physical activity on inflammatory outcomes in PLWH. Most of these showed a reduction of soluble markers of inflammation, such as high-sensitivity C-reactive protein, Interleukin-6 and Interleukin-8 . AdditioPLWH may show a degree of musculoskeletal impairment and an increased risk of sarcopenia, likely resulting from a combination of HIV-associated chronic immune activation, metabolic complications and advancing ageing. Different physical activity interventions have shown to be effective to increase muscle mass and function in this population. However, no study has so far addressed sarcopenia as specific target of physical exercise intervention in PLWH. Nevertheless, physical activity, in its various blends, increases aerobic capacity and muscle strength and this is associated with improvement in body composition and inflammatory outcomes. The information from longitudinal trials of physical exercise is relevant in order to prescribe cost-effective tailored exercise interventions and protocols that focus specifically for prevention and treatment of sarcopenia in PLWH.


A key embryonic process that occurs early in ocular development is optic fissure closure (OFC). This fusion process closes the ventral optic fissure and completes the circumferential continuity of the 3-dimensional eye. It is defined by the coming together and fusion of opposing neuroepithelia along the entire proximal-distal axis of the ventral optic cup, involving future neural retina, retinal pigment epithelium (RPE), optic nerve, ciliary body, and iris. Once these have occurred, cells within the fused seam differentiate into components of the functioning visual system. Correct development and progression of OFC, and the continued integrity of the fused margin along this axis, are important for the overall structure of the eye. Failure of OFC results in ocular coloboma—a significant cause of childhood visual impairment that can be associated with several complex ocular phenotypes including microphthalmia and anterior segment dysgenesis. Despite a large number of genes identified, the exact pathways that definitively mediate fusion have not yet been found, reflecting both the biological complexity and genetic heterogeneity of the process. This review will highlight how recent developmental studies have become focused specifically on the epithelial fusion aspects of OFC, applying a range of model organisms  and utilizing emerging high-resolution live-imaging technologies, transgenic fluorescent models, and unbiased transcriptomic analyses of segmentally-dissected fissure tissue. Key aspects of the fusion process are discussed, including basement membrane dynamics, unique cell behaviors, and the identities and fates of the cells that mediate fusion. These will be set in the context of what is now known, and how these point the way to new avenues of research. Tissue fusion is an essential process in vertebrate development. It requires the growth and coming together of groups or sheets of cells, typically epithelia, and their joining to create continuous bodies with lasting structural integrity . Fusion The developmental formation of the eye is broadly conserved among vertebrates and requires a complex sequence of coordinated morphogenetic events  coloboma (OC) and manifests as open clefts at any point along the ventral PD axis of the eye . OCs canKnowledge of coloboma-gene function in eye development is well supported by animal models using knock-out or engineered disruptions to disease genes. The vast majority of these loci encode transcription factors or signaling molecules that drive the early phases of eye development, such as patterning and growth, thereby implicating these genes for roles in setting up the eye to be competent to fuse . In manyMuch of our current knowledge of OFC progression or coloboma causation has come from studies in fish, avians, and rodents, with the vast majority of these most recently carried out using zebrafish or mouse . These hMouse studies of specific gene functions predominated when gene knock-out technologies or mutagenesis screens were developed, but these were largely superseded through the use of morpholino gene knock-down, and latterly gene-editing approaches in zebrafish. In this context these genetically tractable species have been vitally important to determine the conserved patterning events at the ventral retina and optic stalk, and the proportional growth of the optic cup that occurs to facilitate the correct apposition of the OF margins prior to the onset of fusion . They hain ovo development of chick embryos lends itself to experimental and transient genetic manipulations , it has lagged far behind zebrafish and mouse for germline or high-throughput genetic studies. Coincident with the emergence of gene editing, major improvements have been made in transgenic biotechnology, potentially enabling rapid and cost effective generation of transgenic chick embryos  at the medial-proximal region of the optic cup, then extends bidirectionally along the P-D axis  6 (HH stage 28), and that closure involved simultaneous BM dissolution and cell mixing  was not observed in these cells  and their fate in the fused margins, and whether these are conserved across all species. It will also be important to reveal their origin, and how and when during eye development they are specified. Their identification has the potential to be transformative in OFC research, as they represent the emergence of a tractable system that can be manipulated to resolve fusion-specific mechanisms within the complex environment of the developing eye at unprecedented resolution.The architecture and molecular status of the epithelia in the area of fusion initiation is important in the context of accurately determining the underpinning biological mechanisms that mediate the process. In all species, neural retina cells occupy the inner  aspect of the edges of the pre-fusion fissure margin, whereas the RPE is positioned at the outer aspect  . In humad margin . This suse cells . Althougebrafish  and thiso along the sub-retinal space in fused tissue  and actin-remodeling factors (TWF1 and LCP1) have also been identified in patients with isolated colobomas , this may reflect limits of sensitivity in these assays, a tissue-specific EMT gene expression profile, or the degree of EMT-MET within the OFC process. As neither processes have been formally analyzed at the molecular level in any of the OFC model systems, it will be important to determine the contribution of EMT pathways and related molecules to fusion in this context, for example the localization and balance of E- and N-cadherins, and the specific suite of integrins expressed in pioneer cells and within the fusion plate and fused retina.The changes to cell shape and apical-to-basal polarity during OFC are reminiscent of epithelial-mesenchymal transition (EMT) , which hTransforming growth factor beta (TGFβ) signaling has a prominent role in the regulation of EMT processes in multiple developmental and disease contexts  and mutafadd) gene and maintain the balance between cell death and proliferation. In this context, it was the necrosis pathway rather than the apoptotic pathway that controls cell death. Deletion of FADD, a key regulator of apoptosis, is suggested to cause coloboma in humans  are highly cross-linked and dense sheet-like structures of extracellular matrix molecules that border all epithelia . The conin situ hybridization, and confirmed the expression of collagen IV a1 and a2; laminin a1, a4, b1a, c1, and c3; nidogen 1a, 1b, and 2a; and perlecan during OFC. Of these genes, only nidogen expression was observed to be down-regulated at the onset of fusion, with nidogen protein becoming absent from the BM prior to laminin removal and fusion . SMOC proteins are localized to BMs in various developmental contexts  domains, contain laminin EGF-like domains, and can form heterotypic complexes with laminins and other laminin-interactors at cell surfaces, such as integrins and dystroglycans . The disolobomas . These aolobomas . Therefocontexts , 2003, acontexts . SMOC1 i studied . However studied .in situ hybridization studies  and provided the first available list of relative gene expression levels in the fissure margins coincident with fusion. It took another 10 years before the use of RNA sequencing was similarly applied to provide unbiased and fully quantitative gene expression during OFC, in this case with zebrafish and chick OFs , STRA6 and SMOC1 were found to among the genes that were significantly enriched in both chick and zebrafish datasets at the OF during fusion , and Netrin-1. Cadherins are important for epithelial cell-cell adhesion via adherens junctions, whereas Integrin clustering and integrin-mediated cell-anchoring to fibronectin-rich basement membrane at fusion sites were recently been shown to be essential during neural tube closure (Netrin-1 (NTN1) gene was one of the highest expressed and most fissure-specific gene throughout the fusion process in both studies. Ntn1 had also been previously identified as up-regulated in mouse OF  has been developed for the detection and characterization of spatial abundance patterns for a range of molecule classes in a wide spectrum of tissues. These include small molecule metabolites, lipids, and proteins, and even histone modifications, that can be visualized directly in histological sections of tissues . AdvanceBcl2 or adding caspase-inhibitors in the OF (The requirement for apoptosis or cell death in OFC is still unclear. The available evidence suggests that it is required to maintain an appropriate cell number in the ventral eye, but only in chick eyes are apoptotic foci clearly observed in the fissure margins during normal fusion . It wouln the OF . SimilarThrough the recent advances described in this review and the promises of powerful tools to augment targeted research, it is now reasonable to predict that the mechanisms for fusion during OFC will be largely elucidated in the near future. Once the main characteristics of this process have been determined at the genetic, molecular and cell-behavioral level, it will be necessary to use this information to determine the other causes of fusion defects—the contribution of environmental factors such as vitamin or nutrient deficiency, maternal illness, or substance abuse . Having BC and MM contributed to the content and edited the manuscript. JR conceived and wrote the manuscript and prepared the figures. 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.


Oral diseases are highly prevalent and a global burden. Accordingly, their prevention appears essential. Recently, different strategies have been developed, mainly focusing on the presence of singular oral diseases or conditions. This article aims to construct a contemporary concept of individualized preventive care in dentistry whereby the focus is switched from viewing oral health in isolation to viewing the patient as a whole. The basis for individualized prevention measures is the case-oriented profile, including the synthesis of risk- and need-oriented parameters. The risk profile comprises different risk factors within the fields of systemic diseases, medications, and lifestyle that inherently pose a potential risk of complications  and/or oral diseases . The needs profile includes factors originating from the aspects of oral diseases, dental restorations/appliances, and dental results with a potential risk of pathogenesis  and/or the potential progression of oral diseases . Based on these parameters, the general framework and content of prevention measures, as well as the maintenance interval, should be adapted to the individual patient. The implications of this concept might increase the safety, effectiveness, and efficiency of prevention in dental care. A further area of focus is primary prevention, that is, a focus on the preservation of oral health instead of a disease-related approach. However, clinical validation is needed to prove the benefits of the model presented. Individualized prevention promotes a shift from a disease-focused model to a whole-patient-focused model and provides a potential approach for establishing a contemporary concept for preventive care in dentistry. Oral diseases are one of the most prevalent diseases in the world, resulting in an enormous global health problem . The incAnother aspect that is often insufficiently addressed within dental prevention is the presence of general diseases and conditions. Various links between oral and systemic health are known; e.g., the bidirectional relationship between periodontitis and diabetes has already been well described . HoweverAccordingly, a contemporary understanding of prevention in dentistry must include and combine the aspects of oral disease-specific, general health-specific, and individual patient-specific aspects. Novel concepts must be assessed in different dimensions, such as “effectiveness,” “efficiency,” and “safety,” as required in the context of personalized medicine .This narrative review aims to construct a contemporary, individualized prevention concept, including different aspects of oral and systemic diseases and conditions. The definitions and classifications of risk and need factors presented here should allow the current approach to be adjusted to focus on prevention from the perspective of the patient as a whole and not from the perspective of singular oral diseases or conditions. This paper only focuses on prevention and is not exhaustive but should illustrate a comprehensive concept of individualized preventive care.The basis of patient-centered dental care is the understanding of the complex and individual needs, risks, and perceptions of a patient. Before appropriate risk and need classifications can be applied, a sufficient doctor-patient or dentist-patient relationship remains a mandatory prerequisite; this includes the integration of the patient's values and preferences in the context of shared decision-making . This derisk factor, which is defined by the World Health Organization (WHO) as an “attribute, characteristic or exposure of an individual that increases the likelihood of developing a disease or injury” [risk of complications and a risk of oral diseases. The risk of complications includes the increased likelihood of systemic complications during or following prevention measures, e.g., the risk of infectious complications (endocarditis and sepsis) after professional tooth cleaning. The risk of oral diseases is the increased likelihood of the occurrence of an oral disease or condition caused or influenced by a general health condition. The increased risk of periodontitis in patients suffering from different general diseases, such as diabetes or rheumatoid arthritis, can be considered in this context [risk profile  and “effectiveness”  should be addressed. A  injury” , provide context , 18–22.  profile .Different systemic diseases can be seen as risk factors. In the context of the risk of complications, on the one hand, the risk of systemic infections related to bacteremia caused by prevention measures exists , 23–25. Moreover, the risk of oral diseases can be influenced by systemic diseases and conditions. Depending on glycemic control (HbA1c: <7% or ≥7%), diabetes mellitus has an influence on periodontitis; accordingly, diabetes mellitus is a parameter for the grading matrix of periodontitis , 29, 30.Alongside, or combined with, systemic diseases and conditions, the intake of different medications entails potential risk factors. For example, systemic infections within the potential complications can occur in relation to immunosuppressive medications . AnotherLifestyle choices contribute to the complexity of different risk factors within risk-oriented prevention. However, lifestyle parameters rarely affect the risk of complications. Alcohol and/or drug abuse can influence the immune system and might be related to complications during dental therapy and prevention . HoweverSimilar to the risk factors within risk-oriented prevention, oral conditions include specific need factors. These need factors describe oral health-related conditions, which can include both a risk of pathogenesis and a risk of oral disease progression. The risk of pathogenesis describes the increased likelihood of the de novo development of an oral disease due to a specific need factor. The origin of root caries on the exposed root surfaces of periodontally diseased teeth is an example of an increased risk of pathogenesis . The risThis field includes the patient's oral health history. Primarily, the preservation of oral health, as defined by the FDI , should Similarly, the new classification of periodontal diseases and conditions with a staging and grading matrix describing the stage of the periodontal burden as well as the risk of progression , 30, comThe complexity becomes clear when caries and periodontitis are linked within the needs profile: a higher stage of periodontitis can be related to an increased risk of pathogenesis for root caries on exposed dentin areas . In addiDifferent aspects of need-oriented prevention are related to the presence of dental restorations, prosthodontic treatment, or appliances in the oral cavity. Tooth-borne restorations (fixed and/or removable) require a maintenance regime , as therThis field has the largest dynamic within the needs profile and represents the diagnosis following a clinical assessment of oral tissues. However, the targeted assessment of dental and periodontal findings as well as oral hygiene indices are the most important measures for adequately estimating the risk of pathogenesis and the progression of oral diseases. In particular, the accumulation of dental biofilm (plaque) on tooth surfaces and gingival inflammation must be seen as important parameters in the etiopathogenesis of caries and periodontitis , 58. MorThe synthesis of risk-oriented and need-oriented prevention into one model is necessary to fulfill the demand for a contemporary, individualized prevention concept. Consequently, the four risks defined above, including the risk of complications and risk of oral disease within specific risk factors as well as the risk of pathogenesis and risk of progression within corresponding need factors, lead to a case-specific patient profile . To alloThe concept of individualized prevention as presented in this manuscript combines patient-specific risk- and oral-health-specific needs profiles and shifts the focus from oral diseases/tooth-related parameters to the whole patient. The joint prevention of different oral diseases, including caries and periodontitis, considered together is not, however, a new approach , 60. MorFurthermore, some of the diseases and conditions listed above also include a risk of oral diseases. Oral diseases, especially caries and periodontitis, are multifactorial , 4. AvaiThe second main aspect of the individualized prevention concept is that of the patient's needs profile. Recent strategies have primarily focused on arresting/stabilizing disease processes such as caries and/or periodontitis and halting the progression of the existing oral disease , 6. AlthThe dynamic aspect within the needs profile is the assessment of current dental findings. Basic diagnostics , especially including oral hygiene indices, should be applied , 58. DepFurthermore, the dimension of “efficiency” can also be recognized because unnecessary diagnostic and prevention measures might be avoided with a case-oriented adaptation of the prevention cycle. Prevention in dental practice should follow a structured setup . Each seThis manuscript primarily focused on the singular consideration of different risk factors; some of these factors might be additive, but they may also be synergistic, influencing health or diseases more than the added weight. Accordingly, available risk models combining different indicators should be considered for future risk assessment and classification approaches. These factors could include, e.g., the interrelation between cardiovascular and periodontal health with a modified risk ratio affected by smoking, diabetes, C-reactive proteins, interleukins level, and other factors , as wellHowever, several limitations can be listed. On one hand, cost-effectiveness is an important topic related to maintenance therapy , 84. TheA contemporary, individualized prevention concept for dental maintenance should recognize the whole patient within a case-oriented profile. At the same time, the risk of complications and/or oral diseases originating from systemic diseases, medications, and lifestyle choices must be included. Furthermore, the risk of pathogenesis and/or progression of oral diseases based on the assessment of existing oral diseases, dental restorations/appliances, and recent dental results must be determined. This might allow safe, effective and efficient prevention based on the patient's individual risks and needs within an individual prevention cycle. Clinical evaluation of the described construct is necessary to prove its practical benefits.


Atherosclerosis is associated with acute cardiovascular conditions, such as ischemic heart disease, myocardial infarction, and stroke, and is the leading cause of morbidity and mortality worldwide. Our understanding of atherosclerosis and the processes triggering its initiation is constantly improving, and, during the last few decades, many pathological processes related to this disease have been investigated in detail. For example, atherosclerosis has been considered to be a chronic inflammation triggered by the injury of the arterial wall. However, recent works showed that atherogenesis is a more complex process involving not only the immune system, but also resident cells of the vessel wall, genetic factors, altered hemodynamics, and changes in lipid metabolism. In this review, we focus on foam cells that are crucial for atherosclerosis lesion formation. It has been demonstrated that the formation of foam cells is induced by modified low-density lipoprotein (LDL). The beneficial effects of the majority of therapeutic strategies with generalized action, such as the use of anti-inflammatory drugs or antioxidants, were not confirmed by clinical studies. However, the experimental therapies targeting certain stages of atherosclerosis, among which are lipid accumulation, were shown to be more effective. This emphasizes the relevance of future detailed investigation of atherogenesis and the importance of new therapies development. Atherogenesis is a complex process that involves numerous cellular mechanisms and pathways, such as inflammation, lipid accumulation, oxidative stress, and others. Our understanding of the processes underlying atherosclerosis is constantly improving. Over the past decades, clinical and basic research provided evidence of the immune system involvement at all stages of atherosclerosis, from lesion initiation and progression to the advanced plaque formation and destabilization precipitating acute clinical symptoms.Atherosclerosis is the underlying condition of most of the cardiovascular diseases (CVD) that remain the leading cause of adult morbidity and mortality worldwide ,2. AmeriAccording to current understanding, both resident blood vessel wall and circulating immune cells are involved in atherosclerosis progression. Furthermore, such factors as genetics, hemodynamics of the blood flow and plasma lipoproteins have an impact on the disease progression, and the interplay of aforementioned aspects may form atherosclerotic clinical pattern ,5.In this review, we focus on foam cells that are crucial for atherosclerosis lesion formation. It has been demonstrated that the formation of foam cells (cellular lipidosis or cellular lipid retention) is induced by modified low-density lipoprotein (LDL) .The concept of the inflammatory nature of atherosclerosis pathogenesis promoted testing of a range of drugs for anti-atherosclerotic activity. Anti-inflammatory and antioxidant drugs were investigated for their potential to reverse or at least slow down the atherogenic processes. Many of them, however, failed clinical trials. For example, methotrexate could not reduce cardiovascular events , beta caBy contrast, some promising results were obtained in the CANTOS (Canakinumab Anti-Inflammatory Thrombosis Outcomes Study) clinical trial that demonstrated the efficacy of canakinumab (150 mg), a monoclonal antibody that inhibits inflammation by neutralizing Interleukin-1β, for reducing the primary endpoint: non-fatal myocardial infarction (MI), stroke, or cardiovascular death . Thanks The experimental therapies targeting certain stages of atherosclerosis, among which are lipid accumulation, were shown to be more effective than unspecific anti-inflammatory treatments. This emphasized the relevance of future detailed investigations of atherogenesis and the importance of new therapies development .Foam cells are characterized by multiple lipid inclusions in the cytoplasm that gives them a specific look reflected by the commonly used name. The emergence of the foam cells in the arterial intima is considered to be one of the earliest manifestations of atherosclerosis, referring to the preclinical atherogenesis. Moreover, the underlying extra- and intracellular lipid deposition is a crucial trigger of atherosclerotic lesion development ,17.It has long been considered that all foam cells of human atherosclerotic lesions derive exclusively from macrophages and monocytes. This misconception was based on the results coming from studies on murine models, in which macrophages represent a prevalent source of foam cells. However, studies of human arteries demonstrated the prominent role of resident cell types present in the arterial wall, among which are vascular smooth muscle cells (VSMCs) and pericytes, or stem/progenitor cells (SPCs) ,19. The Internalization of lipid particles by macrophages in the subendothelial space is mediated by a range of scavenger receptors (SRs), each of which complies with an exact LDL modification. For example, receptors of the SR-A class, SR-AI and SR-AII, have a strong affinity for acetylated LDL and extensively oxidized LDL . NoteworAtherogenesis is associated with serious disruption of lipid metabolism, including both increased cellular cholesterol uptake and reduced cholesterol efflux. Potential causes of these alterations may be represented by upregulation of the SRs that mediate modified LDL binding. By contrast, inhibition of the expression of cholesterol pumps results in normalization of cholesterol efflux ,27. ThisNewly formed foam cells accumulate in the arterial intima and initiate the formation of atherosclerotic lesions, which at the early stages are called fatty streaks . This prEven though both macrophages and SMA-positive cells are able to internalize native LDL, the majority of lipids taken up during foam cells formation are coming from modified LDL. Studies in cultured cells indicated the inability of native LDL to cause intracellular accumulation of lipids and the consequent formation of foam cells, while significant accumulation of intracellular lipids was observed when native LDL was replaced with in vitro chemically modified lipoprotein particles ,34.Oxidized LDL (oxLDL) has long been considered to be the only type of modified LDL that is significant for atherogenesis, and indirect signs of oxLDL presence in the blood of atherosclerotic patients supported this suggestion . HoweverA hypothesis of multiple modifications of the same LDL particle was formulated based on the properties of several LDL types that could be isolated and characterized by physical-chemical methods. At least three modified atherogenic LDL forms have been observed, namely small dense LDL, electronegative or LDL(–), and desialylated LDL, each of which was shown to possess specific features, including susceptibility to oxidation ,38.Noteworthy, LDL oxidation occurs at the very end of the modification cascade, which is confirmed by the experiment in which LDL isolated from healthy individuals was incubated with plasma-derived serum obtained from atherosclerotic patients. After 1 h of incubation of this mixture at 37 °C, LDL became atherogenic, and presence of desialylated LDL was observed. After 6 h, lipid content and particle size appeared to decrease and particles became more electronegative. Only after incubation for 48–72 h, a decrease of the antioxidant content and increase of LDL susceptibility to oxidation was observed . Several mechanisms explaining the atherogenic properties of modified LDL have been described, including: (1) self-association of LDL particles; (2) LDL association with the extracellular matrix components; and (3) formation of LDL-containing immune complexes. It was shown that desialylated LDL is characterized by the enhanced cellular uptake of particles characterized by a slower degradation rate within the cells. That may explain the increased atherogenicity of this type of modified LDL. Association of modified LDL particles with collagenase-resistant arterial debris, collagen, elastin, and proteoglycans was shown in human aortic intima. This also promoted cholesterol accumulation in cultured cells. On the contrary, isolated electronegative LDL showed no ability to promote foam cells formation by itself, but its atherogenicity was enhanced by the high susceptibility to association . Another interesting type of complexes isolated from the blood of atherosclerotic patients is represented by LDL-containing circulating immune complexes that promote cholesterol accumulation in cultured cells more potently than multiple modified LDL alone. Moreover, the atherogenic effect of these complexes was also increased by their ability to induce proinflammatory cytokines secretion and promote apoptosis in macrophages ,42.One of the most intriguing aspects of foam cells formation is the cellular signaling and interaction of genes that is especially important because of its potential significance for identification of novel diagnostic markers and therapeutic targets.Currently, signaling pathways involved in lipid accumulation are well known and have been thoroughly reviewed ,44,45,46It is well established that Toll-like receptors (TLRs) signaling plays an important role in atherosclerosis pathogenesis and foam cell formation . TLR2 acA recent study has reported 10 master-regulator genes that could be the main regulators of the foam cells formation: IL7R, TIGIT, ITIM, CXCL8, F2RL1, EIF2AK3, IL7, TSPYL2, ANXA1, DUSP1, and IL15 . NotablySeveral recent studies investigated the role of mollified LDL ,54. NatiThe obtained results indicated that common pathways may exist between latex beads and modified LDL effects. Such pathway could be stimulation of phagocytosis. As is known, the stimulation of phagocytosis is a trigger for the proinflammatory response of the macrophage, which is accompanied by the secretion of proinflammatory cytokines. Perhaps the identified common signaling pathways for latex beads and associates of modified LDL taken up by phagocytosis can be involved in the proinflammatory cellular response see .A recent study revealed the genes encoding inflammatory molecules that were up-regulated upon treatment of macrophages with atherogenic modified LDL and were involved in the development of inflammation . FurtherThe latter possibility was explicitly confirmed by another study. Some interleukins, including IL-34 and IL-32, were recently shown to be related to foam cells formation. Liu et al. in 2018 demonstrated that IL-34 can act as a promotor of the lipid accumulation in bone marrow-derived macrophages through the up-regulation of CD36 expression . EarlierPrimary human monocyte-derived macrophages were cultured in serum-supplemented medium with cytokines and multiply-modified LDL isolated from atherosclerotic patients. After 24 h, cellular lipids were extracted and total cholesterol was measured.According to the revised hypothesis, the proinflammatory response could be an inductor of the intracellular lipid accumulation  57]. Th. Th57]. During recent years, many aspects of atherosclerosis development have been revised, improving the general understanding of the disease. First, atherosclerosis was found to be more complex than just a chronic inflammation of the vessel wall in response to damaging stimuli, which was supported by the growing body of evidence about the significance of genetic and metabolic factors.It was shown that macrophages are not the exclusive source for foam cells formation. Pericytes and VSMCs were also shown to have the ability to transform to foam cells. Moreover, studies of modified LDL helped formulate the hypothesis of the modification cascade that provides a new model of multiple modifications and reduces the significance of oxLDL, which is formed at the end of the modification cascade.However, perhaps the most crucial update in our understanding of atherogenesis is the conception of the proinflammatory response as an inductor of intracellular lipid accumulation, which appears to be the cause rather than a consequence of atherosclerosis progression. The most recent research on signaling pathways and identification of master-regulator genes helped transform the conception of the consequence of the events that lead to intracellular lipid accumulation. This consequence now has to be presented as follows: associates of modified LDL interact with cells and thus stimulate phagocytosis, which, being the initial event of the innate immunity reaction, induces the release of proinflammatory cytokines that, in turn, cause or enhance intracellular lipid accumulation.Inflammation and lipid accumulation are considered to be key processes of atherosclerosis development. Thus, the correct causal link between these processes needs to be established. All listed updates have the potential to become a basis for effective therapeutic approaches, but further research is needed. The most promising strategy for the development of effective treatment is targeting of particular signaling pathways that underlie one of the crucial stages of the disease. This is the most straightforward approach that leads to identification and design of both effective and safe compounds for atherosclerosis treatment.


The forthcoming fifth-generation networks require improvements in cognitive radio intelligence, going towards more smart and aware radio systems. In the emerging radio intelligence approach, the empowerment of cognitive capabilities is performed through the adoption of machine learning techniques. This paper investigates the combined application of the convolutional and recurrent neural networks for the channel state information forecasting, providing a multivariate scalar time series prediction by taking into account the multiple factors dependence of the channel state conditions. Finally, the system performance has been analyzed in terms of prediction accuracy expressed as absolute deviation error and mean percentage error, in comparison with an alternative machine learning method recently proposed in the literature with the aim at solving the same prediction problem. The emergence of the need for new network technologies and architectures has been launched by the diffusion of novel services and applications demanding high data rates and reliability, low latency, and congestion levels. Augmented reality, automatic driving, and tactile internet are only a few of the examples of fifth-generation (5G) applications. Furthermore, new era applications require remarkable improvements with regards to the intelligence core of the network, giving rise to self-organizing ecosystems able to contextualise awareness in all its different forms and facets. In fact, one of the most challenging issues in 5G networks is the empowering of the learning processes, recreating on devices actual human neural network behavior to obtain effective solutions to new era networks problems.From previous network generations, the analysis and design of innovative technologies and methodologies to move towards wireless systems able to optimally exploit radio resources and actuate proper decision policies has gained attention. During the years, cognitive radio (CR) approaches have played a crucial role, aiming at designing context-driven strategies. In this regard, the advent of the 5G networks, which involve the usage of the millimeter wave (mmWave) band has resulted in a large variety of obstacles such as pathloss, blockage, and high oxygen absorption . All theGenerally speaking, the channel prediction is an impressive approach to obtain accurate CSI values. Differently, during years, many CSI estimation methods have been proposed , from thTypically, these methods involve matrix operations as the matrix inversion or the eigenvalue decomposition, resulting in procedures characterized by high computational costs. Due to such hardness in channel condition knowledge or the presence of remarkable overhead costs associated with the traditional channel state estimation methods, the design of novel strategies has become a necessity in reference to upcoming 5G networks. In fact, 5G environments have to face highly complex scenarios, in which CSI prediction is a large-scale channel problem, due to the presence of massive multiple-input-multiple-output (MIMO), orthogonal frequency division multiplexing (OFDM), and mmWave communications . As consRecently, the machine learning (ML) techniques have gained attention for their application in numerous research fields such as channel prediction , detectiThis paper proposes the application of the ML principles to the CSI prediction, by formulating that problem in terms of scalar time series forecasting .Overall, the class of the prediction problems can be classified into short-, medium-, and long-term forecasting. In the first one, the predictions are performed over horizons from a few minutes up to a few days ahead, and the second one deals with time horizons from few days to few months ahead. Finally, predictions from quarters to years ahead  are nameDesign and development of an ML-based framework involving the usage of the convolutional neural networks (CNNs) and the recurrent neural network (RNN) to predict the CSI behavior;Extensive numerical simulations to provide the performance evaluation analysis as regards the prediction accuracy expressed as mean absolute deviation error and mean percentage error between the actual CSI value and those predicted;Testing of the proposed framework under different application scenarios, i.e., outdoor and indoor conditions, in comparison with the deep learning strategy proposed in  and thatThe rest of the paper is organized as follows. The CSI prediction problem has been largely studied and, during the years, different approaches have been pursued. Recently, the involvement of ML techniques for estimating the CSI has gained momentum. Authors in  address Differently, in the paper  highly mG represents the product of the antenna field radiation pattern 16]. As i


The retina is a highly metabolically active tissue with high-level consumption of nutrients and oxygen. This high metabolic demand requires a properly developed and maintained vascular system. The retina is nourished by two systems: the central retinal artery that supplies the inner retina and the choriocapillaris that supplies the outer retina and retinal pigment epithelium (RPE). Pathological neovascularization, characterized by endothelial cell proliferation and new vessel formation, is a common hallmark in several retinal degenerative diseases, including age-related macular degeneration (AMD). A limited number of studies have suggested that microglia, the resident immune cells of the retina, have an important role not only in the pathology but also in the formation and physiology of the retinal vascular system. Here, we review the current knowledge on microglial interaction with the retinal vascular system under physiological and pathological conditions. To do so, we first highlight the role of microglial cells in the formation and maintenance of the retinal vasculature system. Thereafter, we discuss the molecular signaling mechanisms through which microglial cells contribute to the alterations in retinal and choroidal vasculatures and to the neovascularization in AMD. The retina is the nervous tissue that sits at the back of the eye and is responsible for converting light into electrical signals and sending these on to the brain for visual recognition. Retinal development is a long and complex process that, in humans, begins during the fourth week of embryogenesis and continues into the first year of life . During Microglia, the resident immune cells of the retina, are not derived from retinal progenitors cells but are derived from primitive yolk sac progenitors , and theWith approximately 4.5 million cone and 90 million rod photoreceptors, the human retina is the most metabolically expensive tissue in the human body. The retina is fed nutrients and oxygen from a unique dual blood supply that divides the retina into outer and inner layers . DysreguThe retina has the particularity of being the only tissue in the human body for which deep vasculature can be visualized directly and in a non-invasive way using, for example, optical coherence tomography angiography (OCTA), presenting a unique opportunity to study vascular alterations in eye diseases ,18.Several studies demonstrated the importance of microglial cells in retinal development and degeneration. However, recently, some studies suggested that microglia also play an important role in the development of retinal vasculature ,20,21,22The development of the vasculatures of the embryonic and fetal human eye is an orchestrated and synchronous process that is dependent on the demand for oxygen. The choroidal vasculature is the first to develop followed by the hyaloid vasculature and then the retinal vasculature .The choroid is a thin, highly vascularized and pigmented tissue positioned under the neural retina that constitutes the posterior part of the uveal tract . The inner limit of the choroid is the Bruch’s membrane on which the retinal pigment epithelium (RPE) lies . The vasThe main function of the choroid is to nourish the outer retina, RPE, the foveal avascular zone and the optic nerve , maintaiThe development of human choriocapillaris is unusual when compared with other capillary beds in the body . ChoriocAlterations in choroidal structure or impaired blood flow result in degenerative changes and neovascularization, such as choroidal coloboma and AMD ,32. The Retinal blood vessels are organized in two planar layers that are restricted to the inner layers of the retina. The central retinal artery enters the eye through the centre of the optic nerve. The artery then branches in the inner retina to form three capillary layers. The retinal vessels provide blood to the inner two-thirds of the retina . The retEarly in eye development, the oxygenation of the retinal tissue is provided by the vascular networks of the choroid and hyaloid. Hyaloid vessels progressively regress by apoptosis while the development of the retinal vasculature occurs on an astrocyte scaffold . FailureAngiogenesis is the process by which proliferating endothelial cells form new vessel sprouts and extend the vascular network from pre-existing vessels. Vasculogenesis is defined as the de novo formation of blood vessels from isolated vascular endothelial precursor cells that coalesce into cords and then form a lumen. Historically, there has been controversy regarding the development of retinal vessels. Some authors support that retinal vasculature is formed by angiogenesis ,36,39,40164 isoform  leads to leukocyte adhesion in pathological, but not the physiological, neovascularization [164, and in combination, may be sufficient to promote normal physiological neovascularization. Despite the high expression of VEGF by retinal ganglion cells in the developing macula [Non-endothelial cells, such as astrocytes and retinal ganglion cells, provide key molecular and structural cues for the developing retinal vasculature. Astrocytes, retinal ganglion cells and cells in the inner nuclear layer express vascular endothelial growth factor (VEGF) . In partrization . The comrization ,51, suggg macula , the migg macula . This feg macula . Axon gug macula .In the retina, vascular patterning is essentially dependent on astrocytes in physiological conditions . AstrocyMicroglia are the resident innate immune cells of the central nervous system with crucial functions during development, and in normal and pathological conditions . In the Microglia, present at sites of endothelial tip cell anastomosis, stimulate vessel sprouting. However, physical contact between microglial and endothelial cells is not essential for the angiogenic stimulatory effects of microglia, suggesting that the effects of microglial cells on retinal vasculature are mediated via soluble factors secreted by these cells. Indeed, microglia secrete soluble factors that shape vascular growth and branching , such asIL-1β, IL-6 and TNF, implicating microglia as the cellular player by which perinatal inflammation causes visual deficits [Interestingly, systemic inflammation in the neonatal period is known to impair vessel development by decreasing vessel extension, reducing capillary density and inducing localized overgrowth of abnormal retinal vessels . In addideficits .Age-related macular degeneration is a leading cause of vision loss among the elderly population in developed countries . The gloAge-related macular degeneration is a leading cause of vision loss among the elderly population in developed countries . The gloAlthough drusen biogenesis is not fully understood, some authors have suggested that drusen result from the RPE or choriocapillaris damage. The specific mechanisms that connect RPE and choroidal endothelial cells pathology and drusen formation may include oxidative injury from light exposure or systemic factors, like compounds associated with smoking, lipofuscin accumulation, complement activation, Bruch’s membrane-induced dysfunction and ischemia ,82,83,84Besides subretinal drusenoid deposits found in AMD, several histopathological studies reported the presence of yellowish lesions in the fundus, which can be viewed using blue light. Although these reticular pseudodrusen have some similarities in their composition compared to the subretinal deposits, such as the presence of vitronectin, complement proteins, apolipoprotein E and unesterified cholesterol, they lack immunoreactivity for protein markers of RPE, Müller glial and photoreceptor cells ,94. InteWith the help of OCTA, that allows the study of retinal and choroidal microvasculature and en face visualization of the blood flow at different anatomic retinal layers, without the need for the dye injection , it is bWith distinct approaches, several studies demonstrated the association between choroidal vascular changes with ageing and early AMD. Fluorescein angiography showed a prolonged choroidal filling phase in patients with early AMD , which iIn the case of neovascular AMD, the disease is hallmarked by choroidal neovascularization and associated manifestations such as pigment epithelial detachment, retinal pigment epithelial tears, disciform scarring and intraretinal hemorrhages . The choIn normal retinas, the continuous surveillance for the detection of noxious stimuli is performed by microglia, which are mostly confined to the plexiform layers where they exhibit complex ramified processes sensing the local retinal microenvironment ,119. The2O3) and peroxynitrite (ONOO−), can be generated in the presence of concomitant oxidative stress [Several mechanisms are known to be involved in endothelial dysfunction in the retina and choroidal neovascularization in AMD, such as oxidative stress and chronic inflammation . Nitric e stress . This cae stress ,126. Oxie stress ,130,131.The retina is particularly prone to oxidative stress since it is the most oxygen-consuming tissue in the body  and mostIn an environment of oxidant stress, as it occurs in aged RPE, production and accumulation of advanced glycation end product (AGEs) are enhanced, as well as, activation of AGEs receptors (RAGE) , which aIt is known that drusen components, such as Aβ peptide 1–40 may be responsible for the increased expression of inflammatory molecules and inflammasome components in the retina and RPE in AMD. Furthermore, several cytokines ) have an important role in CNV . A recenThere is substantial evidence suggesting that changes in microglial cells are not merely associated with secondary phenomena in AMD. Besides the contribution of RPE dysfunction to the formation of the subretinal drusenoid deposits, an alternative or concurrent mechanism for drusen genesis has been proposed; an impaired recruitment of macrophages through a CC chemokine ligand 2 (CCL2) and CC chemokine receptor 2-dependent (CCR2-dependent) pathway from the choroidal circulation may hamper the clearance of age-related accumulation of debris in Bruch’s membrane .+ macrophages/microglia in the retina and choroid in AMD eye sections. These data are in agreement with observations that macrophages and microglia recruitment in the macula are strongly associated with both early and advanced AMD [Both CCL2- and CCR2-dependent macrophage recruitment plays a crucial role in the development of experimental CNV, and ocular-infiltrating macrophages present a direct angiogenic ability . Based onced AMD .−/−) [−/− mice present retinal degeneration, with photoreceptor cell death, upregulation of TNF and breakdown of the outer blood-retinal barrier. Moreover, these animals exhibit drusen-like deposits and the presence of Aβ and Cryab, two abundant proteins that are present in Bruch’s membrane and choroidal tissues of AMD patients [In AMD the complement system activation is compromised at the level of retinal microglia/macrophages, thus contributing to the onset and progression of AMD. Deposition of complement, including C3, in affected areas of RPE/Bruch’s membrane, is associated with the expansion of atrophic lesions. Interestingly, intravitreal injection of small interfering RNA (siRNA) can suppress the local production of C3 by macrophages, increasing retinal complement activation and degeneration . Complem−/−) . Aged CXpatients ,168. Aβ patients ,169.Disturbance of the innate immune system in AMD is associated with dysregulated complement and inflammasome activation and reactive microglia . Increasδ/δ mice) showed that CNV is almost totally prevented [+CD45+CD11b+, which represent circulating cells responding to the early stage of an experimental model of laser-induced CNV, and VEGFR1&2−Iba1−, which represent the microglia in the retina. However, it remains to be clarified whether the activation of the former is needed to recruit the latter to injured sites [Studies carried out on mice lacking the hypoxia response element (HRE) in the VEGF promoter (Vegfrevented . Althouged sites .In this review, we summarized the pieces of evidence supporting the contribution of microglial cells and microglial-driven inflammation to neovascularization. Since inflammation and neovascularization are closely related, several research teams tested if modulation of microglia activity might prevent retinal neovascularization.Ablation of microglial cells via PLX5622 administration, an inhibitor of the colony-stimulating factor-1 receptor, results in a faster decrease of the CNV lesion size , suggestWhile it is still difficult to be certain about the best strategy to modulate microglial cells and to prevent neovascularization, the treatment of neovascular AMD is currently performed by routine intraocular injections of anti-VEGF agents, and to some extent by photodynamic therapy and thermal laser . The widIn this review, we summarized the substantial research effort to identify and study new molecular pathways responsible for the vascular alterations observed in AMD. Although our knowledge is limited, it is becoming clear that the resident immune cells of the retina, the microglial cells, do mediate processes related to neovascularization in AMD. Evidence suggests that the contribution of microglial cells might be either indirectly through the secretion of pro-inflammatory cytokines or directly via the release of angiogenic factors. A better understanding of the role of microglia in neovascularization will allow us to develop new therapeutic modalities based on the modulation of microglia physiology and reactivity. It is then crucial to clarify how, when and for how long we should modulate microglial activity, and only new studies in innovative disease models will allow us to answer these questions.The development of groundbreaking innovations in diagnostic technologies, such as OCTA, that allows unprecedented high-resolution visualization of alterations in the choroidal and retinal vasculature, in combination with new therapeutic approaches, some of them based on the modulation of microglial cells, open new perspectives for early disease detection methods and treatment options thus allowing the prevention of macular degeneration and consequent vision loss.


Water is a matter of vital importance for all developed countries due to the strong impact on human health and aquatic, wetlands and terrestrial environments. Therefore, the monitoring of water quality is of tremendous importance. The enormous advantages that Surface-enhanced Raman scattering (SERS) spectroscopy offers, such as fingerprint recognition, multiplex capabilities, high sensitivity, and selectivity or non-destructive testing, make this analytical tool very attractive for this purpose. This minireview aims to provide a summary of current approaches for the implementation of SERS sensors in monitoring organic and inorganic pollutants in water. In addition, we briefly highlight current challenges and provide an outlook for the application of SERS in environmental monitoring. In recent years, water quality has become a critical concern of most developed countries due to the strong impact on human health and aquatic, wetlands, and terrestrial environments. The growth of human populations, the expansion of industrial and agricultural activities and climate change have been identified as the main threats to cause declining water quality. Therefore, actions for detection, identification, and quantification of pollutants and toxins in water are urgently required. Recently, different agencies such as the European Environment Agency (EEA) or Environmental Protection Agency  have established legal obligations to protect and restore the quality of water. For instance, the Water Framework Directive (WFD) is the most substantial and ambitious piece of legislation dealing with the protection, monitoring, and management of water quality . For WFD backed up by REACH regulation, which defines the chemical status by environmental quality standards of 41 priority substances.Scientific findings show that major water pollutants are complex mixtures of chemicals of different categories . Analytical determination of these pollutants is typically carried out by sampling, extraction, and separation of the chemical compounds from the aqueous matrix by high-performance liquid chromatography or gas chromatography, coupled to selective detection methods such as modern mass spectrometry techniques. Generally speaking, these methods have high sensitivity, good specificity, and outstanding precision. However, all of them require complex equipment and laborious operations, which may lead to inaccurate results because the water samples may undergo chemical and physical transformations. Also, there is an urgent requirement for sensitive detection methods which are simpler and portable for on-site analysis. Thus, the development of improved systems for environmental analysis has attracted a high interest in industry and the research community. Among them, surface-enhanced Raman scattering (SERS) has been extensively applied in various types of ultrasensitive chemical detection in a wide variety of fields ], by-products of natural or artificial processes , among others.Direct SERS analysis in natural and contaminated waters is often impaired by the non-specific co-adsorption onto metallic nanostructures of other species in the matrix solution. This can significantly increase the complexity of the vibrational assignment or even completely prevent the interaction with the target analyte, thereby decreasing the sensitivity of the detection assay. In order to circumvent these issues, Marino-Lopez et al.  developeThe feasibility for the detection of antibiotics in environmental aquatic samples has been addressed in recent studies  is a potential tool for ultrasensitive detection, the strong signal fluctuations at ultralow concentration regimes often limit its expectations as a quantitative analytical technique. Recently, de Albuquerque et al.  developeThe detection efficiency of pollutants in environmental aquatic samples can be significantly improved employing non-wetting phenomena to concentrate analyte molecules within SERS-active regions  have been explored as potential adsorbents for aqueous-phase sorptive removal of emerging environmental contaminants  and heavy metal  cations are major environmental contaminants. With the aim to quantify trace amounts of such possible contaminants, environmental monitoring has generated a need for innovative and improved approaches with ever-increasing sensitivity and selectivity for the detection of these hazardous chemical species. In general, oxyanions, especially those with moderate Raman cross-sections  can be detected by their vibrational signatures. In contrast, direct SERS detection of monatomic metal ions is more challenged due to their small scattering cross-section   and arsenite (As3+), which usually exist in the environment as 2+ ions with a detection limit of 0.2 ng/L. The analysis of the intensity changes of the SERS signal from polyaniline at 1,560 cm−1 was shown to be strongly dependent on the concentrations of Hg2+. Carbon nanotubes over a porous anodic alumina membrane were used for the trace detection of Hg2+, Cd2+, and Pb2+  was self-assembled onto the surface of a nanostructured gold substrate for Hg2+ sensing , and dimethylarsinic acid (DMAV) (see 2+ speciation into methylmercury (CH3Hg+), Guerrini et al.  for the detection of four common arsenic species, MAV) see . The comi et al.  fabricat2+ and Ag+ using triple Raman-encoded Au NP trimers with LODs of 3.4 and 0.92 ng/L, respectively   optical and chemical properties of SERS platforms should be further optimized; (ii) the reproducibility in the synthesis of the SERS substrates in different batches and from different labs is still an issue; (iii) the lack of standardized protocols does not facilitate the comparison of electromagnetic enhancement factors between different laboratories; (iv) most of these new sensing tools are validated only in the lab with model analytes characterized by high Raman cross-section and not actual pollutants; (v) despite the delicate design and finely tuning of SERS substrates their performance in real samples is usually not as good as expected, mainly due to an insufficient consideration of environmental factors that can influence the measurements; (vi) it is also desirable to develop sensing approaches with reusability and recyclability capabilities to make them more cost-effective. Finally, many factors influence the SERS signal such as the strength of the local electromagnetic field, the nature of the analyte, its concentration, the chemical affinity to the nanoparticle surface, as well as the stability of the analyte-nanoparticle among others. Whereas, the interplay between these factors opens up a wide range of possibilities, the rational design of the plasmonic substrate for a specific application is often mandatory, thereby limiting the generalization of its use.The broad interest in SERS together with the improved control over substrate fabrication, as well as the development of new related-instrumentation has resulted in the continuous development of advanced plasmonic platforms , as well as emerging surface-enhanced signal amplification techniques (e.g., TERS, SEIRA, EC-SERS, SESORRS, etc.; Langer et al., GB and IP-S have contributed equally to the writing process of this minireview.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In recent years, gold compounds have gained more and more attentions in the design of new metal anticancer drugs. Numerous researches have reported that gold compounds, in addition to their widely studied cytotoxic antitumor effects, also reverse tumor immune escape and directly facilitate the functions of immune cells, leading to enhanced anticancer effects. This review mainly summarizes our current understandings of antitumor effects of gold drugs and their relationships with various aspects of antitumor immunity, including innate immunity, adaptive immunity, immunogenic cell death, and immune checkpoints, as well as their roles in adverse effects. Some recent examples of anticancer gold compounds are highlighted. The property of gold compounds is expected to combine with anticancer immunotherapy, such as immune checkpoint inhibitors, to develop new anticancer therapeutic strategies. From the accidental discovery of cisplatin's antitumor activity by Rosenberg and his coworkers, platinum-based complexes have been used as standard chemotherapeutic agents more than 40 years in clinical practice  see  Chen an. Innate L1 on cancer cells leads to the anergy and exhaustion of T cells, which restricts CTL from effectively targeting cancer cells  see . For exaA lot of evidence from experimental and clinical studies suggest that the antitumor mechanisms of gold compounds are extremely complex and diverse. Recently, it has been reported that gold compounds may have a potential relationship with anti-tumor immunity. Gold chemotherapy drugs, in addition to their widely studied cytotoxic antitumor effects, might reverse important aspects of tumor immune escape and directly affect several types of immune cells, leading to enhanced anticancer effects. In this article, we will summarize the relationship between gold compounds and various aspects of antitumor immunity, including innate immunity, adaptive immunity, immunogenic cell death, and immune checkpoints.in vitro and in vivo studies have shown that gold compounds can not only promote direct immune cell-mediated destruction, but also synergistic promote the T cell-based anticancer immunity cycle via DC (see 3Au (S2O3)2·2H2O, in vitro. The results showed that sub-lethal concentrations of the three gold compounds could differentially modulate activation of monocytes. Among them, the effect of auranofin was slightly stronger  signaling pathways play a key role in the process of activation, differentiation, and maturation of myeloid and lymphatic cells. NF-κB is a key transcription factor involved in the expression of many inflammatory genes and plays an important role in oncogenesis, which is associated with the proliferation of multiple types of tumors  compounds have been clinically used to treat rheumatoid arthritis due to their anti-inflammatory properties, which appears contradictory to its immune stimulatory activity. This intrigues clinicians and toxicologists for many years. Anyway, the gold paradox is not unique, steroids are also known to have both immunostimulatory and immunosuppressive effects. Radka et al. reported a series of gold(I)-triphenylphosphine compounds  with hyp+ T cells  during immune evasion. Several researches have shown that metal-based treatment (like platinum) might increase the expression of MHC class I in cancer cells  . Their study provides a novel combination of cancer therapy using auranofin in combination with anti-PD-L1 targeting therapy, which has limited clinical efficacy in TNBC patients with monotherapy  and cytotoxic T lymphocyte antigen 4 (CTLA-4) are the most commonly studied. PD-1 is mainly expressed on the surface of activated T cells, B cells, as well as NK cells. The activation of PD-1 suppresses the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, leading to the inhibition of survival and proliferation of T cells (Pedoeem et al., in vivo and in vitro support the hypothesis that these antitumor drugs might be ideal combination partners for immunotherapeutic interventions. Among the various immunotherapeutic strategies, the treatment based on immune checkpoint inhibitors is a highly efficient anticancer therapy and has shown unparalleled efficacy in patients with advanced cancer. A central challenge in modern systemic cancer treatment research is to identify and develop personalized and multitargets combination therapy strategies to improve the efficacy of modern immunotherapies. In Raninga's research, auranofin combined with immune checkpoint PD-L1 inhibitor achieved excellent anti-tumor efficacy in breast cancer cell lines and in mice, demonstrating the promising prospect of combined immunotherapy based on gold derivatives (Raninga et al., Metal-based anticancer drugs are widely used in the clinical treatment of various types of tumors. This situation shows that the systemic anticancer treatment based on metal drugs has high activity and quality, and it is urgent to guide the development of anticancer metal drugs by multidisciplinary approaches. Based on the promising prospect of the anticancer activity of gold derivatives, a large number of articles have been published in recent years (Yeo et al., The immune system plays an important role in the side effects of gold compounds. Interestingly, the effects of gold compounds on the immune system can be broadly divided into two categories. On the one hand, some adverse effects can be related to immune-stimulating reactions, such as various forms of dermatitis, glomerulonephritis, and enteritis. The most common toxicity of gold compounds includes allergic reactions of skin and mucous membranes, such as skin rash, pruritis, contact dermatitis, stomatitis, as well as conjunctivitis. Some patients developed proteinuria during treatment, and the most common renal histologic lesion was membranous glomerulonephritis (Tonroth and Skrifvars, In this review article, we systematically summarize the unique modulatory effects of gold compounds on antitumor immunity, including the enhancement of antigenicity and immunogenicity of tumor cells, the effects on immune cells and immune checkpoints, as well as the induction of ICD. Elucidating these important issues will greatly improve the identification and development of anticancer gold derivatives. In the future, gold compounds may have great application prospects in the combination therapy, potentiating the efficacy of targeted therapies and immunotherapy, especially immune checkpoint inhibitors therapy. Further studies are required to understand the detailed mechanisms of the regulation of anticancer immune response by gold derivatives in order to promote their clinical application.SW and HL: had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis, concept, design, and critical revision of the manuscript for important intellectual content. SY and ML: acquisition, analysis, or interpretation of data and administrative, technical, or material support. SY and SW: drafting of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.