Basic and translational research in reproductive medicine can provide new insights with the application of scanning probe microscopies, such as atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM). These microscopies, which provide images with spatial resolution well beyond the optical resolution limit, enable users to achieve detailed descriptions of cell topography, inner cellular structure organization, and arrangements of single or cluster membrane proteins. A peculiar characteristic of AFM operating in force spectroscopy mode is its inherent ability to measure the interaction forces between single proteins or cells, and to quantify the mechanical properties  of cells and tissues. The knowledge of the cell ultrastructure, the macromolecule organization, the protein dynamics, the investigation of biological interaction forces, and the quantification of biomechanical features can be essential clues for identifying the molecular mechanisms that govern responses in living cells. This review highlights the main findings achieved by the use of AFM and SNOM in assisted reproductive research, such as the description of gamete morphology; the quantification of mechanical properties of gametes; the role of forces in embryo development; the significance of investigating single-molecule interaction forces; the characterization of disorders of the reproductive system; and the visualization of molecular organization. New perspectives of analysis opened up by applying these techniques and the translational impacts on reproductive medicine are discussed.  Basic research in the reproductive medicine field has led to the development of the assisted reproductive techniques (ARTs) that can overcome human infertility. Like other biology areas, this research field benefits from implementing new technological approaches  and advaThe visualization of cell ultrastructure, macromolecular organization, and protein dynamics in living cells provides relevant insights to identify and then clarify the molecular mechanisms involved in biological processes of the reproductive system. Several imaging techniques based on material optical properties  or on fluorescent dyes  have been exploited to characterize the structural organization of gametes and embryos. Essential contributions to the knowledge of these cells were also provided by nonlinear optical approaches ,7. OtherWhile the resolution offered by typical optical microscopies is sufficient to study, e.g., whole-cell or tissue morphology, many subcellular structures and processes remain elusive, hindered by the diffraction limit. For this reason, the biophysical community has directed enormous efforts to developing new super-resolution optical and fluorescence approaches for live imaging, such as total internal reflection fluorescence (TIRF) and stimulated emission depletion (STED), which improved the study of localization and dynamics of biomolecules and organelles in cells at the nanoscale level , as in eMoreover, to improve the knowledge of the molecular mechanisms involved in the reproductive system, it is crucial to analyze other biological properties and processes. The emerging field of mechanobiology, at the interface of biology, physics, and engineering, has highlighted the importance of mechanics in biological processes . With a In this framework, AFM and SNOM appear to be valuable tools for the reproductive biologist. Unlike standard or novel imaging techniques, AFM and SNOM are versatile platforms that can acquire multiple pieces of information: topography, high-resolution imaging, and biomechanics. This information allows a better understanding of how morphological, biochemical, and mechanical properties come together to guide cell processes. This review collects more recent findings obtained employing these techniques in several conditions affecting human fertility. The potentiality of these microscopies in the analysis of molecular mechanisms by single-molecule manipulation is examined. Finally, the translational relevance of these findings in view of developing diagnostic tools and therapeutic treatments is discussed. During the past 30 years, scanning probe microscopies (SPM) quickly gained widespread attention, from hard materials science to biology . SPM encIn AFM, the probes  have a radius of curvature that ranges from a few nanometers for very sharp tips up to 50 nm, and it is located at the end of a flexible cantilever made of silicon or silicon nitride. Forces acting between the probe and the sample surface cause a tiny cantilever bending that is detected with a position-sensitive detector, which measures the deflection of a laser beam focused on the backside of the cantilever A. By mulIn imaging mode, the tip is scanned across the sample surface to obtain a topographical reconstruction. AFM Imaging differs mainly in how the tip moves over the cell sample: contact, intermittent, or non-contact modes: the last two are together referred to also as dynamic modes. In contact mode, the tip is in constant repulsive interaction with the sample surface. This can lead to friction effects, which may drag single proteins or locally modify or damage the cell surface. In dynamic mode, the cantilever is driven at its resonant frequency while the oscillation amplitude and phase are monitored, thereby minimizing the tip-sample interactions. In particular, in non-contact mode the tip vibrates close to the sample surface always in the attractive regime of forces and never touches the surface; in intermittent contact mode the tip oscillates between repulsive and attractive forces, tapping the sample while scanning, but still with reduced friction effects while contouring the biological sample. All these imaging modes can provide nanometric spatial resolution images of cells or single proteins in fluid conditions . The devOne of the most relevant advantages of AFM with respect to other microscopic techniques resides in the possibility of sensing and applying a wide range of forces (from pN up to a few μN) corresponding to the ones that dominate biological phenomena, from the single protein to the single cell . In forcSNOM combines AFM-like topographical information with super-resolution optical images of the sample. SNOM can operate in both aperture and apertureless modes . In aperApertureless SNOM, on the other hand, is based on light scattering from the tip, and its intensity depends on the tip-sample interaction. Without the need for a fiber aperture and relying on scattered, rather than transmitted light, the tip size can be downscaled to a few nanometers with improvements in spatial resolution . Since tThe application of electron microscopy has allowed describing in detail the ultrastructural anomalies of the human spermatozoa  ,35 and oHigh-resolution analysis of surface topography by amplitude  and spatial roughness has been demonstrated to help quantify morphological alterations of the spermatozoa and describe the effects of new male contraceptives . A similIn the oocytes AFM imaging is used to characterize the topography of zona pellucida (ZP). The ZP is a porous network of glycoproteins with a thickness of 10–15 μm that surrounds mammalian oocytes. This peculiar layer has several functions: it regulates the sperm interaction and binding, prevents polyspermy after fertilization, and protects early embryos moving in the female reproductive tract. The penetration of the ZP matrix by spermatozoa is a crucial step in oocyte fertilization, and their inability to penetrate it inevitably leads to infertility. ZP dysmorphology, like dark ZP, large perivitelline space, or irregular-shaped ZP, is associated with reduced implantation and pregnancy rates following IVF compared to normal oocytes ,45,46. AThe mechanics of oocytes deserve particular attention not only for their relevant role in the maturation stages of the oocyte and in the fertilization process, but also for the appealing opportunity to use mechanical features in the screening and selection of oocytes in the assisted reproduction practices. Several papers have reported that the cell’s mechanical phenotype is correlated with its physiological or pathological status ,49,50. TIn ARTs, trained embryologists select oocytes to fertilize or the embryo to transfer, mainly by visual inspection via bright field optical microscopy, subjecting the selection to personal interpretation. To improve this selection method, several non-invasive methodologies based on optical imaging techniques have been proposed ,52,53. SThe mechanical properties of the oocytes could represent non-invasive, quantitative, and objective parameters that might help embryologists with selecting the most competent oocyte for fertilization.Mammalian oocytes go through a maturation process to develop the competencies needed to be fertilized. Oogenesis occurs during the fetal period, resulting in the formation of oocytes arrested in prophase I of meiosis I. Later, after hormonal stimulation , the maturation proceeds to form secondary oocytes (MII stage) that can be fertilized by a sperm cell. The ZP surrounds the oocytes in all of these maturation stages, and it changes its mechanical properties after fertilization, with a process called “zonal hardening.” This phenomenon was confirmed by mechanical measurements on whole oocytes  and isolDifferent approaches are used to investigate the mechanical properties of whole oocytes, such as micropipette indentation and aspiration ,62,63,642 in size), as shown in For that reason, AFM appears as an ideal tool, since it allows fine control of both the applied forces (pN-nN range) and the probe displacement (nm range). Moreover, as already highlighted for somatic cells ,65, probThe E values obtained by AFM indentation on the whole oocyte are lower than those obtained with other techniques see . This trMacro-cantilevers have enabled some to investigate and evaluate the viscoelastic properties of the whole oocyte B. StressIn the mechanical analysis of the oocyte, some authors draw attention to another peculiar structure: the cumulus cell–oocyte complex matrix . This stDuring embryonic development, mechanotransduction phenomena are crucial, as are chemical signals, to control morphogenesis and tissue patterning . InvestiThe physical features of the microenvironment regulate differentiation and self-renewal of pluripotent stem cells , and celIn this context, AFM can be used to investigate the effects of forces in embryo development. The inner pressure and the E values of different blastocyst regions are measured using laser-assisted magnetic tweezers in combination with AFM. During the progression of a mouse blastocyst, from embryonic day E3.0 (16 cells) to E4.5 (>100 cells), the inner pressure of the embryo increases from 100 to 500 Pa; E values of the trophoblast from 300 to 600 Pa; and from 50 to 250 Pa for the inner cell mass. All of these variations are related to the actin organization . More recently, Thompson et al. performed time-lapse in vivo atomic force microscopy (tiv-AFM) to obtain time-resolved measurements of developmental tissue mechanics. Tiv-AFM combines sensitive upright epi-fluorescence imaging of opaque samples and iterated AFM indentations of in vivo tissue at a time resolution of tens of minutes . On thisLike for oocytes, there are significant efforts to improve embryo selection, and many of the optical and AI approaches proposed for oocytes are also considered valid for this choice ,53,55. TSeveral pathologies or inflammation of the female reproductive system severely affect the women’s fertility. The investigation of their hallmarks is of relevance to better understand these diseases. Mechanics are a fundamental feature of the female reproductive system. The organs of this system exhibit astonishing mechanical performance, given their remarkable strength, and undergo huge deformations in their physiological activity. It is also well known that mechanical alterations of the uterus and cervix are associated with pathologies and can induce preterm delivery . AdvanceOvarian cancer is one of the most frequent gynecological malignancies. In total, 12% of ovarian cancer is diagnosed in fertile women, and an early-stage detection of the pathology is vital to increase the survival rate and allow the patients to find a way to preserve fertility . HoweverThe biomechanical characterization of non-malignant immortalized ovarian surface epithelium cells (IOSE) and four ovarian cancer cell lines, performed by AFM, demonstrated that cancer cells exhibit a lower mean stiffness than non-malignant ones. Moreover, it was observed that increases in invasiveness and migratory behavior are both correlated with a significant reduction in cell stiffness , similarFurthermore, AFM was used to mechanically characterize four human ovarian tissues distinct pathological conditions: mucinous and serous cystadenoma, mature teratoma, and endometriosis . As expeA further challenging development of the mechanobiological approach in reproductive medicine research could be the investigation of the mechanical properties of the uterus tissue. The uterus elasticity analysis could shed some light on several issues, from embryo implantation troubles and miscarriage, to the painful endometriosis pathology. Biomechanical investigations of the interested tissues may offer a deeper understanding of the phenomena and provide alternatives for early diagnosis and follow-up treatments.For twenty years, AFM-SMFS experiments, with an exceptionally high spatial and temporal resolution, have been extensively utilized to investigate the intramolecular and intermolecular protein interactions in a large variety of biological processes: folding/unfolding; force-dependent kinetics in molecular interactions; and protein–protein, protein–DNA, and protein–drug interactions ,88,89. TSMFS remains poorly exploited in the reproductive medicine field, although its application could clarify central issues of the field such as the initial sperm–oocyte interaction, binding, and gamete fusion. Three specific glycoproteins in mice and four in humans are the principal components of the ZP; specifically, ZP2 is involved in sperm binding ,91. ThesThe SMFS application could also be extended to investigate the interactions of the adhesion proteins involved in tumor pathologies of the female reproductive system to achieve new information about the molecular mechanisms that trigger and guide tumor cell migration and metastatic process.Advancements in the description of biological processes of interest in reproductive medicine can be achieved by integrating topological information with the visualization of the inner cellular organization up to single molecules. By using aperture-SNOM, a detailed ultrastructural description of the human spermatozoa was achieved. With this technique peculiar structural features of these cells have been disclosed without staining procedures. As shown in Besides the high optical resolution, SNOM opens the opportunity to access nano-spectrochemical information of single proteins, which would broaden our knowledge about the relation between their structure and function . In thisIn the cervical tumor, the histopathology classification is made using four discriminatory features: (1) the ratio of nuclei to the cytoplasm, (2) the diameter of nuclei, (3) the shape factor, and (4) the compactness of nuclei. Once these features are identified in the cervical biopsy images, they are classified into normal, pre-cancer , and malignant. However, the diagnosis has some flaws related to subjective visual inspection, as with other tumor screenings. Recently, Halliwell et al. reported how the combination of scanning near-field optical microscopy with an infrared free-electron laser (SNOM-IR-FEL) aided in distinguishing between normal and squamous low-grade and high-grade dyskaryosis, and between normal and mixed squamous/glandular pre-invasive and cervical adenocarcinoma lesions, by considering specific wavelengths associated with DNA, amide I/II, and lipids . Their fSNOM has been demonstrated to be helpful in visualizing and localizing adhesion molecules in prostate cancer cells. Understanding how individual constituents of cell surfaces localize and assemble is crucial, since the spatial organization and interactions of the cell surface machinery tightly control their functions in physiological and pathological conditions. In this sense, the application of SNOM in combination with innovative labeling strategies such as quantum dots enables the molecular localization of membrane proteins at a high resolution of epithelial prostate cancer cells. Specifically, SNOM acquisitions with a double-labeling allow protein co-localization on the cell surface, as shown in A further important issue in the assessment of male and female infertility is the analysis of chromosomal aberrations. In this framework, SNOM covers the resolution gap between far-field light and electron microscopy on the supra-molecular morphological analysis of meiotic chromosomes. SNOM combined with immunofluorescent staining enables the visualization of the morphology of the telomere end of meiotic chromosomes and identifies structural and functional proteins located in this region . TelomerAlthough SNOM bears appealing perspective in reproductive medicine and biology, its application is still poor due to some limitations. Optimizing staining protocols is essential in some cases. Besides, the high resolution is difficult to maintain in the liquid when scanning in the aperture mode configuration. Owing to tip-sample interactions, the apex aperture can easily widen, resulting in a super optical resolution loss. Furthermore, in shear-force mode and other contact operations in general, SNOM requires long scan times for high-resolution imaging. However, the instrument is constantly evolving to achieve the best configuration to work in biology. Recent advances in the technique propose a combination of high-speed AFM and SNOM. By attaching gold nanoparticles to a home-made tip built on a standard AFM cantilever and a total internal reflection system, the authors achieved an imaging rate of ~3 s/frame for SNOM, more than 100-times higher than the typical SNOM imaging rate. The same group also demonstrated a ~39 nm resolution in high-speed SNOM imaging of fluorescently labeled DNA in solution . Given the growing number of couples turning to reproductive medicine and the not fully satisfying IVF success rate, there is a need to improve assisted reproductive methods. One of the major issues encountered in IVF is selecting the competent oocyte and embryo to achieve a positive outcome. Many studies focused on identifying biomarkers that can improve IVF outcomes are currently ongoing, but objective, accurate, and noninvasive assessments are still missing in clinical practice. Moreover, there is still 25% idiopathic infertility, whose causes are unknown. Likely, they are associated with abnormalities or altered molecular mechanisms which are not detected or described by current methods. All these issues demand new methodological analysis and advanced techniques to solve and overcome these problems. In the basic research of reproductive medicine, AFM and SNOM instrumentation can help clarify molecular mechanisms. However, they cannot be directly applied in clinical practices: they still are time-consuming, expensive, not automatized, and require well-trained operators, but the findings obtained by these techniques are of great relevance to improving our knowledge about gametes and disorders related to reproductive organs. Like optical and spectroscopic features, the mechanical parameters can be considered additional and might be exploited in diagnostic assays and ARTs. Furthermore, due to the enormous potential of such microscopies, numerous efforts are devoted to developing automated systems, such as AFM, which can perform the acquisition of numerous force-distance curves to measure the biomechanical properties of large cell populations or tissues . The combination of biological analysis with biophysical methodologies in the future will contribute to improving our knowledge about molecular mechanisms and open up new possibilities for screening, diagnosis, surgical, and medical treatments in reproductive medicine.


The skin exerts several fundamental functions that are the first physical, chemical and immune barriers to the human body. Keratinocytes, the main cell type of the epidermis, provide mechanical defense, support skin integrity and actively endorse cutaneous immune responses. Not surprisingly, considering these crucial activities, alterations in keratinocyte functions are associated with different inflammatory skin diseases. Recent findings indicate that the skin should not only be regarded as a target for hormones but that it should also be considered as an endocrine peripheral organ that is directly involved in the synthesis and metabolism of these chemical messengers. Sex hormones have multiple effects on the skin, attributed to the binding with intracellular receptors expressed by different skin cell populations, including keratinocytes, that activate downstream signaling routes that modulate specific cellular functions and activities. This review is aimed at reorganizing the current knowledge on the role exerted by sex hormones on keratinocyte function in five different inflammatory skin diseases: Hidradenitis suppurativa; Acne vulgaris; Atopic dermatitis; progesterone hypersensitivity; psoriasis. The results of our work aim to provide a deeper insight into common cellular mechanisms and molecular effectors that might constitute putative targets to address for the development of specific therapeutic interventions.  The skin possesses a well defined tripartite structure that, from the uttermost to the deepest layer, is provided by the epidermis, the dermis and the hypoderm. This complex and intricate structural organization is strictly required for the accomplishment of a wide variety of activities including ,2: immunKeratinocytes are the principal cell type of the epidermis, and their primary activity includes the maintenance of the functional, structural and physical integrity of the skin. Nevertheless, over recent years, consistent evidence revealed that keratinocytes play supplementary roles that go beyond providing a sole protective barrier against external agents. Indeed, upon stimulation, these cells are known to actively participate in the initiation and regulation of cutaneous inflammatory reactions by synthesizing, releasing and responding to different inflammatory, immunomodulatory and immunosuppressive mediators . MoreoveIn this intriguing context, a thorough characterization of the mechanisms underlying the complex interplay existing between defects in functional and immune properties of keratinocytes, cutaneous immune dysregulation and endocrine alterations should be further investigated since they may be closely associated with a continuously expanding spectrum of human immune-mediated skin diseases . TherefoThe physical and functional integrity of the skin is tightly linked to its specific structural partition into three layers ,6: the eSpecifically, the epidermis is composed of the interfollicular epidermis (IFE) and associated skin appendages, collectively organized to form the principal interface between the external environment and the host. The major functions of the epidermis encompass : innate The IFE is composed both by actively proliferating and progressively differentiating keratinocytes that are arranged into four layers that, from the deepest to the most superficial one, compose the basal layer, the spinous layer, the granular layer and the stratum corneum . The mitIn this district, a strict synergistic balance between keratinocyte differentiation and proliferation programs is required; indeed, deregulations in these processes are associated with the pathogenesis of various inflammatory skin diseases, including Hidradenitis suppurativa , Acne vuThe skin possesses a crucial role in host defence by providing a passive protective mechanical barrier that is tightly integrated with active immune surveillance mechanisms aimed at creating a robust and intricate defence network. Cutaneous immune responses are primarily processed by cellular populations that act as sentinels for the recognition of dangerous stimuli and include keratinocytes and resident immune cells encompassing macrophages, dermal DCs, Langerhans cells (LCs) and dermal mast cells . Briefly, during the early stages of innate cutaneous immune responses, keratinocytes together with activated LCs, macrophages and dermal DCs are involved in the rapid synthesis and release of pro-inflammatory cytokines, antimicrobial peptides (AMPs) and chemotactic molecules. These responses are primarily mediated by the activation of pathogen recognition receptors (PRRs) that are involved in the recognition of conserved pathogen components, namely the Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), and by the production of inflammatory and immune molecules that trigger further downstream signalling pathways ,11. In this context, keratinocytes are well known to orchestrate the initial responses to different cutaneous local stimuli by initiating and regulating the early innate immune responses by producing cytokines including the potent inducer of local immune functions interleukin-1α (IL-1α) and interleukin-1β (IL-1β), tumor necrosis factor (TNF), other cytokines, chemokines and AMPs. Next, the pro-inflammatory and immunomodulatory molecules synthesized and released by keratinocytes activate cutaneous DCs, thus stimulating the expression of other immune mediators that further sustain the development of local inflammatory responses and favor the in situ recruitment of immune cells from the bloodstream ,12,13.Activated cutaneous DCs, including LCs and dermal DCs, are involved in the priming and initiation of cutaneous adaptive immune responses by prompting both the sensitization and the elicitation processes. Following antigen recognition and engulfment in the skin, during the sensitization phase, activated DCs migrate to skin-draining lymph nodes, where they mature and encounter naïve T cells in order to promote their differentiation in effector T cells that express homing receptors for the skin, hence redirecting the immune response towards the initial cutaneous inflammatory site. Once in the skin, recruited effector T cells start to accumulate both in the dermis and epidermis, sites in which they encounter a wide variety of costimulatory signals, including inflammatory mediators generated by innate immune responses and other activated antigen-presenting DCs, and are stimulated to produce cytokines and chemokines that promote the development of antigen-specific immune responses (elicitation phase) ,14. MoreThe necessity to consider keratinocytes, the most abundant cellular component of the epidermis, not only as passive constituents composing the physical barrier of the skin but also as crucial actors in early cutaneous immune defence and tolerance mechanism is currently widely accepted. Indeed, keratinocytes are considered as the principal initiators of inflammatory processes since these cells can synthesize and respond to a wide variety of cytokines, chemokines and growth factors, which are involved in the direct synthesis of AMPs and express a wide array of PRRs isoforms ,17. In aThe initiation of immune responses in keratinocytes is driven by the recognition of pathogen-associated molecular patterns (PAMPs) to membrane-bound TLRs and NLRs that subsequently induce the activation of pro-inflammatory signalling cascades. Indeed, the activation of TLRs in keratinocytes is usually associated with the induction of pro-inflammatory responses that lead to the production of inflammatory cytokines, including IL-1β, IL-6, IL-8, IL-18 and interferons (INFs), principally, through the activation of the NF-кB pathway; actually, the activation of the NF-кB signalling cascade collectively promotes the migration of LCs to skin-draining lymph nodes and to the recruitment of effector T cells that express homing receptors for the skin. Moreover, the activation of TLRs also stimulates keratinocytes to further release cytokines  and chemokines , thus favoring the activation of tissue-resident (αβ) CD8+ memory T-cells . Next, tGiven the fundamental role of keratinocytes in skin immunity, the presence of modifications in their immune properties is associated with the pathophysiology of various inflammatory skin disorders . The impThe endocrine system is composed of an intricate and well-organized network of glands and hormone-producing tissues distributed throughout the body whose primary function is the synthesis and secretion of hormones, chemical messengers that are involved in the regulation of a wide spectrum of physiological activities, including growth, developmental and reproductive processes, the maintenance of a stable internal environment by strictly balancing the homeostasis of electrolytes and of nutrient metabolism and the integration of body responses to external stimuli ,25. In the case of human skin, recent findings strongly indicate the necessity to reconsider the strict interactions between the cutaneous district and hormones. Indeed, the skin is not only regarded as a target site of several hormones that exert well-established functions primarily involved in the regulation of cutaneous development and the maintenance of the homeostasis of the skin tissue , but alsTarget districts generally respond to endocrine hormones following hormonal binding to intracellular or to cell surface receptors, interactions that result in the formation of hormone-receptor complexes that trigger the activation of intracellular routes, ultimately leading to the modulation of specific cellular functions and activities .Hormones synthesized by hormone-producing glands exercise well-characterized pleiotropic regulatory effects on different skin cell types due to their expression of high-affinity membrane-bound, cytoplasmic or nuclear hormone-receptors .Located on the cell membrane of various skin cell populations are receptors specialized in the recognition of neurotransmitters and peptide hormones that function by activating hormone-specific second messengers and specific signal transduction routes in target cells .Conversely, cytoplasmic and nuclear hormone receptors, primarily targeted by steroid and thyroid hormones, are defined as soluble molecules that act as transcriptional regulators following the recognition of specific DNA sequences, namely hormone-responsive elements (HRE) . Under bThe progressive characterization of the skin has clarified that this district fulfils the functional requirements to be considered as an independent peripheral endocrine organ. Indeed, recent evidence indicates that the skin is actively involved in the synthesis and metabolism of several hormones, including sex steroids, vitamin D, calcitriol and insulin-like growth factor (IGF) ,4. Steroidogenesis is the process involved in the conversion of cholesterol into active steroid hormones that can be classified into glucocorticoids, mineralocorticoids and sex steroids, the latter including androgens, estrogens and progestogens. This process occurs primarily in endocrine organs such as the adrenal gland and the gonads though it can also prevail in human skin. Specifically, the cutaneous district expresses all the major enzyme systems that are required for the de novo synthesis of androgens ,30. Human skin is a relevant source of vitamin D, being the sole site involved in the synthesis of cholecalciferol. This hormone is known to regulate skin homeostasis by mediating the proliferation and differentiation of the epidermis and epidermal appendages . MoreoveIn addition, dermal fibroblasts and melanocytes can synthesize IGF-1, while epidermal keratinocytes, melanocytes, hair follicles and endothelial cells are shown to produce adrenocorticotropic hormone (ACTH) and the melanocyte-stimulating hormone (MSH) [The metabolism of hormones, aimed either at the activation or inactivation of these chemical messengers, is known to occur in human skin through complex mechanisms that require strict functional coordination amongst different cutaneous cellular populations and yield pathways primarily involved in the transformation of sex steroids, retinoids and vitamin D ,33. In particular, the metabolism of sex hormones includes the hydrolysis of dehydroepiandrosterone (DHEA)-sulfate, a compound produced in the adrenal glands that is then released in the circulation, into DHEA through the enzymatic activity of the steroid sulfatases expressed by sebocytes and the cells of the dermal papilla ,30. NextSkin cells are also able to convert testosterone into the more active metabolite 5α-dihydrotestosterone (DHT) in a reaction catalyzed by 5-α-reductase, an enzyme highly expressed in keratinocytes . MoreoveSex steroids, herein referred to as sex hormones, are represented by androgens, estrogens and progestogens and are molecules presenting multiple effects on human skin. Their function is exerted following their binding to intracellular cytoplasmic hormone receptors that, upon activation, translocate to the nucleus and act as transcriptional regulators for several target genes ,34.Androgens receptors (ARs) are expressed in the cytoplasm of different skin cell types, including keratinocytes, sebocytes, fibroblasts, endothelial cells, sweat gland cells and cells of the dermal papilla. The binding of androgens to ARs is known to induce several effects on various skin cell populations, such as: the regulation of the function of the sebaceous glands by controlling the proliferation of sebocytes and sebum secretion ; the modThe specific role of androgens on keratinocyte function is still controversial and under investigation. It was shown that testosterone stimulates keratinocyte proliferation during wound healing and epidermal barrier formation ; neverthIntracellular estrogens receptors (ERs) exist in two isoforms, namely ER-α and ER-β, that are both known to be expressed in various skin cells . EstrogeProgesterone receptors (PRs) are present in two isoforms, PRA and PRB. PRA possesses nuclear localization, while PRB shows both nuclear and cytoplasmic distribution . ProgestConsidering the role of keratinocytes in skin immunity and the impact of sex hormones on keratinocyte function in terms of regulation of proliferation and differentiation processes, we focus on summarizing the recent findings related to the associations between deregulations in androgens, estrogens and progesterone levels and keratinocyte functions in inflammatory skin disorders.In order to properly address this issue and delineate an overview on the current information available on this topic, we initially queried the NCBI PubMed database by utilizing the keywords “inflammatory skin disease AND keratinocyte AND (androgen OR estrogen OR progesterone)”. Interestingly, this search allowed the identification of five distinct disorders: Hidradenitis suppurativa (HS); Acne vulgaris (AV); Atopic dermatitis (AD); progesterone hypersensitivity (PH); psoriasis (PSO). Hidradenitis suppurativa (HS) is a chronic and debilitating inflammatory follicular occlusive skin disease  possessiNCSTN, PSENEN and PSEN1 genes), aberrant immune responses, environmental agents and hormonal imbalance [Current evidence suggests that the pathogenesis of HS is complex and multifactorial hence showing a strict interplay between different etiologic factors, including genetic background  is a common chronic inflammatory skin disease affecting approximately 9.4% of the world’s population, exhibiting the highest prevalence in adolescents. AV primarily affects the pilosebaceous unit, hair shaft and sebaceous glands and is characterized by the development of comedones and inflammatory lesions such as papules, nodules and pustules on the face, neck, trunk, or in the proximal upper extremities of the body ,68. Propionibacterium acnes in the pilosebaceous unit [The pathogenesis of AV is complex and involves different processes, including alterations in sebaceous gland function correlated to increased sebum production (hyperseborrhea) and abnormal sebum composition, hormonal imbalances, follicular hyperproliferation and hyperkeratinization resulting in follicular plugging and rupture and cutaneous dysbiosis associated with the proliferation of ous unit ,68,69. TTo date, the role exploited by androgens in the pathogenesis of AV is well established; indeed, AV is commonly defined as a hyper-androgenic disorder in which DHT, DHEA-sulfate and testosterone levels are found to be higher in patients when compared to healthy controls . Sebaceous glands constitute the principal site of the skin compartment involved in steroidogenesis. Sebocytes are known to express the central enzymes required for the conversion of inactive or less active circulating androgens to potent hormone-derivates that, upon binding to intracellular receptors, trigger the transcriptional activation of downstream target genes . EnhanceIn a study conducted by Kumtornrut et al., a correlation between androgens, keratinocytes and dermal fibroblast in AV emerged. Dermal fibroblasts express significant levels of ARs, and in vitro analysis showed that growth factors released by these cells upon hormone stimulation could suppress keratinocyte differentiation, thus demonstrating a possible indirect effect of androgens on keratinocytes . Atopic dermatitis (AD) is a chronic, relapsing and remitting inflammatory skin disease. In its acute phase, AD is clinically characterized by cutaneous pruritic and erythematous lesions, while red or brown flares with cracked or squamous skin appear during the chronic state that is also associated with the development of lichenification and prurigo nodules . AD affeFLG) gene encoding for a structural protein of the skin [AD presents a multifactorial aetiology accounting for genetic variations, immune dysregulation and environmental factors. AD patients encompass epidermal barrier dysfunctions linked to altered epidermal differentiation processes that are commonly associated with mutations in filaggrin  is generally more common in females [AD is more frequent in males with respect to females during childhood, but after puberty, this trend is inverted. While considering all ages, intrinsic AD  that is a rate-determining protein for steroid synthesis and the metastatic lymph node 64 (MLN64) that is a cholesterol transport protein, were found to be altered in skin biopsies derived from AD patients when compared to healthy controls. Specifically, in AD cases, the StaR protein that is normally expressed in the basal layer was found to be completely absent. Similarly, the production of MLN64 in the suprabasal layer was found to be reduced .Progesterone hypersensitivity (PH), also known as autoimmune progesterone dermatitis, is a very rare inflammatory skin disease that occurs periodically in correspondence with the peak of progesterone production during the menstrual period. PH is characterized by acute symptoms including urticaria erythema, rash and vesiculobullous lesions, as well as by delayed hypersensitivity signs leading to chronic dermatitis . In mostIn this context, it is speculated that women develop sensitization to progesterone following its release at a systemic level  . On the Psoriasis (PSO) is a chronic inflammatory skin disease given by recurrent inflammatory episodes that lead to an impairment in the proliferation and differentiation of keratinocytes. PSO exhibit primary cutaneous manifestations together with a relevant genetic predisposition and possess a worldwide prevalence of about 2% .PSO is characterized by lesions called plaques and papules, displaying epidermal hyperplasia and infiltration of immune cells such as dendritic cells, T-cells, neutrophils and macrophages. The initial phase in the development of the psoriatic lesion involves the keratinocytes of the outermost skin layer that, upon activation triggered by pathogens or mechanical damages, start to interact with other cell types of the deeper layers resulting in the activation of innate immunity mechanisms followed by the onset of the adaptive immune response. Subsequently, the perpetuation of an inflammatory environment promotes the chronicity of the disease . In women affected by PSO, menstruation, pregnancy and menopause influence the progress of the disorder, hence suggesting a possible role of sexual hormones on the pathogenesis of the disease. It was observed that estrogens, mainly E2, affect the severity of the disorder by directly impacting inflammation and keratinocyte function as well as angiogenesis and oxidative stress responses . In the TH1 and TH17 cells are upregulated in psoriatic plaques, sites in which they release inflammatory cytokines that stimulate inflammatory responses mediated by keratinocytes. Estrogens can suppress the activity of pro-inflammatory cytokines, as well as stimulate TH2 cells to produce anti-inflammatory molecules. Moreover, estrogens promote T-cell conversion into T-regulatory cells, a population actively involved in the modulation of immune responses. In this context, estrogens attenuate PSO, as observed in women during pregnancy .Moreover, in PSO, estrogens tightly affect keratinocyte functions. Indeed, several studies demonstrated that E2 promotes keratinocyte proliferation and migration by inducing the expression of cyclin D2 ,93. ThesThe complex interactions existing between alterations in functional and immune activities of keratinocytes, cutaneous immune dysregulation and endocrine alterations should be further investigated since they might be associated with several immune-mediated skin diseases . CollectThe levels of progesterone and estrogens during the menstrual period, pregnancy and menopause highly influence the onset, progression and severity of flares in women affected by HS, AD, PH and PSO. In HS cases, high levels of progesterone and estrogen give rise to an exacerbation of flares during the premenstrual period and an amelioration of symptoms during pregnancy. Conversely, a decrement in estrogen and progesterone during menopause is associated with a remission of symptoms. In AD, the low cutaneous progesterone/estrogen ratio during the luteal phase negatively impacts cutaneous barrier functions by increasing skin permeability, and similar effects were detected during the luteal phase in PH cases. Increased progesterone and estrogen levels during the premenstrual phase and pregnancy exacerbate AD conditions by promoting inflammation and IgE production. Interestingly, in women affected by PSO, progesterone and estrogen appear to both aggravate symptoms by promoting keratinocyte proliferation and migration and reducing cytokine production by keratinocytes hence limiting cutaneous inflammation.Androgenic imbalances are registered in women affected both by HS and AV, disorders in which hyperandrogenisms are associated with higher rates of occurrence of PCOS, hirsutism and irregular menstruation cycles. In addition, in HS lesions, an enrichment for genes regulated by a deregulated activation of ARs was observed and presumably favors proliferation and differentiation programs in keratinocytes. In AV affected cases, high levels of circulating androgens excessively stimulate the proliferation of sebocytes and keratinocytes, impair keratinocyte differentiation and desquamation and also promote lipid synthesis by sebocytes hence resulting in hyperseborrhea. Finally, in AD affected males, low serum concentrations of androgens, in particular of DHEA and testosterone, lead to reduced hormonal suppressive effects on immunity and therefore to an aggravation of AD phenotype . The study of these complex cross-talks gave rise to crucial similarities and contradictions amongst the different analyzed disorders. We are convinced that the application of multi-OMICS studies on large groups of clinically well characterized patients is envisaged in order to characterize these complex interactions and to clarify the possible associations between hormonal imbalances and clinical features. Furthermore, the integration of clinical and OMICs findings might provide a better-defined answer relative to the mechanisms involved in HS, AV, AD, PH and PSO, therefore giving novel insights for the definition of tailored therapeutic interventions.


In the developing brain, new neurons are generated at specific locations before migrating to their final destination, where they perform their adult functions. The cerebral cortex comprises neurons born in different regions and at different developmental times, but these are eventually organized into six layers according to their birth date. During this process, excitatory neurons undergo dynamic changes in their morphology and migration modes, including multipolar migration, locomotion, and terminal translocation. During development, neuronal migration impairment causes abnormal organization of the neocortex, resulting in various functional disorders such as epilepsy and neurological disability. Understanding the precise mechanisms underlying neuronal migration is a fundamental basis for understanding both neocortical development in the healthy brain and pathophysiological mechanisms underlying developmental neurological disorders.During migration, neurons sense and respond to a broad range of extracellular environmental signals, including biochemical and mechanical cues, which serve as scaffolds to guide and support them. Genetic and molecular analyses have revealed signaling networks that precisely control the biochemical cellular microenvironment during neocortical development, including extracellular matrix (ECM) proteins, cell surface proteins, and gradients of diffusible guidance cues. Recent studies using live imaging and physical measurement techniques, such as atomic force microscopy (AFM), have revealed the essential role of the mechanical environment, including tissue stiffness, dynamic forces, and scaffolds, in neuronal migration. This Research Topic aims to highlight the critical relevance of the extracellular environment to various modes of neuronal migration in the mammalian brain under both physiological and pathological conditions.Ferent et al. review the extracellular factors controlling the formation and maintenance of RG scaffolding. The first step of neuronal migration is cell delamination, in which neuronally differentiating cells generated from apical RG cells retract their apical processes and depart from the ventricular surface. Kawaguchi reviews the cellular and molecular mechanisms that regulate this process.Radial glial (RG) cells lining the ventricular surface function as primary neural progenitor cells, generating various neurons and glial cells. RG cells also serve as a physical scaffold supporting the radial migration of newborn neurons. In this Research Topic, Martinez-Garay summarizes the function of cadherins during cortical migration, mainly focusing on CDH2, which plays critical roles in multiple steps of migration, including the apical delamination of newborn neurons, acquisition of polarity in the intermediate zone, RG-guided locomotion, and terminal soma translocation. More specifically, Peregrina and del Toro focus on reviewing the functions of the FLRT family of cell adhesion proteins to control the radial migration and tangential spread of migrating neurons by regulating the balance between cell adhesion and repulsion. Down syndrome cell adhesion molecules (DSCAMs), a small group of transmembrane proteins of the immunoglobulin superfamily, are also known to be involved in neuronal migration during cortical development. In their original article, Mitsogiannis et al. show that an increased dosage of DSCAMs, which is observed in human disorders, affects morphology and migration of cortical interneurons.Cell adhesion molecules located on the cell surface play essential roles in the interaction between migrating neurons and their environment. The review by Yamagishi et al. and Gonda et al. review the involvement of two well-known axon guidance families of proteins, Netrin and Robo/Slit, respectively, in neuronal migration during neocortical development and further discuss their participation in human developmental pathologies.Extracellular signaling components, which are known as axon guidance molecules, also regulate neuronal migration. Hatanaka and Hirata summarize the critical roles played by multiple microenvironmental factors, including the ECM, Cajal–Retzius cells, RG cells, and neighboring neurons, during the terminal phase of neuronal migration.The termination of neuronal migration is the final essential step for the formation of the fine laminar structure of the cerebral cortex, where neurons detach from the RG scaffold and initiate differentiation. Minegishi and Inagaki discuss the relevance of forces created between neurons and their surrounding environment and those produced within cells in controlling neuronal migration and molecular mechanisms underlying these forces. Novel insights into these forces are provided by Iwashita et al. who present a novel strategy of measuring tissue stiffness using glyoxal as a fixative combined with AFM. They then used this method to investigate the link between mechanical properties and species-specific brain structures, comparing mice, chicks, turtles, and ferrets. In addition to mechanical forces, the electrical properties of migrating neurons and their cellular environments also influence neuronal migration. Medvedeva and Pierani review the fascinating mechanisms underlying the electric field-mediated control of neuronal migration and maturation.In addition to the signaling action of molecular factors, recent studies have begun to reveal the central importance of mechanical forces in controlling neuronal migration during cortical development. In their review, Amin and Borrell review the role of ECM molecules in progenitor cell proliferation, neuronal migration, and the evolution of cortical folding. Cortay et al. provide new data from ex vivo analysis of the embryonic macaque cortex, demonstrating that the radial migration speed and trajectory vary significantly between cortical areas and layers in primates, exhibiting unique features in an area- and layer-specific manner.The evolution of the mammalian cerebral cortex is considered fundamental for the acquisition of higher brain functions. Accumulating evidence indicates that the specific adaptation of progenitor cell proliferation and neuronal migration mechanisms play critical roles in neocortical evolution. Hansen and Hippenmeyer. They discuss the different extracellular elements that define the microenvironment that radially migrating neurons encounter, as well as experimental assays and methods to study non-cell-autonomous mechanisms of neuronal migration and brain development.Our Research Topic ends with a review by By assembling these articles together, this special issue provides an updated picture of the field and highlights the importance of the interplay between extrinsic signals and migrating neurons during neocortical development. We are confident that this collection will be of interest to both new and established scientists and help promote research in this fascinating field.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


It is a key issue for the Chinese government to improve eco-efficiency and realize green development. As a spatial organization mode of industrial labor division, industrial agglomeration has a complex impact on eco-efficiency. However, it is still debatable which industrial agglomeration modes have a positive impact on eco-efficiency. This paper employs a panel threshold model, enterprise micro-level data, and relevant economic environment data from 283 cities in China from 2004 to 2012. It tests the nonlinear effects of specialized, related diversified, and unrelated diversified agglomeration on industrial eco-efficiency. The results show that the impact of specialized and related diversified agglomeration on industrial eco-efficiency is first inhibited and then promoted. The unrelated diversified agglomeration has a significantly negative impact on industrial eco-efficiency, but the negative impact weakens when agglomeration reaches a certain level. Furthermore, the impact of the three agglomeration modes on industrial eco-efficiency depends on city size. The impact of specialized agglomeration on industrial eco-efficiency is insignificant in small- and some medium-sized cities, but it has a significant inhibitory effect on industrial eco-efficiency when the city surpasses medium size. The role of related diversified agglomeration in promoting industrial eco-efficiency is further enhanced with the growth of city size. The impact of unrelated diversified agglomeration on industrial eco-efficiency gradually changes from negative to positive, but it plays a promoting role only when the city reaches the scale of super-large and mega-cities. Finally, this paper suggests that policymakers should formulate differentiated agglomeration policies according to changes in industrial agglomeration level or city size to improve industrial eco-efficiency. After the Chinese economic reform in 1978, China’s economy expanded rapidly and became the world’s second-largest economy. Simultaneously, China ranks as one of the countries with the most consumption of resources and energy and the highest emission of various pollutants in the world. As the pillar of the national economy, the industrial sector is the main source of China’s economic growth. However, industrial development causes high consumption and emissions, making it the main source of resource and energy consumption and environmental pollution . For thiAs a spatial organization mode formed by the deepening of the industrial division of labor, industrial agglomeration concentrates enterprises of the same or different industries in a specific region, resulting in the formation of external economies of scale . With thTheoretically, industrial agglomeration may have both positive and negative effects on eco-efficiency. On the one hand, industrial agglomeration promotes labor productivity and economic growth through the positive effects of knowledge spillover and scale economies. In addition, industrial agglomeration reduces energy consumption and pollutant emissions through the spillover of clean and environmental protection technologies, the sharing of pollution control facilities, and the development of a circular economy . On the The contribution of this paper mainly includes four aspects. Firstly, the paper distinguishes the impact mechanism of different agglomeration modes on eco-efficiency. Secondly, it calculates the specialized, related diversified, and unrelated diversified agglomeration index of Chinese cities using the micro-level data of the China Industrial Enterprise Database, and measures the industrial eco-efficiency of each city using the super-efficiency SBM-DEA model with undesirable outputs. Thirdly, it employs a panel threshold model to test the nonlinear impact of specialized agglomeration and related and unrelated diversified agglomeration on industrial eco-efficiency. This study also uses the static and dynamic spatial panel to examine the robustness of the nonlinear relationship. Finally, it discusses the impact of city-size heterogeneity on the relationship between the three agglomeration models and industrial eco-efficiency.The rest of this paper is organized as follows. The next section is a literature review. The third section analyzes the influence path. The fourth section explains methodology, variables, and data sources. The fifth section gives the empirical results and discussion. Finally, the last section provides the conclusion and policy implications.Since Marshall proposed the concept of industrial agglomeration, scholars have widely discussed the effects of industrial agglomeration on economic growth, labor productivity, and technical innovation. Most studies believe that industrial agglomeration produces scale economies and knowledge spillover effects, which promotes economic growth ,10,11,12Furthermore, researchers divide industrial agglomeration into two modes of specialized and diversified agglomeration, according to whether the externality of agglomeration comes from the same industrial type. According to Marshall , the MARAlthough many studies confirm Jacobs’ view, a large number of studies show that diversified agglomeration does not bring knowledge spillover or growth. In fact, two completely unrelated industries establish an ineffective knowledge spillover channel. Only industrial sectors with complementary elements and knowledge can effectively produce knowledge spillover and promote regional economic growth. Therefore, Frenken et al.  first di2 emissions, and Wang and Wang [As environmental pollution has become more and more prominent in recent years, numerous scholars have studied the impact of industrial agglomeration on environmental pollution. However, there is serious disagreement on the relationship between industrial agglomeration and environmental pollution. One view is that industrial agglomeration intensifies environmental pollution since the expansion of production scale caused by agglomeration increases pollutant emissions. Virkanen  believesand Wang  confirm 2 emission intensity, while the impact of unrelated diversification is obscure.Despite the increasing number of studies on the impact of industrial agglomeration on environmental pollution, only a few studies discuss the impact of different agglomeration modes on environmental pollution. Deng and Xu  argue thAlthough there is a limited amount of research on the impact of industrial agglomeration on eco-efficiency, there are profound disagreements among their findings. Some studies show that industrial agglomeration improves eco-efficiency significantly. Otsuka et al.  found thTo sum up, there are relatively few studies on the impact of industrial agglomeration on eco-efficiency, and they mainly focus on the linear relationship between industrial agglomeration and eco-efficiency. The research on the nonlinear relationship between them is insufficient and limited to some industries or regions. More importantly, the existing research ineffectively distinguishes the impact of different agglomeration modes on eco-efficiency, especially the impact of related and unrelated diversified agglomeration. In addition, the current research ignores the role of heterogeneous factors, such as city size, in the impact of industrial agglomeration on eco-efficiency. Specialized agglomeration improves innovation ability and reduces production costs and pollution emissions through the scale economies of labor, intermediate inputs and infrastructure, and the knowledge spillover effect. These factors promote the improvement of eco-efficiency. When a specific space gathers the specialized labor force, enterprises can easily obtain the required labor force according to their own needs, and the highly skilled workers in the region can also find jobs matching their skills, which reduces the labor search cost and improves labor productivity . The intHowever, specialized agglomeration produces many negative externalities, which hinder the improvement of eco-efficiency. On the one hand, specialized agglomeration expands production capacity and aggravates energy consumption and pollutant emissions . On the Related diversified agglomeration improves eco-efficiency through scale economies by sharing labor and infrastructure, the spillover effect of knowledge and technology, and the circular economy effect. Related diversified agglomeration provides labor with different skills, which reduces the search cost and improves the matching efficiency of the labor market. Furthermore, it helps enterprises to share all kinds of environmental protection infrastructure and promotes the formation of a specialized pollution control market, which finally produces the scale economies effect of environmental governance . Due to As a mode of agglomeration without obvious economic and technological links, unrelated diversified agglomeration results in the limited sharing of labor and infrastructure such as pollution control facilities, which hinders the exertion of scale economies. Due to the lack of complementary factor inputs among enterprises in different industries, enterprises in industrial agglomeration areas lack input-output linkages, which is detrimental to the formation of the circular economy effect. In the short term, there are few opportunities for environmental protection knowledge and technology exchange among enterprises due to the great cognitive distance between enterprises. It limits the spillover of various progressive technologies. In the long run, however, unrelated diversified agglomeration achieves breakthrough innovation and improves eco-efficiency since the reorganization of unrelated technologies leads to the emergence of new operating principles, functions, and applications . Moreovem, r1, and r2 denote the number of input, desirable output, and undesirable output indicators, respectively; j in year t; j in year t; j in year t; This paper adopts Data Envelopment Analysis (DEA) to measure the industrial eco-efficiency of Chinese cities. The DEA model treats each city as a Decision-Making Unit (DMU) and mainly uses linear programming technology to make a productive evaluation of the DMU. Compared with the parametric frontier approach, such as the stochastic frontier analysis (SFA), the DEA model not only does not need to provide prior weight information or set definite function form, but it can also easily deal with the efficiency measurement problem under the condition of multi-inputs and multi-outputs. Therefore, the DEA model has become an effective tool to evaluate eco-efficiency in various studies. However, the traditional DEA model, such as the CCR  and BCC  models, ignores the relaxation of input and output and ineffectively measures the efficiency with undesirable output. Therefore, Tone  proposedi and t, respectively, are individual and time; itY is the explained variable; itE shows the core explanatory variable; itT denotes the threshold variable; itX is the control variable; itX; I(·) is the characteristic function, which is 1 when the conditions in the parentheses are met and 0 when they are not fulfilled. To generate an estimate of the parameters, we first need to deduct the group average from the observed values of each variable in Equation (2), to transform it into:This paper empirically tests the dynamic impact of industrial agglomeration on industrial eco-efficiency using the panel threshold model proposed by Hansen . The panWe use the Ordinary Least Square (OLS) method to estimate 0:Hp-value.Then, we test the threshold effect and estimate. First, we check the significance of the threshold effect. The linear constraint 0H：The next step is to test the authenticity of the threshold estimate. The null hypothesis The distribution of the statistic This research conducts the above parameter estimation and hypothesis test methods regarding the single threshold model. The multi-threshold model is an extension of the single threshold model .itIee represents industrial eco-efficiency; itSpe is specialized agglomeration; itRd and itUd are related and unrelated diversified agglomerations, respectively; and itX represents a group of control variables.According to Equation (2), this paper takes the indicators of specialized, related diversified, and unrelated diversified agglomeration as threshold variables to study the impacts of different industrial agglomeration models on industrial eco-efficiency. Taking the existence of a threshold as an example, the model is set as follows:itUs represents city size.Then, this paper investigates if city size affects the relationship between industrial agglomeration and eco-efficiency. To this end, our research constructs a panel threshold model with the city size (city population) as the threshold variable to explore the impact of city-size heterogeneity on the relationship between different agglomeration models and industrial eco-efficiency. Taking a single threshold as an example, the model is as follows:2, and industrial soot (dust) in each city.The determination of input, desirable output, and undesirable output variables is necessary to measure the industrial eco-efficiency of cities using the super-efficiency SBM-DEA model. The relevant indicators are as follows. (1) Capital input: the net fixed assets of industrial enterprises above designated size in each city as a proxy for capital investment. (2) Labor input: the annual average number of employees in industrial enterprises above the designated size in each city as the labor input indicator. (3) Energy input: the annual industrial electricity consumption of each city. (4) Desirable output: the gross industrial output value of industrial enterprises above the designated size in each city. (5) Undesirable output: the total volumes of industrial wastewater, industrial SO(1)Specialization (Spe)LQ), Concentration Ratio (CR), and Krugman Specialization Index (KSI) to measure the specialization level of industries. Considering our data and research purpose, we use KSI to measure the specialized level of the urban industry. The calculation formula for KSI is as follows:i is the city; j is the industrial sector; ijS denotes the output value of j industrial sector in city i; iS and jS denote the gross output value of the industry in city i and industrial sector j in all sample cities, respectively; and S denotes the gross output value of the industry in all sample cities. KSI ranges within 0–2, and the greater the value of KSI, the higher the level of specialized agglomeration in a city.Previous studies employed the Location Quotient ((2)Related diversification (Rd) and Unrelated diversification (Ud)jP represents the output proportion of industrial sector j to the gross output value of the industry in a city.Following Frenken, Van Oort and Verburg , this paj is small, and all the small industrial sectors Ej  can be aggregated into a smaller number of large industrial sectors S1,…, SG in such a way that each small industrial sector falls exclusively under a large industrial sector Sg . The share of the large industrial sector gP is the summation of the share of small industrial sectors jP: Assumedly, the industrial sector Finally, the industrial diversification index of the city is decomposed as follows:Based on the output value data of industrial enterprises above designated size in the China Industrial Enterprise Database, this paper calculates the specialized, related diversified, and unrelated diversified agglomeration indexes of city industry. Following Lin’s  method, (1)Environmental Regulation (Er)2 and the treatment rate of industrial wastewater of each city to construct the comprehensive index of environmental regulation.Strict and appropriate environmental regulation reduces the scale of polluting industries and enhances the innovation of environmental protection technology in enterprises, which reduces the intensity of pollution emissions and improves eco-efficiency. Using the entropy method, the paper selects the removal rate of industrial soot (dust) and SO(2)Industrial structure (Is)The increasing proportion of pollution-intensive industries expands the scale and intensity of pollution emissions, which affects eco-efficiency adversely. This paper defines pollution-intensive industries according to Guanghui et al.  and expr(3)Technology innovation (Tech)Technology innovation is not only an important driving force of economic growth, but also an important way to reduce environmental pollution and improve resource utilization efficiency, which has a positive impact on eco-efficiency. Considering that invention patents can objectively reflect the original innovation capacity of a region or enterprise, this paper uses the authorization amount of invention and utility model patents to reflect technology innovation capacity.(4)Foreign direct investment (Fdi)Foreign direct investment provides advanced environmental protection technology for local enterprises, which improves industrial eco-efficiency. However, foreign direct investment causes environmental pollution due to the pollution haven effect , lowerin(5)Economic development level (Edl)Various modes of social production at different levels of economic development have heterogeneous levels of resource utilization, which have an important impact on industrial eco-efficiency . Per cap(6)Government intervention (Gi)The government’s limited intervention in the market effectively alleviates the problems of information asymmetry and monopoly caused by market failure, which optimizes the allocation of resources and improves industrial eco-efficiency. However, if the government intervenes excessively, it leads to the inefficiency of resource allocation and reduces industrial eco-efficiency. Therefore, this paper uses the proportion of the city’s fiscal expenditure to GDP to measure the degree of government intervention.(7)OtherIt should also be noted that specialized, related diversified, and unrelated diversified agglomeration indexes are also alternately regarded as control variables.Considering the adjustment of administrative division and data accessibility, this paper selects 283 prefecture-level and higher-level cities in China except for Chaohu, Bijie, Tongren, Lhasa, Longnan, and Zhongwei as the samples. Because the current data from the China Industrial Enterprise Database is only updated to the year 2013, and the standard of the national economic industrial classification has been significantly adjusted in 2003 and 2012, this paper sets the research period from 2004 to 2012 to ensure the consistency of the data. This period plays a key role in transforming China’s industrialization from accelerated development to a mature stage. In this period, not only did industrial agglomeration develop rapidly, but also the problem of resources and environment became prominent. Therefore, research on the relationship between industrial agglomeration and eco-efficiency is typical in this period. The data in this paper are mainly derived from the China Industrial Enterprise Database, China Urban Statistical Yearbook, and China regional economic statistical yearbook. The patent data come from the patent retrieval database of the State Intellectual Property Office (SIPO). To eliminate the impact of inflation, the gross industrial output value and per capita GDP have been adjusted into comparable prices according to the producer price index of industrial products in each province, and the net value of fixed assets have been adjusted into comparable prices according to the price index of fixed asset investment in each province. The base year is 2004 for all the variables, and the price indexes come from the China Statistical Yearbooks 2005–2013. Finally, the descriptive statistics show that the numerical distribution of each variable presents a relatively stable trend in To highlight the characteristics of industrial eco-efficiency, this paper analyzes the industrial eco-efficiency from two aspects: the whole country and cities of different sizes. According to the standard of city size classification, Chinese cities are divided into four categories: super-large and mega-cities with a population of more than 5 million, large cities with a population of 1–5 million, medium-sized cities with a population of 0.5–1 million, and small cities with a population of less than 0.5 million. Firstly, from the perspective of nationwide characteristics see , the avep-value, and critical values of the three panel threshold models  of Column (2) of Column (3) of Based on columns (1), (2), and (3) of The spatial correlation gradually increases among cities in the case of the free and frequent flow of production factors. Various economic and environmental activities of a city have spatial spillover effects on other cities. Therefore, variables  have a strong spatial correlation. Based on this analysis, our paper uses a spatial panel model for the robustness test. The commonly used spatial weight matrices employ geographic adjacency, geographic distance, or economic distance matrices. Since the spatial correlation among regions comes from both the aspects of geography and economy, this paper constructs a comprehensive spatial weight matrix to integrate geographical and economic distance based on Xinshuo et al.  method.Following Elhorst , this paAccording to This paper explores the heterogeneity impact of city size on the relationship between different agglomeration modes and industrial eco-efficiency. To this end, it takes city population as the threshold variable and tests the models with specialized, related diversified, and unrelated diversified agglomeration as the core explanatory variables. As shown in Based on the threshold value, our research estimates the above panel threshold model. Column (1) of Column (2) of Column (3) of The conclusions of this paper are as follows:(1) The results show a nonlinear relationship between the three industrial agglomeration modes and industrial eco-efficiency. The impact of specialized agglomeration and related diversified agglomeration on industrial eco-efficiency presents a U-shape pattern of first hindrance and then promotion. The unrelated diversified agglomeration has a significantly negative impact on industrial eco-efficiency, but it weakens when agglomeration increases to a certain level. (2) From the perspective of city-size heterogeneity, if the city size is smaller than the threshold of 0.799 million persons, the impact of specialized agglomeration on industrial eco-efficiency is insignificant. However, the specialized agglomeration has a significant inhibitory effect on industrial eco-efficiency when the city size crosses this threshold, that is, after the city surpasses a medium size. The impact of related diversified agglomeration on industrial eco-efficiency increases as the city size grows. Moreover, when the city size reaches 3.822 million people, the promoting role of related diversified agglomeration is further enhanced. The impact of unrelated diversified agglomeration on industrial eco-efficiency gradually changes from negative to positive as the city size increases. However, its positive effect appears only in the super-large and mega-cities. Therefore, it is only the related diversified agglomeration that improves industrial eco-efficiency in cities of different sizes. Only the super-large and mega-cities benefit from unrelated diversification agglomeration, while improving industrial eco-efficiency in smaller cities does not necessarily benefit from specialized agglomeration.Based on the above conclusions, this paper proposes the following policy implications:(1) The relationship between different agglomeration modes and industrial eco-efficiency should be rapidly adjusted according to the dynamic change of agglomeration level. The impact of specialized agglomeration and related diversified agglomeration on industrial eco-efficiency is first suppressed and then promoted. Therefore, the government should promote the specialized agglomeration and related diversified agglomeration to cross the inflection point by adopting the market economy and related regulatory policies for the cities with less than the inflection point. In addition, the government should formulate relevant measures such as appropriate environmental regulations to avoid the phenomenon of “pollution first, treatment later”. For cities that have already passed the inflection points, the government should encourage technology innovation and optimize the industrial structure. It transforms the development of clustered industries into high-quality development with high income and low energy consumption and pollution. Moreover, the government should also appropriately control the level of agglomeration to avoid the negative externalities caused by excessive agglomeration. Due to the inhibitory effect of unrelated diversified agglomeration on industrial eco-efficiency, the government should cultivate leading industries according to its advantages and promote the vertical or horizontal extension of the industrial chain, which improves the correlation level among industries. Moreover, the government should strengthen the intensity of environmental regulation to eliminate backward production capacity and force the innovation of cleaner production technology.(2) Differentiated policies of agglomeration should be adopted according to the size of cities. Small and some medium-sized cities should improve infrastructure and supporting services, such as environmental protection, and carry out technical training for workers to increase the supply of specialized labor. Based on the comparative advantages, such as resource endowment and the local industrial base, small and some medium-sized cities should concentrate limited resources on developing the key industries in a region. In addition, they should actively extend a clean industrial chain around the leading industries to improve industrial eco-efficiency. Large cities should encourage the coordinated development of multiple industries with considerable relevance and promote the transformation of clustered industries into high value-added and green industries. Due to the large urban capacity, super-large and mega-cities can also reduce the impact of economic fluctuations and promote breakthrough innovation by developing industries with low correlation, which improves industrial eco-efficiency.Due to the availability of data, this paper only uses data from 2004 to 2012. Extending the research period to recent years will help to enhance the robustness of our research results and provide more enlightenment for the green transformation of China’s industry. This can be left as a possible issue for future research.


Central itch syndrome has been previously described in conditions such as stroke. The neurophysiology of central itch syndrome has been investigated in non-human primates but remains incompletely understood.We report an observational study of a rare case of severe central itch following thalamic deep brain stimulation and postulate the location of the central itch centre in humans.The patient was a 47-year-old female, with congenital spinal malformations, multiple previous corrective spinal surgeries and a 30-year history of refractory neuropathic pain in her back and inferior limbs. Following multidisciplinary pain assessment and recommendation, she was referred for spinal cord stimulation, but the procedure failed technically due to scarring related to her multiple previous spinal surgeries. She was therefore referred to our centre and underwent bilateral deep brain stimulation (DBS) of the ventral posterolateral nucleus of the thalamus for management of her chronic pain. Four weeks after switching on the stimulation, the patient reported significant improvement in her pain but developed a full body progressive itch which was then complicated with a rash. Common causes of skin eczema were ruled out by multiple formal dermatological evaluation. A trial of unilateral “off stimulation” was performed showing improvement of the itchy rash. Standard and normalized brain atlases were used to localize the active stimulating contact within the thalamus at a location we postulate as the central itch centre.Precise stereotactic imaging points to the lateral portion of the ventral posterolateral and posteroinferior nuclei of the thalamus as critical in the neurophysiology of itch in humans. Itch is an irritative sensation, usually originating in the skin, but also in transitional tissues, such as conjunctiva or mucosa . It is uItch can be generally classified as systemic, infectious, dermatological, medication-related, psychiatric or neuropathic . InteresAcute itch is experienced when a pruritogenic stimulus contacts the surface of the skin and is relieved by scratching, which activates spinal interneurons to inhibit itch-transmitting neurons , 3, 8. CHere, we report a rare case of severe central itch following thalamic deep brain stimulation for the management of neuropathic pain. By means of detailed stereotactic imaging available and review of state-of-art works on the neurophysiological mechanisms underpinning pain and itch, we postulate the location of the thalamic central itch centre in humans.The patient was a 47-year-old female, with congenital spinal malformations and multiple previous corrective spinal surgeries during adolescence. She had a 30-year history of complex regional pain syndrome with refractory neuropathic pain in her both legs, particularly in the left lower limb, and failed-back surgery syndrome following spinal stabilization procedures. The patient would describe two types of pain: (i) a moderate to severe (VAS 7–9), continuous, stabbing and burning pain in the lumbosacral region, which increased by standing and walking, and did not settle completely when resting; (ii) permanent numbness associated with a shooting pain in the lower limbs, mainly on the left side and down to her feet. Clinical and neuroimaging investigation ruled out any neuronal compression, infection, or any signs of instability of the lumbar spine. She was on high dose analgesic medications, including opiates. Apart from chronic pain, she had type 2 diabetes mellitus and asthma. Despite her moderate disability due to refractory back and leg pain, she was mobile with walking-aids and able to perform routine domestic tasks with help. Following multidisciplinary pain assessment and recommendation, she was referred for spinal cord stimulation, but the procedure failed technically due to scarring related to her multiple previous spinal surgeries. She was therefore referred to our centre and underwent bilateral deep brain stimulation (DBS) of the ventral posterolateral nucleus of the thalamus for management of her chronic neuropathic back and leg pain..Bilateral octopolar DBS electrodes  were implanted under local anaesthesia using stereotactic MRI and the Leksell G frame . The electrodes were connected to a single rechargeable implantable pulse generator  placed subcutaneously in the infraclavicular region in a single-stage operation. The final position of the electrodes was verified by fusion of the postoperative stereotactic CT with the pre-operative stereotactic planning MRI scan. Targeting for DBS electrode placement was planned based on the somatotopic organization of the homunculus within the VPL, from medial—head representation, to lateral—lower-extremity, with the upper extremity between based on the Schaltenbrand and Wahren Atlas . Since pFour weeks after switching the stimulation on, the patient reported beneficial effect on her pain control, with at least 50% overall improvement on VAS scale and also of her mobility due to better pain control. Nevertheless, concomitant with pain relief, the patient reported significant itch on her arms and legs, which had appeared quite suddenly, following stimulation Fig. . The iniThe patient was regularly assessed and objectively evaluated by neurologists, neurosurgeons and dermatologists during the trial, and the evolution of the skin lesions documented by medical photography. The Pruritus Severity Scale (PSS) and the Dermatology Life Quality Index (DLQI) were applied in every follow-up consultations , 21. Thewww.github.com/netstim/leaddbs) .Interestingly, the neuroradiological analysis of the position of the electrodes in the sensory thalamus, revealed the active contacts to sit within the targeted ventroposterior/ventrobasal thalamic complex. On the left side, it was bordering the posterior part of the ventral posterior lateral (VPLp), ventral posterior inferior (VPI), ventral posteromedial (VPM) and the reticular nucleus of the thalamus. On the right side, the active contact was on the lateral border of the ventral posterior lateral nucleus, close to the posterior limb of the internal capsule Fig. a–d. FurtDue to limited pain control during the stimulation-off trial, decision was taken to switch the DBS device back on. Optimization of the stimulation settings, avoiding spread of current beyond the ventral posterior thalamic complex, allowed considerable relief of pain, without recurrence of pruritus.Central itch may pose a challenge both for attending dermatologists and neurologists. This is partly due to the rarity of the condition and partly as a result of late presentation to specialist services, with many patients having already received a range of empirical therapies which might mask or complicate the initial signs and symptoms. In our patient, for several years, the presentation was inaccurately interpreted by the local team as a primary skin rash which had turned itchy, rather than the primary sensation of an irritating itch driving scratching and the subsequent rash.Although not very often seen as a manifestation of neurological conditions, pruritus of central origin has been previously described in association with both central and/or peripheral nervous system injuries of diverse etiologies, such as infection, tumors, demyelinating disorders, and more frequently, stroke , 15–17. Acute pain and itch represent two distinct sensations, with different underlying neural networks and behavioral responses (scratching versus withdraw). Although, evidences from preclinical works support the existence of neural discrimination between itch and pain, the neurobiology of chronic itch and pain share largely similar mechanisms –10, 13, In the present study, the access to detailed brain imaging provided a unique opportunity for the investigation of the relationship between these systems in humans, shedding light on the discussion regarding plausible mechanisms implicated in the manifestation and perpetuation of chronic neuropathic itch and pain.It has been consistently observed that painful stimuli suppress itch , 9, 33. The pain/itch network crosstalk is also well illustrated through the mechanisms of action of opioids . The admThe underlying neural mechanisms for itch sensation are still not completely understood . AlthougAlthough advances in the understanding of the neurobiology of itch have been achieved, questions regarding the mechanisms involved in chronic neuropathic itch remain to be further elucidated , 31. ThuThus, in our patient, the mechanism implicated in the development of severe itchy rash was pruritus triggered by stimulation of the lateral aspect of the ventral posterolateral and ventral posterior inferior nuclei, which led to scratching and chronic inflammation of the skin. In addition, subsequently, intracutaneous inflammation caused by incoercible scratching might have led to further neurogenic inflammation and perpetuation of chronic itch, in a vicious cycle , 31, 34.Several brain regions have been implicated in the pathophysiology and shown to be activated during an itch , 9, 10. Thus, another potential mechanism for our observation may relate to the thalamo-cortical projections and somatotopic organization of ascending fibers within the posterior limb of the internal capsule and thalamic peduncles. As previously discussed, specialized itch ascending STT lamina I neurons synapse with thalamic third neurons within the VPL and VPI nuclei, which then project to somatosensory cortical regions, cingulate, insula and anterior cingulate. The thalamic peduncles consist of ascending and descending fibre bundles that detach from the corona radiata and internal capsule to connect thalamic nuclei not only to the cerebral cortex, but to the brainstem, spinal cord and cerebellum . The antDespite of extensive neurological and dermatological investigation, close clinical follow-up and systematic documentation, this observational study has limitations. Firstly, due to the severity of her skin lesions, the patient was kept on previous pharmacological treatment throughout the evaluation period, which might have partially contributed to improvement of itch. Secondly, the evaluation of itch symptoms and skin lesions might have been biased by the open-label design of the study, where both patient and medical staff were aware of which electrode had been temporarily switched off. Finally, regarding limitations intrinsic to the method of image processing and analysis of the position of the active contacts within the thalamus, the neural structures identified by the application of standard and normative stereotactic brain atlas might not correspond exactly to the patient’s specific neuroanatomy.Pathophysiology of central itch remains complex. A better understanding of the underlying mechanisms involved in the genesis and interactions between pain and itch, may contribute to the further development of future analgesic and antipruritic therapies. Targeted neuromodulation of the peripheral and/or central nervous system has already proven to be highly effective in selected conditions associated with refractory neuropathic pain . PreviouThalamic neuromodulation as cause of neuropathic central itch is a rare condition. We postulate that the lateral portion of the ventral posterolateral and ventral posteroinferior nuclei of the thalamus, play an important role in the neurophysiology of itch in humans. Our findings shed light on the neurobiology of central itch, providing an opportunity to further investigate the sensory thalamus as a potential locus for neuromodulation in the management of chronic neuropathic itch.


There has been a recent, unprecedented interest in the role of gut microbiota in host health and disease. Technological advances have dramatically expanded our knowledge of the gut microbiome. Increasing evidence has indicated a strong link between gut microbiota and the development of cardiovascular diseases (CVD). In the present article, we discuss the contribution of gut microbiota in the development and progression of CVD. We further discuss how the gut microbiome may differ between the sexes and how it may be influenced by sex hormones. We put forward that regulation of microbial composition and function by sex might lead to sex-biased disease susceptibility, thereby offering a mechanistic insight into sex differences in CVD. A better understanding of this could identify novel targets, ultimately contributing to the development of innovative preventive, diagnostic and therapeutic strategies for men and women. Despite advances in prevention strategies, as well as pharmacological and technology-based cardiovascular (CV) therapies, CV diseases (CVD) remain a major health burden, as they are the leading cause of morbidity and mortality . NotablyThe human gut hosts tens of thousands of microorganisms, up to 100 trillion microbes, which are collectively referred to as the gut microbiome , 12. TheAlterations in gut microbiota influence the CV system and can lead to the development of CVD . The intBacteroides acidifaciens compared with mice fed a control diet  is a devastating syndrome with poor prognosis. Gut microbiota and their metabolites produced from dietary metabolism have been linked to HF. To this extent, gut dysbiosis with decreased microbial richness and increased gut permeability has been reported in patients with HF , 58, as There are pronounced differences between men and women in the epidemiology, manifestation, pathophysiology, treatment and prognosis of CVD. As these have been reviewed recently elsewhere , 76, 77,Human and animal studies have shown that biological sex has an impact on gut microbial composition . In partA large body of literature demonstrates that sex hormones modulate CV physiology and pathology. Of relevance, among the various factors underlying sex differences in CVD, such as sex chromosomes and (epi)genetic factors , 106, seAlong this line, among various types of hormones with regulatory effects, sex hormones have a key role in the regulation of the distribution of the gut microbiome. In this context, sex hormones affect gene expression and other processes of gut microbiota, thereby influencing intestinal microbial composition and function. In parallel, the microbial community also alters sex hormone levels, thereby regulating disease development and outcome , 72. ThePotential differences in microbial composition, diversity and function between humans and animal models might limit the translation of experimental studies with animals. Furthermore, given the major impact that environmental factors have on gut microbiota, it needs to be highlighted that most environmental conditions of humans differ significantly from those of laboratory animals. In addition, estrous cycle-related changes in gut microbiota in rodents might not reflect any changes due to menstruation in women. Nevertheless, the degree of any inconsistencies remains to be determined. Lastly, abundant species do not necessarily confer abundant molecular functions, underscoring the importance of a functional analysis to understand microbial communities .The role of sex has yet underestimated consequences for physiology and pathology . A betteMoreover, the gut has been shown to be a target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In fact, gastrointestinal manifestations of SARS-CoV-2, such as anorexia, nausea, vomiting, diarrhea and hepato-cellular injury (transaminitis), among others, have been widely reported , 143. SAThe mechanisms by which the gut microbiome affects CV (patho) physiology are incompletely understood. From several human and animal studies, it is clear that the gut microbiome exerts sex-biased effects in health and disease. At least in part, sex hormones account for these sex-dependent effects in a complex bi-directional interaction with the microbial community. Gut microbiota may become a novel target for pharmacological or dietary interventions as part of new preventive and therapeutic strategies in CVD. A better understanding of the effects of biological sex and considering its role in such novel approaches will improve clinical care and management via new strategies for the prevention, diagnosis and treatment of disease in both men and women.GK conceived the work. SL and GK wrote the manuscript. Both authors contributed to the article and approved the submitted version.GK acknowledges lab support provided by grants from the Icelandic Research Fund (217946-051), Icelandic Cancer Society Research Fund and University of Iceland Research Fund.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.