The neurovascular units (NVU) are the minimal functional units of the blood–brain barrier (BBB), composed of endothelial cells, pericytes, astrocytes, microglia, neurons, and the basement membrane. The BBB serves as an important interface for immune communication between the brain and peripheral circulation. Disruption of the NVU by the human immunodeficiency virus-1 (HIV-1) induces dysfunction of the BBB and triggers inflammatory responses, which can lead to the development of neurocognitive impairments collectively known as HIV-1-associated neurocognitive disorders (HAND). Methamphetamine (METH) use disorder is a frequent comorbidity among individuals infected with HIV-1. METH use may be associated not only with rapid HIV-1 disease progression but also with accelerated onset and increased severity of HAND. However, the molecular mechanisms of METH-induced neuronal injury and cognitive impairment in the context of HIV-1 infection are poorly understood. In this review, we summarize recent progress in the signaling pathways mediating synergistic impairment of the BBB and neuronal injury induced by METH and HIV-1, potentially accelerating the onset or severity of HAND in HIV-1-positive METH abusers. We also discuss potential therapies to limit neuroinflammation and NVU damage in HIV-1-infected METH abusers. At the end of 2019, there were an estimated 38 million people living with human immunodeficiency virus (HIV) globally ,5,6. AltMethamphetamine (METH) is a highly addictive psychostimulant drug, the abuse of which has reached epidemic proportions worldwide, with its use being particularly high among people living with HIV-1 . METH abThe current review aims to summarize the findings from recent human and animal studies which might provide new insights into the pathogenesis of HAND in the context of METH dependence. We will not exhaustively review the literature regarding the impact of HIV-1 infection or METH alone on the NVU, because several excellent reviews summarizing the effects of these factors have recently been published ,27,28,29The NVU incorporates cellular and extracellular components involved in regulating cerebral blood flow and BBB function . The stuThe BBB is a complex structure composed of a tightly sealed monolayer of brain microvascular endothelial cells (BMECs) endowed with barrier properties, the basement membrane, and other NVU cells, including astrocytes and pericytes that wrap the abluminal capillary surface and provide physical support and stability to the BBB . One of To enter the CNS, METH and HIV-1 must first break through the BBB. Interestingly, METH and HIV-1 appear to compromise BBB integrity in a synergistic manner . Recent The presence of numerous luminal and abluminal protein transporters, solute and ion transporters, and efflux transporters strictly controls the entry and exit of molecules and metabolic by-products across the BBB. It has been shown that HIV-1 Tat protein and METH synergistically impaired the functions of two major glucose transport proteins, GLUT1 and GLUT3 in the brain endothelium of tree shrews . This reClinical evidence shows that exposure of endothelial cells to HIV-1 is linked to endothelial dysfunction . MoleculIn vivo studies have failed to demonstrate HIV-1 replication in endothelial cells, suggesting that the mechanism accounting for endothelial damage may partially rely on the indirect action of molecules released to the microenvironment by other infected cells in the brain , such asPericytes are a type of vascular cell that wrap the cerebral capillary walls and play a critical role in the regulation of the BBB . SignalsThe toxic effects of METH on pericytes have been previously reported in several in vitro studies. For example, METH has been shown to increase the migration of pericytes from the endothelial basement membrane via activation of p53 upregulated modulator of apoptosis and sigma-1 receptor . Another+ T-cells in vitro [A number of previous studies have shown that METH has the ability to disrupt immune homeostasis, affecting diverse leukocyte subsets, and thereby making METH users more susceptible to HIV-1 infection . In humain vitro ,103, thein vitro . METH hain vitro . A recenin vitro . MMP-9 iin vitro , basemenin vitro , and dysin vitro . Cyclooxin vitro ,110 and in vitro . NeverthWhile neuronal injury and loss are at the heart of HAND, HIV-1 does not directly injure neurons by productive infection, but rather by indirect or inflammatory factors released from infected cells in the CNS . HIV-1 iRecent studies have demonstrated that chronic exposure to METH exacerbates Tat-induced neurotoxicity, partially due to causing excessive ROS production and impairing antioxidant systems . For exaNeural tissues depend on mitochondria to produce ATP via the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation (OXPHOS) required for mechanisms that ultimately determine neuronal performance, such as excitability and synaptic signaling . ExcessiAlterations in mitochondrial dynamics maintained by a balance of fission and fusion is believed to contribute to neurodegeneration. Indeed, any disbalance in mitochondrial dynamics may ultimately lead to the accumulation of either fragmented or hyperfused mitochondria. Dynamin-related protein 1 (DRP1) is the central molecular player that mediates mitochondrial fission. METH has been found to increase mitochondrial fragmentation through translocation of DRP1 in HIV-1-treated human primary neurons . The impAlterations in the dopaminergic system have long been associated with HIV-1 infection in the CNS . METH, iThere is substantial evidence that inflammatory responses mediated by the activation of microglia are major drivers for the development and progression of HAND as well as METH-induced neurotoxicity ,53,114. Astrocytes provide trophic support for neurons and are involved in the regulation of transmission and synaptic activity, therefore an impairment in astrocyte function can negatively impact neurons. The mechanisms by which HIV-1 and METH lead to astrocytic neurotoxic activity are not entirely clear. A recent study demonstrated that acute treatment with HIV-1 led to the opening of mitochondrial permeability transition pores in the inner mitochondrial membrane in METH-cotreated primary human astrocytes . AnotherIt has been proposed that METH use disorder exacerbates HAND partly through amplifying glutamate dysregulation of astrocytes, resulting in excessive release of glutamate to the CNS microenvironment. Chronic excess of extracellular glutamate acts as a neurotoxin, triggering neuronal oxidative stress and DNA damage . When HISustained attention and vigilance are key components of situational awareness and underlie other cognitive functions, such as working memory and learning. While METH use has been found to be associated with sustained attention and vigilance deficits in humans, no interaction between HIV-1 and METH effects were found in a recent study . FurtherNeurocognitive deficits are thought to substantially impact the ability to engage in the vital tasks of daily living. Although comorbid HIV-1 infection and METH dependence have been associated with worsening cognitive performance, additive effects of HIV-1 and METH were not observed for everyday functioning . ResultsWith the great variety of impacts on the brain caused by HIV-1 infection, and further amplification when coupled with METH abuse, many novel treatments have arisen and continue to be developed to treat specific or multiple impairments. Depression was found to be decreased following consistent administration of mirtazapine , as wellThe combined effects of HIV-1 infection and METH use on the NVU can be deleterious and contribute to the clinical complexity of HAND. Indeed, HIV-1 infection and METH use disorder have a striking synergistic impact on NVU dysfunction and BBB damage, by inducing vascular oxidative stress, increasing the degradation of TJs and basement membrane proteins, and inhibiting nutrient and drug transporters. The impairments in astrocytic glutamate clearance and in the release of proinflammatory mediators and neurotrophic factors are potentiated in HIV-1 and METH comorbidity, and may lead to compromised synaptic functions along with neuronal cell death. The synergistic induction of mitochondrial dysfunction and apoptosis by HIV-1 and METH occurs in both neurons and astrocytes, despite the fact that these NVU cell types preferentially use different metabolic pathways. Disruption of the BBB by the combination of METH and HIV-1 may be related to the release of proinflammatory and neurotoxic mediators not only by glial cells but also by pericytes. Thus, future studies examining the mechanisms of BBB disruption and neuroinflammation mediated by pericytes during co-morbid METH abuse and HIV-1 infection are warranted.A focus on the NVU for potential drug targets may be helpful to develop novel NVU-targeted therapies in HAND aggravated by METH use. In this regard, TRPM2 channels in brain endothelial cells may serve as a promising drug intervention target to reduce BBB injury and neuropsychiatric symptoms in HIV-1-infected METH abusers. Targeting the EAAT2/TAAR1 pathway in astrocytes could also represent a promising strategy to reduce the accumulation of extracellular glutamate and ameliorate neuronal injury. There is a strong need for in vivo studies addressing the association not only between cognitive impairment and neuronal injury induced by the combination of METH use and HIV-1 but also between BBB breakdown and cognitive disturbances in this pathology. Although most of the studies analyzed in this review were performed in the absence of ART, it is imperative for future studies to characterize the interactive effects of not only METH and HIV-1, but also of antiretroviral therapeutics.The coupling of METH misuse with HIV-1 infection is characterized by worsening neurocognitive deficits in executive functions, working memory, and to a lesser degree in learning. Impairment in executive functions such as planning or self-regulation may contribute to medication nonadherence, whereas the association between METH use and risky sexual behavior can be mediated by impaired executive functions in HIV-1 infection. Moreover, alterations in dopamine receptor density or availability that are synergistically induced by METH use and HIV-1 may be especially harmful because they can promote addictive behaviors, such as impulsivity or cravings, and facilitate the development of depression symptoms. The findings from these studies lay the groundwork for future longitudinal research, examining whether frailty and disrupted sleep predict onset of neurocognitive decline in individuals with comorbid HIV-1 and METH use disorder. Furthermore, psychosocial treatments as well as the use of a variety of emerging technologies, such as online social networking and mobile technologies, should be utilized in conjunction with pharmacotherapy to target METH use and improve cognitive functioning in HAND.


Machine learning methodology using a random forest classifier confirmed significant differences between the treatment groups. Conclusions: MPD and TCZ improved outcomes  among hospitalized COVID-19 patients, but confounding variables such as the date of admission during the COVID-19 pandemic should be considered in observational studies.Background: The evidence for the efficacy of glucocorticoids combined with tocilizumab (TCZ) in COVID-19 comes from observational studies or subgroup analysis. Our aim was to compare outcomes between hospitalized COVID-19 patients who received high-dose corticosteroid pulse therapy and TCZ and those who received TCZ. Methods: A retrospective single-center study was performed on consecutive hospitalized patients with severe COVID-19 between 1 March and 23 April 2020. Patients treated with either TCZ  and methylprednisolone pulses (MPD-TCZ group) or TCZ alone were analyzed for the occurrence of a combined endpoint of death and need for invasive mechanical ventilation during admission. The independence of both treatment groups was tested using machine learning classifiers, and relevant variables that were potentially different between the groups were measured through a mean decrease accuracy algorithm. Results: An earlier date of admission was significantly associated with worse outcomes regardless of treatment type. Twenty patients died (27.0%) in the TCZ group, and 33 (44.6%) died or required intubation ( Coronavirus disease 2019 (COVID-19) has evolved into a global pandemic with a profound impact on public health. Approximately 20% of cases may develop severe COVID-19 infection with pneumonia, which could potentially lead to hypoxic respiratory failure, acute respiratory distress syndrome, and/or septic shock ). This hAccordingly, immunomodulatory drugs such as glucocorticoids have been proposed as a treatment for patients with severe COVID-19 to tackle hyperinflammation and immune dysregulation . Indeed,Therefore, the aim of our study was to compare, by means of machine learning methodology, the outcomes of hospitalized patients with COVID-19 who received TCZ compared with those who received TCZ and high-dose glucocorticoid pulse therapy.We performed a retrospective study at a 1000-bed university tertiary care hospital located in Salamanca (northwestern Spain). Consecutive patients admitted due to severe COVID-19 from 1 March to 23 April (peak of the first wave in our center) were analyzed for this study. Patients with COVID-19 were considered to have severe disease because of respiratory failure or other organ dysfunction assessed by Sequential Organ Failure Assessment (SOFA) score > 2, which received per protocol in our center TCZ and/or corticosteroids. TCZ was administered according to our hospital protocol and the treatment consisted of two doses until 26 March (administered at a dosage of 8 mg/kg (max 800 mg) by two consecutive administrations 8 h apart) and one dose thereafter (600 mg (400 mg for weight < 70 kg)), due to the recommendation issued on this date by the Spanish Agency of Medicines and Medical Devices (AEMPS) based on drug shortage. High-dose corticosteroid pulse therapy was administered according to the hospital protocol after 26 March (250 mg of intravenous methylprednisolone (MPD) for 3–5 days) and previously at the physician’s discretion. Patients who received at least 125 mg of intravenous MPD  for 2 to 5 days and intravenous TCZ composed the corticosteroid and TCZ (MPD-TCZ) group, and patients who received only intravenous TCZ composed the TCZ group. Patients within these groups were also able to receive hydroxychloroquine and lopinavir/ritonavir according to the protocol. These groups of patients (MPD and MPD-TCZ) were deemed comparable after stratification by our own validated prognosis score . Thus, wWe collected data on demographic variables, the patients’ individual comorbidities and Charlson Comorbidity Index scores, chronic medical treatments, clinical characteristics, physical examination parameters, and the laboratory findings available at hospital admission, as previously described . Regardit-test for continuous variables and Fisher’s exact test for binary variables. If treatment was assigned randomly to all patients and the measured variables were independent, the p values of these tests would be distributed uniformly. Consequently, we would expect approximately six variables showing differences with statistical significance (p < 0.05) between both treatment groups in univariate analysis. In addition, Kaplan–Meier curves were used to compare mortality rate between groups (long rank test).The differences in the measured variables between both treatment groups were analyzed using Student’s The relevance of the collected variables in the prognosis of the patients was previously analyzed in the same cohort of patients , where a2)/fraction of inspired oxygen (FiO2) ratio, patient age, estimated glomerular filtration rate, procalcitonin levels, C-reactive protein levels, updated Charlson comorbidity index scores, and lymphocyte levels. We considered these predictors separately in the multivariate analysis, as differences in these variables between the treatment groups could have likely impacted our results. Additionally, due to the changing epidemiological situation and evolution of the COVID-19 pandemic, we performed a comparison of the mortality and severity in both treatment groups matched to both the basal risk score of the patients and the date of admission of the patients. This comparison was performed as a subclassification matching with five subclasses divided along quintiles [As previously described, the final model of this COVID-19 Salamanca Risk Score identified the following variables as predictors of severity in both our cohort and the external validation cohort : the peruintiles , to asseTo test the independence between both treatment groups in a multivariate analysis, we developed a machine-learning algorithm for predicting whether a patient belonged to the TCZ or MPD-TCZ group. Machine learning methods were chosen because they encompass classical statistic models (such as logistic regression) while providing the benefits of regularization and cross-validation, and also offer different categories of models of greater discrimination power in case of non-linear relationships and interaction effects between the variables. The performance of this algorithm was given by the area under the ROC curve and the corresponding 95% confidence interval. The null hypothesis  was rejected if the value of 0.5 for the area under the ROC curve fell outside the confidence interval. This analysis was performed both using all clinical variables and using only the variables included in the abovementioned COVID-19 Salamanca Risk Score .In brief, machine learning methodology was described as follows : data frBetween 1 March and 23 April 2020, 918 patients were admitted to the University Hospital of Salamanca because of severe COVID-19 with PCR-confirmed infections. Of them, 74 patients received only TCZ, and 136 patients received TCZ and high-dose corticosteroids . Survival analysis and Kaplan–Meier curves also showed a significant effect (p = 0.002) of MPD-TCZ in mortality  in the TCZ group and the combined event of death and need for invasive mechanical ventilation occurred in 33 patients (44.6%), whereas 15 patients died (11%), and 29 patients died or required intubation (21.3%) in the MPD-TCZ group. These improved outcomes in the MPD-TCZ group were significant after univariate analysis , which means that patients from both groups have a similar risk of severe outcomes according to this score and therefore that these groups may be considered comparable due to similar baseline risk. In addition, no statistically significant differences were found in any of the variables composing this score between both treatment groups. The comparison of the mortality and severity between the treatment groups matched to the score risk of patients is shown in p = 0.004) and the MPD-TCZ group (p = 0.007). Therefore, this finding shows that patients had worse outcomes in the first weeks of the analyzed period regardless of treatment type.The average COVID-19 Salamanca Risk Score, as previously described , was 30.2)/fraction of inspired oxygen (FiO2) ratio, patient age, the estimated glomerular filtration rate, procalcitonin levels, C-reactive protein levels, updated Charlson comorbidity index scores, and lymphocytes levels) was regularized logistic regression, which obtained an area under the ROC curve of 0.49 (0.40–0.57). However, the best model built with all the clinical variables was the random forest classifier, which obtained an area under the ROC curve of 0.60 (0.51–0.69), thus detecting statistically significant differences between both groups. Therefore, multivariable analysis including all potentially relevant variables confirmed a statistically significant difference between both treatment groups regarding outcomes. The most relevant variables used for this multivariable analysis as calculated by the mean decrease accuracy algorithm can be found in Therefore, univariate analysis and stratification according to baseline risk score calculated by means of COVID-19 Salamanca Risk Score and date of admission showed that patients who received tocilizumab and glucocorticoids had better outcomes than those receiving tocilizumab alone, without significant differences in baseline risk score but with differences regarding date of admission. In order to include other variables potentially associated with outcomes, machine learning models were built to test differences between the two treatment groups by means of multivariable analysis. In addition, we compared whether the date of admission was more relevant than prognostic scores to differentiate between treatment groups. In our study, we found that the combination treatment with glucocorticoids and TCZ, when compared with TCZ alone, is associated with both reduced mortality and a reduced risk of a composite endpoint of invasive mechanical ventilation and death. To allow comparison, we selected two groups with similar baseline risk according to our previously validated risk score, which made the presence of potential unknown confounders related to patient characteristics unlikely . AdditioOur results are in line with data from other observational studies showing that this combination therapy is associated with better outcomes when compared with either TCZ or glucocorticoids alone ,22,23,24In line with this, although we found a significant difference between the treatments after machine learning methodology, we also found that mortality improved over time as an independent variable associated with better outcomes. In our study, the choice of treatment for each patient was made according to the protocol at the time of admission: patients treated in the early stages of the pandemic received only TCZ, while in later stages, the protocol for treatment changed to include both TCZ and corticosteroids. Mortality improvement over time may have therefore been due, at least in part, to different treatment efficacies, as the random forest classifier also confirmed, but a potential confounding effect for the date of admission has to be considered. Indeed, improved mortality over time has already been described in other series of COVID-19 patients and may also be explained by the increased knowledge of the disease, different population groups being affected by the pandemic at different time points, and reduced hospital burden, regardless of treatment type . This woAnother potential limitation of our study is the different sample sizes between groups and the use of different types and doses of steroids. Although the Recovery trial ,28 used In conclusion, our data support the potential utility of the combination of glucocorticoids with TCZ, in line with previous findings but outcomes were influenced by date of admission regardless of treatment type and thus this variable as a potential source of bias should be considered in observational studies on this topic. Rigorous randomized trials are needed to assess the efficacy of TCZ and corticosteroids combined and to evaluate the optimal corticosteroid dose for treatment.


Low protein diets are associated with increased lifespan and improved cardiometabolic health primarily in rodents, and likely improve human health. There is strong evidence that moderate to severe reduction in dietary protein content markedly influences caloric intake and energy expenditure, which is often followed by a decrease in body weight and adiposity in animal models. While the neuroendocrine signals that trigger hyperphagic responses to protein restriction are better understood, there is accumulating evidence that increased sympathetic flux to brown adipose tissue, fibroblast growth factor-21 and serotonergic signaling are important for the thermogenic effects of low protein diets. This mini-review specifically focuses on the effect of low protein diets with variable carbohydrate and lipid content on energy intake and expenditure, and the underlying mechanisms of actions by these diets. Understanding the mechanisms by which protein restriction influences energy balance may unveil novel approaches for treating metabolic disorders in humans and improve production efficiency in domestic animals. Energy balance is a fundamental biological process that is dependent on a complex interplay of calories consumed as macronutrients , and energy expended and stored. A dysregulation of the mechanisms that sense and signal dietary nutrients in the gut may predispose to obesity and metabolic complications. Among the macronutrients, the intake of protein is tightly regulated and dietary protein restriction is purported to extend lifespan, improve energy balance and cardiometabolic health ; howeverThe “protein leverage” hypothesis posits that protein intake is tightly regulated in several species including rodents, small animal pets, birds and humans –4. When in vitro and with amino acids, we  which in turn phosphorylates eukaryotic translation initiation factorα (eIF2α) leading to activation of activating transcription factor 4 (ATF4) and CCAAT/Enhancer-binding protein homologous protein (CHOP) to inhibit protein synthesis and increase fibroblast growth factor-21 (FGF21) expression and secretion –25. Furtcids, we  and othecids, we –30 showecids, we  suggesticids, we , 33.Accumulating evidence indicates that moderate protein restriction in rodents  stimulates FGF21 secretion from the liver which acts on β-klotho receptors in the brain to promote hyperphagia –36 Figu. We haveProtein or total amino acid restriction increases thermogenesis in rodents , 48–61. Given the concurrent increase in EE and energy intake in response to protein restriction , 56, 60,Due to high carbohydrate content of low protein diets , 60, 61 Despite intense efforts to gain insights into the hyperphagic responses driven by protein dilution, the underlying mechanisms of changes in EE received less scrutiny. The thermogenic effects of low protein diets have been associated with (i) increased sympathetic flux to brown adipose tissue (BAT) via β-adrenergic receptor (β-AR) signaling , 72, 73,The BAT plays an important role in diet-induced thermogenesis –79. An iFibroblast growth factor-21 is released in response to nutrient deficiency and stimulates browning of white adipose tissue to regulate adaptive thermogenesis , 74. Inf2 and EE were shown to be decreased by administration of a non-selective 5-HT receptor antagonist, metergoline (The role of serotonergic neurons in regulation of thermogenesis and BAT activity has been previously documented , 101. Inergoline  and 5-HTergoline  in rats Dietary protein restriction results in reduced concentration of most essential amino acids in the circulation, which play a role in metabolic adaptations to protein deficient diets. Among the amino acids studied, methionine , 105–108Dietary protein restriction orchestrates a whole organismal physiological response to stimulate energy intake and EE. The low protein induced thermogenesis is largely driven by dietary protein content and less likely by the concurrent hyperphagia. Though the liver and brain appear to be the primary sites for sensing protein deficiency, the coordination of these tissues with intestinal sensing mechanisms to promote hyperphagia and thermogenesis remains poorly understood. Although liver driven FGF21 seems essential for stimulating EE in response to protein or amino acid restricted diets, the role of local FGF21 synthesized and released from BAT, skeletal muscle and gut in the thermogenic responses to low protein diets remains to be determined. Further, the roles of systemic and local amino acid concentrations, as well as sympathetic and serotonergic signaling in brain, adipose and muscle tissues to coordinate intake and expenditure responses to dietary protein restriction remains largely unexplored. A deeper understanding of the neuroendocrine mechanisms by which dietary protein dilution modulates energy balance may lead to the development of novel strategies for preventing and treating obesity and associated comorbidities in humans, as well as for improving production efficiency in domestic animals.AP and PC wrote the article and agree to be accountable for the content of the work. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Follicle stimulating hormone (FSH) and its receptor (FSHR) have been reported to be responsible for several physiological functions and cancers. The responsiveness of stem cells and cancer stem cells towards the FSH-FSHR system make the function of FSH and its receptors more interesting in the context of cancer biology. This review is comprised of comprehensive information on FSH-FSHR signaling in normal physiology, gonadal stem cells, cancer cells, and potential options of utilizing FSH-FSHR system as an anti-cancer therapeutic target. Gonadotrophs of the anterior pituitary gland synthesize and secrete follicle stimulating hormone (FSH) in response to stimulation by the hypothalamic gonadotropin releasing hormone [GnRH also referred to as luteinizing hormone releasing hormone (LHRH)] in vitro studies. In an earlier study, Timosi et al. Functional specificity of FSH is provided by its β-subunit that ensures FSH interaction specifically with FSH receptor (FSHR) th amino acid position of a hinge loop in the signal-specificity sub-domain is responsible for the hormonal activity Biological functions of FSH are activated through its interaction with its specific receptors (i.e. FSHR1). Human FSHR1 protein is composed of 695 amino acids polypeptide including the signal peptide. A mature FSHR1 protein of 76 kDa molecular mass is expressed in Sertoli and granulosa cells In humans, the FSHR gene spans 52 Kbp on chromosome 2 and consists of eleven exons and ten introns. FSHR gene exhibits four alternatively spliced forms . Among these spliced forms, FSHR1 and FSHR3 are more recognized for their participation in various functions of FSH, including: cell growth, proliferation, and steroidogenesis Among the four alternatively spliced isoforms of FSH receptors, FSHR1 transcript contains all the ten exons and is known as the major isoform that actively participates in steroidogenesis, follicular development, and spermatogenesis. It has a large N-terminal extracellular domain, seven α-helical transmembrane domains interconnected with alternating extracellular and intracellular loops, and an intracellular C-terminal tail FSHR1 is a member of the G-protein-coupled receptors (GPCRs) family and activates the G-protein (Gs), resulting in the production of cyclic adenosine monophosphate (cAMP), which subsequently leads to the induction of various signaling pathways after the initial binding to FSH i, instead of the stimulatory Gαs protein. Further research remains to be performed to understand the physiological significance of the FSHR2 splice variant. FSHR3 possesses a distinguishing feature from FSHR1. It functions independently of cAMP-controlled pathways 2+) influx via modulation of Ca2+-dependent channels modus operandi similar to that of insulin-like growth factor (IGF)-1-binding proteins FSHR2 is a truncated form of FSH receptor and this truncation disrupts the intracellular portion of the receptor and negatively affects the downstream signaling process Existing literature suggests the presence of spermatogonial stem cells (SSCs) in the mammalian testis. Further, recent reports demonstrate that stem cells are localized in adult ovaries and undergo postnatal oogenesis, similar to spermatogenesis in the testis In reproductive biology, a general dogma exists which states that the mammalian ovary contains a fixed number of follicles that are depleted progressively with the advancement of age and culminates in menopause. However, this belief has been challenged by the existence of stem cells in adult ovaries. Various groups have demonstrated the presence of ovarian stem cells in adult mammalian ovary and put forth a hypothesis in favor of ovarian stem cells (OSCs) for their role in neo-oogenesis and primordial follicular (PF) assembly, similar to that of SSCs in spermatogenesis. According to this hypothesis, the presence of OSCs leads to the regular assembly of follicles on ovarian surface epithelial/cortical regions and that the process of primordial follicle assembly from stem cells is regulated by FSH in vivo conditions needs to be developed. Recent studies have demonstrated the localization of stem cells in adult mammalian ovary surface epithelium. The very first study that challenged the concept of a fixed reserve of follicles in females was reported by Tilly and his group in vitro conditions in vitro without any specific requirement of growth factor/ cytokine and thought to be developmentally equivalent of primordial germ cells - the natural precursor of gametes Menopause probably occurs due to an age-related compromise in the somatic niche and, under this situation, OSCs are unable to undergo differentiation, thus leading to menopause et al.et al.Both FSHR1 and FSHR3 were found to be expressed in sheep OSCs, whereas the epithelial cells were distinctly negative for any type of FSHR. An active transcription of FSHR3 was observed in OSCs in response to FSH within 3 h, although FSHR1 levels remained unaffected in vivo models to understand FSH functioning in physiological context contributed significantly towards our understanding of the modus operandi of this critical molecular partnership The evidences prove the role of FSH in stimulating OSCs. Nevertheless, direct action of FSH on ovarian stem cells and its physiologic function can be demonstrated only through knockout/down and overexpression studies on FSHR. To the best of our knowledge, there is no study that deals with the knockout/down of FSH/FSHR to establish the physiological role of FSH/FSHR in ovarian stem cells. However, the results obtained from earlier animal knock down studies of FSH/FSHR clearly demonstrate the obligatory role of FSH-FSHR in the female reproductive system The process of spermatogenesis originates from SSCs and involves several sequential steps of cell proliferation and differentiation, which lead to the development of spermatozoa In vitro studies have been conducted to delineate the role of FSH in the development of Sertoli stem cells. Both in vitro and in vivo studies suggest that FSH acts as an important mitogenic factor for Sertoli cells and stimulates their proliferation. Additionally, FSH also stimulates the synthesis of certain growth factors, like Glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (bFGF2), in Sertoli cells that supports the proliferation and colonization by type A SSCs Conversely, testosterone interacts with androgen receptors in Sertoli cells and regulates several responses involved in spermatogenesis in vivo using mouse testicular section based on the nuclear OCT-4 immunolocalization in vivo-/CD45-/SCA-1+]. FSH plays a crucial role in spermatogenesis and it has been confirmed that both VSELs and SSCs express FSHR, and further they respond to FSH treatment. According to this study, pluripotent VSELs are the most primitive stem cells in the testis and FSH acts directly on them through FSHR3, not through canonical FSHR1, to stimulate their asymmetric cell division to undergo self-renewal. These VSELs then further develop into SSCs through symmetric cell division, undergo clonal expansion, and eventually terminally differentiate into sperm. Consistent with these observations, a similar positive impact of FSH on sheep testicular stem cell stimulation was also observed by Bhartiya and her group et al.Similar to VSELs reported in adult human ovaries, these cells have also been documented in the testis of humans and mice Cancer stem cells drive the cancer progression through modulating signalling pathways and regulating miRNAs to offer therapy resistance and promote metastasis The major transcriptional regulatory pathways controlling stem cell signaling, cellular reprogramming that are responsible for cellular pluripotency are: Hedgehog, Ephrin, WNT, and certain microRNA pathways +/CD117+ cells exhibiting stemness FSH-FSHR upregulates OCT4 expression in ovarian cancer stem cells, most likely through its regulation of GSK3β/β-catenin and PI3K/Akt effector pathways 307Thr, and Ser680Asn in exon 10 of FSHR gene alone or in combination with polymorphisms in the promoter region (-114 T/C and -29 G/A) are reported to be associated with a reduced risk of the disease 307/Ser680 alleles have been observed to increase the risk and recurrence of ovarian cancer Polymorphisms in the FSHR gene have been found to modulate the risk of testicular germ cell tumor formation. Polymorphisms in the coding region, such as: AlaA few, but conflicting reports are available regarding the FSH-FSHR signaling system pertaining to tumor malignancies As mentioned above, the involvement of FSH-FSHR signaling in malignancies in tissues other than gonadal cancers is limited. However, gradually accumulating evidence points towards the involvement of FSH-FSHR signaling in cancers of various tissues other than gonads. In this regard, a recent trial reporting FSHR expression in vascular endothelial cells in prostate, breast, and urothelium cancers are reported For almost over two decades, FSH-FSHR has been believed to be acting through the Gαs/cAMP/PKA signaling pathway to mediate its biological actions in the target cells The well-studied Gαs pathway shows that FSH-FSHR interactions lead to coupling of the receptor to Gαs subunit inducing adenylate cyclase activity to produce cAMP Starting off as a pathway for desensitizing and recycling FSHR, β-arrestin pathway, gradually received the recognition of one with adapters and transducers of FSH-FSHR signaling Involvement of PI3K/mTOR pathway as an effector route for FSH-FSHR mediated signaling has been substantiated through several studies. All the pathways show PI3K activation and PIP3 accumulation modus operandi to modulate gene expression, cytosolic PKA binds to retinoic acid receptor alpha to inhibit its nuclear translocation, thereby, modulating the expressions of the genes under the regulation of this transcription factor Modulation of expressions of genes involved in steroidogenesis by FSH is a well-established Until now we elaborately described about the pathways that get activated mostly through second messengers due to the interaction between FSH and FSHR. FSHR being a member of the GPCR family, this mode of activating signaling cascade is expected. Interestingly, FSH-FSHR interaction can also adopt alternative pathways to transduce signals and this largely, involves direct physical interaction of certain proteins with FSHR Figure . A numbe189Val mutation, known for misrouting the receptor and retaining it in the endoplasmic reticulum In vitro observations revealed a possibility of slowing the growth of FSH-responsive tumors through siRNA-mediated silencing of FSHR3 in mouse ovarian cancer cell lines The association of identified mutations in FSHR with specific cellular functional has enabled this information to be utilized in identifying therapeutic targets and designing of novel therapeutics. For example, pharmacophore (Org 41841) was designed to bind FSHR at a site other than that used by FSH and improve the membrane localization of the receptor in the presence of an AlaThe elevated FSH levels of ovarian cancer corroborate with the increased expression of FSHR in several cancers. Upregulated endothelial FSHR levels in cancers are associated with vascular remodeling and tumor angiogenesis, whereas, its epithelial counterpart facilitates cell proliferation, migration and invasion in vitro functional studies provides valuable information on FSH-FSHR interactions at the molecular level. Few mutations have been reported in either subunit of FSH and may lead to pathological implications. Comparatively, FSHR has a greater number of mutations, some resulting in gain-of-function, while others resulting in a loss-of-function. These mutations affect hormone binding ability or hormone-induced signaling ability by introducing structural changes in the receptor. Various polymorphisms of FSHR have been associated with risks of different types of cancers. Finally, the therapeutic implications of this information have been documented with a positive future perspective on its potential applications.FSH/FSHR signaling controls several crucial biological pathways, therefore, malfunctioning of this signaling system is bound to culminate into several disorders, including cancer. This review mainly covers the connection between FSH and FSHR signaling and its role in cancer. Attempts were made to provide a comprehensive understanding of the various aspects under the control of the FSH/FSHR signaling system. This included the effect on stem cell maintenance and propagation, as well as sources of variations in the functioning of FSH or FSHR (or both). Studies reporting various mutations, polymorphisms, and alternatively spliced variants of FSHR have correlated these products with their respective phenotypes, clearly suggesting that such genetic alterations may result in infertility. Discussion on Many aspects of the FSH-FSHR signaling system require better insight and clarifications, for which detailed studies are needed. Moreover, along with the understanding of the alterations in the signaling mechanisms that affect the prognosis of cancer, it is important to devise protocols of translating this foundational knowledge to clinical applications. This review has put together several aspects of FSH-FSHR signaling which are highly informative and provide quick reference to facilitate the designing of elaborate studies with the objectives dedicated towards harnessing the therapeutic aspects and potentials of FSH in fertility and disorders especially cancer.


The treatment of hypoxic tumours continues to be one of the main challenges for radiation therapy. Minibeam radiation therapy (MBRT) shows a highly promising reduction of to-xicity in normal tissue, so that very heavy ions, such as Neon (Ne) or Argon (Ar), with extremely high LET, might become applicable to clinical situations. The high LET in the target would be unrivalled to overcome hypoxia, while MBRT might limit the side effects normally preventing the use of those heavy ions in a conventional radiotherapeutic setting. The work reported in this manuscript is the first experimental proof of the remarkable reduction of normal tissue (skin) toxicities after Ne MBRT irradiations as compared to conventional Ne irradiations. This result might allow for a renewed use of very heavy ions for cancer therapy. (1) Background: among all types of radiation, very heavy ions, such as Neon (Ne) or Argon (Ar), are the optimum candidates for hypoxic tumor treatments due to their reduced oxygen enhancement effect. However, their pioneering clinical use in the 1970s was halted due to severe side effects. The aim of this work was to provide a first proof that the combination of very heavy ions with minibeam radiation therapy leads to a minimization of toxicities and, thus, opening the door for a renewed use of heavy ions for therapy; (2) Methods: mouse legs were irradiated with either Ne MBRT or Ne broad beams at the same average dose. Skin toxicity was scored for a period of four weeks. Histopathology evaluations were carried out at the end of the study; (3) Results: a significant difference in toxicity was observed between the two irradiated groups. While severe da-mage, including necrosis, was observed in the broad beam group, only light to mild erythema was present in the MBRT group; (4) Conclusion: Ne MBRT is significantly better tolerated than conventional broad beam irradiations.  The treatment of radioresistant hypoxic tumours is still one of the major challenges in radiation therapy . AccumulThe remarkable reduction of normal tissue toxicities observed after both X-rays and proton minibeam radiation therapy (MBRT) as compared to conventional broad beam irradiations ,11,12,13MBRT uses narrow beams (0.5 to 1 mm) to create a distinct spatial modulation of the dose, featuring alternating regions of high dose (peaks) and low dose  . MBRT wiA further improvement could be to use (very) heavy ions , thanks to their superior relative biological effectiveness and reduced oxygen effect as compared to X-rays and protons, provided that normal tissue toxicity stays below the tolerance limit. Advantageous dose distributions for normal tissue sparing were obtained in our recent Monte Carlo (MC) studies ,22 on heWe performed the first in vivo evaluation of very heavy ions MBRT to confirm our hypothesis. In particular, we chose to start with Ne ions. since our Monte Carlo calculations indicated that, among all heavy ions, they offer the best balance between having a high peak-to-valley dose ratio and peak-to-valley-linear energy transfer ratio in normal tissues and high linear energy transfer values (close to 100 keV/µm) in the target region . 2. The dose rate was 2 Gy/min.The irradiations were carried out in the biology room at Heavy Ion Medial Accelerator (HIMAC) of the National Institute of Radiological Sciences, Chiba, Japan . A beam 2 field size) with an ionisation chamber (PTW Markus chamber). The relative dosimetry was realised by means of two high spatial resolution detectors: a PTW 60019 microdiamond detector [Dosimetry was performed using a two-step protocol, which was similar to previous works . Absolutdetector  and gafcThe PTW microdiamond was cross-calibrated in the Ne beam towards the Markus ionisation chamber in broad beam conditions. Subsequently, the possible impact of quenching effects in Ne ion beams was assessed. The quenching effects are caused by a local (microscopic) saturation, which occurs around highly ionizing ion tracks. The result is a reduction of the dose actually measured by the detector. The effect depends on the linear energy transfer (LET) of the radiation, and it has been reported in films and microdiamond detectors exposed to heavy ions ,27. The A large area chamber  was also used to evaluate the scatter factors, as in previous works .Ethics statement: all animal experiments were conducted in accordance with the animal welfare and ethical guidelines of our institutions. They were approved by the National Institute of Radiological Sciences Institutional Animal Care and Use Committee (permit no. 19–2004). The legs of C57BL/6J mice  were irradiated and the skin response was evaluated in order to assess the potential reduction of normal tissue toxicity of Ne MBRT as compared to broad beam irradiations. The mice were anaesthetised for irradiation with pentobarbital Sodium Salt (~0.5 mg/10 g body weight). Two groups of animals were considered: (i) one group received broad beam (BB) conventional irradiation ; (ii) a second group received Ne MBRT with the same mean dose as the BB group . Non-irradiated legs served as controls. The doses reported here are physical doses and they were delivered in one fraction. Relative biological effectiveness (RBE) values of between 1.5 and 2 have been previously reported in the plateau region of Ne beams ,30. TherGafchromic films were placed on top of the legs to assess the quality of the irradiations, see The animals were followed up for four weeks. The skin reaction was scored every two to three days using the arbitrary score that is shown in At the end of the study the animals were sacrificed, and the legs were cut and immersed in formol 10%. After 24 h, the legs were embedded in paraffin; 4-μm-thick sections were cut and stained in haematoxylin and eosin (HE). All the samples were image scanned by an Olympus Slideview VS200 slides scanner using an Olympus U-TV1XC camera and 20× objective. The histopathological (double-blinded) evaluation was carried out by board certified pathologists (European college of veterinary pathologists (ECVP)). Histological changes were described according to the distribution, severity, and morphologic character. The severity scores were assigned grade 1 to 5, as described in The irradiated animals gained weight at the same rate as the controls. The skin from control (non-irradiated) legs did not show any relevant abnormalities, see The skin exposed to MBRT irradiation presented multifocal epidermal hyperplasia in low severity, which was characterized by an increase in thickness of the epidermal layer from single-cell layer to 2–3 multilayers. Often, there were minimal infiltrates of inflammatory cells and/or decrease in the presence of annexal structures , see The skin that is irradiated with broad beam presented relevant changes of epidermis and dermis, epidermal necrosis, associated with inflammation, oedema, and loss of the annexal structures, see Despite major advances in RT in the last decades, the treatment of hypoxic tumours remains elusive. In this context, the use of very heavy ions for therapy , which are less dependent on the oxygen effect than X-rays, was explored in the past . Between 1975 and 1992, 433 patients were treated with C, N, O, Ne, Si, and Ar ions  for variThe work that is presented in this paper aims to explore a new RT approach that might allow for a renewed and optimal use of such very heavy ions for therapy. In this first evaluation, we were able to demonstrate that the alliance of MBRT and Ne ions leads to a net reduction of toxicity as compared to conventional broad beam irradiations in the plateau region . In our evaluation, severe toxicity was observed after conventional Ne broad beam irradiations, including radionecrosis. The onset of severe damage was significantly earlier (14 days versus 21 days) than higher doses (40 and 80 Gy) deposited in X-ray irradiations (28). In contrast, only light to mild erythema was present after MBRT, despite peak doses of 60 Gy  delivered in one sole fraction. Additionally, the damage seemed reversible, as reflected by the reduction of the damage score from the 20th day after irradiation. Thus, the damage may be considered adaptive and of low clinical significance. It should also be highlighted that the peak dose deposited in this experiment (60 Gy) is significantly higher than the ones employed in previous skin irradiation experiments while using conventional (seamless) exposures and that led to severe skin toxicity. In particular, doses higher than 24 Gy (X-rays) in one fraction have been previously reported to lead to a high risk for severe and irreversible skin toxicity . A dose It is also worth noting that the average length (487 µm) and spacing (1.1 mm) among lesions do not directly correspond to the lengths of the peak and valley dose regions. Consequently, this disregards a simple dose effect. Instead, the pattern of the lesions points to a more global or collective tissular effect. It might be due “beneficial” bystander effects leading to tissue restoration, already observed in previous studies in skin ,38. ThosThe minimal toxicity observed after Ne MBRT irradiations is coherent with the results of our previous theoretical works ,22, whicThe gain in normal tissue preservation opens the door to a potential renewed use of those very heavy ions for therapy. Several works have reported an equivalent or superior tumour control with both x-rays and protons MBRT than with BB ,17,18,19Moreover, there are some indications that heavy ions cans potentiate the immune response in a more effective way than photons, or even proton beams . This miThe results of this first experiment support the continuation of the exploration of very heavy ions for therapy. A first evaluation of tumour control effectiveness was planned in March 2021, and it is now postponed due to COVID-19 crisis. This manuscript reports the first experimental evidence of the significant gain of normal tissue preservation that is provided by Ne MBRT in comparison with broad beam irradiations. The mice leg’s skin exposed to MBRT showed low local effects, which were mostly characterized by multifocal epidermal hyperplasia, minimal inflammatory reaction, and a decrease in annexal structure numbers. At this timepoint, these local effects were considered to be adaptative and of low clinical significance. In comparison, mice exposed to broad beam exhibited severe side effects. Therefore, Ne MBRT treatments are remarkably better tolerated than broad beam. This result could allow for a renewed use of very heavy ions, whose high linear energy transfer in the target would be unrivalled to overcome tumour hypoxia.


Graphene has been extensively investigated in advanced photodetection devices for its broadband absorption, high carrier mobility, and mechanical flexibility. Due to graphene’s low optical absorptivity (2.3%), graphene-based photodetection research so far has focused on hybrid systems to increase photoabsorption. However, such hybrid systems require a complicated integration process and lead to reduced carrier mobility due to heterogeneous interfaces. Crumpled or deformed graphene has previously been reported in electronics and optoelectronics. However, a depth study on the influence of the morphology of nanofilms  related to light absorption in photodetection devices has not been demonstrated yet. Here, we present an interesting study in terms of the effect of the deformable surface and the smooth surface of a nanofilm transferred onto Si through two transfer strategies using isopropanol injection and nitrogen blowing (to form a deformable nanofilm surface) and deionized water injection and van der Waals interaction (to form a smooth nanofilm surface). As a result, optical detection in the case of the deformable nanofilm surface was enhanced significantly (~100%) compared with that of the smooth nanofilm surface in the visible laser wavelength (532 nm). In addition, evidence from the computational simulation also firmly affirms an advancement in the optical detection of deformed nanofilm-surface-based photodetection devices compatible with the experimental results. As seen in 2)) of the deformable surface and the smooth surface, calculated as 2 × 10−11 and 4 × 10−11 W/(Hz)1/2, respectively.To highlight the effect of the deformable nanofilm surface on light detection, the photodetection devices were fabricated on the SiO devices . ConsequDeformable surface/ASmooth surface) × 100 in percentage, where ADeformable surface and ASmooth surface are the absorptivity in the deformable and smooth surface structures, respectively, and the result is depicted in To confirm our experiments above and reveal the enhancement mechanism in the visible spectra, both smooth and deformable surface structures were simulated using the 3D FDTD technique with Lumerical FDTD Solutions tools. Illustrations of the simulation model are shown as simulated nanofilm surfaces in In summary, we demonstrated a simple nanofilm transfer method to enable the enhancement of photodetection. Consequently, increasing the areal deformable density of the nanofilm surface resulted in an order-of-magnitude enhancement of the optical extinction, which led to photoresponsivity enhancement (100%). This study will allow the realization of enhanced, strain-tunable, and wavelength-specific optoelectronics, as well as the enhancement and tuning of photodetection in future high-performance integrated optoelectronics. Although optical detection based on a deformably transferred nanofilm is enhanced, further improvement of the interface contact between the nanofilm and Si is required to obtain a more stable device structure with better contact. Our work could pave the way for other applications in electronics and optoelectronics in terms of ultraviolet-visible wavelength regions using other 2D thin films  integrated with this deformable transfer technique.


Prostate cancer (PCa) is the second most common neoplasm in men and the fifth leading cause of death worldwide . DespiteThe ability of a cell to maintain genome stability is pivotal to control tumorigenesis. Somatic mutations occur as a result of errors in DNA repair mechanisms or as a direct response to stress, and normally occur during the ageing process: despite being involved in the carcinogenesis of sporadic cancers, these mutations are not passed down to the offspring through gametes, and consequently are not responsible for cancer familiarity. Conversely, a germline mutation involves gametes by definition and is therefore passed down to the offspring. Consequently, the presence of an inherited alteration in oncosuppressors could increase the risk of developing hereditary cancer. The presence of these mutations and their proper identification, both before and after the development of cancer, is relevant to the prevention, diagnosis and even treatment of affected patients .BRCA1-2 germline mutations affect the susceptibility of individuals to carcinogenesis through the two-hit hypothesis: the first hit owing to the inherited pathogenic mutation, and the second hit owing to the somatic inactivation of the second wild-type allele of the oncosuppressor gene. The failure of the BRCA HRR mechanism is due to the combination of germline and somatic mutations that favour the activation of alternative and less effective DNA repair pathways such as base excision repair, nucleotide excision repair and mismatch repair . In addiThe Cancer Genome Atlas Research Network identified several germline mutations of DNA damage repair (DDR) genes in 333 patients with primary PCa, comprehending BRCA2 (mutated in 13% of cases), ATM (7.3%), MSH2 (2%), and BRCA1 (0.3%) . AlthougRecognizing the importance of BRCA germline mutations in PCa is particularly relevant in the clinical setting for several reasons, mainly because carriers present an increased lifetime risk of developing a PCa compared to non-carriers, and because BRCA-related PCa is characterized by worse clinical outcomes. Furthermore, BRCA gene alterations have recently been evaluated as predictors of response to both poly ADP-ribose polymerase (PARP) inhibitors and platinum chemotherapy in the metastatic setting. Carrying a BRCA1 mutation is associated with a PCa standardised incidence ratio of 2.35 in comparison to the overall population, while carrying a BRCA2 mutation increases this ratio to 4.45. Similarly, the standardised mortality ratio is increased by a 1.75 and 3.85 factor for BRCA1 and BRCA2 mutation carriers, respectively, in comparison to non-carriers . AnotherIn addition to disease incidence and survival outcomes, the presence of BRCA germline mutations affects the effectiveness of treatments. BRCA2 carriers are indeed more likely to present a tumour requiring active treatment: however, despite radical surgery, 5- and 10-years MFS was, respectively, 89% and 67% for patients withBRCA1 and 2 germline mutations, compared to 97% and 91% for those with the wild-type alleles. Clinical outcomes appear to be even worse when radiotherapy was chosen as the primary treatment, with 57% and 39% of BRCA1-2 carriers free from metastasis at 5 and 10 years compared to the 91% and 80% of non-carriers . MoreoveDue to these premises, specific treatment strategies are required in order to improve clinical outcomes for BRCA germline mutations carriers. Platinum-based therapy has long been a cornerstone in the treatment of BRCA positive metastatic tumours due to the intrinsic susceptibility of tumour cells to DNA damaging agents. In standardized protocols for PCa, platinum-based chemotherapy has been proposed only for neuroendocrine differentiation, even though different studies have reported an increased sensitivity of BRCA mutated PCa to this agent. Indeed, 75% of BRCA2 carriers have reported a PSA decline > 50% within 12 weeks of treatment initiation compared to 17% of non-carriers, with overall prolonged survival in the first cohort of 18.9 months versus 9.5 months [Another effective treatment strategy in the BRCA-mutated population comprehends the inhibition of the PARP enzyme complex involved in DNA repair, thus exploiting the deficiency of HRR mechanisms in these tumours to disrupt DNA integrity and cause cell death. Current PARP inhibitors that have been approved for the treatment of mCRPC with BRCA germline mutations are Olaparib and Rucaparib; however, other PARP inhibitors, as Niraparib and Talazoparib, are currently being investigated in this setting. The TRITON2 phase II trial, which evaluated the efficacy of Rucaparib in a population with DDR defects, reported PSA and radiographic responses in 48% and 45%, respectively, in BRCA2 carriers . SimilarDDR defects are present in prostate cancer at a higher prevalence than previously recognized. In particular, BRCA germline mutations have implications not only when assessing the risk of developing PCa, but also in the prognosis and the management of this disease. Most of the time, DDR mutation carriers lack a personal or family history of cancers that could be suggestive of an inheritable predisposition. Understanding the real prevalence of BRCA germline mutations could substantially change clinical practice and help to improve therapies and diagnostic protocols in a tailored therapy setting. Routing genetic testing and molecular characterization of patients with either mCRPC or locally advanced PCa could therefore be a feasible approach to select patients who are more likely to respond to targeted agents, minimizing toxicities from unnecessary therapies and tailoring a more efficient patient-based treatment. A multidisciplinary approach should also be considered in these patients to guarantee proper genetic counselling to the carriers. Although the approval of PARP inhibitors in mCRPC is the first step towards a targeted therapy in this subpopulation, further efforts are required to properly evaluate and exploit DDR mutations in PCa.


Erythropoiesis is a process of enormous magnitude, with the average person generating two to three million red cells every second. Erythroid progenitors start as large cells with large nuclei, and over the course of three to four cell divisions they undergo a dramatic decrease in cell size accompanied by profound nuclear condensation, which culminates in enucleation. As maturing erythroblasts are undergoing these dramatic phenotypic changes, they accumulate hemoglobin and express high levels of other erythroid-specific genes, while silencing much of the non-erythroid transcriptome. These phenotypic and gene expression changes are associated with distinct changes in the chromatin landscape, and require close coordination between transcription factors and epigenetic regulators, as well as precise regulation of RNA polymerase II activity. Disruption of these processes are associated with inherited anemias and myelodysplastic syndromes. Here, we review the epigenetic mechanisms that govern terminal erythroid maturation, and their role in human disease. Erythropoiesis is the process of making red blood cells. At each stage of maturation, erythroblasts are both phenotypically distinct and have a unique transcriptomic profile  and chroDuring the maturation of a committed progenitor to a reticulocyte, the nucleus condenses to ∼1/10th of its original volume prior to enucleation.. This drAcetylation of histone tails is generally associated with open chromatin structure and transcriptional activation, and regulation of histone acetylation is critical for the establishment appropriate patterns of erythroid gene expression and chromatin structure. Studies of friend leukemia transformed erythroblasts suggested that global deacetylation of histone residues is necessary for erythroid chromatin condensation.. Mass spAlthough a global decline in histone acetylation does not accompany human erythropoiesis, deacetylation of select histone modifications likely plays an important role. Several histone deacetylases (HDACs), including HDACs 2, 3, and five are highly expressed in maturing erythroblasts and have essential roles in erythropoiesis.. HDAC5, Dynamic changes in histone methylation also occur during erythropoiesis, with both histone methyltransferases, such as NSD1  and SETDHistone marks that reflect active transcription through gene bodies, such as H3K36me2/3 and H3K72me2 also vary dynamically during terminal maturation, , and plaIn vivo deletion of factors that regulate the transcription cycle including HEXIM1, TIF1γ, TFIIs, JMJD6 and TRIM28, results in severe anemia,  to shape the H3K27me3 landscape.. GATA faKLF1 is essential for both chromatin condensation and enucleation during terminal erythropoiesis.. KLF1 prOther transcription factors also play a role in regulating the erythroid epigenome and chromatin architecture. For example, the transcription factor FOXO3 is upregulated during the late-stage of terminal erythroid maturation and promotes the expression of genes essential for terminal erythroid maturation, with loss of FOXO3 leading to multilobed nuclei and deficient enucleation . The traMutations of transcription factors and epigenetic regulators can lead to both inherited and acquired erythroid disorders. In addition, Genome Wide Association Studies have demonstrated that genetic variants associated with red cell phenotypes often occur in enhancers or other regulatory elements critical for the proper expression of the erythroid transcriptome.. Table 1Mutations in GATA1, or GATA1-target sequences, cause anemia, which can be accompanied by thrombocytopenia, consistent with its role both megakaryopoiesis and erythropoiesis. Highlighting the essential nature of the cross talk that occurs between transcription factors and epigenetic modifiers, mutations that impair the ability of GATA1 to interact with co-activator and co-repressor complexes can lead to severe clinical phenotypes. For example the R216Q mutation, which impairs the ability of GATA1 to recruit the TAL1 activating complex, , leads tMutations in GATA1-binding sites that lead impaired expression of the target gene have been linked with a number of hematologic disorders, , includiDecreased GATA1 protein levels, have also been associated with human disease. DBA is an inherited erythroid failure syndrome that is associated with short stature and predisposition to cancer, most commonly caused by mutations in the ribosomal protein genes.. In DBA,KLF1 is also an essential erythroid transcription factor. . MutatioMutations in epigenetic regulators have also been associated with inherited anemias. As noted above, a point mutation of the histone methyltransferase ASH1L has been associated with a beta thalassemia-like phenotype.. MutatioAcquired mutations in GATA1 and other erythroid transcription factors and epigenetic regulators can also lead to hematopoietic disorders. Intriguingly, the GATA1s mutation is commonly acquired in infants with Down Syndrome, and results in transient myeloproliferative syndrome , highligThe epigenetic and transcriptional control of terminal erythroid maturation is complex, with each stage of erythropoiesis tightly regulated by various interdependent epigenetic mechanisms. Mutations that disrupt the transcriptional and epigenetic control of erythropoiesis lead to inherited anemias, highlighting the critical nature of these interactions. Understanding the fundamental mechanisms that govern erythroid maturation is essential to understanding the how mutations or other genetic perturbations result in disordered erythropoiesis, and to designing rational therapies.


In the process of cancer EMT, some subgroups of cancer cells simultaneously exhibit both mesenchymal and epithelial characteristics, a phenomenon termed partial EMT (pEMT). pEMT is a plastic state in which cells coexpress epithelial and mesenchymal markers. In squamous cell carcinoma (SCC), pEMT is regulated, and the phenotype is maintained via the HIPPO pathway, NOTCH pathway and TGF-β pathways and by microRNAs, lncRNAs and the cancer microenvironment (CME); thus, SCC exhibits aggressive tumorigenic properties and high stemness, which leads collective migration and therapy resistance. Few studies have reported therapeutic interventions to address cells that have undergone pEMT, and this approach may be an effective way to inhibit the plasticity, drug resistance and metastatic potential of SCC. Epithelial-mesenchymal transition (EMT), which refers to the biological process through which epithelial cells transform into cells with a mesenchymal phenotype and plays an important role in cancer progression, embryonic development, inflammation, tissue reconstruction, and fibrosis The pEMT phenotype plays an important role in the tumor progression, process of organ branching morphogenesis, diabetic kidney disease and wound healing Squamous cell carcinoma (SCC) accounts for most esophageal cancers (upper esophagus) pEMT is usually evidenced by simultaneous expression of epithelial and mesenchymal protein markers in a single cell +/-/CD51-/CD61- phenotype, late pEMT status is characterized by a CD106+/-/CD51+/CD61- phenotype, and full EMT tumor cells exhibit a CD106+/-/CD51+/CD61+ phenotype Pastushenko et al. pEMT may be a transient state in the EMT process, but the pEMT state can be stabilized or maintained through several mechanisms TFs bind to and inhibit the transcription of genes encoding adhesion junction and tight junction molecules, thereby triggering EMT. These TFs include Snail1/2, zinc finger E-box binding homeobox (ZEB) 1/2, Twist and lymphoid enhancer-binding factor 1 (LEF-1) FAT1 is a transmembrane protein involved in the regulation of EMT, cell growth and actin dynamics and plays a key role in tumorigenesis and development A ligand  binds to a receptor (NOTCH1-4) to activate Notch signaling, thereby initiating the intercellular communication system Numb, which regulates the endocytosis of adhesion molecules (such as E-cadherin), is important for epithelial cell-cell and cell-matrix interactions Transforming growth factor β (TGF-β) is a main driver of EMT. TGF-β is a secreted cytokine that can regulate cell proliferation, migration and differentiation of many different types of cells pEMT depends on the TGF-β pathway and is involved in lymph node metastasis in NSCLC patients Nuclear factor E2-related factor 2 (NRF2) can prevent adequate EMT during wound healing Epigenetic regulatory factors  and posttranslational modifications  regulate the expression of EMT-TFs, thereby regulating pEMT and subsequent metastasis, stemness and therapeutic resistance high/ZEBlow, miR-200low/ZEBhigh, and miR-200medium/ZEBmedium cells have epithelial, full EMT and pEMT phenotypes, respectively miR-200 is considered a marker of cancer cells and a determinant of the epithelial phenotype miR-151a promotes the contact and barrier properties of endothelial cells and promotes endothelial cell movement and angiogenesis by inducing Snail2, which is frequently amplified in solid tumors, including lung tumors Based on a comparison of pEMT cells with non-pEMT cells by single-cell sequencing, Snail2 is the only activated EMT-TF in pEMT cells + tumor epithelial cells and cells in the early mixed EMT state and prevents further progression of EMT to a fully mesenchymal state The cancer microenvironment (CME) surrounding tumor cells contributes to the emergence, stabilization and regulation of the pEMT phenotype, thereby promoting tumor progression Hypoxia is a powerful driving force for the disruption of normal tissue homeostasis and tumor-stromal interactions The extracellular pH of most solid tumors is acidic due to the high lactate production rate and poor perfusion. Acidosis induces genomic instability, promotes local tumor invasion and metastasis, and inhibits antitumor immunity and therapeutic resistance, thereby promoting tumor progression Metastasis is a process by which localized cancer becomes a systemic disease; cancer spreads due to the migration of individual cells of the primary tumor. However, new evidence found in many types of cancers, such as breast cancer, lung cancer, and mesenchymal tumors, indicates that tumor metastasis can also occur via the spread of large, cohesive cell populations that accumulate in adjacent tissues A higher pEMT score is related to HNSCC lymph node metastasis and higher lymph node staging Cells in migrating clusters are usually organized into two groups: leader cells and follower cells. Leader cells are responsible for sensing the microenvironment, generating traction to move other members of the group and reshaping the matrix through proteolysis to create a path through which the group can navigate EMT is associated with an increase in tumor stemness The mechanism by which pEMT regulates stemness in SCC is still largely unknown. However, some evidence has helped us understand the connection between pEMT and stemness. In NSCLC, TGF-β1 promotes the expression of CD133 in pEMT cells, which in turn leads to the conversion of non-stem cells to CSC high/EpCAMhigh/CD24low) is sensitive to cisplatin, paclitaxel and salinomycin; the mesenchymal cell subset (CD44high/EpCAMlow/CD24low) is sensitive to cisplatin and salinomycin; and the pEMT subgroups (CD44high/EpCAMlow/CD24high) are resistant to all three drugs NSCLC patients usually benefit from treatment with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib. However, EGFR resistance is likely to occur and is at least partially mediated by EMT Drug resistance is often associated with the EMT process and CSC. Increasing evidence shows that traditional therapies often fail to eradicate cancer cells that have been activated by EMT programs and that have become CSC, thereby allowing CSC-mediated clinical recurrence Through EMT studies on breast, pancreatic and ovarian cancer, the existence of pEMT was confirmed Although we have noted the markers of each stage of EMT in SCC, which is very important for the study and separation of pEMT subgroups, there is no clear consensus on the current definition of pEMT based on the coexpression of selected mesenchymal and epithelial markers in SCC. Therefore, determining the model of epithelial and mesenchymal marker coexpression to standardize the characterization of pEMT remains a main challenge in this field. In addition, developing strategies to design treatments for pEMT subgroups is a direction worthy of consideration. The drug resistance and plasticity of pEMT subgroups, the tendency of SCC to metastasize and the poor prognosis of SCC patients with metastasis emphasize that traditional treatment methods cannot effectively overcome the poor prognosis conferred by pEMT.


During the development of atherosclerosis and other vascular diseases, vascular smooth muscle cells (SMCs) located in the intima and media of blood vessels shift from a contractile state towards other phenotypes that differ substantially from differentiated SMCs. In addition, these cells acquire new functions, such as the production of alternative extracellular matrix (ECM) proteins and signal molecules. A similar shift in cell phenotype is observed when SMCs are removed from their native environment and placed in a culture, presumably due to the absence of the physiological signals that maintain and regulate the SMC phenotype in the vasculature. The far majority of studies describing SMC functions have been performed under standard culture conditions in which cells adhere to a rigid and static plastic plate. While these studies have contributed to discovering key molecular pathways regulating SMCs, they have a significant limitation: the ECM microenvironment and the mechanical forces transmitted through the matrix to SMCs are generally not considered. Here, we review and discuss the recent literature on how the mechanical forces and derived biochemical signals have been shown to modulate the vascular SMC phenotype and provide new perspectives about their importance. Cardiovascular diseases are one of the leading causes of global death in developing countries . More thDuring life, cells in the vasculature are continuously exposed to different mechanical forces that regulate their function and homeostasis. These forces include fluid shear stress, cyclic stretch, and hydrostatic pressure . Fluid sGiven the critical role of SMC phenotypic modulation in the pathogenesis of vascular diseases, understanding how SMCs sense and transduce mechanical forces into biochemical signals and their interactions with surrounding ECM proteins is critical to prevent the development and progression of vascular diseases. However, the importance of mechanical forces for controlling the cellular phenotype in vascular cells has largely been neglected in previous in vitro research, particularly on SMCs. Furthermore, unlike the native vessel, the substrate (a rigid and static plastic culture plate) used in most studies of in vitro culture lacks a physiological microenvironment. Therefore, in this review, we discuss recent 2D in vitro studies that evaluated the effect of mechanical forces (cyclic stretch) on vascular SMCs. We particularly spotlight the effects of these forces on their interaction with ECM proteins, the expression of smooth muscle (SM) contractile and synthetic marker genes, and other key aspects of SMC function, such as migration and proliferation.It is widely accepted that, when SMCs are removed from their native environment and placed in a culture, they undergo a similar shift in cell phenotype as observed in several vascular diseases . This phACTA2), calponin (CNN1), smoothelin (SMTN), smooth muscle myosin heavy chain (MYH11), transgelin (TAGLN), etc. A reduction in the expression of SMC contractile markers is often accompanied by increased cell migration and proliferation [In vitro, the loss of the SMC contractile phenotype has, in most studies, been measured as reduced mRNA or protein levels of classical contractile marker genes, such as actin alpha 2 smooth muscle , vimentin, matrix metalloproteinases, and inflammatory cytokines, in comparison to stiff (1.2 kPa) substrates [CD68, galectin 3 (LGALS3), and inflammatory interleukin 6 (IL-6) and interleukin 1 beta (IL1β) markers compared to cells on medium stiffness substrates [MYH11 expression, contrary to previous findings, was found upregulated on hard, compared to soft, substrates, thus suggesting that moderate stiffness, a condition closer to the physiological parameters, could be beneficial to SMC function.An example of how cells can respond to different stiffness conditions was shown by experiments with human SMCs cultured on soft (2 kPa) and stiff (20 kPa) surfaces for 24 h. The expression of SM contractile marker genes such as oft ones . In contbstrates . Interesbstrates . They obbstrates . NotablyInterestingly, the effects on the SMC phenotype elicited by the combination of distinct cues such as different stiffnesses and changes in the ECM proteins associated with stiffening have not been systematically evaluated. Most of the studies have only used gels coated with collagen I or fibronectin to mimic the in vivo microenvironment that SMCs experience in arteries with increased stiffness. For example, a recent study showed that the ECM protein used to coat the gels can differentially affect the SMC phenotype . In thisAs mentioned above, there are important discrepancies in the results of studies examining the influence of substrate stiffness on the SMC phenotype B. It is Under normal physiological conditions, SMCs within the vascular wall are primarily exposed to mechanical forces due to the pulsatile nature of blood pressure. To counteract these forces, the vascular tissue stretches circumferentially and longitudinally . SMCs anHowever, when the blood pressure is increased, the degree of stretch also increases, at least until chronic remodeling of the arterial wall changes the average diameter or increases the stiffness of the vascular tissue . In prac® tension system operated through a vacuum pump and regulated by computer software. A uniaxial or equibiaxial strain can be applied to cell monolayers cultured on soft-bottomed silicone elastomer membranes. In contrast to SMCs within blood vessels, cells in a culture are randomly oriented. Since the cellular biochemical responses to uniaxial stretching depend on the cell‘s orientation relative to the direction of stretching [Researchers have created diverse in vitro technologies during the last years that, by applying different stretches to SMCs, can mimic in vivo wall distension. Perhaps one of the most widely used systems is the Flexcellretching , the in retching . Other tretching .Multiple previous studies have evaluated the effects of cyclic stretch on SMC modulation using gene expression as the readout . In most of these, SMCs were subjected to stretch using flexible-bottomed plates coated with collagen I. In interpreting these studies, it should therefore be noted that collagen I itself can contribute to the dedifferentiation of SMCs , and theSurprisingly, very few studies have examined the effects of the physiological levels of stretch on human SMCs ,45,46,47CNN1, TAGLN, ACTA2, and myocardin (MYOCD) [KLF4), another marker gene associated with the synthetic phenotype of SMCs, was also upregulated [(IL-8), IL-6, IL1β, vascular cell adhesion protein 1 (VCAM-1), and intercellular cell adhesion 1 (ICAM-1) [Other studies have looked at high stretch stimulations (>10%) of human SMCs, which have generally been shown to induce a dedifferentiated phenotype. For instance, human aortic SMCs cultured on collagen I coated-flexible membranes showed a decreased expression of the SM contractile markers  (MYOCD) . The Krüegulated . Similaregulated . Reduced(ICAM-1) . The cur(ICAM-1) ,52. TherCnn1 and Smtn and increased levels of the synthetic marker Opn were observed [The vast majority of stretch studies found in the literature have used primary aortic rat SMCs  can induce SM phenotypic modulation.Given the importance of ECM proteins for the behavior of SMCs, there is a knowledge gap regarding the effect of the stretch on cells cultured on other ECM substrates, which are thought to retain the SMCs in the contractile phenotype, such as laminin and collagen IV or others (recombinant engineered proteins) ,53. MoreCNN1 and TAGLN compared to cells stretched with the simple and widely used sinusoidal waveform [Currently, there is a limited understanding of how the different local distension patterns that occur through different vascular beds and during disease conditions affect the SMC functions. Lee and colleagues have suggested that the type of waveform can modulate the human SMC phenotypic state. For instance, a 24-h cyclic stretch with an amplitude of 7.5% and a waveform that mimics the pressure generated by the heart can induce the upregulation of waveform . It woulIL-6 [®), which were subsequently exposed to a 15% stretch for 4 h or left static [Cxcl1) and (C-X3-C motif) ligand 1 (Cx3cl1) on the cells exposed to a stretch compared to the cells under static conditions.Other in vitro studies that applied a stretch on SMCs have focused on exploring inflammatory genes as a surrogate for dedifferentiation markers. Primary mouse aorta SMCs stimulated by a 15% stretch for 3 h on Flexcell plates coated with collagen I showed increased expression levels of IL-6 . Anothert static . The genSimilarly, another study performed a microarray analysis of human aortic SMCs after being exposed to a 20% supraphysiological stretch for 24 h compared to cells on static cultures . Their sIn normal, healthy vessels, the majority of SMCs are found in a differentiated or contractile state, are quiescent, and do not migrate. However, during the development of vascular diseases, increased migration and proliferation are important signs of a SMC phenotypic switch that often accompanies the decreased expression of contractile SM marker genes.The effects of a physiological or supraphysiological stretch on SMC migration has been examined in several studies, but the results have not been consistent .One recent study investigated the migratory capacity of human aortic SMCs exposed to stretching by the scratch wound-healing assay. The scratch was performed on confluent monolayers before the cells were either subjected to stretching  or left in static conditions, and the gap closure area was measured at 12 h after the static or stretch stimulation . They reThe effects of a supraphysiological stretch have been examined in primary mouse aortic SMCs seeded on collagen I-coated membranes and stimulated with a supraphysiological stretch . After 3 h, the stretch was stopped, and the scratch was performed. An increased cell migration was seen in stretched compared with static controls . It is pThe effect of physiological stretching (< 10%) has been shown to reduce the proliferation of human and rat aortic SMCs compared to the static controls in different studies ,61,62. HOn the other hand, a non-physiological stretch stimulation (> 10%) of either human or rat aortic SMCs has been reported to increase cell proliferation compared to static conditions. For instance, human aortic SMCs that were stretched (> 10%) on collagen I-coated membranes for 12 or 24 h showed increased proliferation compared to cells under static conditions ,65,66,67Overall, the combined results indicate that a physiological stretching force is quiescent in SMCs, while high-intensity or pathological stretching induces SMC proliferation .Further studies are required to understand the potential interactions between different types of stretching, including waveforms, and different ECM proteins on SMC proliferation.Apoptosis or programmed cell death has been identified as an essential process contributing to cardiovascular disease development . It has The exposure of human aortic SMCs to high-intensity stretching >15% for 12 h showed similar results to those exposed to low-stretch levels. The apoptosis and proliferation were increased compared to the static cultures ,66. AddiIn general, studies with human, pig, rat, or mouse SMCs concluded that mechanical stretching increases the level of apoptosis independent of the stretch intensity, duration, ECM coating, and origin of the SMCs .Different laboratories have investigated the mechanisms by which high intensity stretching could induce apoptosis. Some results indicate that the p53 upregulated modulator of the apoptosis protein (PUMA) is induced by interferon-gamma (IFN-γ), the c-Jun N-terminal kinase (JNK), and the interferon regulatory pathway 1 (IRF-1) pathways in response to stretching . A recenDuring vascular diseases or surgical interventions such as angioplasty or endarterectomy, vascular endothelium damage can occur, directly exposing SMCs to different patterns and intensities of shear stress . An earlStudies investigating the effects of shear stress have mainly been performed on cultured monolayers of ECs seeded on flat and stiff substrates . The sam2) for 24 h and compared to the cells under static conditions. Among the top regulated were the genes involved in the cell cycle and death, cell adhesion, and ECM. In the same study, they confirmed by BrdU labeling that laminar shear stress promotes human SMC proliferation compared to static controls [2) for extended periods of time (15–24 h) reduced the expression of some classical SM markers when compared to static controls, as summarized in 2) for 6, 12, and 24 h resulted in the time-dependent downregulation of Acta2 and Tagln, while matrix metalloproteinase 2 (Mmp-2) and tumor necrosis factor-alpha (Tnf-α) were upregulated. Phenotypic switching in this study was also accompanied by the enhanced proliferation and migration of SMCs after shear stress [Early studies have used DNA microarrays to determine the global expression profile of human aortic SMCs under fluid shear stress . Cells ccontrols . Unfortur stress . Thus, t2) for 24 h compared to the static controls [2) for 24 h decreased the cell proliferation and migration activity [2) for 24 h [However, not all observations fit this concept. One study found that proliferation was decreased rather than increased in rat aortic SMCs exposed to laminar shear stress (14 dynes/cmcontrols . Unfortuactivity  comparedfor 24 h . Unfortu2) for 3 or 5 days compared to the static controls [Overall, the physiological relevance of the in vitro responses of SMCs to laminar shear stress is unclear. The patterns of shear stress at sites of endothelial cell injury in vivo do not necessarily mirror the continuous laminar shear stress addressed by several studies. Disturbed or turbulent shear stress patterns have been shown to induce atherosclerotic plaque formation in vivo and activate inflammatory signaling on endothelial cells in vitro . Howevercontrols , but the1R) can be activated by excessive mechanical stretching (20%) and induce ERK signaling, which leads to increased migration and protein synthesis [The cellular process of converting mechanical cues into biochemical signals is known as cellular mechanotransduction. This aspect has been reviewed extensively in other vascular cells . Howeverynthesis ,95. Diffynthesis . Cyclic ynthesis . FurtherDifferent studies have investigated the potential intracellular pathways induced by mechanical stretching on SMCs. For example, transforming growth factor-b (TGF-β) signaling has been implicated. The TGF-β1 levels were increased in the supernatant of rat SMCs exposed to 10% stretching for 24 h compared to the static controls . TogetheThese changes in the expression of stretched cells accompanied a reduced migration compared to the static controls . The othEffectors of the Hippo pathway, YES-associated protein (YAP), and the transcriptional coactivator with a PDZ-binding motif (TAZ) are also involved in the stretch-induced phenotypic modulation of SMCs. YAP/TAZ activation after 24 h of cyclic stretching (13%) was linked to an increase in proliferation and proinflammatory gene expression  in human umbilical artery SMCs compared to the static controls . Human aThese data exemplify the high degree of complexity among the intracellular pathways induced by stretching and highlighting the need for more research to identify new mechanoreceptors and regulators . AlthougIdentifying the mechanisms that control SMC phenotypes is crucial to develop new drugs against vascular diseases characterized by the presence of highly modulated SMCs and for improvements in the tissue engineering of vascular tissues ,104. A bDespite clear evidence that mechanical forces can modulate SMC phenotypes, there are still many discrepancies in the results from different in vitro studies . Some ofFinally, the lack of thorough and unbiased approaches to investigate the effects of mechanical stimulation on SMC phenotypes limits the current research, which mostly relies on a few contractile markers. Recent studies using single-cell transcriptomics have demonstrated SMC heterogeneity in healthy and diseased arteries ,108,109.Considering the importance of stretch-induced changes in SMC phenotypes for physiological functions and disease processes and the many remaining knowledge gaps, clearly, more research in this area is needed.


Cancer Research as a new concept in cancer chemotherapy. My review here describes the history and heterogeneity of the EPR effect, which involves defective tumor blood vessels and blood flow. We reported that restoring obstructed tumor blood flow overcomes impaired drug delivery, leading to improved EPR effects. I also discuss gaps between small animal cancers used in experimental models and large clinical cancers in humans, which usually involve heterogeneous EPR effects, vascular abnormalities in multiple necrotic foci, and tumor emboli. Here, I emphasize arterial infusion of oily formulations of nanodrugs into tumor-feeding arteries, which is the most tumor-selective drug delivery method, with tumor/blood ratios of 100-fold. This method is literally the most personalized medicine because arterial infusions differ for each patient, and drug doses infused depend on tumor size and anatomy in each patient. Future developments in EPR effect-based treatment will range from chemotherapy to photodynamic therapy, boron neutron capture therapy, and therapies for free radical diseases. This review focuses on our own work, which stimulated numerous scientists to perform research in nanotechnology and drug delivery systems, thereby spawning a new cancer treatment era.This Special Issue on the enhanced permeability and retention (EPR) effect commemorates the 35th anniversary of its discovery, the original 1986 Matsumura and Maeda finding being published in  Congenital deficiency of NADPH oxidase results in chronic granulomatous disease (CGD), particularly in infant and children because of the lack of H2O2 or O2− to kill bacteria, and constant or chronic infections will lead to CGD. We therefore prepared PEGylated DAO to deliver PEG-DAO to inflamed sites and thus supply ROS, in parallel with administration of D-proline or D-alanine, the DAO substrates. When H2O2 is generated, it will be converted to the more powerful bactericidal molecule. HClO is generated by neutrophils in the presence of both myeloperoxidase and chloride ion, which will kill bacteria [2O2 and SOD for O2−.Hbacteria ,83. NormMany cancer cells lack these anti-oxystress enzymes or have downregulated levels of these enzymes, so they are vulnerable to oxystress. Many advanced cancer cells propagate well under anaerobic conditions, and antioxidant enzymes may be lost ,12,81,82This Special Issue commemorates my 35th year after discovery of the EPR effect ,2, and tThe ultimate purpose of personalized medicine is to provide the best benefits for individual patients. The EPR effect is a ubiquitous phenomenon found in almost all solid tumors, with sizes from less than 1 mm to larger than 10 cm; this effect also occurs in inflamed tissues and applies to biocompatible macromolecules. To utilize the EPR effect or the related drug delivery system more effectively, vascular blood flow must be restored and maintained. Nanomedicines are of prime importance for receiving the benefits of the EPR effect. The issues of vascular flow in tumor tissues is a relatively recent issue in cancer therapy ,63,86,88Various advantages of the unique properties of nanomedicines as well as selective drug targeting to tumors and inflamed tissues were easily demonstrated via pharmacokinetics and pharmacodynamics and imaging; the use of EPR effect enhancers exhibit fewer adverse effects and improved therapeutic results are thus expected when combined with nanomedicines compared with conventional medicines in the future. Nanomedicine is therefore worthy of study and challenges for the benefit of patients. Wider applications of PDT and BNCT as well as strategies to control the ubiquitous undesirable molecules like ROS/RNS are future lines of study . The gr