Clinical observation and experimental studies have long suggested that the perifollicular nerves have nutritional and regulatory effects on the growth, development, and physiological cycle of hair follicles (HFs), even though the concrete mechanism remains obscure. Recently, with the progress of immunohistochemistry and molecular biology techniques, more innovation has been made in the study of the follicular sympathetic nerves and its nerve-effect factor norepinephrine affecting hair follicle stem cells. This review highlights the progress in the regulation of the sympathetic nervous system toward the growth of HFs. Immature hair follicles (HFs) are divided into three enclosed epithelial cylinders as highly sensitive, dynamic micro-organs developed from the embryonic epidermis. The largest cylinder in the center forms the hair shaft (fiber), and the outermost cylinder forms the outer root sheath (ORS), which separates the intact structure from the dermis. In the middle, the inner root sheath (IRS) shapes the hair shaft and induces its outward growth . Mature Presenting normally as an indispensable part of the physiological environment of HFs, the sympathetic nerves play an important role in the growth of HFs.Our review focuses on the biological characteristics of the sympathetic nerves, along with the application of its nerve-effect factor, norepinephrine (NE), in HF growth.HFs undergo various stages of morphogenesis, which is consistent with a certain state of innervation . From thLocated between blood vessels and HFs, the sympathetic nerves form a tight connection with the arrector pili muscles (APMs) and vascular smooth muscle. In the dermis, sympathetic nerves converge as a single nerve fiber. Enwrapped and penetrated by sympathetic nerves, APMs provide stable anchors that maintain sympathetic innervations to the HFs. Meanwhile, such a reticular network at the dominant site surrounds the hair follicle stem cells (HFSCs) and forms synapse-like connections with HFSCs. Adjacent to the site where APM inserts into the connective tissue sheath of HFs, sympathetic nerve fibers intermingle but extend beyond the APMs, thus composing a spatial connection with the HFSCs at different positions throughout the outer bulge region and the hair germ , 1999.In addition, sympathetic fibers are wrapped by the endoneurium composed of specialized collagen and Schwann cells. As the sympathetic nerves approaches HFSCs, the bundled endoneurium opens only on the side that faces HFSCs, exposing nerve fibers to HFSCs, thus preferably enhancing the diffusion of neurotransmitters such as NE-containing vesicles toward HFSCs. This suggests that the sympathetic nerves innervate not only APMs, but also HFSCs in the extra-follicular bulge area and hair germ layer .Because of the insufficiency of melanocytes in the epidermis, the pigmentation in the trunk of C57BL/6 mice depends completely on the melanin-producing melanocytes in their HFs, whereas the production of melanin in HFs only remains active during their growth period. Also, the color-synchronized growth phases of C57BL/6 mice allow for the isolation and analysis of HFs at specific stages induced by hair extraction . TherefoConsidering the characteristics of uniform hair growth waves induced by shaving the back of C57BL/6 mice, These findings suggest that the formation and sequential appearance of various types of nerve endings, including sympathetic nerves, are determined by HFs rather than programmatically produced by the dorsal root ganglia .Proopiomelanocortin (POMC) is an upstream regulator of the sympathetic nerves in the skin that regulates the secretion of various hormones, including adrenocorticotropic hormone (ACTH), melanocyte-stimulating hormone (MSH), and β-endorphin. The skin not only serves as the target of POMC but also expresses the POMC gene to promote the secretion of related peptides . The traThese changes are accompanied by a parallel expression of adrenergic neurotrophic factor and beta 2-adrenoceptor (β2-AR), -the specific adrenergic receptor on HFs. Likewise, keratinocytes containing β2-AR can be gradually detected in the non-follicular circulating epithelium  during the early anagen . MoreoveIn summary, the periodic physiological distribution of sympathetic nerves in HFs and the diversity of adrenoceptor expression show that the growth of sympathetic nerves is related to the growth cycle of HFs. Furthermore, searching for the periodic expression level of sympathetic nerve-related peptides and upstream and downstream regulatory factors in HFs exploit new ideas regarding of the effect of sympathetic nerves on HF growth and the treatment of pathological situations of HFs.As a unique and sensitive mini-organ, the HF represents not only the target of stress hormones and autoimmune responses, but can also secrete related stress factors that affect skin conditions . The sysRecently studies have shown that a functional stress response system acts independently of the central HPA axis in the HFs, which performs and coordinates the peripheral stress response . EmerginEarly studies have shown that both primary and secondary lymphoid tissues are innervated by postganglionic sympathetic neurons, which secrete NE as primary neurotransmitters, while immune cells express adrenergic receptors, which bind to epinephrine and norepinephrine, enabling the immune system to respond directly to signals from the autonomic nervous system . When th+ T cells, but also indirectly limits the extent of CD8+ T-cell initiation by inhibiting the cross-presentation of dendritic cells  and sympathetic nerves form a dual-component niche to modulate hair follicle stem cell (HFSC) activity, which means sympathetic neurons directly regulate stem cells with NE through synaptic-like structures, while the APMs maintain sympathetic innervation of stem cells. Such a tri-lineage unit formed in the skin is stimulated by cold, causing not only sympathetic excitation and contraction of pili muscles, manifesting as goosebumps, but also activates HFSC. In the absence of NE signaling, HFSCs enter deep quiescence by down-regulating the cell cycle and metabolism and up-regulating the resting regulatory factors Foxp1 and Fgf18. With the progression of the hair cycle, descendants of HFSCs secrete SHH to guide the formation of APM sympathetic niches, which control the regeneration of adult HFs . The triResearchers have reported that a low concentration of 750 nM NE injected into the back of C57BL/6 mice significantly promotes the proliferation of corneal epithelial cells, while 100 μM NE has a toxic inhibitory effect . We suspRelated to the clinically observed phenomenon called “overnight white-head,” which means the rapid destruction of hair pigmentation, a study indicated that acute stress leads to hair graying through the rapid depletion of melanocyte stem cells. Loss of pigmentation was independent of immune attack or adrenal stress hormones but resulted from activation of the sympathetic nerves, which changes the melanocyte niche. The motionless melanocyte stem cells proliferate and differentiate rapidly, then migrate outward from the normal survival area, and become permanently exhausted, causing irreversible whitening of black hair .Abnormal sympathetic nerves distribution was observed in the skin lesions of patients with alopecia areata . In thisA large amount of experimental data has confirmed the reciprocal communication between the sympathetic nerves and HFs, which provides novel insights into the mechanisms of physiology progression on hair growth. Shwartz et al. discovered that the sympathetic nerves secreted NE to activate hair growth of mice , and ZhaThe action of HFSCs is controlled by its niche. This microenvironment incorporates the daughter cells of the HFSCs in the bulge, which enact their self-recovery ahead of schedule and in the late anagen stages . MaintaiWithout the sympathetic signaling, HFSCs enter deep quiescence by down-regulating the cell cycle and metabolism while up-regulating quiescence regulators Foxp1 and Fgf18. During development, HFSCs progeny secretes Sonic Hedgehog (SHH) to direct the formation of this APM-sympathetic nerve niche, which in turn controls HFs regeneration .Hair growth was also regulated prevalently by the Wnt biomolecular pathway and ERK activation in which HFSCs and growth factors are involved. Specifically, it has been additionally demonstrated that Wnt/β-catenin signaling is necessary for the growth and upkeep of the mesenchymal stem cells (MSCs) . The surIf the sympathetic nerves support hair growth, sympathetic neuropeptides must be secreted at a certain level to maintain the stability of the internal environment of HFs. However, the understanding of the aspect of sympathetic neuropeptides in both stable physiological state and pathological conditions of HFs is still limited.Recently, the use of autologous platelet-rich plasma (PRP) was determined to provide substantial help in hair regeneration owing to the platelets’ ability to advance neo-angiogenesis, cell expansion, and separation , 2013. PConsidering the short half-life of neuropeptides and the fast regulation of sympathetic nerves, local application of related drugs may reduce the side effects associated with systemic administration. In recent years, drug carriers such as liposomes, exosomes, and extracellular vesicles (EVs), have been used to effectively deliver neuropeptides into the skin. In a clinical model review, micro-needling is a minimally invasive dermatological procedure in which fine needles are rolled over the skin to puncture the stratum corneum. This therapy is used to induce collagen formation, neovascularization and growth factor production of treated areas. Micro-needling has been used in a wide range of dermatologic conditions, including AGA and alopecia areata . NeuropeThus, given these factors, the concrete mechanism of sympathetic nerves taking effect on hair growth will be key to elucidating the complex regulation of HFs, and will facilitate the identification of druggable molecular targets for hair disease intervention. It makes sense for further experimental or clinical studies to investigate the translational value or clinical applications of the sympathetic nerves in hair follicles.JZ was responsible for manuscript writing. RC, LW, ZF, and YG were responsible for manuscript modifying. YM was responsible for the conception and design. ZH was responsible for the final approval of the manuscript. RC was responsible for the figure preparation and manuscript modifying. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Phylogenetic relationships within the magnoliid order Piperales have been studied extensively, yet the relationships of the monotypic family Lactoridaceae and the holoparasitic Hydnoraceae to the remainder of the order remain a matter of debate. Since the first confident molecular phylogenetic placement of Hydnoraceae among Piperales, different studies have recovered various contradictory topologies. Most phylogenetic hypotheses were inferred using only a few loci and have had incomplete taxon sampling at the genus level. Based on these results and an online survey of taxonomic opinion, the Angiosperm Phylogeny Group lumped both Hydnoraceae and Lactoridaceae in Aristolochiaceae; however, the latter family continues to have unclear relationships to the aforementioned taxa. Here we present extensive phylogenomic tree reconstructions based on up to 137 loci from all three subcellular genomes for all genera of Piperales. We infer relationships based on a variety of phylogenetic methods, explore instances of phylogenomic discordance between the subcellular genomes, and test alternative topologies. Consistent with these phylogenomic results and a consideration of the principles of phylogenetic classification, we propose to exclude Hydnoraceae and Lactoridaceae from the broad circumscription of Aristolochiaceae, and instead favor recognition of four monophyletic and morphologically well circumscribed families in the perianth-bearing Piperales: Aristolochiaceae, Asaraceae, Hydnoraceae, and Lactoridaceae, with a total of six families in the order. Aristolochia , Saururaceae , Asaraceae , Lactoridaceae (Lactoris), Hydnoraceae , and Aristolochiaceae . The former two families are the perianth-less Piperales and the latter four are the perianth-bearing members of the order , Thottea was missing in their sampling.Results of molecular phylogenetic analyses of Piperales in previous studies are generally congruent with respect to the placement of Lactoridaceae. Ignoring the placement of Hydnoraceae, Lactoridaceae are typically recovered as the sister group of Aristolochiaceae, including the studies by Aristolochia, Lactoris, a clade of Asarum and Saruma, as well as a clade of Hydnora and Prosopanche (the two genera in Hydnoraceae). A six-gene analysis (two nuclear and four plastid loci) in the same study placed Lactoridaceae as the sister group of Hydnoraceae, with Aristolochiaceae then sister to this clade, again with poor support (Thottea and Asaraceae were not included in the sampling). Note that in the study by Lactoris as the sister group of Aristolochia and Thottea received poor support in the maximum likelihood (ML) analysis, as did the sister relationship of Hydnora to this clade. Prosopanche was not included in their study. The very short estimated branches separating the families in perianth-bearing Piperales are noticeable, and are in close proximity to the extremely long branch leading to Hydnora. To date, there has been no phylogenetic study that includes all genera of Piperales.Studies that included the holoparasitic Hydnoraceae led to the recovery of multiple different topologies within the perianth-bearing Piperales . For exaLactoris in the order, without consideration of Hydnoraceae. Only a single expert noted the phylogenetic evidence on the placement of Hydnoraceae at that time. Despite this, Apart from these uncertainties on the relationships within the order, the composition of Piperales in terms of its constituent families has also fluctuated in recent angiosperm-wide classification schemes. For example, At the time of the survey only three studies had sufficiently sampled the aforementioned families, and each recovered contradictory and poorly supported topologies concerning their interrelationships . Even igLactoris  methods, using data from all three subcellular genomes. We then test for potential phylogenomic discordance of inferences based on different genomic compartments, analyze and compare the topological results of the largest sampling of loci for Piperales to date, and conduct several topology tests to evaluate the recovered topologies. Finally, considering our phylogenomic results in perianth-bearing Piperales we discuss arguments for the reconsideration of their classification in light of the principles described by Zippelia begoniifolia, Manekia incurva, Peperomia griseoargentea, Verhuellia lunaria, Anemopsis californica, Gymnotheca chinensis, Houttuynia cordata, Thottea sumatrana, and Saururus cernuus was collected at the Botanical Garden in Dresden, Germany, cut into smaller fragments and dried in silica gel. Genomic DNA was extracted using the protocol of Thottea sumatrana, ∼4 M. 150 bp PE reads were sequenced on an Illumina NextSeq500 platform with 500 bp insert size. Genome skimming data of Lactoris fernandeziana was created based on material used by Hydnora visseri were provided by Prosopanche americana by Aristolochia fimbriata were supplied by Yuannian Jiao  as part of a yet unpublished paper.Fresh leaf material of Drimys  and Canella (nuclear) were merged for the concatenated analyses. We are not trying to resolve phylogenetic relationships within the outgroup orders, therefore, this merging is not expected to have an impact on the ingroup results, given that the Canellales terminal serves to anchor that order.Publicly available repositories such as GenBank  and the de novo assembly function in CLC Genomics Workbench  using the MAFFT alignment algorithm v.7.450  and then6 generations, after which chains converged (assessed using the estimated sample size ESS) and a burn-in of 2 × 106 was chosen. In addition to the above analyses, each complete single genome and concatenated nucleotide data set was considered with Hydnoraceae excluded . With regard to the recovered number of loci and the overall locus coverage, the nuclear data set was the most variable , with only a single locus available for Schisandra and Prosopanche (Assembly of newly generated next generation sequencing (NGS) data and database-mined loci of interest recovered varying amounts of data per accession and genome . 83 plasvariable . Completsopanche .Manekia + Zippelia is sister to Peperomia + Piper, and Verhuellia is sister to this entire clade. Within Saururaceae, the clades comprising Gymnotheca + Saururus, and Anemopsis + Houttuynia, have 100% support in all analyses, except in the analysis of nuclear data alone (Anemopsis is sister to the clade of Gymnotheca + Saururus with low support (BS 47%) and Houttuynia sister to the entire clade (BS 100%). Within perianth-bearing Piperales, relationships are identical between the plastid, mitochondrial, and concatenated data-based analyses  was generally well-supported for inferences based on loci from all three subcellular genomes combined , concate studies , short b studies , but als Hydnora . These d Hydnora . All inf Hydnora . In cont Hydnora , in cont Hydnora . The grele locus . These fle locus .The sister relationship of Lactoridaceae + Hydnoraceae, also inferred in the six-gene analysis of Overall, inferences based on mitochondrial data alone proved to be the most consistent across analyses with regards to topology. Topologies within Piperales were identical, regardless of analysis type , data reduction  and partitioning approach. Inferences based on the concatenated three-genome data recovered an identical topology to the predominantly recovered one based on plastid data alone (clade II), though with much higher support for branches within perianth-bearing Piperales. Across all performed analyses, generally the use of gene partitioned ML analyses  tended to lead to the highest support values. Removing data subpartitions that are rapidly evolving (the 3rd codon position) or using amino-acid data and amino-acid substitution models (amino-acids evolve slower than nucleotide data) were unsuccessful in the sense that they yielded poorly supported trees with in some cases altered topology  ASP; thiConsidering all the evidence, the most likely topology for relationships within perianth-bearing Piperales is the one recovered for the concatenated three genome analysis , with stdoes not result in a gain of information” and they argue to instead follow more traditional family circumscriptions until the proposed argument for higher practicability in Aristolochia, Asarum, Saruma, and Thottea), also demonstrated in previous studies  is generally undesirable, and the latter was recommended by ications , 2009. Wwould result in the least amount of disruption” and “…would recognize the morphological distinctions among the members.” The primary principle of monophyly of maximizing the ease of identification”  is widespread in the fossil record, from Late Cretaceous deposits from Namibia (Turonian-Campanian) to India, Australia, and North and South America .The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: TreeBase (SW: conception of the study. MJ, M-SS, IM, SG, and SW: data generation. MJ: analyses and visualization of results. MJ and SW: writing of the first draft. All authors reviewed and edited the draft and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The critical role of the coronary microvascular compartment and its invasive functional assessment has become apparent in light of the significant proportion of patients presenting signs and symptoms of myocardial ischemia, despite the absence of epicardial disease, or after the adequate treatment of it. However, coronary microvascular dysfunction (CMD) represents a diagnostic challenge because of the small dimensions of the coronary microvasculature, which prevents direct angiographic visualization. Several diagnostic tools are now available for the invasive assessment of the coronary microvascular function, which, in association with the physiological indices used to investigate the epicardial department, may provide a comprehensive evaluation of the coronary circulation as a whole. Recent evidence suggests that the physiology-guided management of CMD, although apparently costly and time-consuming, may offer a net clinical benefit in terms of symptom improvement among patients with angina and ischemic heart disease. However, despite the results of several observational studies, the prognostic effect of the physiology-driven management of CMD within this population is currently a matter of debate, and therefore represents an unmet clinical need that urgently deserves further investigation. The invasive assessment of coronary artery disease (CAD) has historically focused on understanding the epicardial coronary anatomy and function. More sophisticated tools have been developed since it became clear that angiography was not sufficient to appropriately evaluate the composition and the ischemic potential of coronary atherosclerotic plaques. Intravascular imaging techniques and intracoronary physiology tests are now widely used to aid in diagnosing and managing CAD. However, there is much more to coronary circulation beyond epicardial arteries, including the microvasculature, which is often neglected, and which still represents a “black box” for clinical and interventional cardiologists. Coronary microcirculation is a complex and structured system of small vessels , which adapt their function in order to sustain the myocardium’s physiological needs. The clinical importance of this vascular compartment has become apparent in light of the significant proportion of patients presenting signs and symptoms of myocardial ischemia, despite the absence of epicardial disease, or even after the adequate treatment of it . These pWithin the definition of CMD , two majNevertheless, several other non-endothelium-dependent mechanisms may equally lead to CMD. In particular, the microvascular wall remodeling, due to the increased wall-to-lumen ratio , with or without the loss of the myocardial capillary density (capillary rarefaction), may cause a decrease in the microcirculatory conductance, with an impaired oxygen delivery capacity ,9. Of noIrrespective of the underlying pathophysiological mechanism, CMD seems to be associated with traditional cardiovascular risk factors. A large body of evidence highlights a higher incidence of CMD among females who smoke  , or amonMicrovascular angina (MVA) represents the more specific and reliable symptom of a broad and heterogeneous spectrum of disorders known as INOCA (ischemia with nonobstructive coronary arteries), of which CMD is a relevant component. According to the COVADIS definition  Table 2Table 2, Among the causes of INOCA, vasomotor tone abnormalities of both the epicardial and the microvascular districts play a crucial role. The sudden occurrence of chest pain due to coronary spasm, so-called “vasospastic angina” (VSA), should be suspected according to the COVADIS criteria  if the fBlood flow across coronary vessels consists of a highly dynamic phenomenon, aimed at delivering an adequate blood supply to the myocardium, either at rest or during exercise. The ability of the entire coronary bed  to actively adapt its size to satisfy any increased myocardial oxygen demand is invasively and noninvasively assessed with a physiological index known as the “coronary flow reserve” CFR, . AlthougThe CFR may be measured invasively using thermodilution or Doppler flow velocity , or noniCFV rest . The meaCFV rest ,20,21; iCFV rest . On the CFV rest ,24, theras found ,26, thusurements ,28, and urements ,30.Of note, several other noninvasive methods (PET and CMR) used for assessing the CFR have been considered with encouraging results: both PET and CMR seem to provide independent prognostic information in patients with known or suspected CAD ,32,33. AA comprehensive assessment of coronary circulation concerning the functional relevance of epicardial CAD using hyperemic and non-hyperemic indexes and the CFR grants a detailed analysis of ischemic heart disease . More spAccording to Ohm’s law, the vascular resistance (R) is equal to the driving pressure (∆P) divided by the flow rate (Q): R = ∆P/Q. ∆P is the pressure difference across the myocardium (Pd – Pv), and Q represents the coronary flow, which is known to be inversely related to the Tmn (Assuming that the venous pressure is close to zero (Pv = 0), the final equation will be: Pd × Tmn. Therefore, the index of coronary microvascular resistance (IMR) is calculated with thermodilution as the product of the distal coronary pressures (Pd) and the Tmn during maximal hyperemia  39]. Of. Of39]. Although the IMR is independent of the hemodynamic state and the coronary flow, many studies have shown a relevant overestimation of the microvascular resistance with critical epicardial stenosis. In the presence of severe epicardial stenosis, both the coronary flow and the collateral flow contribute to the overall myocardial flow; therefore, assuming the IMR as the product of the Pd and the Tmn during maximal hyperemia, one or more epicardial flow-limiting stenoses may significantly prolong the time needed to travel down the coronary artery, thus determining a higher IMR . HoweverTheoretically similar to the thermodilution-based IMR, hyperemic microvascular resistance HMR– is a Dopsistance . The HMRsistance . Howeversistance , while osistance .Although a gold standard for coronary microvascular assessment has not yet been established, several studies have compared the thermodilution-derived IMR and the Doppler-derived HMR, aiming to determine the level of agreement between the IMR and the HMR, and to compare the ability of the IMR and the HMR to predict the independent measures of CMD. However, only a modest correlation was found between them, thus suggesting that these measurements cannot be considered equivalent predictors of CMD .Minimal microvascular resistance mMR– is anothThe resistive reserve ratio (RRR) is a thermodilution-derived index that integrates both the coronary flow and the pressure as the ratio between the basal and hyperemic microcirculatory resistances BIRIMR, . As a coThe RRR has been initially investigated as a potential predictor of post-PCI complications in acute coronary syndromes (ACS). In a population of ST-segment elevation myocardial infarction (STEMI) patients, an RRR ≤ 1.7 was associated with the coronary microvascular obstruction (CMVO) extent, myocardial hemorrhage, the infarct size, and adverse clinical outcomes . The proBoth the CFR and the IMR are thermodilution-based physiological indexes in which the coronary flow and the microvascular resistances are indirectly estimated via the Tmn of a manually injected saline bolus, thus implying that its measurement depends, to a certain degree, on an operator injection technique. Moreover, both the CFR and the IMR require the achievement of adenosine-induced stable hyperemia.The principle of absolute coronary flow (Q) and microvascular resistance (R) has been proposed to overcome these limitations through the adoption of a technique that requires a continuous infusion of saline and thermodilution . ContinuBoth the absolute coronary flow and resistance  have beeThe main limitation of continuous thermodilution is that it does not provide enough information regarding the amount of myocardial mass subtended by the coronary flow. However, the association of continuous thermodilution with noninvasive methods of myocardial mass quantification (computed tomography or magnetic resonance imaging) has been proposed to compare the standardized values of either the CFR or the IMR from different myocardial territories and patients .In order to outline a surrogate of microvascular function that is based on the operator-independent measurements of the absolute flow and pressures, as well as one that is unrelated to the epicardial resistance, the autoregulation principle, and the myocardial mass and, therefore, that is specific to coronary microcirculation, the microvascular resistance reserve (MRR) was proposed by De Bruyne et al. as a novel index of the microcirculatory function. In an analogy to the FFR concept, the MRR has been described as the ratio between the resting and hyperemic microvascular resistances, theoretically depicting a quantitative assessment of the extent of the potential microvascular resistance decrease resulting from a hyperemic stimulus. In its general form, the MRR can be expressed as the ratio of the CFR to the FFR, corrected for the driving pressures. However, the MRR cut-off values, as well as the clinical and prognostic relevance of the MRR compared to the other existing indexes of microvascular function, are not yet currently established .Initial exploratory results about the potential clinical significance of both the absolute coronary flow and resistance have been reported by Konst et al. : in a coThe invasive functional assessment of coronary microcirculation represents an essential resource for deeply understanding the broad spectrum of ischemic cardiopathy mechanisms and improving the treatment and management of these patients. Although CMD investigation has been initially restricted to cardiovascular research, several studies support the hypothesis of the potential clinical benefit of its adoption in daily practice, particularly in patients with CAD in the absence of flow-limiting epicardial stenosis . The rouMoreover, the prognostic impact of CMD among patients who underwent PCI has been largely investigated. When already present before the intervention, microvascular dysfunction may predict periprocedural outcomes in patients with stable CAD ,65, thusIn addition, several pieces of evidence supporting the key prognostic role of the postprocedural abnormal IMR are currently available. High post-PCI microvascular resistances are significantly predictive of poor procedural-related and long-term outcomes for patients undergoing both elective ,67 and pCoronary microcirculation has also been extensively assessed in the setting of an ACS. CMD and CMVO, a well-known complication of coronary interventions in the presence of a high thrombotic burden, occur in up to half of the patients undergoing otherwise successful primary PCI . In factSeveral preventive measures have been investigated with regard to the wide range of mechanisms contributing to PCI-related microvascular dysfunction and myocardial injury. Nicorandil, a potent cardioprotective agent, which acts by selectively opening the mitochondrial adenosine triphosphate-dependent potassium channels, improves coronary perfusion in both the epicardial and microvascular compartments . InteresSimilar perioperative results have been observed after the periprocedural administration of verapamil. In particular, post-PCI improvements of the microvascular function appear to be reasonably attributable to the beneficial effect of intracoronary verapamil in preventing , or reveThe available evidence with regard to perioperative myocardial injury prevention measures have been further expanded with the ProMicro (PROtecting MICROcirculation during coronary angioplasty) study. In this randomized placebo-controlled clinical trial, administering a single bolus of enalaprilat before PCI improved coronary microvascular function, and protected the myocardium from procedure-related injury in patients undergoing elective PCI . Among sHowever, some contrasting evidence is currently available. In the randomized controlled Strategies of Loading With Prasugrel Versus Clopidogrel in PCI-Treated Biomarker Negative Angina (SASSICAIA) trial , a pre-PAlthough appearing costly and time-consuming, the invasive microvascular assessment allows for the achievement of crucial information to establish more appropriate decision-making processes and therapeutic strategies for patients with ischemic heart disease. In fact, despite all of the recent research and clinical efforts, the global burden of myocardial ischemia persists at unacceptably high levels, resulting in a worse quality of life and a poorer prognosis , particu


Various nutrients can change cell structure, cellular metabolism, and cell function which is particularly important for cells of the immune system as nutrient availability is associated with the activation and function of diverse immune subsets. The most important nutrients for immune cell function and fate appear to be glucose, amino acids, fatty acids, and vitamin D. This perspective will describe recently published information describing the mechanism of action of prominent nutritional intervention agents where evidence exists as to their action and potency. The immune system protects the host against infection from pathological microorganisms and provides constant surveillance for malignant cells that arise over a lifetime. The immune system is able to develop appropriate tolerance to self-proteins, circulating macromolecules, self-cells, and tissues, and to harmless environmental molecules .Individual heterogeneity regarding the intensity of immunological responses exists, largely dependent on genetics, environment, lifestyle, nutrition, and the interaction of these factors. Nutritional immunology is a field of immunology that describes the influence of nutrition on the immune system, antiviral activity, and associated protective functions . DeficieThis perspective will describe recently published reports describing the mechanism of action of prominent nutritional intervention agents where evidence exists as to their action and potency . This perspective has concentrated on describing the molecular and/or metabolic basis of their immunological effect.Glutamine is the most abundant amino acid in the blood and in the free amino acid pool in various tissues . The con2, aspartate, alanine, and lactate in a process called glutaminolysis , whereas glutamine is converted into glutamate, aspartate, and alanine by undergoing partial oxidation in the TCA cycle to CO in ref. –7. Thus, in ref. .Different populations of macrophages have now been identified, including M1 and M2 macrophages –11. M1-lGlutamine metabolism and supply of metabolic intermediates have been proposed to be essential to the antiviral activity of the immune system, although many of the mechanisms are as yet unknown .l-arginine is 0.077 mM, approximately tenfold less than glutamine. Arginine is particularly important in the liver, where it is an intermediate in the urea cycle, thus is an intermediate in amino acid catabolism. Arginine is also very important in a number of immune cells, as a substrate for nitric oxide synthase (NOS), thus is a substrate for the production of nitric oxide, a potent immunoregulatory mediator that is cytotoxic to tumor cells and many microorganisms. Macrophages can produce relatively large amounts of nitric oxide, especially the M1 inflammatory macrophage, via increased concentrations of the enzyme inducible NOS (iNOS). The activated macrophage can actually release the enzyme arginase to deplete local concentrations of arginine (indeed arginine is required for tumor growth). It has been reported that mouse peritoneal resident and Bacillus Calmette–Guerin (BCG)-activated macrophages and human monocytes are capable of utilizing glutamine at high rates, contain sufficient activity of the enzymes required to convert glutamine to citrulline  to account for observed rates of nitrite synthesis (derived from NO) in the absence of extracellular l-arginine, and will release nitrite when exposed to intermediates of the proposed glutamine --> arginine pathway [l-arg and indeed may play a role in the suppression of the antitumor response [The plasma concentration of  pathway . Thus thresponse .Fatty acids (FAs) play several essential roles in the homeostasis and structure of the cell and various tissues and organs. FAs are the main components of all biological membranes and are built into sphingolipids, phospholipids, glycolipids, and lipoproteins. They are also the major source of energy stored in the triacylglycerols. In addition, various metabolites of FAs serve as essential intracellular and extracellular lipid mediators and hormones. Therefore, FAs have many possibilities to modulate immune cell function by influencing their structure, metabolism, and function, acting through surface proteins , nuclear receptors, or membrane transporters . Much haVesicular stomatitis virus [Many viruses, including coronaviruses, are enveloped viruses with a nucleocapsid mainly made of phosphorylated nucleocapsid protein embedded inside a phospholipid bilayer envelope. This envelope plays an important role in virus assembly and release from the host cell, and it is critical for viral pathogenesis. The viral surface consists of the spike protein of the virus, which is essential for the attachment to the host cell receptor and for initiating infection. Given the indispensable nature of lipids in multiple stages of viral replication, lipid synthesis and metabolism in host cells have been recognized as essential for the assembly of coronaviruses. Bioactive lipids with antiviral properties include certain long-chain monounsaturated fatty acids (MUFAs) and medium-chain fatty acids (MCFAs), specifically lauric acid (LA), the primary fatty acid of coconut oil. It is a saturated MCFA with potent antimicrobial properties . Long-chis virus .The active form of vitamin D  is usually abbreviated as vitamin D. Vitamin D receptors have been identified in most immune cells . MacrophA recent study reported for the first time the potential influence of vitamin D status on bioenergetics and metabolism in freshly isolated peripheral blood mononuclear cells. The data indicated a close relationship between Vitamin D levels and immune cell bioenergetic responses. Low vitamin D levels were clearly associated with a pattern consistent with increased oxidative metabolism and inflammatory activation that was reflected in altered bioenergetics of PBMCs .Studies on the effects of nutrient combinations, whole foods, and diets on human immune cell metabolism and function, in vivo and ex vivoNutritional supplementation and potential prevention or slowing of immunosenescence by altering immune cell metabolism and functionInvestigation of the role of obesity in altering immune cell metabolism and function in humans, including antiviral activityThe role of the gut microbiota in shaping host immune maturation, metabolism, and functional responses in animals and humans including antiviral activityDetermination of the role of glutamine metabolism and supply of glutamine derived metabolic intermediates in the essential antiviral activity of the immune system (adapted from ref. ).


Nosocomial pneumonia is one of the most frequent infections in critical patients. It is primarily associated with mechanical ventilation leading to severe illness, high mortality, and prolonged hospitalization. The risk of mortality has increased over time due to the rise in multidrug-resistant (MDR) bacterial infections, which represent a global public health threat. Respiratory tract microbiome (RTM) research is growing, and recent studies suggest that a healthy RTM positively stimulates the immune system and, like the gut microbiome, can protect against pathogen infection through colonization resistance (CR). Physiological conditions of critical patients and interventions as antibiotics administration and mechanical ventilation dramatically alter the RTM, leading to dysbiosis. The dysbiosis of the RTM of ICU patients favors the colonization by opportunistic and resistant pathogens that can be part of the microbiota or acquired from the hospital environments (biotic or built ones). Despite recent evidence demonstrating the significance of RTM in nosocomial infections, most of the host-RTM interactions remain unknown. In this context, we present our perspective regarding research in RTM altered ecology in the clinical environment, particularly as a risk for acquisition of nosocomial pneumonia. We also reflect on the gaps in the field and suggest future research directions. Moreover, expected microbiome-based interventions together with the tools to study the RTM highlighting the “omics” approaches are discussed. The healthy respiratory mycobiome is enriched in environmental fungi from the phyla Ascomycota and Basidiomycota, the most common also in the GIT , as it has been extensively documented for the intestinal microbiome , and poor adaptive immune response , and/or carbapenemases, and methicillin-resistant Staphylococcus aureus (MRSA), which are often selected and spread in the ICU environment  or paranasal viruses established in the oral-nasopharyngeal cavity, which can colonize and eventually cause respiratory infections  by competence-activated killing factors (bacteriocins). The killing leads to HGT by acquiring exogenous DNA from the dead cells, including ARGs , lyses and acquires genes through HGT from neighboring Escherichia coli cells, including ARGs  or by the stochastic propagation of bacteria  seems to work through systemic dissemination of beneficial anti-inflammatory and antimicrobial metabolites, such as short-chain fatty acids The gut-lung axis might become harmful in circumstances of famine and during critical illness. In the ICU, the customary use of anti-acid therapy  might contribute to gut-respiratory transmission. The abdominal cavity (peritoneum) and the thoracic cavity (pleura) are neighbor spaces separated by a dynamic and not totally hermetic membrane (diaphragm), and then pneumonia is frequently associated with peritonitis  to contain a minimum microbial load. Similarly, the area involved in oxygen acquisition by the respiratory tract has evolved to be very poorly colonized . Whether a normal indigenous RTM in the LRT persists over time or is constant community immigration from the URT is a matter of debate and challenge. Until the development of high-throughput sequencing approaches, the LRT colonizers in the absence of infection have been disregarded. This fact was primarily due to the failure to grow lung microbes in routine microbiological cultures  are poorly understood. Furthermore, the identification of “keystone” microorganisms  and their microbial interactions (network's edges), generally involving mechanisms such as metabolite exchange, metabolite conversion, signaling, or genetic exchange, remain largely unknown. Identifying the RTM composition, the microbial interactions, and the mechanisms involved in CR are becoming relevant to define a “healthy microbiome” along the respiratory tract and to understand the impact of clinical and environmental factors that lead to dysbiosis. The development of accurate, personalized microbiome-based treatments would significantly remodel the management of patients with respiratory diseases.The detection of microbial eukaryotes (fungi and protozoa), archaea, and viruses have been neglected in RTM research. However, through the application of “omics,” the RTM composition of non-bacterial microorganisms is starting to be described  have been applied to restore the gut microbiome in several conditions associated with antibiotics treatments, although studies of its efficacy have yielded heterogeneous but moderately positive effects  is a promising approach that has been successfully applied to restore the microbiome of patients under recurrent As the FMT stimulation of the intestine activates the immune lung responses, another way to cure respiratory infections via the gut-lung axis is by stimulation of the production of gut microbe-derived metabolites, such as the SCFAs or polyamines that have positive systemic effects , and the Instituto de Salud Carlos III (PI18/1942) co-funded by the European Regional Development Fund . Also supported by InGEMICS-CM (B2017/BMD-3691), funded by Comunidad de Madrid (Spain) and CIBER  and , integrated in the Spanish 2013-2016 I+D+i State Plan and funded by Instituto de Salud Carlos III. AEP-C is recipient of a Grant for the Attraction of Talent within the Comunidad de Madrid (Grant No. 2019-T2/BMD-12874).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Through this editorial, we aim to present the main aspects related to the scientific contributions that can be found in the Special Issue dedicated to the topic “Investigation of Polymer Nanocomposites’ Performance”. By going through this editorial, the readers will have an idea of the potential of polymer nanocomposites in some cutting-edge applications.It is well-known that nanotechnology represents one of the most promising areas of current research and development in several fields. In the case of polymer science, the investigations have been mainly focused on novel technologies such as nanoelectronics, polymer-based biomaterials, nanoparticle drug delivery, miniemulsion particles, layer-by-layer self-assembled polymer films, electrospun nanofibers, imprint lithography, polymer blends, and nanocomposites ,2. For pAfter the editorial process of analysis and in-depth verification by reviewers, six contributions were accepted, coming from researchers working in universities located in five different countries.The articles included in this Special Issue focus on the importance of polymer nanocomposites in some emerging technical fields. This Special Issue reports only some examples of the potential of nanocomposites in modern technology. Given the complexity of the discussed topic and the wide variety of technological applications of these materials, we have not been very restrictive in setting specific themes because nanocomposite technology can be approached from a multitude of angles.Given the recent environmental concerns about plastic pollution, in the last years, there has been a worldwide increase in societal interest in using environmentally friendly plastic materials, which has in turn increased the number of polymers being researched and developed. Among them, great attention has been devoted to bioplastics, i.e., plastics derived from renewable resources and/or biodegradable materials . These mw/v) can strongly improve the barrier properties and the tensile mechanical behavior of the resulting films. The materials could be therefore considered as good candidates for the production of novel biodegradable packaging films.The paper of Calambas et al., entitled “Physical-Mechanical Behavior and Water-Barrier Properties of Biopolymers-Clay Nanocomposites”, reports on the preparation of novel biodegradable starch-polyvinylalcohol (PVA)-nanoclay nanocomposites films . DiffereIn the last decades, bioderived polysaccharides, such as chitosan (CS) and pectin (PC), have attracted the attention of researchers and industries, as they can be applied in the biomedical and packaging field . MoreoveAnother interesting group of biopolymers that has recently attracted much academic interest is that of polys (PAFs). These novel polymers can be synthesized from the polycondensation between an alkylene glycol and 2,5-furandicarboxylic acid (FDCA), which has been recently listed among the top 12 high-value-added chemicals from the biorefinery of carbohydrates by the United States Department of Energy. In this sense, PAFs represent the most promising bioderived alternative to fossil-based poly (PATs), as they show thermo-mechanical and gas barrier properties similar or superior to those of the corresponding PATs . The papAmong the biocomposites containing small amounts of inorganic nanofillers, those including selenium nanoparticles (SeNPs) could be particularly interesting because of their high bioavailability, low toxicity, and therapeutic properties . In factAs discussed earlier, the scientific interest in polymer nanocomposites is not only aimed at improving the mechanical performance, but also at increasing the multifunctionality of the resulting materials. A clear example of this approach has been reported in the paper of Bertani et al., entitled “Improving the Antimicrobial and Mechanical Properties of Epoxy Resins via Nanomodification: An Overview”, which summarizes the potential of multifunctional nanomodified epoxies coupling improved antimicrobial activity and enhanced mechanical properties . These mThe application of novel approaches for the formulation of multifunctional rubber-based nanocomposites could be particularly interesting for the modern tire industry. Considering that rubber materials generally exhibit poor thermal transfer, the addition of carbon-based or inorganic thermally conductive fillers at elevated amounts could represent an effective solution to achieve satisfactory heat dissipation performance . HoweverIn our opinion, the purpose of this Special Issue has been well achieved. Going through the six papers grouped in this Special Issue, the readers can have some valuable examples of the real potential of polymer nanocomposites in modern society. Interestingly, in these works, the investigation of the physical behavior of the presented systems has been often accompanied by the definition of novel experimental protocols.As a future goal, we would like to extend the proposed approach by editing another Special Issue in which the multifunctional properties of polymer nanocomposites could be applied to energy saving/storage/management applications. In fact, many examples of polymer nanocomposites for electrical and thermal energy storage can be found in the open literature, as well as novel nanocomposite systems for thermal insulation. Considering the recent environmental concerns about global warming, a Special Issue on this topic could meet the interest of many industrial and academic readers.


In the space of four decades, Brazil has faced two serious pandemics: human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) and Coronavirus disease 2019 (COVID-19). The country’s response to HIV/AIDS was coordinated by several stakeholders and recognised the importance of scientific evidence in guiding decision-making, and a network offering monitoring and antiretroviral treatment was provided through coordinated efforts by the country’s universal health system. Conversely, the lack of a centrally coordinated strategy and misalignment between government ministries regarding the COVID-19 pandemic response, together with the denial of scientific evidence, promotion of ineffective treatments and insufficient vaccination efforts, have all led to the uncontrolled spread of infection, the near-total collapse of the health system and excess deaths. In 1982, Brazil reported its first cases of acquired immunodeficiency syndrome (AIDS) as the country was emerging from a military dictatorship (1964-1985). In 1986, the Ministry of Health (MoH) established the National STD/AIDS Program, which involved extensive stakeholder participation.HIV/AIDS and Coronavirus disease 2019 (COVID-19) are diseases with distinct epidemiological, biological and clinical profiles. However, when faced with both infections, the scientific community responded promptly by identifying and sequencing causal agents, developing diagnostic tests, characterising each disease, setting up clinical trials to evaluate potential treatments and providing clinical facilities to care for infected individuals. Concerning COVID-19, effective vaccines were quickly developed using innovative technologies. Considering its experience acquired during the AIDS pandemic, the extensive reach of SUS and the presence of a robust national immunisation program (PNI) capable of vaccinating millions per day, Brazil had all the necessary tools to effectively respond to the COVID-19 pandemic. Unfortunately, despite activating the national Emergency Health Operations Centre in January 2020, prior to diagnosing the first domestic case,Many factors can help explain Brazil’s antithetic response to COVID-19 in comparison to HIV/AIDS. Disparities among Brazilians throughout the continental-sised country demanded coordinated efforts in both cases. For example, the alignment between the MoH and the Ministry of Foreign Affairs enabled Brazil to have a prominent position in international forums and to express leadership in negotiations to expand universal access to antiretrovirals. In contrast, since the beginning of the COVID-19 pandemic, there has been disarticulation regarding the acquisition of inputs, such as diagnostic kits and masks, between these same ministries. The lack of appropriate strategies has also hampered the acquisition of raw materials essential to vaccine production, as well as to vaccines themselves. In addition, misguided and often negligent decisions were also taken due to deficient coordination between the MoH and the Ministry of Economy, Finance and Planning, resulting in inadequate financial support to facilitate adherence to social distancing measures.It is important to note the autonomy of the MoH and the National AIDS Program with regard to ideological preferences or outside influence from private groups, which permitted the establishment of bold price negotiations in the purchasing of antiretrovirals, as well as the establishment of progressive health campaigns aimed at AIDS prevention. On the contrary, the government’s response to the current COVID-19 epidemic has been characterised by an absence of autonomy, as technical and expert committees have been marginalised at the expense of feeble solutions and ineffective treatments.Brazil also played an essential role to the approval to the use of compulsory licensing of drugs during pandemics, such as HIV/AIDS, as a basic human right.Denying the severity of COVID-19 and issuing recommendations for ineffective treatments has facilitated non-adherence to preventive measures.The restrictions imposed by EC95 weakened SUS and evaporated financial support necessary to spur scientific and technological development. All this is also in contrast to the AIDS epidemic measures.,,All of these aspects may have potentially increased morbidity and mortality, especially among the most vulnerable populations,The HIV/AIDS epidemic in Brazil prompted clear political decision-making and the establishment of strong partnerships among several stakeholders, including government leaders, civil society, universities, researchers and health professionals. Collaboration was also very clear and effective between ministries. As already mentioned, a good example was the alignment of the MoH and Foreign Affairs which facilitated the prominent position in various international fora, especially World Health Oraganization/Joint United Nations Programme on HIV/AIDS (WHO/UNAIDS), in defense of human rights, against discrimination and for a worldwide expansion of access to antiretrovirals. To effectively combat COVID-19, the lessons previously learned from the Brazilian experience with HIV/AIDS must be applied to counteract isolationism, boost international solidarity and cooperation, adequately finance science and quality public health services, confront anti-science rhetoric and anti-vaccine movements and prioritise egalitarian access to technological progress.


Nidovirales (The members of the coronavirus (CoV) family refer to infectious disease, which refer to CoVflu, CoV pneumonia, or a respiratory syndrome. COVID-19 causes various contagious diseases,such as the Middle East respiratory syndrome (MERS), severe acute respiratory syndrome (SARS),which causes common strains of the cold. CoVs are of the new diseases of the respiratorysystem , which iovirales . The terovirales .The corovirales .The outbreak of the infectious virus first time appeared in Wuhan, China, in late December,2019, and later this pandemic (COVID-19) rapidly spread in other cities of China and over 200countries worldwide by end of 25 April 2020 . The IraEconomic factors: the financial inability of the government of Iran to protect the nationfinancially during quarantine was not sufficient, which in turn caused the majority of people,who usually wing their bread, and remain active in many crowded places and business centers.It made a descendant course of the COVID-19 infectious disease and its control impossible associal distancing, which is one of the most critical principles to reduce the risk ofcontracting the virus, specified a disruption . BesidesSocial factors: although most people observed self-quarantine at homes; however, it developedempathy and intimacy among family members at a larger scale. These social factors causedpsychological problems and violence by creating differences among people as well as verbal andphysical disputes. Besides, staying at home for a long time may lead to depression, lethargy,and exacerbation of previous illnesses like hypertension, stress, and anxiety. The othersocial factor refers to the lack of adequate consumption of antiseptics at home, which leadsto other problems, such as damage to the respiratory system. People have reacted to thispandemic and became sensitive in interacting other individuals at large .Cultural factors: it becomes necessary to abuse certain social conditions and to endanger thesafety and health of people by some people, leading to hoarding and smuggling of basic healthproducts and food .Legal factors: the lack of participation of the private sector in the performance ofsemi-clinical services (laboratory and radiology) in the outpatient unit and inpatient wardsof patients with COVID-19 in the hospital indicates that there are no clear and specific rulesin this area .BesidesPolitical factors: although clerics have cooperated in advising communities to remain athomes and follow lockdown policy of the government and performed religious obligations athomes, such as Friday prayers. Besides, people did not practice a suppression strategy, andthe government did not suspend visits to pilgrimage locations, which was a contradicted policyof the Irani government. It caused a rapid spread of the COVID-19 in all provinces in Iranbecause of lacking control over disease transmission. It is vital to note that this issue wasfirst limited, then managed and controlled. The other challenge was to diagnose patients withthe disease and whose polymerase chain reaction (PCR) tests indicated virus-positive, theywere considered as halo, and those with symptoms were not reported on computed tomography (CT)scans for it, which in turn led to a discrepancy between official and actual reports .As mentioned above, the factors directly correlate with the mental health of ordinaryindividuals from one end of the disease; however, there is a severe fear that thousands ofdeaths are from the other end. The COVID-19 outbreak has adversely affected the economy, andpeople have lost their jobs, which has failed in gaining a minimum income level to runlivelihood. The fear of economic loss has increased stress and leads to mental health problemsamong people worldwide.The government of Iran has taken protective measures and policies to reduce anxiety among thepublic to tackle mental health problems. The government of Iran established specialized healthcenters to treat CoV patients after discharge from the leading hospitals. This motive washelpful to keep COVID-19 patients separate from their families until the full recovery, whichwas beneficial to prevent further spread of the deadly disease. The government offered utilitybills delay for 3 months, and payment in installments was helpful to provide comfort topatients' families, which reduced mental stress.The officials paid full attention to reduce anxiety, stress, and mental health issues byproviding necessary measures. The government has initiated schemes to encourage support donorswho cooperated with poor and low-income individuals with financial support. Online andtelephonic psychological counseling services to cater to CoV patients and other healthy peopleare useful through free-of-cost initiative of the government. It has helped to reduce thecommunity’s mental stress. The government has started loans at the low-interest rate for needypeople, controlled social network sites to identify and prevent spreading sensational andfalse information about CoV disease. It helped to manage the terror of fake facts aboutCOVID-19 in the community.The government launched social support programs, and psychological protocol services for thesurvivors of COVID-19 through screening, and identifying suspects of this disease with mentalhealth surveys. Plans were launched Covid-19 dissemination in the cyberspace, education, andcorrect informing was provided to the mass public through media to prevent fear and terror inthe society. The government launched special services for CoV patients for the funeral processas it is one of the cultural factors in society. The government launched programs to educatethe public about the spread of CoV disease and reduce the public mental stress in thecommunity. The protective measures helped to build confidence among people, which helped toovercome fear, anxiety, stress, and mental health problems.The health experts recommended solutions to slow down, sustain and overcome the transmissionof COVID-19 problems as mention below:Priority for investment in the health sector compared to other areas such as military,weapons, and armaments.Allocation of subsidies to all citizens with private business, low-interest loans tolarge and small medium enterprises.Specific attention to vulnerable and elderly people and actively diagnosis of thedisease.A comprehensive view of the health sector and the participation of all agencies andinstitutions such as the government and non-governmental sectors.


Molecular biophysicists and microbiologists working on antimicrobial peptides (AMPs) are traditionally worlds apart. While biophysicists prefer detailed structural and functional approaches in systems they have total control of, such as lipid vesicles, microbiologists trade the details of molecular interactions for the “biorealism” of working with live bacteria. These two types of studies seemed impossible to reconciliate, until now. In recent years, biophysical techniques started to be applied in the quantitative investigation of the interaction of AMPs with target and host cells, providing several novel insights in the mechanism of action of these molecules. The articles of this Research Topic provide a comprehensive overview of the current state-of the-art in this area.Top researchers devoted to AMPs, translating molecular biophysical approaches to bacterial microbiology, have gathered in this Research Topic publication to (i) review how imaging and spectroscopy techniques can be used to unveil AMP action directly in live bacteria, and (ii) present the fundamentals of the mechanism of action of AMP obtained with such techniques, including the influence of intracellular factors, synergy, and cell density. Structure-function relationships and a vision for future applications of antimicrobial surfaces are also present.Gelmi et al. present a general overview of the experimental approaches that provide information on peptide-cell interaction, including fluorescence and calorimetry to quantify peptide-cell association, circular dichroism, and NMR to investigate the secondary structure of cell-bound peptides, infrared and NMR spectroscopies to study peptide effects on membrane structure and dynamics, zeta potential experiments to clarify the electrostatic aspects of the interaction, microscopies, and cytofluorimetry to study peptide localization and effects on bacterial growth and perturbation of cell membranes, scattering techniques to study peptide effects on cell shape and photocrosslinking coupled with mass spectroscopy to identify potential protein targets.Clayton reviews the applications of fluorescence microscopic techniques to follow in real time the main events involved in bacterial killing. These studies are starting to illustrate the complexities of peptide-cell interaction: for instance, they showed that the timescale of AMP effects on bacteria is orders of magnitude slower than that required for the perturbation of artificial membranes, identified preferred sites of attack in cell membranes and demonstrated that peptide sequestration by dead cells can protect the rest of the bacterial population from the action of AMPs.Booth focuses on deuterium solid state NMR studies of intact bacteria treated with AMPs. This approach provides information on peptide-induced perturbation of cell-membranes. For instance, it showed that some AMPs can cause disruption of the lipid bilayer at peptide:lipid ratios lower than those needed for killing, suggesting that membrane disruption may not be the only mechanism by which they harm cells.Separovic et al. address in-cell solid-state NMR studies of AMPs, too, providing an overview of the different molecules that can be investigated with this technique, including peptidoglycans, lipopolysaccharides, phospholipids, DNA and AMPs themselves. The power of this non-invasive technique is exemplified by a recent study, which revealed a peptide-induced disruption of the molecular packing of both bacterial membranes and DNA.Benfield and Henriques focus on the experimental approaches that can determine the mechanism of action of AMPs, and particularly differentiate peptides perturbing cellular membranes from those that interact with intracellular targets. Disruption of membranes can be observed in cells, using fluorophores that detect membrane depolarization or accessibility of the intracellular space. By comparing membrane perturbation and cell viability, membranes can be confirmed or excluded as the main target of AMPs. Peptide uptake into bacterial cells can be investigated using flow cytometry and fluorescence microscopy. Finally, intracellular protein targets of AMPs can be identified using bacterial proteome microarrays.Bechinger et al. analyze the crucial property of synergy in AMPs: often, two (or more) peptides together have a higher activity than the single components. The molecular mechanisms of this effect are still heatedly debated. For the synergism between magainin 2 and PGLa, different hypotheses have been put forward, involving direct interaction of the peptides or changes in peptide orientation, but experiments performed with lipid mixtures correctly mimicking bacterial membranes support lipid mediated effects that favor peptide/membrane binding for the AMP mixture. The authors also show that a major effect of a synergistic combination is to increase the steepness of bacterial killing as a function of peptide concentration. This finding is consistent with a previous study on other AMPs  in E. coli and lipid vesicles. In particular, they determined both an upper limit on the number of surface-adsorbed peptides and total number of peptides partitioned into the bacteria These data indicate that 95–99% of LF11-215 and LF11-324 molecules accumulate inside cells, suggesting that these AMPs might act on intracellular targets.Schefter et al., in their original work, discuss several parameters that influence AMP activity and selectivity: density of target and host cells, competitive peptide association to the two cell populations, and peptide sequestration by association to intracellular targets. The authors propose a biophysical model, based on chemical equilibria, leading to a prediction of the cell-density dependence of peptide activity and selectivity, and allowing the correct design and interpretation of selectivity measurements.Strandberg et al. review the specific class of artificial AMPs constituted by sequences of alternating cationic and hydrophobic residues, such as [KL]n, or [RW]n. When bound to membranes, these peptides attain a beta-like structure. Longer peptides can also form beta-aggregates in solution. Medium length peptides of 8–10 amino acids appear to be the optimum compromise for a high activity and low toxicity.Mullen et al. present their perspective on the use of peptides and other molecules to design antimicrobial surfaces. This is a view into the future of peptide-based products and technologies to fight bacterial threats, mainly biofilms, which are probably the next big challenge ahead for AMP developers.After bridging molecular biophysics to microbiology, it is important to bridge these fields to bacterial biofilm biomedicine. AMPs are up to the challenge. Are we, too?Manuscript content was discussed and decided by all authors. MC and LS drafted the manuscript. All authors revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Retinal neurodegeneration affects an increasing number of people worldwide causing vision impairments and blindness, reducing quality of life, and generating a great economic challenge. Due to the complexity of the tissue, and the diversity of retinal neurodegenerative diseases in terms of etiology and clinical presentation, so far, there are no cures and only a few early pathological markers have been identified. Increasing efforts have been made to identify and potentiate endogenous protective mechanisms or to abolish detrimental stress responses to preserve retinal structure and function. The discovering of the intracellular monomeric globin neuroglobin (NGB), found at high concentration in the retina, has opened new possibilities for the treatment of retinal disease. Indeed, the NGB capability to reversibly bind oxygen and its neuroprotective function against several types of insults including oxidative stress, ischemia, and neurodegenerative conditions have raised the interest in the possible role of the globin as oxygen supplier in the retina and as a target for retinal neurodegeneration. Here, we provide the undercurrent knowledge on NGB distribution in retinal layers and the evidence about the connection between NGB level modulation and the functional outcome in terms of retinal neuroprotection to provide a novel therapeutic/preventive target for visual pathway degenerative disease. Globally, in 2015, it was estimated that blindness and moderate/severe vision impairments affect more than 36 and 216 million of people, respectively, and the number has raised along with the increase in population and age . DespiteThe retina is a high-differentiated nerve light-sensitive tissue that has been estimated to be responsible for the 80% of all sensory information in humans ,10. FuncRetina metabolism is the highest of the total central nervous system and the lack of oxygen under hypoxic conditions is thought to have a direct detrimental effect on visual function and to be pivotal for retinal disease ,15,16. TThe complex structural and functional nature of the retina makes such tissue extremely vulnerable to aberration due to any kind of injury ,5. In faRetinal neurodegenerative diseases are different pathologies in terms of etiology and clinical presentation . AffectiDespite differences in terms of etiology across the retinal neurodegenerative disease, among them, the molecular and cellular mechanisms of degeneration show a large pattern of similarity ,4,5. Ind50 value ranging from 0.9 mmHg to 10 mmHg for human NGB and from 0.9 mmHg to 2.2 mmHg for the murine globin . However, the discovery of NGB as a monomeric intracellular globin, that has been so named for its preferential expression in the central and peripheral nervous system, has raised the possibility of a myoglobin-like function of NGB ,25. NGB  globin  strain which shows defects of mitochondrial respiratory chain complex I and RGCs loss [However, beyond the possible interspecific differences of NGB in the retina, the peculiar property of the globin as an inducible protein whose levels and intracellular distribution is highly modulated by physiological states ,49, hormGCs loss . On the GCs loss  and plasGCs loss  of patieGCs loss  has beenConsequently, since the inducible nature of NGB and the proposed function in the rapid stress response aimed to preserve retinal tissue ,60, NGB The preferential expression of NGB in the retina suggests that it can be sensitive to multiple retinal diseases. Based on the time-dependent modulation of retinal NGB levels under stressing conditions , the gloHq mice, overexpressed NGB has been demonstrated to restore the mitochondrial function and improve RGCs survival, to maintain nerve fiber integrity, reduce the glial cell activation and, in turn, protect the visual functions [Despite studies reporting no effects of lack of NGB on light-induced cellular stress response  have raiunctions . Furtherunctions .In addition to the transgenic-induced overexpression of NGB, an alternative approach using the injection in the mouse eyes of a chimeric human-zebrafish NGB with the ability to cross the plasma membrane, demonstrated that high levels of intracellular NGB led to a threefold increase in survival of RGCs accompanied with a significant axon-regenerating outgrowth after optic nerve injury . SimilarDespite the efficacy of genetic and molecular approaches in up-regulating intracellular NGB levels and enhancing its neuroprotective effects, the identification of exogenous or endogenous inducers of NGB has been considered a valuable way to understand the molecular pathways regulating NGB levels and, in turn, to deepen the NGB neuroprotective functions aimed to preserve neuronal functionality under stressing conditions. In vitro and in vivo evidence have demonstrated the positive effects of stressing condition in regulating NGB levels, justifying a possible role of the globin as a key compensatory factor in the endogenous neuroprotective pathway(s) devoted to promoting adaptive response and resistance to death in the brain neurons ,72,73,74Evidence of NGB function in preserving the neuronal cell death and visual function when ectopically overexpressed (see above) along with the documented rapid increase in its endogenous levels under acute stress condition ,47,59,60The discovery of the intracellular globin NGB has suggested its function as an oxygen supplier and carrier protein in high metabolic active neurons including those of retinal tissue ,34,39. ABased on the common mechanisms of neurodegeneration across different retinal neurodegenerative diseases including oxidative stress, inflammation, and apoptosis, several therapeutic approaches are aimed at restoring an adequate retinal environment to promote cell viability by using compounds able to impact several biochemical pathways . Among t


This review presents the analysis and systematization of modern data on the molecular mechanisms of autism spectrum disorders (ASD) development. Polyetiology and the multifactorial nature of ASD have been proved. The attempt has been made to jointly review and systematize current hypotheses of ASD pathogenesis at the molecular level from the standpoint of aberrant brain plasticity. The mechanism of glutamate excitotoxicity formation, the effect of imbalance of neuroactive amino acids and their derivatives, neurotransmitters, and hormones on the ASD formation have been considered in detail. The strengths and weaknesses of the proposed hypotheses have been analyzed from the standpoint of evidence-based medicine. The conclusion has been drawn on the leading role of glutamate excitotoxicity as a biochemical mechanism of aberrant neuroplasticity accompanied by oxidative stress and mitochondrial dysfunction. The mechanism of aberrant neuroplasticity has also been traced at the critical moments of the nervous system development taking into account the influence of various factors of the internal and external environment. New approaches to searching for ASD molecular markers have been considered. Autism spectrum disorders (ASD) is a comprehensive problem of modern psychology, neurology, and related sciences which is being much discussed . This isIn 80s of the XX century, autism was considered a relatively rare disease. In recent decades, however, the percentage of ASD diagnosing is growing steadily –9. AccorThe term “autism spectrum” was introduced by L. Wing in 80s of the XX century. It implied a continuum of the diseases accompanied by the disorders in communication, social interaction, social understanding, and imagination. Presently, the concept includes genetically and clinically heterogeneous psychic disorders united by the sign such as impaired social interaction. This is a group of the heterogeneous complex disorders of the nervous system development , 23.The classification of the American Psychiatric Association  uses the term “spectrum” in the “Autism spectrum disorders” section (299.00 (F84.00) code) , since tThe main symptoms of autism are distinguished as follows , 27:decrease of communicative and social skills including speech communication;stereotypy in behavior and speech; restricted, specific interests.The described triad of symptoms can be accompanied by a weak visual contact, sensory dysfunction, impairment of cognitive and motor functions of various degree , self-agAs a rule, autism spectrum disorders are diagnosed at the age of 3–5 years, it is possible to reveal them at 1.5 years and in some cases at 6–12 months; the symptoms are preserved in adolescence and adulthood . CurrentThe mechanism of ASD development is rather complicated, it includes, inter alia, heredity (64–91%) and genetic predisposition. More and more reports appear in the literature about new candidate genes , 38. In The aim of the present review was to systematize, summarize and describe in greater detail the main hypotheses of ASD development. The primary task was to reveal potential targets which could be used for the development of the novel methods for ASD diagnosis, prevention, and treatment.The pathogenesis of autistic disorders is presently not clear enough. There exist a number of theories: genetic concepts, impairments of brain development associated with the impact of perinatal, neurochemical, immune, and other factors.For a long time, hereditary causes of autism have been considered dominating . DespiteThe risk of ASD development in siblings is known to be 20 times higher than in the general population. Twin studies have shown that family accumulation of autistic traits is a consequence of high heritability (h2:0.8). Patients were found to have changes in the number of chromosomes in separate cells, nucleotide substitutions in the structural genes, an abnormally changed gene copy number –49. TherThe majority of ASD-associated genes determine functioning of the nervous system and the activity of certain proteins influencing the reproduction of genetic information , 53. MahStudying the literature devoted to the genetic component of autism, two important facts were discovered.Firstly, the effect of these mutations starts still in the process of the nervous system formation in the perinatal period and in early childhood as well. A number of mutations converge on the common path of the nervous system maturation in neurogenesis, axon development, and synapse formation  and thesSecondly, there appear first attempts to prove the existence of interconnection between genetic, ecological factors and epigenetics , 58. AccColle et al.  have estGender , metabolic and chromosomal diseases  are referred to other factors.Thus, according to the present concept, ASD develops under unfavorable exo- and endogenous factors in the critical periods of CNS ontogenesis when structural and functional qualitative changes providing the formation of more complex functions take place.However, the mechanisms realizing the impact of these factors are not yet fully studied. Numerous genetic investigations and proposed diagnostic criteria are unfortunately not often reproducible and need further improvements.This direction in the study of ASD is related to the established disorders of the nervous system development at the early ontogenetic stages. Using current diagnostic methods  and histological investigations of the nerve tissue samples, abnormalities of the brain development have been detected , 82. It Spread of diffuse changes in the brain results in disrupted interneuronal contacts, commissural and associative connections. For example, an alteration of functional coordination of cerebrocerebellar circuits in ASD in presence of the excitation/inhibition imbalance (glutamate + glutamine/glutamate + GABA ratio) has been shown , 84. AllThe role of external factors provoking the CNS developmental disturbances not only at the genome level but also in the process of neuroontogenesis has been determined , 89. TheNeurochemical hypotheses consider mainly ASD genesis from two sides: in connection with abnormal formation of neurotransmitter systems  related to the deviations in the exchange of some amino acids and with the development of the oxidative stress .Literature analysis has revealed the emergence of dysfunctions in mediator systems due to the exchange of amino acids and their derivatives  participating in the processes of synaptic plasticity .Glutamate (glutamic acid) is one of the main excitatory neurotransmitters responsible for learning, memory, behavior, motion, and sensation. Glutamate regulates induction of synapses and their interconnection with astrocytes, cell migration, synaptic spatial organization of the cerebellum, cell differentiation, and apoptosis.Gamma-aminobutyric acid (GABA) is responsible for synaptic inhibition  being thAccording to the data presented by Al-Otaish et al. , increasNot only glutamate concentration plays an important role but also a prolonged imbalance in the excitatory and inhibitory mechanisms of GABA and glutamate , 97. GABThe ratios of the listed metabolites in the blood are proposed as ASD biochemical markers .Serotonin (tryptophan derivative) in the nervous system is responsible for cell proliferation and differentiation; migration; apoptosis; synaptogenesis; maturation of the prefrontal cortex, parts of the limbic system, corpus callosum; regulation of emotions, mood, memory, and learning. Muller et al. [r et al.  have notToo high efficacy of the serotonin reuptake transporter (SERT) in autism was also reported , 101, thCatecholamines (tyrosine derivative) in the nervous system are neurotrophic factors, neurotransmitters, modulators of the neural and humoral regulation. Dopamine, in particular, is responsible for attention, emotional reward, sensory and motor functions, communication skills which are impaired in autism [DβH. A low activity of this gene in the perinatal period creates the conditions for autism development [n autism . The risn autism . Thus, melopment . It is celopment .Obviousness of implication of some amino acids and biogenic amines in ASD development should be recognized. Taurine (inhibiting neurotransmitter), arginine (nitric oxide donor), methionine (choline formation) have also been noted in maintaining the balance between the inhibitory and excitatory systems regulating neuroplasticity .Stroganova et al.  suppose Having obtained the results on an ASD animal model Wang et al.  proposedOXTR) in individuals with ASD diagnosis. This epigenetic modification may contribute to the formation of autistic social and behavioral phenotypes.Oxytocin and vasopressin, hormones secreted in hypothalamus and stored in the posterior lobe of hypophysis, are considered among the factors participating in ASD development. The interest to them was not accidental. Oxytocin is an important modulator of human associative behavior including social skills, pair bonding, parental affection and friendship, confidence formation . OxytociDysfunction of dopamine-oxytocin systems is supposed to lead to the lack of striving for rewards in patients with ASD, therefore the reward system during treatment may be ineffective .Carson et al.  suggest Describing neurochemical mechanisms of ASD development, glutamate excitotoxicity should be distinguished as one of the prime causes .Excessive amount of glutamate, aspartate, and other excitatory neurotransmitters leads to overexcitation of ionotropic glutamatergia due to NMDA (N-methyl-D-aspartate) receptors and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) resulting in the increase of calcium concentration in cytosol. Calcium ions activate the inducible isoform of NO synthase, increasing nitric oxide concentration which, in its turn, activates phosphorylation of protein kinase C and phospholipase A2.A succession of these events leads to the enhancement of lipid peroxidation (LPO). Free radicals may inhibit oxidative phosphorylation decreasing ATP production in mitochondria and triggering apoptosis cascade. The ATP products such as 4-hydroxynonenal (4-HNE) may interact with synaptic proteins and disturb transport of glucose and glutamate, increasing sensitivity of the nerve cells to excitotoxicity .The imbalance of excitation and inhibition at the cell level causes the appearance of new properties in the neuronal network. Levin, Nelson  believe It is notable that the child’s brain is more vulnerable to excitotoxic effects as the developing nervous system contains more synaptic receptors of glutamate than at birth and their quantity decreases with age . TherefoThe vitality of the hypothesis is confirmed by the fact that mitochondrial dysfunction and oxidative stress, as the links of pathogenesis, may influence social and cognitive impairments in autism , 114. PaAt the same time, abnormally high concentrations of lipid peroxides were determined in these areas of the brain being evidence of oxidative stress induction .+/ K+-ATPase activity increases the glutamate receptor sensitivity which, in its turn, leads to depolarization and increased excitation of the nervous system in the regions connected with motor activity and promotes calcium output. The enumerated processes are part of glutamate excitotoxicity, thus neuroinflammation, oxidative stress, and glutamate excitotoxicity are part of the signaling system associated with ASD. Hypoperfusion, the extent of which correlates with autism severity, is also considered to be a cause of this dysfunction. Chauhan et al. [Patients with ASD and animal models were noted to have increased number of reactive oxygen species (ROS), LPO activation, and accumulation of its products . At the n et al.  have sup12 usually accompanies folate deficit causing neurological disorders and congenital defects [12 deficit is in inverse proportion to the homocysteine level which is known to be a modulator of the lipid metabolism [3 in childhood therefore its additional intake is also recommended as a method of treatment and prevention of ASD [To correct mitochondrial dysfunction and oxidative stress, various types of supplements to the diet including antioxidants  are proposed by some authors , 120. Ch defects . В12 deftabolism . Increastabolism . In one tabolism , the roln of ASD .Melatonin (tryptophan derivative) is functioning as an electron donor reducing electrophilic radicals. There is experimental evidence that it acts as an indirect antioxidant stimulating antioxidant enzymes. Several studies have shown that melatonin diminishes chronic and acute inflammation, NO amount, and the level of malonic dialdehyde .Thus, the oxidative stress and mitochondrial dysfunction occupy an intermediate position between several factors of external environment, glutamate excitotoxicity, and neuroinflammation in pathochemistry of ASD.Immune hypothesis suggests –127 the Additional investigations  demonstrIL-6 is a neuropoietic cytokine influencing neuronal proliferation, synapse formation, differentiation, and migration. It is supposed that activation of the immune system which may be connected with ASD development in children depends on mother’s IL-6. Besides, IL-6 plays a critical role in the development and modulation of autistic behavior due to the disorders in neuronal network formation .The recent study  has discThe investigation carried out by Bryn et al.  has showIn recent years, disruptions of endocannabinoid signaling pathway in the formation of neuroinflammation have been explored .The growing interest in the role of chronic inflammation in neurological disorders is connected to the fact that generalized alterations in oxidative/antioxidant status, as well as immune, endocrine systems, and neuromediator transmission in the brain take place in inflammation . Immune The emergence of this theory which got the name “exorphine hypothesis of autism” is linked to the discovery of casomorphins (products of milk casein hydrolysis), gliado- and glutenomorphins (products of grain crop protein hydrolysis) which are dietary-derived opioid peptides. Data on the interconnection of autism with the impairment of the intestine barrier permeability and the decrease in the activity of proteolytic enzymes breaking down proteins and peptides underlie the hypothesis.These disorders result in an increased level of the non-spliced peptides, caso-gliado- and glutenomorphins, in the blood. The generated exorphins penetrate the brain with the blood current where they affect the opioid and related neurochemical systems resulting in the development of autism spectrum symptoms , 137. GlShank3. Some ASD may be connected with the dysfunction of neuroligins and neurexins. Angelman syndrome (happy puppet syndrome) is associated with the excess of target-proteins, Arc in particular, in the postsynaptic space. Its function is the internalization of AMPA-subtype glutamate receptors [Synaptic plasticity is considered to be implemented by protein biosynthesis changes. Thus, Phelan–McDermid syndrome is associated with the loss of functionality by gene eceptors .There is an opinion about the role of microRNA in the formation of aberrant neuroplasticity since microRNA is capable of inhibiting protein synthesis by transcription repression and destabilization and destruction of mRNA. The necessity of continuing investigations in this direction is also noted by the authors .A local translation is a finely regulated process; the central role in the regulation initiation is played by mTOR  signaling pathway which integrates numerous intracellular and extracellular signals including growth factors, nutrients, stress, infections and is also implicated in the formation of memory and long-term synaptic plasticity. The disruption of separate links of this pathway leads to the development of autism spectrum disorders , 141. ImFragile X syndrome is linked to mTOR hyperactivation. After the application of mTOR inhibitors  on the models of the mutant mice with increased mTOR activation, an improved condition of the animals was observed . NotablyIt is interesting to note that low levels of mTOR activity are associated with Rett syndrome . BDNF prArmeanu et al.  report tThe conducted analysis of the current literature sources on the mechanisms of autism spectrum disorders proves polyetiologicity, complexity, and multifactorial nature of this group of diseases related to the disorder of brain development and functions. The majority of researchers today are convinced that autism is a genetically determined illness. However, not every individual possessing candidate genes develops autism spectrum disorder, and if the disease develops it may have different degree of severity. The epigenetic factors and factors of external environment are able to activate by various ways the conversion of the nervous system to the autistic type. These factors must act at the early stages of the nervous system formation — at the perinatal and early postnatal periods, i.e. actually we may speak about aberrant neuroplasticity in the critical moments of the nervous system development. However, the mechanisms by which these factors influence the development of aberrant neuroplasticity in autism spectrum disorders need further studies and clarification.In 2013, Essa et al.  showed tIn this connection, the possibility of using glutamate, GABA, oxytocin, serotonin, dopamine; combination of IL-6 and serotonin, ratios of various interleukins; enzyme activity of mitochondrial electron transport chain and, so on, is considered as diagnostic criteria, meaning that there is no unified biosensor at present time which would be highly selective and sensitive in autism spectrum disorders. This is extremely important as medical rehabilitation must start at the very early stages of autistic disorder formation. Additional investigations, meta-analysis of data using specialized databases (“dry biochemistry”) are required to form a complete picture of the ASD nature. Their high prevalence demands stirring up researcher activity in this direction. This will help elaborate a more effective therapeutic strategy in regeneration of physiological homeostasis in patients as well as in creation of unified generally recognized diagnostic criteria within the frames of evidence-based medicine.