Both earthquakes, but especially the later one resulted on severe damage of the regions and caused severe disruptions in  health service delivery. In these circumstances, the RECOVER-E project , which was ongoing in Croatia from 2018-2022 aimed to implement flexible assertive community treatment as the new health care service for persons with severe mental illness (SMI). Since the pandemic and earthquakes have significantly influenced the life circumstances of all RECOVER-E project participants with SMI, we wanted further to examine the impact of health care delivery on mental health and the response to stress caused by a pandemic and earthquake in the patients with SMI involved in the project, in the first and second wave of the COVID-19 pandemics. Additionally, using case series, we will demonstrate the community mental health teams’ contributions in managing SMI after a double disaster in providing feasible, comprehensive, and accessible mental health services.1During the ongoing COVID-19 pandemic, two regions in Croatia, Zagreb and Petrinja in the Sisak-Moslavina County experienced strong earthquakes, on the March 22No significant relationships.


Biological Resource Banks (BRB) or Genetic Resource Banks (GRB) are critical tools for the conservation of animal biodiversity. According to the International Union for Conservation of Nature, more than 38,500 species are threatened with extinction, out of a total of 138,300 surveyed species. These banks are repositories of biological samples and data recovered and preserved for the long term by zoos, universities, research centers and other conservation organizations. In recent years, BRB have increasingly included ovarian and testicular tissues as additional options to rescue and propagate wild species, especially those at risk of extinction. After in vitro culture or grafting, gonadal tissues are potential sources of matured gametes that can be used for Assisted Reproduction Technologies while informing about gametogenesis or mechanisms involved in infertility. It therefore is crucial to properly recover, cryopreserve, and culture these tissues using species-specific protocols. Developing BRBs is currently one of the strategies to preserve species from the Caatinga biome - an exclusively Brazilian biome with a rich wild fauna that suffers from anthropogenic activities. Among wild species from this biome, studies have been primarily conducted in collared peccaries, agoutis, cavies, and armadillos to preserve their ovarian and testicular tissues. Additionally, domestic species such as the domestic cat and donkeys have been proposed as models for wild species that are phylogenetically close. This review addresses the main technical aspects involved in obtaining BRB derived from gonadal tissues in some wild species of the Caatinga biome. It reports recent advances and perspectives to use these biological materials for wildlife conservation. Although the extinction of species is an irreversible process and is the result of natural evolution, anthropogenic activities have considerably accelerated extinctions of many species . Accordiin vivo and in vitro embryo production, as well as providing reproductive cells for in vitro toxicity testing. Moreover, studies related to gonadal tissues allow to understand the developmental biology of gametes, as well as understanding mechanisms involved in infertility.The BRB or Genetic Resource Banks are considered sources of gametes, embryos, gonadal tissues, and somatic cells after recovery, processing, and cryopreservation for long term storage . Though in vivo or in vitro culture and characterization of these tissues, which are all species-specific (Pecari tajacu), red-rumped agoutis (Dasyprocta leporina), Spix’s yellow-tooth cavies , and six-banded armadillos (Euphractus sexcinctus). Preservation of ovarian and testicular tissues have already been attempted in those species. Additionally, domestic species such as the donkeys  of the oocyte reserve of the ovary is found in these follicles, making it possible to preserve genetic material from a single female, (i) before it goes to atresia, (ii) from individuals at any stage of development  and (iii) individual status  (The cryopreservation associated with the -mortem) . This te-mortem) . In addiin vitro and in vivo culture of tissues. In collared peccaries, red-rumped agoutis, Spix’s yellow-tooth cavies, armadillos, and Northeastern donkeys, significant advances have been achieved  and media based on powdered coconut water  were evaluated for short-term storage of PAFs for morphology, ultrastructure, and follicular viability . Under rThe cryopreservation of ovarian tissue has been developed to increase the number of female genetic materials in BRB . One of In collared peccary , two stuDespite being used for the preservation of the female germplasm of domestic  and wildFor red-rumped agouti , preservFor Spix’s yellow-tooth cavy , a pionein vivo and in vitro) of PAFs from wild species, (i) understanding the events involved in the activation, growth, and maturation of PAFs, and (ii) obtaining fully-grown gametes for use in ARTs. Several factors can influence the efficiency of in vitro culture, from those related to the culture system itself, to the reproductive stage of the ovary donor and categories of cultured follicles (in vitro.There are two main applications of the culture (ollicles . One key+) or tissue culture medium-199 (TCM199+) media on the in vitro development of PAFs by 7 days derived from collared peccaries. The FSH promotes the maintenance of the ovarian extra-cellular matrix, contributing to the follicle development. Therefore, authors suggested the use of TCM199+ medium supplemented of FSH for the in vitro development of collared peccaries PAFs under 7-day culturing conditions.In wild species from the Caatinga biome, the first studies have been carried out to identify the culture conditions involved in the development of PAFs. In collared peccaries, three studies exemplify this. Initially, in vitro development of collared peccary PAFs. Therefore, BMP-15 and GDF-9 contribute for follicle activation and development during in vitro culture. These collective results improve the understanding of initial follicle development in collared peccaries and can be used to improve the protocols for in vitro culturing of PAFs.For red-rumped agoutis, initial evaluations have been carried out from cultured non-cryopreserved and cryopreserved ovarian fragments. In this study, authors evaluated the cryopreservation conditions using a culture system of ovarian fragments in TCM-199 plus ITS medium , 0.23 mM sodium pyruvate, 2 mM glutamine, 2 mM hypoxanthine, and 1.25 mg/mL bovine serum albumin - Praxedes et al., 2021). In this species, studies aimed at identifying the most suitable components of the culture medium for PAFs are still necessary, to evaluate follicular activation and development.in vitro culture, ovarian tissue grafting has been used in the evaluation of cryopreservation protocols for PAFs, allowing it to be a tool for assessing the recovery of ovarian function. In red-rumped agoutis, In addition to Among the many challenges in establishing strategies for the conservation of wild species, it is worth highlighting the fact that the reproductive physiology of many of these species remains unknown. Despite this, numerous studies have shown the efforts of researchers to fill this gap. Thus, the continuity of studies focused on the understanding of ovarian physiology and its preservation is justifiable. This perspective is evident, mainly because despite the need for improvements in the protocols for the preservation and culture of ovarian tissue, the birth of mice offspring from frozen material has already been obtained , encouraPreviously, preservation of male germplasm had been limited to well-established protocols for cryopreservation of spermatozoa obtained by ejaculation or retrieval from the epididymis. In recent years, the possibility of safeguarding testicular tissue has emerged as a promising tool for ARTs. According to this technology, the undifferentiated spermatogonia preserved into the testicular tissue could be cryopreserved and cultured, guaranteeing the unlimited production of viable spermatozoa to be used in ARTs. Therefore, the cryopreservation of testicular tissues represents an important technology for the preservation of fertility in wild mammals, enabling essential advances for the knowledge and maintenance of the species .in vitro and in vivo culture of tissues. Significant advances have been achieved and the establishment of protocols has allowed important results in collared peccaries, red-rumped agoutis, and Spix’s yellow-tooth cavies  seems to be essential for the maintenance of cell survival and proliferation (A pioneering study conducted by our collared peccaries research group has established means and supplements for the development of spermatogenesis. In collared peccary, after 28-days feration .Technologies for testicular tissue preservation are relatively recent compared to other ARTs. Despite this, numerous advances have been described around the world, to either develop cryopreservation protocols or to establish adequate culture systems. Birth of viable offspring from frozen testicular tissue and subsequently submitted to grafting protocols has already been described not only in mice , but alsOver the past decades, biodiversity loss has accelerated at a global scale. To maintain and sustain biodiversity, ARTs have been developed and implemented by many researchers around the world. Cryopreservation of ovarian and testicular tissue, when properly performed, can be useful approaches for understanding the biology of germ cells in different species. It allows the evaluation of the mechanisms of contraceptive agents responsible for infertility, the correction of the causes of infertility by editing the genome of these cells, and the obtaining of viable sperm to be used in the embryo production.in vitro culture of PAFs have been important tools for the knowledge of folliculogenesis in different species. Several protocols for cryopreservation and in vitro culture of PAFs have already been developed, and their results vary according to the conditions established for the preservation protocols, in vitro culture systems, media and supplements used, and, mainly, according to the investigated species.In some species from the Caatinga biome, important progress has been made. Regarding the preservation of PAFs, it still has limitations when it comes to being a source of oocytes for ARTs in wild animals. On the other hand, the use of cryopreservation and Although testicular tissues from adult individuals have already been cryopreserved in different species, no protocol is effective enough to produce mature spermatozoa from cryopreserved testicular tissues. This fact highlights the need to continue studies to produce viable sperm and, consequently, live offspring. For this reason, steps to optimize SSV have been developed in collared peccaries and agoutis, wild mammals of the national fauna, with the aim of developing SSV as a safe bioprocess in the conservation of biodiversity.


The mechanical heart valve (MHV) is commonly used for the treatment of cardiovascular diseases. Nonphysiological hemodynamic in the MHV may cause hemolysis, platelet activation, and an increased risk of thromboembolism. Thromboembolism may cause severe complications and valve dysfunction. This paper thoroughly reviewed the simulation of physical quantities  in healthy and dysfunctional MHV and reviewed the non-Newtonian blood flow characteristics in MHV. In the MHV numerical study, the dysfunction will affect the simulation results, increase the pressure gradient and shear stress, and change the blood flow patterns, increasing the risks of hemolysis and platelet activation. The blood flow passes downstream and has obvious recirculation and stagnation region with the increased dysfunction severity. Due to the complex structure of the MHV, the non-Newtonian shear-thinning viscosity blood characteristics become apparent in MHV simulations. The comparative study between Newtonian and non-Newtonian always shows the difference. The shear-thinning blood viscosity model is the basics to build the blood, also the blood exhibiting viscoelastic properties. More details are needed to establish a complete and more realistic simulation. Cardiovascular disease is one of the leading causes of death in the world . Heart vThe previous numerical studies of MHV mainly concentrated on healthy valves with velocity fields, transvalvular pressure drop, blood component damage, and the downstream flow of normal MHV. The studies also attempted to quantify the relationship between hemodynamics and the possibility of thromboembolic complications . The stuNonphysiological blood flow of mechanical valves leads to being patients at risk of complications of thromboembolism. One of the potential complications associated with mechanical valves is valve dysfunction, which is the dysfunction caused by the formation of pannus and blood clots around the valve structure. It impedes the rotation of one or two leaflets in the BMHV . The freIn the previous study, many researchers have assumed blood as an incompressible Newtonian fluid. This assumption is considered valid as the blood behaves as a Newtonian fluid in large arteries , 3, 25, Sotiropoulos and Borazjani  reviewedThis paper addresses an additional scope for numerical studies of the MHV dysfunction and a non-Newtonian blood flow model for MHV simulation. The review of MHV dysfunction mainly focuses on the one leaflet blocked by the clot and reviews the blood flow patterns in that leaflet dysfunction conditions. The non-Newtonian blood flow part will review the non-Newtonian viscosity blood simulated in MHV, and the different shear-thinning non-Newtonian viscosity blood constitutive model also reviewed some basics of the viscoelastic constitutive model. Finally, conclude the current non-Newtonian blood flow simulation issues in MHV .Smadi et al. and Kondo et al. , 27 and The severity of the dysfunction will influence the flow field pattern through MHV . The abnFor the boundary conditions, Smadi et al.  simulatek-ω model which was used with 5% turbulent intensity [k-ω turbulence model [For the MHV dysfunction numerical method, Smadi et al.  set the ntensity , 51. Smantensity  introducntensity . Yun et ntensity , 36 perfce model , 54 was Due to the different opening angles of the dysfunction leaflets, the flow fields in the upstream and downstream of the dysfunction MHV were different from the normal MHV, especially the flow separation and vortex formed more obvious in dysfunction MHV .When the percentage of dysfunction increased, shown in k-ω turbulent model. The SPH method simulation results show the consequence of one dysfunctional valve leaflet on velocity, corresponding to a 76% severity in leaflet dysfunction, which leads to a significant increase in the maximal velocity reaching up to 3.52 m/s. Furthermore, the maximal velocity is not located through the central orifice anymore, as expected in the healthy case, but through the lateral normally functioning orifice shown in Smadi et al.  used theEarlier studies on the flow characteristics of MHV showed that the three orifices of the valve characterized the flow, formed between the sinus and the aortic wall, and formed in the sinus vortex –58. In tConversely, periodic vortex shedding was observed after the valve leaflets , 27. SomFlow disturbance in MHV induces blood constituent activation and damage. Vortex shedding is due to the vortices' complex flow . Vortex Further downstream, the vortex becomes turbulent , 64. VorHuang et al.  performeWhen the percentage of dysfunction increased MHV, the downstream flow fields became more vortical . In a paWith the number and scale increased of vortices, the valve's blood residence time downstream will increase. As a result, this will increase the level of platelet activation and thrombosis. Studies have shown that in moderate levels of dysfunction, platelet activation is significantly increased, which may cause thrombosis or worsen thrombosis. Also, leading to a vicious cycle, the abnormal flow fields accelerate the thrombosis formation on the valves, which aggravates valve dysfunction, which is shown in When the blood flow passes through the MHV, it will generate a high-pressure drop, resulting in recirculation and stagnation –70. The Turbulent shear stress (TSS) is generated by valve-induced turbulence, which is usually focused on studying blood hemolysis . Many stWall shear stress refers to the force per unit area exerted by the wall on the fluid in the direction of the local tangent plane . When thSeveral studies –83 suggeIn dysfunction MHV, with the percentage of dysfunction increased, the TSS level and highest value position will change; the TSS-affected regions move to the upper side of the valve . The numFrom the physical quantity review of the dysfunction MHV, it is obvious that the dysfunction changed the blood flow patterns in MHV, increased the maximum velocity and shear stress, and increased the number and scales of vortices. Those results will lead to hemolysis and thrombosis formation or worsen thrombosis, which will also induce a vicious circle. Obvious recirculation and stagnation region will decrease the shear stress, and downstream of complete dysfunction occurred in low-velocity region that is meaningful to assimilate the blood flow as a non-Newtonian flow.Blood is a complex fluid consisting of blood cells suspended in plasma. The shear-thinning behavior depends on the properties of the suspended particles ; the bloSeveral factors can affect blood viscosity, like hematocrit, plasma viscosity, vascular diameter, and temperature –98. The In reality, the blood flow in the aorta is pulsatile and transitional. The flow is very inhomogeneous, which means the previous assumption might be incorrect. The Newtonian model is not suitable for all situations. The transient shear rate over one cardiac period in the aorta . In a caViscosity plays a vital role in induced shear stresses. Various models have proposed  to descrThe Carreau-Yasuda constitutive model was most commonly used to describe non-Newtonian blood due to its reported property of viscosity changes that are more accurate , 114. HaIn the study of the difference between the non-Newtonian and Newtonian models, the Newtonian model generates lower wall shear stress. In the non-Newtonian model, the Cross model shows a significant difference . Doost eThe MHV is an unnatural structure, and small gaps will be formed when it is at its closing position and around its hinges. There are small regions of different flow domains at valve hinges. In the numerical simulation of flow patterns in the hinge regions, the micro size of the hinge gaps should be taken into account. When the physical size is comparable with the characteristic length of red blood cells, the non-Newtonian viscosity blood assumption is important , especiaThere are more than eleven kinds of the non-Newtonian models  applied to define the rheology of blood flow. All of these non-Newtonian shear-thinning viscosity models are the most famous models that can simulate non-Newtonian blood flow.k and n (dimensionless) are selected to fit experimental data. When n = 1 and k = μ, the model reduces to the Newtonian case. For n > 1, the model represents shear thickening, whereas for n < 1, it represents shear thinning [The simplest type of model to account for shear rate-dependent viscosity is the Power Law model. This model has two parameters: the thinning . The limthinning , 122.The viscosity model has two main types. The first is the shear-dependent type, which equation can be written in the general form.Different choices of the function correspond to different blood flow models. The model parameters are easily affected by many factors like hematocrit, temperature, plasma viscosity, age of RBCs, and exercise level or gender. The Cross, Carreau, and Carreau-Yasuda models include this type.Another type is the yield stress type. When the shear stress is less than the yield stress, the fluid exhibits a solid-like behavior. Thus, the fluid flows depending on the yield stress and requires a minimum pressure gradient to drive the fluid. Casson's constitutive equation is the typical yield stress type non-Newtonian viscosity model, which has zero viscosity at an infinite rate of shear and infinite viscosity at zero rates of shear . The QueWhole blood is a concentrated suspension of formed cellular elements, which exhibits non-Newtonian characteristics due to the erythrocyte aggregation at low shear rates. Except for shear thinning, viscoelastic and thixotropic are also important non-Newtonian behaviors of the blood .−1, the elastic nature of the blood is negligible, as evidenced by the combination of oscillation and stable flow viscosity. However, in the circulatory system, use the viscoelastic constitutive equation to model the blood; the finite viscoelastic behavior of the blood should be taken into account. Blood viscoelasticity also is dependent on factors such as temperature, hematocrit, and RBC characteristics [Blood cells are essentially elastic membranes filled with fluid; RBCs can form aggregates, called rouleaux, at low shear rates; the rouleaux aggregates and stores elastic energy, with the fluid flow; the sliding of internal cellular needs input energy; this energy dissipated through friction and later gradually disaggregated into cells at high shear rates . Elasticeristics .There are several different kinds of viscoelastic models used to describe the viscoelastic blood fluid dynamics. There are three types of governing equation building viscoelastic extra stress: statistical models, integral models, and differential models. In which the differential models describe the viscoelastic extra stress tensor by a partial differential equation, it was widely used in numerical simulation due to its easy implementation in current CFD codes; the Johnson-Segalman differential viscoelastic constitutive model is widely used in a blood flow numerical study . The welRecently, the generalized Maxwell model , 128 andHigher shear rate is generated in the large arteries, the blood flow can be assumed as Newtonian flow in many review studies . AlthougThe shear-thinning non-Newtonian viscosity model has been used in many MHV numerical studies, except for shear thinning. Also, viscoelastic is an important non-Newtonian behavior of the blood; the generalized Holroyd-B model can build shear-thinning and viscoelastic characteristics, has successfully extended to simple 3D simulation, and it is still full of challenges for complex 3D structures like MHV.The human aortic wall is compliant; the compliance of the aorta determines its increase in pressure , 134. A Studies with a flexible aortic root can be performed to recapitulate better physiological conditions in the FSI model . The expAdditionally, the simplification of hinge structural integrity in BMHVs and without applied friction will affect the microflow near the leaflet hinges. Hinges with a wide gap (around)  lead to Results are always different in comparative MHV numerical studies between Newtonian and non-Newtonian fluids because there has much difference in the parameters for the non-Newtonian viscosity model and even for the structure of the model itself . The sheThe current review compiled studies carried out on the dysfunctional MHVs and non-Newtonian models of MHV. In the dysfunction condition, the dysfunction model is usually with one healthy leaflet and one dysfunctional leaflet with several different percentages of dysfunction. The results show that the maximum blood velocity increased with the effective orifice area decrease. Dysfunctionality increases the transvalvular pressure gradient, flow separation, growing eddies, stagnation, and recirculation. Heart valves induce flow disturbances that play a role in blood constituent activation. When the percentage of dysfunction security increased, there has a significant increase in the number of vortices and creates conditions for thrombus formation, which will worsen the valve dysfunction.Blood is a complex fluid composed of blood cells suspended in plasma. Due to the presence of these particles, the blood exhibits a non-Newtonian behavior, and its viscosity is vital in the shear stress of the walls and particles. Non-Newtonian characteristics include shear-thinning and viscoelastic behavior. The shear-thinning viscosity models are widely used in MHV blood flow simulation. In the hinge recesses of MHV and the gap between two leaflets during a leakage phase, the non-Newtonian effect of the blood may become apparent. In order to build an accurate blood model, changes in blood viscosity must accurately be observed, and the Carreau-Yasuda viscosity model is most commonly used in shear-thinning non-Newtonian MHV blood flow simulations. However, the viscoelastic model still not be used in the previous study. Some studies also found that simulations of Newtonian fluids and non-Newtonian or between different non-Newtonian models will yield different results, such as higher shear stresses for non-Newtonian fluids. Although the current results are still different, creating more models to simulate and compare conditions will help solve the problem and allow for better studies of the failure mechanism and preventive measures of MHV dysfunction.


The retinal pigment epithelium (RPE) forms an important cellular monolayer, which contributes to the normal physiology of the eye. Damage to the RPE leads to the development of degenerative diseases, such as age-related macular degeneration (AMD). Apart from acting as a physical barrier between the retina and choroidal blood vessels, the RPE is crucial in maintaining photoreceptor (PR) and visual functions. Current clinical intervention to treat early stages of AMD includes stem cell-derived RPE transplantation, which is still in its early stages of evolution. Therefore, it becomes essential to derive RPEs which are functional and exhibit features as observed in native human RPE cells. The conventional strategy is to use the knowledge obtained from developmental studies using various animal models and stem cell-based exploratory studies to understand RPE biogenies and developmental trajectory. This article emphasises such studies and aims to present a comprehensive understanding of the basic biology, including the genetics and molecular pathways of RPE development. It encompasses basic developmental biology and stem cell-based developmental studies to uncover RPE differentiation. Knowledge of the in utero developmental cues provides an inclusive methodology required for deriving RPEs using stem cells. Age-related macular degeneration (AMD) is one of the most prevalent forms of irreversible vision impairment with a multi-factorial etiology comprising genetic, environmental, age-related, immune system-related, and lifestyle factors. Its pathogenesis is characterized by the early appearance of deposits of lipids and proteins (drusen), followed by functional anomalies in the retinal pigment epithelium (RPE) and progressive photoreceptor (PR) degeneration affecting the macular region  Figure . These eLight-mediated ionization of photosensitive retinoids, generation of reactive oxygen species (ROS), and triggering of inflammatory cascade can initiate photoreceptor cell death . Among tCurrently, there is no therapeutic intervention that can fully reverse AMD ,16. HoweHowever, recent studies suggested that the injection of RPE in the subretinal space did not lead to complete epithelization of the degenerated RPE monolayer and diminished reversibility of vision loss. This could be attributed to various reasons, including improper attachment of the RPEs on the underlying Bruch’s membrane (BM), cell clumping leading to the loss of cell-to-cell contact required for the formation of RPE monolayer ,26, lossThe first report describing in vitro development of RPEs involved spontaneous derivation of RPE-like cells in culture of ectoderm-committed embryonic stem cells (ESCs). This process usually takes 4–6 months to achieve sufficient number of cells for in vivo studies ,19,20. HTherefore, in this article, we provide a concise summary of the developmental studies pertaining to the RPE using model-based developmental studies and stem cell-based cell signalling and developmental trajectory studies to uncover the mechanism of eye development through progressive stage-dependent commitment and progression. The RPE is a tightly arranged monolayer of pigmented cells which has an important role in PR maintenance and visual function. The apical side of the RPE faces the PRs and regulates their function through their microvilli-like projections. These microvilli play an important role in the visual cycle between the RPE and the PRs by acting as a binding site for the photoreceptor outer segment (POS) and, thus, are essential for the phagocytic function of the RPE . The basThe whole process of eye development is a complex mechanism that depends on multiple tissues of ectodermal and extraocular, mesodermal origins  Figure . Here, wAnterior neuroectoderm formation: Early embryonic development exhibits the formation of  resulted in small and deformed systems with abnormal RPEs [The global profile of the chromatin accessibility in healthy RPE versus diseased RPE in AMD revealed a decrease in chromatin assembly in case of early AMD, suggesting RPE dysfunction is associated with AMD onset. In the same study, the correlation between a known risk factor (cigarette smoke) and AMD pathogenesis was shown to be similar in the RPEs derived from AMD patients and iPSC-derived RPEs. A global profile of chromatin accessibility using ATAC-Seq between these two groups showed that cigarette smoke-treated RPE recapitulated the chromatin accessibility changes as observed in AMD, thus providing an epigenetic linkage between a known risk factor for AMD and AMD pathogenesis. Overexpression of histone deacetylase (HDAC11) also resulted in decreased chromatin accessibility . The impmal RPEs . All theEpigenetic conditioning or reprogramming has been used to reverse the age-related morphological and functional characteristics of the RPE. In a recent study, it was demonstrated that epigenetic regulation is highly critical not only during the course of RPE development in utero, but also throughout the life span of the organism . The autdnmt2) in zebrafish resulted in mild microphthalmia and RPE malformation [dnmt1 in mice resulted in abnormal RPE morphology and polarization at E15.5. Thus, this study underlines the importance of DNA modifying epigenetic regulatory enzymes in controlling the RPE apicobasal polarity and, in turn, regulating the development of the POS [The importance of the DNA methylating enzymes was shown when morpholino knockdown of DNA methyltransferase 2  to that of an established RPE cell line, i.e., ARPE-19 . The aut reasons . Continuous long-term culturing of the RPE leads to a loss of functions and cell de-differentiation . Such deThe extracellular proteome offers a very important and insightful role in the physiology, function, and interaction with neighbouring cells. It includes the secretome in the form of secreted proteins, exosome-containing proteins, and ECM proteins that form the microenvironment of the cellular niche and help maintain the morphological state of the cell. In another study, the global-scale proteome secreted from RPEs was studied to understand the molecular mechanism of AMD pathogenesis in the context of drusen formation in the eye . The expC. elegans as a small class of non-coding RNA molecules  that regulate the expression of genes at the post-transcriptional level [Micro RNA, commonly abbreviated as miRNA, was first identified in al level ,78. The al level  and Trieal level . The rolal level ,82. The al level . The essal level , Drosha,al level . In a stal level , it was al level .There is a large number of miRNAs that have been identified across species, and these play critical roles in the developmental pathways and functionality of RPEs both in utero and during post-natal development . One groApart from playing a role in the differentiation process both in utero and in vitro, it has been shown that miRNAs regulate cell-to-cell interaction and membrane transporter proteins. Thus, miRNAs are involved in controlling one of the critical features of the RPE, which is the maintenance of its polarity and monolayer arrangement with highly packed and tightly regulated cell-to-cell connections. This feature also helps in maintaining the BRB and in regulating the transport of ions and molecules across the choriocapillaris and the retina . MiR-204The role of the RPE in the visual cycle is very important. Otherwise, the PRs would die rapidly due to the accumulation of photo-oxidized protein products as a process of light adsorption and transmission of signals. This cycle involves the development of POS which is phagocytosed by the RPE. Inside the RPE, all photo-oxidized proteins and phospholipids of the outer segments are processed, recycled, and transported back to the PRs. The process of phagocytosis and cell clearance have been shown to be regulated by a group of miRNAs, such as miR-184. It was observed that inhibition of miR-184 resulted in lowering the phagocytosis of POS and the recycling process. A dysfunction in phagocytosis or an imbalance in the visual cycle-related recycling of POS has also been implicated in the development of AMD. It was further confirmed that the RPEs isolated from AMD patients showed a downregulation of miR-184, thus pointing out the role of this miRNA in the visual cycle as well as AMD pathogenesis . SimilarDevelopmental studies of the eye using animal models and pluripotent stem cells have helped in elucidating the pathways responsible for stage-specific progression of various eye tissues . The finEye field induction is the most important stage in RPE lineage trajectory as the RPE, the neural retina, the lens, and the optic stalk arise from the eye field that originates from the ANE. Few authors have focused on the derivation of eye field-like state from ANE for RPE development. However, critical transcription factors, such as RX ,41 and SXenopus knockdown of a lysine-specific histone demethylase 5C (Kdm5c) resulted in small and deformed systems with abnormal RPEs [Further, epigenetic studies are quite important and should be considered in validating a protocol development apart from RNAseq-based characterization of the differentiation method. Inducing certain stages of RPE development using a cocktail of growth factors and small molecules leads to the activation of signalling pathways, but whether such activation also leads to changes in the epigenetic status of cells are not studied for all published protocols. Moreover, only modifying the epigenetic regulators can have significant effect on the cells. For example, in a study, mal RPEs . The cellular aspect of RPE development is also crucial as it is known that RPE differentiation from the outer layer of the optic vesicle also depends on the factors secreted from the extraocular mesenchyme. It has been shown that in the presence of extraocular mesenchyme, RPE differentiation and maturation is promoted with a marked increase in Mitf expression . HoweverThus, the development of a new methodology for improved and efficient differentiation of RPE should cover epigenetic factors, microRNAs, and secretome in comparable way to the native primary RPE. Such information can be derived from developmental studies as well as studies on the diseases and phenotypes of the RPE. This would also be beneficial in promoting cells for long-term functionality upon in vivo transplantation. RPE derivation using stem cells offers a plausible solution for the treatment of both dry and wet forms of AMD. The development of a defined and efficient protocol for RPE differentiation or the production of a highly pure population of iPSC-derived RPEs from the initial stages of development to the final maturation stage is still warranted. Using the knowledge derived from the basic cell biology of the RPE, and the information produced over the last three decades from developmental biology focusing on RPE and eye development using a variety of cell and animal models, has improved the design and development of strategies for iPSC-based RPE differentiation. Certain limitations still exist in producing functionally mature iPSC-derived RPEs and recapitulating the critical features, such as homogeneous pigmentation and ciliation, which still require further exploration.


There is a growing evidence that social determinants of health influence the health outcomes. These non-medical factors, i.e., social determinants of health / mental health, are defined as the conditions in which people are born, grow, live, work and age, including the factors shaping these conditions. They either have direct effects on health and ill health or work as mediators. In this respect, racial discrimination is a fundamental social determinant of ill health / mental health and health inequalities. A strong correlation between reported experiences of racial discrimination and poor general health and poor mental health has been reported. Besides, racial discrimination may lead to risk taking behaviors increasing poor health / mental health especially in vulnerable disadvantaged populations. A leading factor mediating the negative effects of any biopsychosocial factor on mental ill health is the degree of discrimination. Furthermore, racial discrimination is one of the processes explaining and reinforcing racial disparities in health and ill health. From a conceptual point of view, racial discrimination and its effects on ill health could be discussed in the context of the issue of othering and related dehumanization and violence. Psychiatrists and mental health workers have accumulated considerable knowledge and experience on understanding and overcoming some of the consequences of racial discrimination, especially via anti-stigma studies. The unfair and avoidable influences of racial discrimination on mental health are neither fated nor inevitable. As Gramsci had said, we have the pessimism of the intellect and optimism of the will.No significant relationships.


Traditional Chinese medicine (TCM) has a long history, rich clinical experience, and unique advantages in the prevention and treatment of diseases. The quality and safety of Chinese medicinal materials (CMMs) directly affect the clinical efficacy and development of the TCM industry. However, confused provenance, counterfeiting and adulteration of CMMs hinder the acceptance of its therapeutic benefits in modern society. Therefore, the establishment and improvement of a TCM traceability system would be conducive to the transparency of the CMMs production, distribution, and circulation, thereby improving drug safety and promoting industry development. This review discusses the challenges faced in the development of TCM traceability system, the technologies currently available for tracing CMMs, and the potential application of Barcoded Microbial Spores (BMS) to improve CMMs origin traceability and TCM traceability systems. Traditional Chinese medicine (TCM) is a valuable asset accumulated by Chinese during their long-term struggle against diseases. Based on a unique system of knowledge and therapeutic procedures, TCM practitioners use a wide variety of Chinese medicinal materials (CMMs) from different plants and animals to prevent, diagnose, and treat diseases. The rapid growth in the use and demand for TCM in recent years has led to an increase in the demand for CMMs resources, with a consequent increase in the risk of quality degradation in the planting, processing, transporting, and storing stages. In addition, different environmental factors, such as topography, soil, climate, humidity, and light, can affect secondary metabolites, which are usually the bioactive components of CMMs . Herbal In the process of improving and enriching the TCM traceability system in China, some challenges we have to face and overcome. Firstly, the production of herbal medicine is still dominated by traditional agriculture, with the widely distributed planting sites, inexperienced growers, and region-specific CMMs names increasing the difficultly of traceability . SecondlTCM traceability systems based on Internet of Things (IoT) technology can record information on CMMs and ensure the stability of information flow. With the development of analytical instruments and the introduction of molecular biology technology, several traceability techniques have been developed, including chemical fingerprinting, DNA barcoding, and stable isotope tracing, which can sensitively and accurately identify the species, quality, and origin of CMMs , 4, 5. CChemical fingerprinting can analyze CMMs through chromatographic or spectroscopic techniques to obtain corresponding chromatograms or spectrograms, which accurately identify the different chemical components in CMMs, referred to as TCM fingerprints . InfrarepsbA-trnH complementary locus for identification of planta medica, and COI core barcode and ITS2 complementary locus for identification of animal medica [DNA barcoding enables standard and rapid identification of CMMs by selecting a relatively simple gene sequence with stable genetic variation across a species that can be easily amplified. The gene sequence identification method was first proposed in 2003 by Paul Hebert and his colleagues . DNA barl medica . HoweverStable isotope tracing is based on differences in stable isotope compositions, which can be influenced by local environmental factors such as soil and moisture. Stable isotope technology can be applied to characterize the isotopes of CMMs from diverse geographical origins, and thus determine differences in different production areas to achieve accurate CMMs traceability and authenticity . At presNanomolecular tags are difficult to counterfeit and can be applied to small and numerous objects, thus exhibiting good prospects in the field of TCM traceability. Porcupine is a molecular tagging system based on the presence or absence of synthetic DNA sequences. Using a portable nanopore device, the DNA-based tags can be read within seconds, showing considerable potential for the molecular tagging of different objects . HoweverThe BMS system identifies the provenance of objects within one hour at meter-scale resolution and near single-spore sensitivity and can be safely introduced into and recovered from the environment . The sysBacillus thuringiensis, which is closely related to Bacillus subtilis, is widely used as a pesticide for many commercially available agricultural products [Bacillus thuringiensis can be detected on store-bought products, even after washing, boiling, frying, and microwaving, indicating that the BMS tags will not be lost during CMMs processing. Detection of BMS only requires a swab to collect a sample from the CMMs surface. SHERLOCK analysis can then rapidly and sensitively detect the shared group sequence, thereby facilitating on-site testing for CMMs quality and identification during transport to ensure no adulteration or production of substandard medicines. After the CMMs arrive at their destination, Sanger sequencing is performed on the BMS-specific sequences, and the identified information is compared with the sequence information recorded in the database to determine the source of each CMM.The BMS protocols for CMMs traceability are shown in Fig. products . The genAs an innovative microbial traceability technology, BMS, combined with genetic marker technology, distinguishes released microbial spores from those existing in the wild. BMS technology solves several common challenges that hinder the development of microbial source tracing technology and can improve TCM traceability. Synthetic spores can be mass-produced, thereby reducing the costs of BMS and improving applicability to a variety of CMMs. BMS technology simplifies the collection and verification of information from cultivation to sale, and can accelerate the promotion of TCM traceability nationwide, although validation across a wider range of real-world conditions is required. Furthermore, BMS can realize the transparency of information from provenance to market for both artificial cultivars with relatively concentrated production areas and wild CMMs with relatively dispersed distribution. The mature application of BMS technology shows great potential in standardizing the CMMs market and accelerating the international development of the TCM industry.The establishment of a TCM traceability system aims to achieve openness and transparency of information in the whole process of CMMs circulation, which would be conducive to the global development of TCM. BMS technology can accurately trace CMM provenance, and the use of shared group sequence and specific sequence in tandem provide a nearly infinite set of identification codes, which can meet the requirement that each CMM has its own barcode. The SHERLOCK assay, which can be performed on-site, and Sanger sequencing, which can identify specific sequences, make the entire process fast, convenient, and accurate. Thus, the use of BMS technology could significantly improve TCM traceability, guarantee the quality and safety of CMMs, and promote the sustainable and rapid growth of the TCM industry.


Skull base and sinonasal tumors (SBSNTs) represent a considerable challenge for clinicians in view of their rarity, anatomical complexity of the site of origin, and great histological variety.Survival rates from the 1970s to early 2000s did not significantly improve, approximately presenting a 5-year estimate of 50% [In recent decades, a multidisciplinary approach and histology-driven treatment of SBSNTs has revolutionized the therapeutic management of these cancers. The evolving understanding of SBSNT biology has been paralleled by the investigation of new therapeutic regimens, such as systemic therapy, as induction chemotherapy, systemic targeted agents, and immunotherapy, even if in limited series. An analysis of the clinical data led to the assessment of the radio- and chemosensitivity of different histotypes, and the beneficial effect on the survival rates as a result of multimodality treatments became evident. Increasingly robust evidence supported the relevance of a histology-driven treatment plan, and investigations were addressed towards a precise definition of the best therapeutic strategy for specific groups of histologies. In this context, the collection of specific expertise obtained from different disciplines , the discussion of each clinical case in a tumor board, and the coordination among treating physicians is gaining extraordinary relevance to improve survival rates. A paradigmatic example is represented by sinonasal undifferentiated carcinoma (SNUC), an aggressive neoplasm historically burdened by an ominous prognosis. The introduction of multimodality treatment relevantly improved prognosis to close to 80% at 5 years. Induction chemotherapy was introduced as a prognostic biomarker and treatment modifier to select candidates for radio(chemo)therapy (responders) vs. surgery and adjuvant treatments (non-responders) .Simultaneously, relevant innovations in surgery and radiotherapy were introduced, including the endoscopic endonasal resection of sinonasal cancers, expanded endoscopic approaches to the skull base, and intensity-modulated and heavy-ion radiation therapies. In general, these approaches guaranteed equal (or superior) chances of local control, but remarkably reduced the associated morbidity.The above-mentioned novelties considerably improved the efficacy and quality of treatments in the last two decades. However, a relevant proportion of patients still have poor prognoses despite the intensified regimen of treatments, which seem to have reached a plateau in their anti-tumor activity. In this subset of patients, tumor biology largely drives the outcome. Future evolutions in SBSCTs are likely to rely on refinements of multimodality protocols and an improved profiling of tumor biology. Multi-omics analysis has the potential to greatly improve our understanding of these diseases and provide us with biomarkers to better characterize these tumors on multiple levels, i.e., the diagnosis, prognostic stratification, and prediction of response to treatment.Within the framework of histology-driven treatment, pathological diagnosis is of utmost importance to create a correct therapeutic plan. Owing to the rarity and histological heterogeneity of these tumors, a change in diagnosis after a second revision is frequent (up to 24% in specific histotypes) and represents a negative prognostic factor . The resRadiology has played a pivotal role in diagnosis by improving the definition of tumor local extension and early tumor relapse thanks to the evolution of magnetic resonance imaging. Radiomics is a novel, promising field of investigation that aims to extract quantitative textural information from medical imaging thanks to complex mathematical analyses and artificial intelligence algorithms. Theoretically, it can provide a considerable amount of objective information  that cannot be retrieved or analyzed by the human eye or brain.Recent experiences have shown its promising role in the anticipation of tumor response to treatment. In an Italian series, radiomics signatures predicted the response to induction chemotherapy better than classical RECIST and volumetric criteria . The ideMolecular analyses are probably the most promising field of investigation in tumor biological profiling. Genomics and the tumor mutational landscape has been extensively studied in the literature. Preliminary evidence has demonstrated the possible role of genomic signatures in the prediction of responses to induction chemotherapy  and progMolecular studies can also identify therapeutic targets to inspire new pharmaceutical options. At present, several therapeutic targets  are potentially druggable; however, preliminary clinical data are disappointing, which suggests that the biological profile of these tumors is very complex and far from being fully elucidated. Nevertheless, the thorough definition of tumor molecular alterations is a prerequisite of any targeted therapy; therefore, investigations in this area are needed to improve survival rates in patients who are not curable with conventional therapies.Finally, the tumor–host interplay and immune contexture has been largely studied in the last decade in many cancer types, including head and neck tumors. Due to their rarity, evidence in SBSNT is still sparse. This represents a new frontier to be explored, and would represent an essential prerequisite to include immunotherapy in the therapeutic armamentarium for SBSNTs. To our knowledge, the first phase-II study has been initiated to investigate immunotherapy in the treatment of sinonasal, poorly differentiated carcinomas, by employing the combination of pembrolizumab, docetaxel, and platinum in patients with stages II–IVb squamous cell carcinomas of the nasal cavity/paranasal sinuses (NCT05027633).In conclusion, treatment strategies used for SBSNTs have greatly improved in recent decades, and these patients can now benefit from a greater chance of being cured with reduced toxicity outcomes. Nevertheless, a non-negligible fraction of patients still presents a poor outcome despite the intensified, histology-driven treatments. Historical weapons, such as surgery and radiotherapy, have undergone a great evolution in the last three decades, which has led to a relevant increase in the rate of tumor local control and a simultaneous decrease in morbidity rates. However, this evolution apparently came to a plateau, and further improvements in these fields are unlikely to significantly improve survival rate when considered on their own.On the other hand, multi-omics analyses are the best candidates to achieve a thorough biological profiling of SBSNTs, which, in turn, should lead to an overall improvement in the management of these diseases. From a therapeutical standpoint, molecular analysis can provide the following relevant insights at multiple levels: (a) define targets for new therapeutic agents; (b) describe biomarkers to predict treatment response and/or stratify prognosis; and (c) better depict tumor behavior and chemo-radiosensitivity in a single patient and help in the planning of multidisciplinary regimens. Overall, the final goal is the achievement of a greater personalization of the therapeutic strategy, with the early identification of “bad” tumors where treatment should be intensified, and “good” ones in which deintensification can possibly guarantee equal survival with fewer toxicity outcomes.


Angiogenesis is the formation of new capillary blood vessels from pre-existing arteries, veins and capillaries. This process involves proliferation, migration and differentiation of vascular endothelial cells following the stimulation by specific angiogenic factors. Angiogenesis is a hallmark of several physiological and pathological conditions. It contributes to tissue repair, expansion, and remodeling in physiological processes such as wound healing, ovulation and embryo development. Nevertheless, when dysregulated, angiogenesis contributes to disease progression in different pathologies including cancer, atherosclerosis, cerebrovascular diseases and chronic inflammation. Many of these conditions share the same characteristics, including the occurrence of hypoxia, inflammation or oxidative stress, recruitment of inflammatory cells, angiogenic growth factor production, basement membrane degradation, endothelial cell migration, proliferation and differentiation and modulation of vascular support cells.Targeting the angiogenic process is at the present one of the main goals in pharmacology and there has been a great interest in the development of angiogenesis strategies that could inhibit tumor vascularization, diabetic retinopathy, inflammatory or cerebrovascular diseases. At present, the antiangiogenic therapy utilizes drugs that target either the angiogenic factors or angiogenic factor signal cascade. However, several clinical trials have shown that these angiogenic regulators have clinical limitations including short therapeutic effects, development of resistance and adverse reactions which have led to the decrease in the use of anti-angiogenic drugs and have encouraged the search for alternative strategies to treat angiogenic diseases.The term nutraceuticals refers to dietary components that provide health benefit in addition to their basic nutritional value. In the last years, the diet-based approach for the treatment of angiogenesis is gaining more attention, as testified by several reports showing the anti-angiogenesis effect of food components. A wide variety of bioactive compounds identified specifically in plants are known to possess anti-angiogenic effects by modulating different pathways. Thus, identification of natural food components for preventing or ameliorating angiogenesis-associated complications would be of a greater advantage because of their low toxicity or limited side effects.Pan et al.. Pharmacological properties of flavonoids, terpenoids, saponins, alkaloids, polysaccharides, tannins are described, with a particular focus on their ability to inhibit or promote angiogenesis in different models. These bioactive compounds can regulate the transmission of different angiogenesis-related signaling pathways. In particular, the inhibition of growth and migration of vascular endothelial cells, as well as the regulation of the angiogenic factors is one of the basic anti-angiogenic mechanisms of food active components. The use of functional foods or bioactive food components as regulators of angiogenesis is proposed not only to treat tumors and cardiovascular diseases, but also for the prevention and treatment of other angiogenesis-related diseases, including skin wounds and ocular conditions related to aberrant vessels formation.A comprehensive review of the role of chemical components of foods in the regulation of angiogenesis is provided by Smeriglio et al.). However, it has been shown that fruit juices without additives as Citrus juice retain the majority of bio-active compounds of the parent fruit. Smeriglio et al. have investigated the chemical profile and the biological properties of the juice of Citrus lumia Risso, an ancient Mediterranean Citrus fruit. This original study shows that C. lumia juice has antioxidant, anti-inflammatory and anti-angiogenic properties. These activities are due to the marked presence of phenolic acids, flavonoids and ascorbic acid. This study sheds light on the main biological activities of this plant-derived food, although its potential is limited by the low availability of C.lumia fruits. However, the promising results of this study may pave the way to an increase in crop production of this ancient species of Citrus.Among functional foods, there is a global consensus about the protective role of fruits and vegetables, while there is no clear evidence about the effects of fruit juices consuming, due to their lower fiber content and higher caloric density in comparison with the fresh fruits -mediated PI3K/Akt and MAPK/ERK signaling pathway that may further affects angiogenesis. Overall, in this study the authors gained insight into the mode of action of a combination of two compounds from an originally more complex formulation, which could be beneficial to standardize and simplify its complexity while maintaining efficacy.Shexiang Baoxin Pill (SBP) is an oral formulation of traditional Chinese medicine used for the treatment of coronary heart diseases. It displays pleiotropic roles in protecting the cardiovascular system. However, the mode of action of SBP in promoting angiogenesis, and in particular the synergy between its constituents is currently not fully understood. Pan et al.): 1) current research is limited to the use of cells and animal models, and there is no regulation of clinical trials, thus the translation of in vitro and in vivo results on the clinical setting still represents an obstacle to overcome; 2) most studies on the pharmacological activity of natural compounds only indicate a dose range and no indications are available regarding a precise dosage; 3) the regulatory mechanism of functional foods and their active components is not clear as in most cases they consist of mixtures of compounds.Compared with traditional therapy, the use of functional foods or food-derived products is safer and more easily accepted by patients. Nevertheless, the currently available knowledge on these treatments has many limitations (In conclusion, the above works presented in this special Research Topic highlight the enormous potential of functional foods and bioactive constituents of diet in the prevention and treatment of angiogenesis-related diseases, even though a deeper understanding of their mechanisms of action and better designed clinical trials are needed.


Waste management industries are vital to achieve the Sustainable Development Goals (SDGs) suggested by World Health Organization . BesidesThe scientific community and stakeholders should be also aware of the foreseen increased exposure to microbes and antimicrobial resistance in different environments that will lead to a boost of exposure to bioaerosols (for instance mycotoxins and climate-sensitive infectious diseases) in the waste sector, mainly due to climate change , 4.Kontro et al. found that bioaerosol levels were high especially in the composting compared to bioenergy producing facilities. Endotoxin, A. fumigatus and Streptomyces spp. detected in bioaerosols were also observed in the nasal passages of the workers, indicating that bioaerosols in composting plant has great potential to harm to workers' health.Aiming to assess health risks due to bioaerosol emission and air pollution, four articles contributed with their results. Pascale et al. revealed high contamination levels and large microbial heterogeneity both for PM and bioaerosol samples. They also found Bacillus spp., Saccharopolyspora spp., and Thermomyces spp. may be suggested as indicators of biological contamination in composting plants. In addition, this work showed that using multiple assays is helpful to ensure the needed sensitivity and accuracy of bioaerosol detection.Pan et al. assessed airborne bacterial community in electronic waste dismantling site and a waste transfer station based on culture-dependent and culture-independent methods. Bacterial communities in waste-associated bioaerosols were predominated by Proteobacteria spp. and Bacteroidetes spp.. One-third of the species in these genera were uncultured. Differences community structure existed in airborne bacterial diversity among different sampling sites, showing that waste-associated environments have unique bacterial diversity.Bai et al. used the Taiwan Longitudinal Health Insurance Database and the Taiwan Air Quality Monitoring Database to conduct a retrospective cohort study to investigate whether air pollution increases the risk of uveitis. Overall, 175,489 subjects were linked to their nearby air quality monitoring stations; air pollution was significantly associated with incidental uveitis, especially at high total hydrocarbon (THC) and CH4 levels; and uveitis risk increased with increasing NOx and THC levels.Lin et al. reviewed the effects of water pollution on human health and disease heterogeneity. Eighty-five relevant papers were selected. The results shown that the impact of water pollution on human health is significant, although there may be regional, age, gender, and other differences in degree. Taken together, diarrhea caused by enteroviruses in aquatic environments is the most common disease caused by water pollution. Therefore, some suggestions about strengthening water intervention management and carrying out intervention water quality measures have been put forward.For public health risk assessment of wastewater, Ruppen et al. reported that Hwange, Western Zimbabwe used a community-based monitoring to identify sources of pollution and related these to past and present mining activities in a river downstream of a coal mining area. The primary source of acid mine drainage came from abandoned underground mine sites. Concentrations of Mn, Ni, and As were exceeding national fresh water guidelines and international drinking water standards. Results showed that this community-based monitoring offers a promising approach to establish a high-quality dataset for assessing risks.In addition, for health risk of waste management, The exposure to bioaerosols in solid or liquid waste processing facilities and their surrounding can lead to different types of adverse effects. Although they are well-documented by epidemiological surveys and numerous studies have monitored the air microbial contamination in these settings, a clear link between the environmental microbial pollution and diseases of workers or population is still lacking.To better understand the relations between bioaerosol concentration and composition and risks for human health future research is needed, addressing several points:1) Epidemiological studies and dose-response estimation. The evaluation of relationship between microbial contamination and health problems is very complex, because of the diversity of microorganisms in the bioaerosol and the variability of their distribution according to the area and time, as well as the difference in human susceptibility and reactions. Large and long lasting cohort studies, monitoring the air microbial contamination during the follow up could provide answers, but they are hampered by the complexity and costs. Moreover, the current use of personal protective equipment would hide the real risk.2) Exposure assessment. The choice of appropriate indicators or index pathogens should take in account the aim of the study and their measure should be easy, timely, and representative of risk. For safety purposes total bacterial and fungal counts and fecal indicators are commonly used. Nevertheless, the lack of epidemiological studies hampers the definition of limits for an acceptable risk. On the other hand, the study of microbial composition of bioaerosol with cultural or non-cultural methods can show the presence and concentration of pathogens, allowing a more specific risk assessment , 6. Neve3) Risk assessment. The application of the qualitative or semi-quantitative methods can be useful for risk management, to apply preventive and protective measures, but only the Quantitative Microbial Risk Assessment (QMRA) can allow defining acceptable limits and to simulate and evaluate different scenarios . MoreoveCY, CV, YH, and AC contributed to conception and design of the study, organized the database, performed the statistical analysis, wrote the first draft of the manuscript, wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.


This thematic issue on “Male Gonadal Function and Ageing” addresses an emerging and controversial topic of age-related changes in male gonadal function which may negatively impact the health and wellbeing of older men.With the global increase in average life expectancy to 72 years or more, men are living almost a third of their lives after the age of 50. Increasing longevity however has not been accompanied by an extended period of good health. This has resulted in a burgeoning population of elderly men (and women) living for prolonged periods burdened with disabilities and chronic illnesses. Ageing healthily to break the relationship between chronological age and senescence through preservation of physical and mental quality of life (and avoiding or postponing the development of age-related comorbidities) in the elderly has become a major health target and societal concern .Ageing in men is usually associated with a plethora of functional deteriorations and symptoms such as sexual dysfunction, prostatism, sleep disturbance, declining physical strength/function/mobility, falls/fractures, depression, cognitive impairment and accumulation of chronic diseases, culminating in frailty, dementia and loss of independence.The expectation to defer or mitigate the above scenario of the decrepitude of ageing by preserving individual intrinsic capacity  and exteAgainst this background, longitudinal population-based health surveys such as the Baltimore Longitudinal Study of ageing (BLAS) , MassachFollowing up on some of the themes addressed in the landmark studies cited above, this special issue expands on the pathophysiology and clinical implications of the declining gonadal function in the ageing male. We have assembled an international panel of prestigious authors to critically appraise a wide-ranging collection of topics which addresses not only the latest conceptual and knowledge advances or gaps, but also controversies and debates. These state-of-the-art reviews will provide both trainees and experienced clinicians as well as academics with the best available evidence base with which to guide clinical practice and research in the management of the increasing population of ageing men with symptoms and functional deficits that could be related to low T concentrations. The following contains a brief introduction to the topics to be covered.Martins da Silva and Anderson discuss the changes in endocrine and reproductive functions of men with ageing, focussing on changes in function of the hypothalamic-pituitary-testicular axis, affecting testicular T production and spermatogenesis. The decline in T levels occurs largely due to comorbidities, particularly obesity. The prevalence of sexual dysfunction increase with age while erectile dysfunction is a marker of arterial disease. While the male remains potentially fertile throughout his adult life, there are qualitative alterations in spermatogenesis with significant implications for offspring health.Anawalt and Matsumoto. Whether this ageing-related decline in serum T is a cause or a marker of poor health, and whether ageing-related male hypogonadism exists, are critically appraised to integrate available evidence into a rational clinical approach for the assessment and management of an older man with a low serum T concentration.The physiological mechanisms underlying the ageing-related decline of serum T concentrations, the health and lifestyle factors, the aging-related changes in T metabolism and androgen sensitivity associated with this decline are discussed by Kaufman discusses the challenges of correct diagnosis of LOH. The man should present with a clinical syndrome suggestive of androgen deficiency and have consistently low serum T. However, multiple uncertainties are involved in LOH, such as the nonspecific nature of the signs and symptoms of androgen deficiency in older men, the clinical significance of moderately suppressed T levels, the role of associated comorbidities, and the debatable role of T therapy. A pragmatic and appropriately conservative approach is recommended in the diagnosis of LOH. The overall approach to diagnose the syndrome is in principle similar to the other forms of hypogonadism, but the danger of overdiagnosis should be emphasised above the risk of underdiagnosis.The hypogonadism of ageing men, in the absence of organic causes, is often referred to as functional or late onset hypogonadism (LOH). The review of Snyder discusses the benefits and potential risks of T treatment for LOH as well as clinical decision making arising therefrom.T replacement therapy for older men with low T concentrations is a challenging but important issue faced by many clinicians and patients. Expanding on the data from the TTrials, Corona and Maggi summarize and critically appraise the role of low T and the currently available treatment options in the impairment of sexual function of ageing men.The specific contribution of reduced T levels to the reduced sexual function of ageing men is still incompletely known. Likewise, the role of T replacement therapy, phosphodiesterase type 5 inhibitors, and their combination, in the treatment of erectile dysfunction are still controversial. David, Narinx, Antonio, Evenepoel, Claessens, Decallonne and Vanderschueren. The bone loss and increased fracture risk of older men is associated with the decrease of circulating androgen levels. Osteoporosis remains more often undiagnosed and untreated in men than in women. The authors provide several clinical and preclinical arguments in favour of a ‘bone threshold’ for the occurrence of hypogonadal osteoporosis. Although T replacement therapy increases the bone mineral density in men, no evidence for fracture risk reduction is available. T replacement therapy should not be used as the sole bone-specific treatment in ageing men.Osteoporosis also affects men, and its prevalence is increasing with increased life expectancy, as discussed by Gattu, Goldman, Guzelce, Galbiati and Bhasin.One well-known aspect of ageing is the progressive decrease in skeletal muscle mass, strength, and power. The role of the ageing-related decline of T in sarcopenia and the role T supplementation in improving physical function and quality of life in older adults with functional limitations are discussed by Welén and Damber summarises the existing data on the associations between endogenous T and the development of benign prostate hypertrophy (BPH) and prostate cancer (PC). They conclude that there is no connection, and that androgen therapy could even improve PBH symptoms. Apparently, other factors than androgens are crucial for the development of PBH. In PC, circulating androgens are not either associated with the risk of initiation or progression of the disease. This apparent controversy could be related to the androgen receptor saturation model. In addition, the position of PSA measurements in the diagnosis and follow-up of men with T replacement therapy are discussed.The review by Wittert and Grossmann give a critical appraisal for two clinically-important questions, namely: the benefits and limitation of weight (including bariatric surgery) and lifestyle management for obesity/MS/T2DM in reversing low T or hypogonadism; the possible role for TRT in the management of Ob/MS/T2DM including effects on glucose metabolism (insulin sensitivity and glycaemic control) and body fat distribution.Low T is frequently associated with obesity/Metabolic Syndrome (MS)/Type 2 Diabetes Mellitus (T2DM). Potential mechanisms underlying the association between obesity and low T are complex. Yeap and Flicker discuss whether there is a clinically significant role of sex steroids in the occurrence, prevention, or treatment of cognitive decline and dementia in ageing men.Cognitive decline is an inevitable feature of ageing with major negative impact on quality of life. Hauger, Saelzler, Pagadala and Paizzon discuss the interaction of T levels with depression and the antidepressant effect of T treatment in hypogonadal men with depression. Major depressive disorders have clinically heterogeneous phenotypes with multifactorial and polygenic differences in pathophysiology. They reach the conclusion that the benefits of T replacement therapy on mood in men with clinically defined hypogonadism are uncertain.Gagliano-Jucá & Basaria give a critical and sobering appraisal of this phenomenon and provide helpful evidence-based advice for clinicians.The mass marketing of T products by direct-to-consumer product advertising (DTCPA) for common, non-specific, ageing-related symptoms associated with low T in older men has been regarded by some as disease mongering. Men’s health clinics have sprung up across the world and global T prescription have increased by some 20–fold into a multi-billion$ market in the last two decades. Zhao and Crimmins discuss the reasons for this universal phenomenon, which has not been succinctly characterized. The prevalence of lethal diseases is higher in men, shortening their life expectancy, whereas in women, non-lethal conditions prevail. Gender differences in the risk factors for disease have changed over time, and they are also influenced by gender-related differences in behaviour. Basic molecular and cellular measures related to ageing indicate that men age faster than women, but ageing is finally a combination of biology, behaviour, social and environmental factors.Males live shorter lives than women in all countries, and Thirumalai and Anawalt synthesise the latest information  and provides advice for clinicians regarding starting and continuing T therapy in patients with or without prevalent cardiovascular diseases.As T is increasingly prescribed to older men around the world and it is also an integral part of developing hormonal male contraceptives for younger men, the potential risks of T treatment on cardiovascular disease are of critical clinical importance, but the topic remains highly controversial. Liu and Reddy discusses the possible relationships between sleep deficits and variations in T and other steroid hormones which may have important clinical implications, for example for shift workers. The relationships between obstructive sleep apnoea, obesity and T and the effects of T replacement therapy on obstructive sleep apnoea as well as the effects of continuous positive airways pressure (CPAP) treatment of obstructive sleep apnoea on T levels are also discussed.Changes in sleep quantity and quality occur with ageing in men. Carrageta, Guerra-Carvalho, Spadella, Yeste, Oliveira and Alves review the animal models of male ageing and reproduction. Such studies are needed to complement human data, because relevant experimental approaches are needed to unveil the molecular mechanisms by which ageing affects the male reproductive potential. Such models are advantageous, including e.g. their reduced costs, general ease of maintenance in laboratory conditions, the availability of rigorous manipulation tools, the short lifespan of animals, their known genetic backgrounds, and reduced ethical constraints. The best known and widely applied reproductive ageing models are rodents and non-human primates. The data on testicular ageing, steroidogenesis, and genetic and epigenetic changes in spermatogenesis are detailed. Interestingly, some species challenge the current ageing theories and the concept of senescence itself.


In this review, we will focus on different non-invasive retinal imaging techniques that can be used to evaluate morphological and functional features in full-thickness macular holes with a prognostic purpose. Technological innovations and developments in recent years have increased the knowledge of vitreoretinal interface pathologies by identifying potential biomarkers useful for surgical outcomes prediction. Despite a successful surgery of full-thickness macular holes, the visual outcomes are often puzzling, so the study and the identification of prognostic factors is a current topic of interest. Our review aims to provide an overview of the current knowledge on prognostic biomarkers identified in full-thickness macular holes by means of different retinal imaging tools, such as optical coherence tomography, optical coherence tomography angiography, microperimetry, fundus autofluorescence, and adaptive optics. Idiopathic macular hole, also known as full-thickness macular hole (FTMH), is a foveal defect involving all the neuroretinal layers, from the internal limiting membrane (ILM) to the photoreceptor layer . IdiopatThe most accepted classification is the OCT-based staging proposed by the International Vitreomacular Traction Study (IVTS) group in 2013 , which uPars plana vitrectomy (PPV) with gas or air tamponade is widely accepted as the gold standard technique, with reported closure rates after the first surgery ranging from 78 to 96% . HoweverThe technological innovations and developments in retinal multimodal imaging made in recent years have increased the knowledge of FTHMs by identifying a series of potential biomarkers useful for surgical outcomes prediction. In addition to widely accepted prognostic factors of FTHM, such as stage and size, symptoms duration, and preoperative VA ,13,14, sIn this review, we will focus on non-invasive retinal imaging techniques that can be used to assess morphological and functional features in FTMH, evaluating the contribution of each imaging modality in the identification of prognostic factors of visual outcome. Our review aims to provide an overview of different non-invasive retinal imaging tools in FTMH and the prognostic significance of their findings.The integration of several non-invasive imaging techniques has recently gained importance in the characterization of vitreomacular interface pathologies, including FTMHs. Multimodal imaging allows the integration of images acquired with multiple modalities, such as optical coherence tomography (OCT), OCT angiography (OCTA), fundus autofluorescence (FAF), fundus photography, and adaptive optics (AO). Functional imaging modalities, such as microperimetry (MP), can assess the retinal sensitivity and add measurable parameters of visual outcome. The aim of integrated imaging is to overcome the limitations of a single technique obtaining a more thorough comprehension of the morphological and functional features of the disease.The role of different retinal imaging modalities in the evaluation of FTMHs is summarized in Structural OCT imaging plays an essential role in diagnosing and managing FTMHs. OCT has traditionally enabled an accurate diagnosis and differentiation of vitreomacular pathologies and a potential prediction of visual prognosis after successful surgery. In patients with FTMH, structural OCT has been first used to measure the size of the hole, drawing with the caliper function a horizontal line connecting the two closest components of the retina (minimum linear dimension) . Other mThe minimum linear dimension is a critical factor in determining the anatomical success rate of hole closure after surgery, and is the basis for the international OCT-based classification system for FTMH. The success of the surgery and the postoperative VA is better when the hole diameter is smaller in size ,17,18,19Recently, other OCT structural features as predictors of functional recovery after surgery for FTMH have been investigated.We will discuss the following preoperative OCT biomarkers: external retinal layers status, intraretinal cystic changes, supra-retinal pigment epithelium (RPE) granular deposits, macular holes borders morphology, and epiretinal proliferation.Concerning the external retinal layers, structural OCT allows the delineation of three hyperreflective bands: the external limiting membrane (ELM), the ellipsoid zone (EZ), and the interdigitation zone (IZ) . The EZ It has been reported that idiopathic FTMHs may be associated with cystic changes. Intraretinal cystoid cavities at the edges of an FTMH have been recognized in the earliest OCT images as intraretinal hyporeflective spaces, frequently observed around the edges of FTMHs  29]. It. It29]. Ozturk et al. quantified the cyst and macular hole areas, finding a moderate negative correlation with postoperative best-corrected visual acuity (BCVA) . Roth etAlso, the pathophysiology of intraretinal cystoid spaces in FTMH may involve other pathways distinct from exudation and traction, including degenerative cellular loss and retrograde trans-synaptic degeneration, among others . The proAnother specific morphological characteristic of idiopathic FTMH is the presence of supra-RPE granular deposits , which hIt has been reported that FTHM can be associated with the presence of epiretinal proliferation, also known as atypical epiretinal tissue, identified in structural OCT. Epiretinal proliferation presents as a premacular tissue with homogenous medium reflectivity over the internal limiting membrane on OCT and it is distinct from a hyperreflective tractional epiretinal membrane . AlthougFundus microperimetry (MP) is a precise, repeatable, non-invasive modality that provides a functional evaluation of retinal sensitivity (RS) and can be used as a complementary technique in addition to VA measurement to better assess changes in central macular function in patients with vitreomacular pathologies, including FTMHs ,47,48. MOCTA is a non-invasive tool that allows the visualization and measurement of macular microcirculation without dye injection. It can produce high-resolution images of the retinal vasculature and is useful in the analysis of retinal disorders. With OCTA, it is possible to quantify the vessel density in the superficial and deep retinal capillary plexuses (SCP and DCP) and to evaluate the morphology of the foveal avascular zone (FAZ).Although several studies report no conclusive data, OCTA can be considered an objective and non-invasive tool for the evaluation and monitoring of the retinal microvascular changes and quantitative characteristics in FTMH before and after surgical treatment  49,50,5,54,55,56The main issues in the OCTA study of FTMH are about the segmentation, which may produce image artifacts, with consequent difficulties in data interpretation . For thiThe main vascular alterations detected with OCTA in FTMHs seem to be located in the deep retinal layers ,55,56,57Rizzo et al. evaluated FTMHs before surgery by means of OCTA and en face OCT, detecting the most important vascular alterations in the DCP . The autMichalewska et al. evaluated OCTA images in FTMHs, before and after surgery . The autSavastano et al. found a significative correlation between the BCVA and the vessel density both in the superficial and in the deep vascular plexuses at baseline and 6 months after surgery, with the most significant correlation at 6 months follow-up in the deep capillary plexus .The finding that FTMH surgery may lead to an improvement in VA and to the restoration of the vessel density in deep capillary plexus corroborates the importance of vascular deep plexus impairment as a prognostic factor. Pierro et al. also observed in eyes with FTMH that DCP was the most involved capillary plexus . The autSome studies combined OCTA with functional tests, such as microperimetry, to evaluate the correlation between retinal structure and function in patients with idiopathic FTMH before and after surgery ,57,58,59Bacherini et al. conducted a prospective study on patients with FTMH, which combined the evaluation of OCT structural findings with functional parameters obtained by OCTA and microperimetry integration: absolute scotomas corresponding to the FTMH were observed, while rings of relative scotoma in the perilesional area were detected and correlated to perifoveal cystic spaces associated to FTMH on en face OCT and OCTA . SignifiBaba et al. evaluated in FTMHs the vessel density, the RS, and the inner retinal thickness before and after surgery at the four parafoveal quadrants . They foKunikata et al. evaluated retinal vessel density and RS after FTMH surgery with the superior inverted ILM flap technique . They foRegarding the FAZ diameter in FTHMs, it seems to be slightly larger than the minimum diameter of the macular hole and slightly smaller than the maximum diameter of the macular hole as the size of the FTMH gradually increases from the inner retinal layers to the RPE .Different studies have been conducted on FAZ evaluation before and after surgery for FTMH, but they do not report conclusive data ,61,62,63FAZ area seems to reduce after surgery, and it seems to correlate to an improvement in postoperative BCVA ,59,61,62Wilczyński et al. found an inverse correlation between the preoperative superficial FAZ area in FTMHs and the postoperative BCVA . The autBaba et al. also found a decrease in the superficial FAZ area after MH closure, and they attributed this to a centripetal movement of the foveal tissue postoperatively . The autMichalewska et al. found that the FAZ area decreased after surgery in the superficial but not in the deep retinal layers and that the only factor predicting final visual results was the final area of FAZ at the level of deep retina vessels plexus . It mighKim et al. also found changes in the macular vasculature in eyes after MH closure, with smaller FAZ areas and lower macular parafoveal vessel densities, compared with fellow eyes; in addition they found a correlation between the deep and superficial FAZ areas and the postoperative BCVA .Tsuboi et al. conversely found that after an early postoperative reduction of the FAZ calculated in the inner retinal slab, after FTMH closure, it subsequently increased toward the normal value over time . The incShort-wavelength (488 nm) blue-fundus autofluorescence (B-FAF) is a non-invasive retinal imaging modality useful in the evaluation of a wide spectrum of diseases involving the retina and RPE. The short-wavelength FAF signal mainly derives from the lipofuscin in RPE cells. It can vary depending on different factors, such as the presence and quantity of absorptive pigments and structures ,65,66. LThe disappearance of FAF from the FTMH, that may occur after successful surgical repair, suggests that the RPE is again covered by the retinal and/or glial tissue, as also demonstrated by the OCT images ,72,73. POptical coherence tomography and other imaging modalities, such as scanning laser ophthalmoscopy (SLO), could fail to provide sufficiently detailed images of photoreceptor microstructure, primarily because of aberrations in ocular optics. Adaptive optics (AO) is a technology that improves image quality by reducing the effect of wavefront distortions in an optical system. In eyes in vivo, the aberrations can be compensated by using imaging systems incorporating AO, which consists of a wavefront sensor that repetitively measures aberrations on the surface of the eye and a deformable mirror or a spatial light modulator. AO integrated into retinal imaging systems, such as a flood-illuminated ophthalmoscope, SLO equipment, or fundus camera, could enhance the resolution of retinal images, and has been used to document microscopic retinal characteristics such as photoreceptors and vascular structures ,76. AO-fThe rapid advances in retinal imaging over the recent decades have led to a better characterization of FTMHs and significant improvements in their diagnosis and management. The search for biomarkers predictive of surgical outcomes in this pathology is a fast-evolving field of ophthalmological research.A large number of non-invasive tools for retinal imaging are currently available, allowing the acquisition of useful morphological and functional information. These imaging modalities include OCT, OCTA, MP, FAF, and AO. Each single imaging modality provides different and sometimes unique information and has intrinsic advantages and limitations. Recently, aiming to overcome the limitations of each single imaging modality, a multimodal imaging approach have gained popularity in the study of different retinal pathologies, both of medical and surgical interest. The combination of different retinal imaging modalities provides complementary information , increasing the diagnostic sensitivity and specificity and improving the comprehension of pathophysiological aspects of the diseases. Thus, the application of a different imaging approach could enhance the evaluation and management of vitreomacular interface pathologies, including FTMHs, providing a better characterization of their pre- and postoperative features. Moreover, the non-invasive retinal imaging can be periodically repeated after surgery, evaluating the morpho-functional aspects which can correlate with the final visual outcomes.For vitreoretinal surgeons, identifying reliable morpho-functional biomarkers is meaningful for predicting surgical outcomes and choosing the best therapeutic approach. A preoperative evaluation with multiple imaging modalities may add useful information if properly interpreted.In recent years several potential prognostic biomarkers in FTMHs have been identified by means of various non-invasive structural and functional retinal imaging techniques. In this review, we have summarized the key findings of OCT, OCTA, MP, FAF, and AO imaging in FTMHs characterization. From our point of view, structural OCT biomarkers are the most useful in clinical practice: their interpretation is direct, and the availability of this imaging modality is high. However, a combined analysis of structural OCT, OCTA, and MP overlap could be useful for a more comprehensive understanding of the functional consequences induced by anatomical alterations and for the research of new prognostic biomarkers. A few studies recently evaluated the employment of combined microperimetry and OCTA in FTHM, providing useful information on the correlation between structure and function before and after vitreoretinal surgery. However, conclusive, reliable preoperative prognostic biomarkers identifiable with OCTA are still not available. Moreover, despite the high quality of retinal vascular imaging, OCTA images are still not of simple and immediate interpretation for clinicians, and the detection of retinal vascular details information is often inaccurate, probably due to their recent introduction in clinical practice and the scarce availability of reliable biomarkers.In this perspective, the near future directions aim to the application in clinical practice of an advanced imaging evaluation, possibly based on artificial intelligence-based image analysis, in order to achieve higher accuracy and sensitivity in detecting retinal details information. In particular, promising artificial intelligence-based models have been recently developed in ophthalmology for the prediction of different retinal pathologies, including FTMH. Obata et al. investigated if postoperative VA after FTMH surgery could be predicted by means of deep learning using preoperative OCT images . The autDespite successful surgery, predicting the visual outcomes in FTMH is often difficult, and vitreoretinal surgeons need to identify the most reliable prognostic biomarkers. In this review, we resumed different preoperative factors influencing the final functional outcome, identified with different imaging modalities. We believe that the employment of different imaging modalities, if properly interpreted, could improve the comprehension of the functional effects of morphological alterations and could contribute to find new biomarkers of prognosis after surgery. In a future perspective, the application of artificial intelligence-based models in the clinical setting could support the prediction of functional outcomes in macular hole surgery.