The results indicate that Na4BRA is able to complex Cu2+ in a 1:1 ratio and Pb2+ in a 1:2 ratio, while Na4SRA complexes Hg2+ in a 1:1 ratio. On the contrary, no indications have been observed that either of the resorcin[4]arenes studied complexes the Cd2+ ions. The results suggest that the bonds established between the sulfur atoms located at the lower edge of the SRA4− and the solvent hydrogens could prevent the entry of the guest into the host cavity. However, in the case of Hg2+ ions, the entry is favoured by the interactions between the sulfur donor atoms present on the lower edge of Na4SRA and the Hg2+ ions. Therefore, it can be said that Na4BRA is selective for Cu2+ and Pb2+ ions and Na4SRA is selective for Hg2+ ions.The complexing properties of two water-soluble resorcin[4]arenes resorcin[4]arene, Na Soil contamination by heavy metals poses a great risk to human health, animals, plants, microorganisms and their interactions . In huma2+, Pb2+, Cd2+ and Hg2+, using complexing macrocycles such as the resorcin[4]arenes, to try to apply it in their removal. Although, when Cu is present in the human body in amounts that do not exceed 75–100 mg, it is vital for health, when the concentration of this metal is higher, various disorders occur such as nausea, vomiting, abdominal pain and cramps, headache, dizziness, weakness and diarrhea, among others; such disorders occur mainly in the case of individuals with liver diseases and other pathologies in which the excretion of bile is compromised arene (Na4SRA), whose structures are shown in Resorcin[4]arenes  and theiβƒ ,35. Info4BRA and Na4SRA with the heavy metals of interest are shown in There are several simple experimental techniques that can be used to determine the stoichiometry and stability constants of complex species. Among the most widely used are conductometry, sonometry, ion-selective potentiometry (ISE), acid-base potentiometry, nuclear magnetic resonance (NMR), UV-VIS spectroscopy and isothermal titration calorimetry (ITC). In the present case, the techniques used to study the complexation of NaThe process of recognition and evaluation of the selectivity of the resorcin[4]arenes was carried out in water by using the conductivity technique, and the positive findings were confirmed by other techniques such as ion-selective potentiometry (ISE) and atomic absorption. The results are discussed in terms of the structure and three-dimensional arrangement of each macrocycle.4BRA and Na4SRA were synthesized, purified and characterized according to the procedures described by Sanabria et al. /[Na4BRA] ratio.2+]/[Na4BRA] ratio of approximately 1.2, which could suggest the presence of a complex of 1:1 stoichiometry. Both the presence of the complex and its stoichiometry were confirmed by potentiometric titration with a copper selective electrode (Cu-ISE). The dependence between the potential and the [Cu2+]/[Na4BRA] ratio is shown in As it can be ascertained, a slight change in slope is observed for a 2− complex using the Hyperquad software /[Na4BRA] ratio.2+]/[Na4BRA] ratio, approximately. According to Ashram /[Na4BRA] and [Cd2+]/[Na4BRA] ratios.2+ or Hg2+ complexes with Na4BRA must be ruled out.As it can be seen in both cases, the appearance of a change in slope is not observed in these plots, so the formation of Cd2+]/[Na4SRA] ratio.2+]/[Na4SRA] equal to 1, which clearly indicates the formation of a stable complex of 1:1 stoichiometry  ratio.4SRA-Pb2+, Na4SRA-Cu2+ and Na4SRA-Cd2+ systems show complexation.As it is ascertained, in no case a change in the slope is observed, so it must be deduced that none of the Na4BRA and Na4SRA.4SRA selectively complexes Hg2+, while Na4BRA complexes Cu2+ and Pb2+. The fact that Na4BRA complexes two guests and Na4SRA only one can be explained on the basis of the greater conformational stability of Na4BRA compared to Na4SRA, caused by Van der Waals-type interactions between the lower edge chains of Na4BRA, whereas in the case of Na4SRA, conformational mobility can prevent the entry or facilitate the exit of the guest through the upper edge of the host. Additionally, due to the presence of sulfur at the lower edge of Na4SRA, this can form bonds with hydrogen from water, which could prevent the inclusion of some guests within the host. In the case of the [SRA-Hg]2+ complex, this situation does not occur since mercury can interact with the sulfur donor atoms of Na4SRA.According to the above table, Na4BRA and Na4SRA were studied by conductometry, and the positive results were confirmed by other techniques such as ion-selective potentiometry (ISE) and atomic absorption. The results indicate that Na4BRA is able to complex Cu2+ in a 1:1 ratio and Pb2+ in a 1:2 ratio. Regarding the complex formed by Na4SRA with Hg2+, the ratio found was 1:1. In this latter case, the results suggest that the interaction between the Hg2+ ions and the sulfur atoms present at the lower edge of the SRA4− is greater than that established between these latter atoms and the hydrogens of the solvent (an interaction that would prevent the entry of the guest into the cavity of the SRA4−), which favors the entry of Hg2+ and means that the Na4SRA are selective for these ions.The complexing properties of Na


The aim of surgery for skull base meningiomas is maximal resection with minimal damage to the involved cranial nerves and cerebral vessels. Compared to non-skull base meningiomas, these lesions show a reduced rate of gross total resection (GTR). Therefore, the use of technologies for improved orientation in the surgical field, such as neuronavigation and augmented reality (AR), is of interest. We confirmed in a consecutive series of 39 patients who underwent surgery for skull base meningiomas that automatic registration with intraoperative computed tomography (iCT) showed high registration accuracy and that microscope-based AR largely facilitated the resection by increasing surgical precision and providing improved intraoperative orientation by visualizing the tumor and the critical neurovascular structures in the operative microscope. No injuries to critical neurovascular structures occurred. There were 26 patients (66.6%) who underwent GTR. Additionally, 33 out of 35 patients who lived to follow-up could ambulate.Background: The aim of surgery for skull base meningiomas is maximal resection with minimal damage to the involved cranial nerves and cerebral vessels; thus, implementation of technologies for improved orientation in the surgical field, such as neuronavigation and augmented reality (AR), is of interest. Methods: Included in the study were 39 consecutive patients  who underwent surgery for skull base meningiomas using microscope-based AR and automatic patient registration using intraoperative computed tomography (iCT). Results: Most common were olfactory meningiomas (6), cavernous sinus (6) and clinoidal (6) meningiomas, meningiomas of the medial (5) and lateral (5) sphenoid wing and meningiomas of the sphenoidal plane (5), followed by suprasellar (4), falcine (1) and middle fossa (1) meningiomas. There were 26 patients (66.6%) who underwent gross total resection (GTR) of the meningioma. Automatic registration applying iCT resulted in high accuracy . The effective radiation dose of the registration iCT scans was 0.58 ± 1.05 mSv. AR facilitated orientation in the resection of skull base meningiomas with encasement of cerebral vessels and compression of the optic chiasm, as well as in reoperations, increasing surgeon comfort. No injuries to critical neurovascular structures occurred. Out of 35 patients who lived to follow-up, 33 could ambulate at their last presentation. Conclusion: A microscope-based AR facilitates surgical orientation for resection of skull base meningiomas. Registration accuracy is very high using automatic registration with intraoperative imaging. In order to minimize the morbidity rate in patients with skull base tumors, integration of preoperative image-based segmentation with three-dimensional 3D) reconstruction of critical neurovascular structures with navigation was proposed . InitialD reconstUse of AR in resections of meningiomas has been previously described, with a total of 25 cases in the literature, but only 3 of them have been skull base lesions ,12,13. AThirty-nine consecutive patients  who underwent surgery for skull base meningiomas at our department by a single surgeon (C.N.) between September 2016 and January 2022 using microscope-based AR and automatic patient registration using iCT were included in the study. The definition of skull base meningiomas was based on Al-Mefty et al. . IndicatThe extent of resection was determined on the postoperative MRI 3 months following surgery and was deemed as a gross total resection (GTR) with dural coagulation or removal of adjacent dura and bone . Details about the setup, intraoperative CT scanning protocols and conversion factors for determining the effective radiation dose (ED) have been previously described . Segmentation of the tumor was usually performed in the T1-weighted post-contrast MRI modality, manually or using autosegmentation with an anatomical mapping element (Brainlab). Brainlab was used to delineate the optic nerves, chiasm, ventricles, pituitary gland or brain stem with unique colors being assigned to each object, and with additional manual segmentation for correction of the automatic segmentation and for segmentation of tumor and risk structures. For segmentation of the vascular risk structures, contrast-enhanced T1-weighted magnetic resonance imaging (MRI), time-of-flight (TOF) MRI angiography or computed tomography (CT) angiography was used and then rigidly registered. Rigid registration was performed by an image fusion element . This 3D data set formed the basis for patient registration  Figure . Head fixation following narcosis and positioning for the appropriate approach was established by applying a radiolucent carbon Doro head clamp. Metallic fixation pins were placed outside the area of the intraoperative scan. The reference array with four reflective markers was attached to the head clamp . RegistrRigid registration of the preoperative plan with the registration CT scan was performed by image fusion software, Brainlab elements . Accuracy of the fusion was checked by a spy-glass feature, with visualization of the preoperative images in an insert of the intraoperative CT, which was moved around for detection of registration failure in all views with appropriate anatomical landmarks . For AR support, the HUDs of the operating microscopes  or Kinevo 900 ) were used. Tracking of the microscope was performed by a registration array attached to the microscope. Checking the calibration of AR was performed by centering the microscope above the divot of the registration array, as well as additional markers. In this way, the optical outline and the AR visualization of the reference array could be adjusted and 3D objects could be visualized in semitransparent, solid or outlined mode using the AR display and the microscope application allowed for visualization of these objects in different modes on the microscope video .n = 6), meningiomas of the cavernous sinus (n = 6) and clinoidal meningiomas (n = 6), meningiomas of the medial (n = 5) and lateral (n = 5) sphenoid wing and meningiomas of the sphenoidal plane (n = 5), followed by suprasellar meningiomas (n = 4), falcine meningioma of anterior fossa (n = 1) and middle and posterior fossa/petrous meningioma (n = 1). Sixteen tumors (41%) had a volume more than 10 cm3 with encasement and compression of the neurovascular structures. General characteristics of the patients are summarized in n = 28) and following craniotomy in patients no. 29–39 (n = 11). Automatic registration applying intraoperative CT in patients 1–29 resulted in high accuracy . The mean dose-length product (DLP) of the scout and registration scan was 243.16 ± 392.34 mGy*cm. Patients 1–16 and 29–39 received a registration scan following the scout scan, whereas patients 17–28 received a registration scan without a prior scout scan. The effective radiation dose of the registration CT scans (patients 17–28) and scout scans (patients 1–16 and 29–39) was 0.58 ± 1.05 mSv. Registration accuracy: Registration scanning was performed prior to skin incision in patients no. 1–29  a control iCT scan for exclusion of perioperative complications and control of the extent of resection was performed. Repeat iCT scanning was used for updating navigation. 3) or recurrent tumors. Accuracy of patient registration and microscope registration were the two dependent variables for clinical AR accuracy. Microscope registration accuracy was checked by applying the AR visualization of the reference array outline. Landmark checks were successfully performed, apart from checking the target registration error (TRE), which confirmed high accuracy and excluded errors due to potential shift  as well as in cases of recurrent tumors (patients 24 and 35). No injuries to critical neurovascular structures occurred. The tumor was visualized in all patients, followed by vessels of interest (27 patients or 62.3%) and the optic chiasm and optic nerves in 22 patients, or 56.4%. Individual objects or HUDs could be switched off on the preference of the surgeon, in the case of AR information overflow. If this was the case, further AR support was provided by the standard navigation display and AR display on the video screens, which allowed the assisting staff to monitor the surgery, thereby serving as an educational tool.Case 1: Patient no. 28 was a 77-year-old female patient with right clinoidal meningioma who experienced visual field deficits and visual deterioration. A complete resection of the tumor was performed via right fronto-temporal craniotomy. AR support facilitated the course of the resection with prompt localization of the segmented carotid and cerebral arteries, as well as the optic chiasm, providing surgical precision throughout the procedure. Case 2: A 38-year-old female patient with giant medial sphenoid wing meningioma on the left side presented with vertigo (patient number 9). GTR was performed using iCT-based navigation registration and microscope-based AR. Case 3: A 67-year-old female patient (patient number 19) with visual deterioration and bitemporal hemianopsia. MRI showed a suprasellar meningioma which was resected via right pterional approach. Case 4: The patient was a 55-year-old male with multiple intracranial meningiomas (patient no. 31). He underwent two surgeries and radiotherapy for lateral sphenoid wing meningioma on the left side. Due to tumor progression, he underwent subtotal resection (Simpson Grade III) via left fronto-temporal approach. Histology indicated an atypical meningioma WHO II°, and postoperative particle radiation therapy (carbon protons) was performed. The operative video demonstrates the surgical resection of the tumor using microscope-based AR . Case 5: A 72-year-old patient presented in a comatose state following several days of mental symptoms (patient number 36). He underwent GTR for WHO I° olfactory meningioma via bifrontal approach. Following surgery, the patient experienced prolonged weaning followed by tracheotomy. Furthermore, a hydrocephalus developed, and a subduro-peritoneal shunt was implanted. The patient recovered to an extent that allowed him to walk; however, assistance in everyday life is necessary.Clinical outcome: An overview of clinical characteristics of the patients is provided in Only one patient deteriorated postoperatively, with a new CN III palsy (patient no. 7). CN III palsy in this patient improved during the postoperative course. Eight patients improved following surgery and thirty patients remained unchanged. Perioperative surgical complications occurred in six patients (15.4%) and included cerebrospinal fluid (CSF) leaks which required surgical interventions (patients no. 4 and 23), wound healing deficits which prompted wound revision (patient no. 9), as well as postoperative hydrocephalus with implantations of a ventriculoperitoneal (patient no. 28) and subduro-peritoneal shunt (patient no. 36). Patient no. 20, with giant olfactory meningioma, experienced a massive pulmonary embolism five days following surgery, which caused asystolia, cardiopulmonary reanimation and diffuse brain swelling due to hypoxia. She underwent bifrontal craniectomy and subsequent ventriculoperitoneal shunt implantation. This patient, unfortunately, remained having an apallic syndrome. Further non-operative complications included heart failure due to asystolia with cardiopulmonary reanimation (patient no. 11), a pulmonary embolism which required cardiopulmonary reanimation (patient no. 14), pneumonia (patient no. 16) and sinus venous thrombosis with a pulmonary embolism (patient no. 29). These four patients recovered fully. The mean follow-up time was 26.7 ± 21 months. Four patients died before follow-up. Patient no. 8 died eleven days after surgery due to septic shock caused by pancolitis. Patient no. 18 died three months following surgery due to a pulmonary embolism. Patients no. 26 and 34 underwent surgery for skull base lesions suspected of brain metastases; however, the histology has shown a diagnosis of WHO I° meningioma. Patient no. 26 died 7 months following surgery due to progression of a renal cell carcinoma, and patient no. 34, died 3 months following surgery due to progression of liver failure caused by metastases of malignant melanoma. Patients numbered 1–4, 16, 13, 14, 16, 22, 30, 31, 32, 35 and 39 underwent postoperative particle radiotherapy. Indications for radiotherapy were Simpson Grade III, IV and V resections, tumors with invasion of the cavernous sinus or a WHO II° histology. The average time range from surgery to radiotherapy was 5.62 ± 1.94 months. Tumor recurrence and progression occurred in four patients . Patient no. 2 experienced recurrence 60 months following surgery and was proposed surgical treatment, but refused further therapy. Patient no. 3 underwent two further surgeries due to the tumor’s progression (30 months and 55 months following primary surgery) and subsequent irradiation. Patient no. 16 was followed up with due to asymptomatic progression, which occurred 6 months following radiation therapy. Patient no. 31 experienced a tumor progress in the setting of meningiomatosis and WHO II° histology 16 months following surgery. This patient underwent a re-radiation for tumor progression. At the follow-up, 33 out of 35 patients could ambulate at their last presentation.A skull base location, WHO II° and higher pathology grades, together with a subtotal resection, are independent predictors of unfavorable outcomes in meningiomas . CompareDespite the evolution of different treatment options for skull base meningioma, the most possible, safest extent of resection still plays a central role in the therapy. AR can greatly enhance the safety of the resection, improving the orientation in the operative field and early identification of critical structures. Spatial accuracy of preoperative image segmentation with intraoperative data for skull base surgery has already been investigated . The surWhen clear anatomical landmarks are lacking, for example, in cases of large destructive skull base tumors and reoperations, AR can decisively assist in comprehending the 3D surgical anatomy and improve the comfort, precision and intraoperative orientation of the surgeon . StandarMonitoring of the AR clinical accuracy can be achieved by evaluating intraoperatively anatomical landmarks that can be clearly located on the pre- or intraoperative images . Using vIn order to rely on AR to perform surgery close to neurovascular risk structures, it is mandatory to have a high navigation accuracy. Patient registration using user-independent intraoperative imaging-based methods is highly recommended . The maiThus far, there are several studies that examined the role of neuronavigation and intraoperative imaging in the resection of skull base lesions ,26,27,28Future directions may include surgical rehearsals with AR-templates for complex skull base cases. A recent video article on the resection of clinoid meningioma demonstrated the use of surgical rehearsal in virtual reality (VR) with an AR-template, where the author demonstrated how AR-enhanced navigation was used for the planning of the incision and soft-tissue exposure and for the guiding of the drilling of the sphenoid wing and the extradural clinoidectomy . Use of There are several limitations of this study. This is a single surgeon retrospective study, which makes its results and conclusions only partially reproducible. However, prospective studies with the use of AR, including its use in the educational setting with less experienced surgeons and the possibility of presurgical rehearsal with AR-templates, are needed to objectively evaluate the use of AR for resection of skull base meningiomas. In our study, follow-up time was too short to make adequate conclusions on the course of the disease, yet our primary goal was to demonstrate the use of AR for the surgical resection, whereas further follow-up is needed for assessment of the long-term outcomes. Since the AR was switched off in cases of information overflow, the microscopic-based AR did not always provide support throughout the entire procedure. There is no control group with patients who underwent resection without use of iCT-based navigation and AR. However, a control group of patients who would undergo surgery without optimal conditions is considered unethical and unpractical. A microscope-based AR facilitated surgical orientation for resection of skull base meningiomas. Registration accuracy was very high using automatic registration with intraoperative imaging. AR proved to be very useful in cases of large skull base meningiomas which encase cerebral vessels through early identification of risk structures and in cases of tumors which compressed the optic chiasm and optic nerves, as well as in reoperations. Enhanced understanding of 3D anatomy could be of potential use as an educational tool and assistance for less experienced surgeons.


Androgen receptor (AR) plays a vital role in prostate cancer (PCa), including castration-resistant PCa, by retaining AR signalling. Androgen deprivation treatment (ADT) has been the standard treatment in the past decades. A great number of AR antagonists initially had been found effective in tumour remission; however, most PCa relapsed that caused by pre-translational resistance such as AR mutations to turn antagonist into agonist, and AR variants to bypass the androgen binding. Recently, several alternative therapeutic choices have been proposed. Among them, proteolysis targeting chimera (PROTAC) acts different from traditional drugs that usually function as inhibitors or antagonists, and it degrades oncogenic protein and does not disrupt the transcription of an oncogene. This review first discussed some essential mechanisms of ADT resistance, and then introduced the application of AR-targeted PROTAC in PCa cells, as well as other AR-targeted therapeutic choices. Globally, prostate cancer (PCa) is the second most frequently occurring cancer in men . The humThe AR belongs to the steroid receptor subfamily of nuclear receptors, which also includes glucocorticoid receptor (GR), progesterone receptor (PR), and estrogen receptor (ER) . The AR Since AR and its signalling are of importance in PCa, androgen deprivation therapy (ADT) has been the key treatment for metastatic PCa for over 30 years . The ADTIn this review, we first summarized some mechanisms of classical ADT failure which are AR pre-translational alterations and up-regulation of other nuclear receptors, and then extensively introduced one protein degradation technology named proteolysis targeting chimera (PROTAC) and its application targeting AR protein in PCa. Finally, some alternative AR-targeted therapeutic choices, including AR antagonist (targeting other domains of AR), androgen synthesis inhibitor, AR expression inhibitor, and AR agonist, were also discussed.In the past decades, mechanisms of classical ADT failure were gradually discovered Fig. . AR amplBy increasing the expression of AR protein, AR gene amplification promotes PCa progression and raises the chance of metastasis despite of reduced serum androgen level . Two stuSimilarly, AR mutations were rarely found in untreated PCa patients, and they were often found to emerge in patients who had been treated with anti-androgens for a long period of time . The firIn addition to AR amplification and AR mutation, the presence of AR-V was demonstrated to be a vital factor in ADT failure and AR-Vs were frequently found to be expressed in CRPC patients . The firAmong discovered AR-Vs, AR-V7 is the most common variant found in clinical practice . It is nThe GR, which is activated by glucocorticoids, is also known as nuclear receptor subfamily 3, group C, member 1 (NR3C1). Upon glucocorticoids binding, the GR proteins are translocated to the nucleus and interact with specific DNA regulatory sequences called glucocorticoid response element (GRE) to activate a specific subset of genes, including CDK1, SGK1, and FKBP5 . It was The PR, classified as nuclear receptor subfamily 3, group C, member 3 (NR3C3), is the most closely related steroid receptor to the AR. The PR displays 88% sequence homology to the AR in the LBD. There are two characteristic isoforms of the PR named PR-A and PR-B, both isoforms are stimulated by progesterone. Both receptor isoforms are encoded from a single gene. PR-B isoforms is a larger molecular weight protein having additional 164 amino acids at the N-terminal region compared with PR-A. The PR’s role in PCa tumourigenesis is currently well established. In PCa cells, Grindstad et al. found that high PR-B expression in PCa tumour tissue was associated with a worse prognosis, reflected in both biochemical and clinical failure . AnotherERα is alternatively classified as nuclear receptor subfamily 3, group A, member 1 (NR3A1). There are two distinct ER genes which are termed ERα and ERβ, the latter known as NR3A2. The role of ERα in advanced PCa was first described in 1999 by Bonkhoff and colleagues . Since tIn human cells, the ubiquitin proteasome system (UPS) plays a vital role in AR degradation and regulation. The UPS consists of ubiquitin (Ub), Ub-activating enzymes (E1), carrier Ub-conjugating enzymes (E2), Ub ligases (E3), deubiquitinating enzymes, and the 26S proteasome . Biologiβ−TRCP [The PROTAC is a protein degradation technology that was extensively applied in recent years, it was designed to degrade its targeted oncogene protein through the UPS. Since AR protein mainly undergo systematic protein degradation via the UPS, the E3 ligases in this process can be harnessed by PROTAC. Structurally, PROTAC is comprised of a degradation machinery recruiting unit , a targeted protein-binding unit and a linker region which joins these two components together   is proposed to eliminate a protein of interest through the autophagy–lysosome system. The AUTOTAC is designed to bind a targeted protein and p62 which is an activator of autophagy. In early this year, one AUTOTAC named VinclozolinM2-2204 composed of a p62-binding moieties and vinclozolinM2, was synthesized to degrade AR protein. The vinclozolinM2 is a metabolite of vinclozolin and it prevents androgen from binding the LBD of AR protein. This AUTOTAC had been demonstrated to decrease the level of AR protein successfully at 0.5–10 µM concentrations in LNCaP cells . Unlike The molecular mechanism of PROTAC is different from traditional drugs that usually function as inhibitors or antagonists to disrupt the transcription of an oncogene or competitively bind specific oncogenic protein, resulting in the suppression of tumour cell proliferation and retarding cancer progression. PROTAC, on the other hand, is specifically designed to join its targeted protein and one selected E3 ligase together. More specifically, the PROTAC binds its targeted protein and recruits an E3 ligase via ligand motif after entering the cells  which started from 2019 and 2022. Although PROTAC selectively degrades AR proteins regardless of pre-translational alterations, there are still several challenges such as off-target, potentially resistance to protein degradation, and structural and functional stability. To overcome these challenges, some potential studies need to be done in the future. In one recent study, ARCC-4 was demonstrated to slightly reduce the level of AR-V7 protein in VCaP cells which suggests that AR-targeted PROTAC may have potential ability to degrade AR-V7 as well . Apart fIn short, the effects of three alternative therapeutic choices need to be extensively evaluated in both treatment-naïve PCa and AR-positive CRPC patients in clinical trials and following cases. Other alternative anti-AR therapies, however, may play less-satisfied roles in CRPC. For example, androgen synthesis inhibitor, such as abiraterone, had already been shown to induce resistance . On the


In silico molecular docking to the human androgen receptor (hAR) combined with machine learning techniques were previously applied to 5206 PFASs and predicted 23 PFASs bind the hAR. Herein, the in silico results were validated in vitro for the five candidate AR ligands that were commercially available. Three manufactured PFASs namely (9-(nonafluorobutyl)– 2,3,6,7-tetrahydro-1 H,5 H,11 H-pyranopyridoquinolin-11-one (NON), 2-(heptafluoropropyl)– 3-phenylquinoxaline (HEP), and 2,2,3,3,4,4,5,5,5-nonafluoro-N-(4-nitrophenyl)pentanamide (NNN) elicited significant antiandrogenic effects at relatively low concentrations. We further investigated the mechanism of AR inhibition and found that all three PFASs inhibited AR transactivation induced by testosterone through a competitive binding mechanism. We then examined the antiandrogenic effects of these PFASs on AR expression and its responsive genes. Consistently, these PFASs significantly decreased the expression of PSA and FKBP5 and increased the expression of AR, similar to the effects elicited by a known competitive AR inhibitor, hydroxyflutamide. This suggests they are competitive antagonists of AR activity and western blot analysis revealed these PFASs decreased intracellular AR protein in androgen sensitive human prostate cancer cells. Hence, the findings presented here corroborate our published in silico approach and indicate these emerging PFASs may adversely affect the human endocrine system.Per- and poly-fluoroalkyl substances (PFASs) are used extensively in a broad range of industrial applications and consumer products. While a few legacy PFASs have been voluntarily phased out, over 5000 PFASs have been produced as replacements for their predecessors. The potential endocrine disrupting hazards of most emerging PFASs have not been comprehensively investigated.   PFASs exhibit useful properties including high thermal and corrosion resistance, low friction performance, and stain repellency. As a result, PFASs are used globally in numerous industrial applications  and consumer products  . In addiAdverse health effects resulting from PFAS exposure are a major public health concern. Studies have shown that exposure to PFASs has been linked to prostate cancer , breast in silico study, 5206 PFASs were screened from the EPA’s CompTox Chemicals Dashboard  against different binding sites on human AR (hAR)  . The comAR (hAR) .f]pyridoquinolin-11-one (NON), 2-(heptafluoropropyl)– 3-phenylquinoxaline (HEP), 3-fluoro-4-{(E)-– 4-yl]diazenyl} phenol (FLU), octafluoronaphthalene (OCT) and 2,2,3,3,4,4,5,5,5-nonafluoro-N-(4-nitrophenyl)pentanamide (NNN). These manufactured PFASs are categorized into the fluorinated aromatic substance subclass of PFASs – 2,3,6,7-tetrahydro-1 H,5 H,11 H-pyrano[2,3-of PFASs . Specifi2.2.1.All PFASs were dissolved into dimethyl sulfoxide  from Fisher Scientific  . Mifepri2.2.2. The human prostate cancer cell line (VCaP) which expresses high levels of wild-type AR, was purchased from ATCC. Cells were cultured in phenol red-free Dulbecco’s Modified Eagle Medium  supplemented with 10% FBS  and 1% L-glutamine  at 37 °C in a humidified incubator with 5% CO2. All PFASs had no good leaving groups such as carboxylic group (The triple negative human breast cancer cell line (MDA-kb2) which highly expresses both endogenous AR and glucocorticoid receptor (GR), stably transfected with the murine mammalian tumor virus (MMTV) luciferase reporter gene construct, was obtained from the American Type Culture Collection . Cells were maintained in Leibovitz’s medium  supplemented with 10% fetal bovine serum  at 37 °C in a humidified incubator without COic group  and wereic group .2.3.4 cells/well in clear 96-well plates . After 24 h incubation, cells were treated with PFASs at concentrations ranging from 0.1 to 100 μM in triplicate per treatment. The optical density was assessed at 570 nm with reference wavelength of 650 nm using microplate spectrophotometer . All cytotoxicity experiments were performed side-by-side with the corresponding cell culture assays and repeated at least three times in a separate independent setup.The cytotoxic effect of PFASs was measured by a colorimetric MTT assay as described previously . Briefly2.4.4 cells/well in white 96-well plates  and incubated for 24 h. Cell culture media containing a potent GR inhibitor (RU486) at 100 nM were used in all luciferase reporter gene assays to completely inhibit transactivation induced by glucocorticoids given AR and GR have the same homologous DNA-binding domains and can activate the MMTV promoter (50) testosterone.The MDA-kb2 cells were maintained prior to the luciferase reporter gene assay as described in the previous study . Brieflypromoter . In all To further examine the mechanism of AR inhibition, cells were co-cultured with the highest non-cytotoxic concentration of each PFAS and different concentrations of testosterone ranging from 9.76 pM to 10 nM. After 24 h incubation, luciferase activity was assessed by a microplate luminometer . All experiments were performed in triplicate wells and repeated at least three times in a separate independent setup. The minimum detection limit for luciferase was 19.5 pM testosterone.2.5.6 cells/well in 6-well plates  and incubated 24 h. Cells were then co-cultured with the highest noncytotoxic concentrations of PFASs and 625 pM testosterone for 24 h. After the incubation, total RNA was extracted using the RNeasy Mini Kit . Briefly, 2 μg of total RNA were reverse transcribed to cDNA using the SuperScript IV VILO  and subjected to real-time PCR . The cDNA was amplified in 10 μL of SYBR Green real-time PCR Master Mixes  according to the manufacturer’s protocol. Relative mRNA expression levels of androgen sensitive genes were evaluated using the following primers: PSA forward (5’-TCTGCGGCGGTGTTCTG-3’) and reverse (5’-GCCGACCCAGCAAGATCA-3’); FKBP5 forward (5’-CGGAAAGGAGAGGGATATTCA-3’) and reverse (5’-CCACATCTCTGCAGTCAAACA-3’); and AR forward (5’-CAGTGGATGGGCTGAAAAAT-3’) and reverse (5’-GGAGCTTGGTGAGCTGGTAG-3’), and were normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (using GAPDH forward (5’-GGATTTGGTCGTATTGGG-3’) and reverse (5’-GGAAGATGGTGATGGGATT-3’ primers)). All primers were obtained from Integrated DNA Technologies . The fold change of the target genes was compared to the vehicle control using the 2−ΔΔCt method  and 1% L-glutamine  for 3 days prior to PFAS treatment. Cells were plated at a density of 2.0 × 10t method .2.6.6 cells/well in 6-well plates  and incubated 24 h. Then, cells were treated with indicated concentrations of PFASs and enzalutamide (positive control) for 48 hr. After incubation, cells were gently washed with ice cold phosphate buffer saline and lysed in RIPA lysis buffer  containing 1 × protease inhibitor . Cell lysates were vortexed rigorously and incubated on ice for 30 min followed by centrifugation at 14,000g for 15 min at 4 °C. The protein concentration was determined by the BCA protein assay . The protein lysates (30 μg) were incubated for 10 min at 95 °C with loading dye containing 2% of β-mercaptoethanol. Proteins were separated on the basis of size in sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis followed by electro-transfer onto nitrocellulose membranes . The membrane was probed with the AR monoclonal antibody  and the secondary antibody goat anti-mouse IgG-horseradish peroxidase . Alpha tubulin monoclonal antibody conjugated with HRP  was used as a loading control. SuperSignal West Femto Maximum Sensitivity Substrate reagents  were added 2 min prior to the chemiluminescent blot visualization with the ChemiDoc Imaging System . The intensity of protein bands was quantified with ImageJ software .The levels of intracellular AR protein were investigated in two different conditions: hormone-deprived (10% CD FBS) and normal (10% FBS) conditions. In the hormone deprived conditions, VCaP cells were cultured and treated as described above in the gene expression assay. To assess the effects of PFASs on the AR protein levels under normal conditions (10% FBS), cells were cultured in phenol red-free DMEM supplemented with 10% FBS  and 1% L-glutamine . Cells were plated at a density of 2.0 × 102.7.Data are expressed as means ± SEM of at least three experiments performed independently. Statistical comparisons between treatments and control were performed by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple-comparison analysis and linear regression analysis to assess linear trends at a statistical threshold of p < 0.05 .3.3.1.in silico analysis which identified 23 emerging PFASs as potential AR ligands , perfluorodecanoate (PFDA) and perfluorohexane sulfonate (PFHxS) exerted an antiandrogenic effect with RIC20 values around 10, 44, 24, and 19 μM, respectively  compared to men with lower PFOS and PFOA levels (15.5 million). This striking difference in the number of normal spermatozoa can be explained by the antiandrogenic effects posed by PFOS and PFOA . It is iOA on AR , others OA on AR . For exaectively . Alarminogenesis . Further3.2.50) of 550 pM. As expected, NON, HEP, and NNN caused a rightward parallel shift of the dose response curves with EC50 values of 722, 810, and 849 pM, respectively  and inhibit the intrinsic activity of the receptor. In bioassays, competitive inhibitors typically generate a parallel shift of agonist dose response curves with no prominent effects on the maximal response . In contectively . These ttrations  and E, ctrations ; however3.3.Molecular docking is an efficient computational method used to examine intermolecular interactions at atomic levels between small molecules and nuclear hormone receptors . The 2D p,p’-dichlorodiphenylethane  has been identified as a weak anti-androgen (in silico in which pi-pi stacking was formed with the same residue (PHE764) as OCT  and one of the phenyl rings of OCT. Comparing OCT with other antiandrogens that have a similar binding mode to AR, androgen  and its ) as OCT . Among t) as OCT . Interesto 10 μM ; however3.4.PSA, FKBP5, and AR, which are biomarkers to evaluate functional and biological effects of antiandrogenic chemicals  conducted in the gene expression assay, immunoblotting results revealed that none of the PFASs significantly changed the level of AR protein. This suggests the PFASs do not affect AR at the protein level and instead that the change in gene expression observed was mediated through the direct inhibition of PFASs on hAR (Despite the antiandrogenic effects of NON, HEP, and NNN observed in both the luciferase and gene expression assays, concern has been identified regarding potential ligand-independent mechanisms as some environmental endocrine disruptors can reduce AR luciferase transactivation independent of ligand binding to AR. For example, pyrifluquinazon, a newly developed insecticide, exhibited antiandrogenic effects by promoting the degradation of cellular AR protein but did not directly inhibit the AR binding . Based os on hAR  and B.3.6.Research studies have shown that exposure to environmental or pharmaceutical antiandrogenic chemicals could reduce intracellular AR protein levels . For exa4.in vitro. More importantly, the potency of antiandrogenic effects of these emerging PFASs is relatively higher than their legacy predecessors reported in previous in vitro studies. Future research should investigate the residue levels of these newly identified antiandrogenic PFASs in humans and their associated health outcomes related to AR signaling pathway disruption such as infertility, cancer, and reproductive development.To the best of our knowledge, this is the first report assessing the biological activity of emerging PFASs against hAR, with evidence indicating that these emerging PFASs competitively inhibit the hAR from binding to testosterone in a concentration dependent fashion. Although, there are no studies to date that measure levels of these PFASs in the environmental matrices or humans, our findings imply that these emerging PFASs may affect the hazard of some androgen-related diseases given they competitively inhibit the hAR and alter the expression of androgenic genes at relatively low concentrations Supplementary Table 1


We also demonstrate that AR antagonists, which suppress transcriptional activity by targeting key regions for homotypic or heterotypic interactions of this receptor, hinder foci formation in PCa cells and phase separation in vitro. Our results suggest that enhanced compartmentalization of AR and coactivators may play an important role in the activation of oncogenic transcription programs in androgen-dependent PCa.Numerous cancers, including prostate cancer (PCa), are addicted to transcription programs driven by specific genomic regions known as super-enhancers (SEs). The robust transcription of genes at such SEs is enabled by the formation of phase-separated condensates by transcription factors and coactivators with intrinsically disordered regions. The androgen receptor (AR), the main oncogenic driver in PCa, contains large disordered regions and is co-recruited with the transcriptional coactivator mediator complex subunit 1 (MED1) to SEs in androgen-dependent PCa cells, thereby promoting oncogenic transcriptional programs. In this work, we reveal that full-length AR forms foci with liquid-like properties in different PCa models. We demonstrate that foci formation correlates with AR transcriptional activity, as this activity can be modulated by changing cellular foci content chemically or by silencing MED1. AR ability to phase separate was also validated  Prostate cancer (PCa) is one of the most common cancers in men. Androgen deprivation therapy (ADT) remains the mainstay of systemic therapy for advanced disease. However, progression to castration-resistant PCa (CRPC) over time is inevitable ,2. A majInteractions with transcriptional coactivators, such as MED1, are required for AR activity and increased interactions with these coactivators are associated with metastatic and castrate resistant PCa . Most rein vitro that concentrate the transcription apparatus from nuclear extracts . LNCaP and LAPC4 cells were maintained in RPMI-1640 media containing 10% fetal bovine serum . All the cell lines were routinely tested for mycoplasma and authentication were confirmed by genomic sequencing or STR profiling.RRID:Addgene_54767). AR constructs were cloned at the N-terminus of the mEGFP tag by using the restriction enzyme sites NheI and BamHI. AR mutants were made by QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies). Med1-IDR (residues 948–1574) was cloned in pET28a with N-terminal OFPSpark tag. For purification purposes, a Histidine tag was added at the N-terminus of AR-mEGFP construct and an AR-mEGFP-MBP-His was cloned in pSF-CMV-PURO-COOH plasmid (Sigma Aldrich).Non-dimerizing mEGFP-N1 (containing A206K mutation) was a gift from Michael Davidson , respectively, following the user guide. The siRNAs targeting MED1 were purchased from Applied Biological Materials  and the scramble was from Dharmacon.Cells grown on glass coverslips were fixed with 3.5% paraformaldehyde (PFA) and then permeabilized with 0.5% Triton X-100. After blocking with 3% skim milk, cells were incubated with the primary antibody diluted in 3% milk (1:200) at 4°C for overnight followed with 1 h incubation of the secondary antibody (1:500) at 37°C for 40 min in the dark. Coverslips were then mounted on slides with DAPI-containing mounting medium (VECTOR). Fluorescence staining was visualized with an Olympus FV3000RS confocal microscope  using a 60x UPLAPO oil objective. The images were taken with the FV31S-SW software with the confocal pinhole set to ‘automatic’ under the Z-stack model. For presentation purposes, images were exported as bitmap (BMP) files. The graph was prepared with the GraphPad Prism 9 software.N = 3) and in total 45 cells were quantified. The data is presented as aligned points. For in vitro droplets, images were analyzed by cellSens to determine the number of liquid droplet formation, droplet size, number and co-localization using cellSens software. The fluorescence images within a set of experiments were acquired using identical microscope settings  to ensure consistency across samples and experimental runs. Thresholding was set to a range of 550–60 000 for green channel, 300–60 000 for red channel and 100–60 000 for gray levels, which effectively eliminate the background signal from the analysis. The pixels within these threshold limits were evaluated for total area (μm2), co-localization coefficient and total number of objects in images.To quantify the numbers of foci, an imaging map of the whole coverslip was first created using the 4× UPLXAPO lens of the Olympus FV3000RS confocal microscope , and then at least 10 fields were randomly selected from the map for each coverslip for the next step high resolution imaging. The high resolution imaging was taken using the 60x UPLAPO oil lens with the confocal aperture airy disk set to 0.6, averaging to 3, actual scan size as 0.067 μm/pixel, and the z-section ranged to 15 μm and step size optimized to 0.21 μm. The laser power and voltage were set to the same conditions within each experiment. The image processing and quantification were performed using the cellSens software (Olympus) with a customized in-house Marco script. The maximum intensity z-projection was applied to the green channel, and Adaptive Threshold was employed to segment the images for foci identification and quantification. The filter area was set to zero to include all the foci. All experiments were performed with three biological replicates . The numbers of foci for each ROI (nucleus) were count and measured. The ROIs containing at least 20 foci were regarded as foci-positive. All experiments were performed with three biological replicates and a total of 45 fields were analysed for each sample. The data are presented as aligned points.in vitro droplets, single pulse of laser at 16 ms dwell time was applied to the AR-mEGFP protein in the 488 nm channel. Recovery was imaged on the Zeiss 780 confocal/Olympus FVS3000 microscope using 60x oil immersion objective lens. When recording fluorescence recovery, 512 × 512-pixel images were collected for 50 frames. Fluorescence intensity was measured using Zeiss ZEN software (ZEN2010).FRAP analysis was performed with a Zeiss confocal laser scanning microscope LSM780 using a Plan-Apochromat 63× 1.40 oil immersion objective. The selected condensates were bleached with the argon laser with 100% of laser power, and the fluorescence recovery was monitored with continuous z-stack scanning for 30 seconds. The images of a non-bleached region were also captured simultaneously from the same field for the reference purpose. A total of 21 foci were monitored for the FRAP assay and the half-lives and mobile fractions of AR-mEGFP-rich condensates were analyzed with Zeiss ZEN software (ZEN2010) and graphed using GraphPad Prism 9 software. For FRAP of et al.  as described before . The siget al.  with theBrUTP was transfected into cells with Lipofectin (Invitrogen Life Technologies) following the user guide. The transfection solution containing 10 mM BrUTP was incubated with cells for 30 min at 37°C and then replaced with culture medium. Two h later, cells were fixed with 3.5% PFA and stained with anti-BrdU antibody .LNCaP cells were hormone starved in phenol red free RPMI medium supplemented with 10% Charcoal Stripped Serum (CSS) for 72 h and plated on precoated coverslips with Polyethyleneimine (PEI). Cells were then transfected with AR-mEGFP using TransIT2020 according to manufacture's protocol. Cells were treated, 48 h post-transfection, with either 1 nM DHT or ETOH for 2 h. Cells were then fixed by 4% paraformaldehyde in PBS for 8 min at room temperature. We then washed the cells with PBS three times, permeabilized them with 0.5% Triton X100 for 10 min, and washed again three times with PBS. Cells were then blocked with 4% Bovine Serum Albumin in PBS for 1 h at 37°C and mouse monoclonal anti-AR antibody was added at a concentration of 1:200 for 16 h at 4°C. Cells were then washed with PBS three times and incubated with Alexa-Flour 488 anti-mouse secondary antibody at 1:1000 for 1 h at 37°C. Cells were washed three times with PBS and were then fixed with 4% paraformaldehyde in PBS for 8 min at room temperature. Cells were dehydrated through successive incubations for 2 min each with 70%, 85% and 100% ETOH. Probe mixture was made mixing 2 μl of FKBP5 probe and 8 μl of probe buffer. Five μl of the mixture was placed on glass slides and the coverslips were placed on top where the cell side is toward the probe mixture. Coverslips were sealed using rubber cement. The genomic DNA were then denatured by placing the slides on a hot plate at 75°C for 8 min in the dark. The slides were then incubated at 37°C for 16 h. The coverslips were then removed and washed with 0.3% Igepal/0.4× SSC  for 2 min followed by a final wash with 0.1% Igepal/2× SSC for 2 min. The coverslips were mounted on slides with DAPI-containing mounting medium.RNA extraction and reverse transcription PCR (RT-PCR) were performed as described previously . Target Proteins (20 μg/well) were separated on SDS-PAGE gels and then transferred to a nitrocellulose membrane (BIO-RAD). Membranes were incubated with a primary antibody (1/1000) at 4°C overnight followed by incubation with a fluorescently labelled secondary antibody (1/5000). Proteins were visualized using the Odyssey Imaging System (LI-COR Biosciences). Vinculin was a loading control. Nuclear concentration of AR was estimated by using a standard curve obtained with recombinant AR protein. Increasing amount of recombinant AR protein were run on SDS Page in parallel with the nuclear extract from AR-mEGFP expressing LNCaP cells. By plotting the intensity of recombinant protein bands as function of concentration, a standard curve was obtained. The intensity of nuclear AR band was then measured and the concentration was derived from the standard curve.LNCaP cells were hormone starved in phenol red free RPMI media supplemented with 5% charcoal Stripped Serum (CSS) for 3 days. Cells were then treated with 1nM DHT or ETOH for 2 h and followed by the treatment with 2.5% 1,6-hexanediol or PBS for 30 min. The cells were immediately washed with PBS, cross-linked with 1% formaldehyde, and harvested. Cells were lysed, nuclei extracted, and DNA was sheared to 300–800 base pair (bp) fragments by Bioruptor (Diagenode). After separating 5% of the sample as ‘input’, the crosslinked chromatin was incubated overnight with AR or MED1 antibody conjugated to protein G beads  for immunoprecipitation (IP). Chromatin from both IP and ‘Input’ samples were eluted and de-crosslinked at 65°C and DNA was isolated by PCR and DNA cleanup kit (NEB). Enrichment of AR and MED1 at specified enhancers were quantified by qPCR.3, the animals were sacrificed and tumors were dissected to prepare for the frozen OCT blocks. 5 μm thick tissues were sectioned from the OCT block for the IF staining. The tissues were fixed with ice-cold acetone for 20 min at –30°C and dried at room temperature for 5 min followed with two times washing with Tris-buffered saline (TBS) containing 0.025% Triton X-100 (TBST). The sections were then incubated with background suppressor  for 10 min and 10% normal goat serum  for 2 h to block the non-specific staining at room temperature. The primary anti-AR antibody was diluted in 1% bovine serum albumin (BSA) in TBST and incubated with the tissues at 4°C for overnight, and the secondary antibody was incubated at 37°C for 1 h in dark. The tissues were counterstained with DAPI (5 μg/ml) in dark for 10 min, washed with TBST for three times and then mounted with ProLong Glass Antifade Mountant . Images were taken with a confocal microscope and the percentage of AR foci-containing cells were quantified from 15 fields of each arm from three tumor lines using the method as described above.The patient-derived prostate tumor tissue lines were implanted in male NOD-SCID mice supplemented with testosterone (10 mg/mouse), as previously described . When tu2. For the expression 1 μg/μl DNA and 3 μg/μl of Polyethylenimine (PEI 25K) was used to transfect the FreeStyle 293F cells at a density of 1 × 106 cells/ml and >95% viability. After 24 h of transfection 10 μM DHT was added to the transfection media and the cells were harvested 48 h post-transfection. Harvested cells were stored at −80°C until purification.Full length AR with C-terminal mEGFP and an N-terminal 8xHis tag was cloned in mEGFP-N1 plasmid and AR-mEGFP-MBP-His was cloned in PSF plasmid. Proteins were expressed in FreeStyle 293F cells (Gibco # R790-07), maintained at 37°C and 90 rpm shaking with 8% CO4; 0.05% NP40; 0.2 mM TCEP; 0.1 mM PMSF and 20 μM DHT) supplemented with 0.1 mg/ml DNaseA, 5 mM MgCl2, cOmplete protease inhibitors  and 1 mM PMSF. After sonication and centrifugation, His-AR-mEGFP and AR-mEGFP-MBP-His were purified by affinity chromatography using Ni-NTA agarose resin and amylose resin, respectively. All the fractions were subjected to gel analysis using 10% SDS-PAGE and confirmed the protein of interest by western blot and mass spectrometry. Fractions containing protein of interest were pooled concentrated , and injected on size exclusion chromatography (Superose 6) and pure non-aggregated fractions pooled, concentrated and used to study the in vitro droplet formation.Cell pellets were thawed on ice and resuspended in buffer A  was mixed with 10% crowding agent (PEG-8000). Ten μl protein solution was loaded onto clean rectangular cover glass  and imaged with Olympus FVS3000 confocal microscope with 60× UPLAPO objective and oil immersion. Images were acquired at 2X zoom and 256 × 256 pixels. 10 frames were collected for each image and five different fields were studied and imaged for each drop. The droplet formation was studied at different time points; different concentrations of protein; in presence of different concentrations of crowding agent, salt and inhibitors. Assay buffer and purified recombinant GFP protein were used as negative control in all the experiments. For AR and MED1 in vitro interaction, purified His-AR-mEGFP or AR-mEGFP-MBP-His was incubated with purified MED1-IDR-OFPSpark , in preTurbidity of protein solutions was determined as the absorption at 340 nm using a Nanodrop OneC instrument (Thermo Fisher). Turbidity was measured after 20 min incubation at room temperature. All the readings were taken in presence of 10% PEG-8000 except when studying the effect of different concentrations of PEG.http://chip-atlas.org). To compare AR and MED1 occupancy in super- and non-super enhancers, BigWig signal was captured by bluegill (http://bream.bio) and then RPM normalized.The H3K27ac ChIP-seq data from LNCaP cells treated with DHT were downloaded from GEO (GSM3358196) and reanalyzed to call super-enhancers. Briefly, raw FASTQ files were aligned to hg19 genome-build using BWA-MEM (v0.7.1) default options. MAPQ <30 alignments were filtered out, and duplicates were removed according to the ‘samtools markdup’ (v1.16) manual page. Peaks are called using MACS3 (v3.0.0a7) that was controlled with input DNA (GSM1249450). Finally, ‘.narrowPeak’ files were converted to ‘.gff’ format as input for the ROSE program with custom scripts. AR (GSM3567213) and MED1 (GSM3567216) BigWig files were downloaded from ChIP-atlas  and a mobile fraction of 51.01% of the reference signal   and a mobile fraction of 96.26% of the reference signal   ,26,27. W) Figure . However2–99.88) .These re2–99.88) . The per2–99.88) . This re2–99.88) . We then2–99.88) ,26, on t2–99.88) . A key f2–99.88) . Overallin vitro, we next expressed and purified DHT activated full length AR-mEGFP tagged with an N-terminal poly-histidine tag or a C-terminal maltose binding protein (MBP)-His tag, the latter allowing for higher purity levels  , DBD 14 or the L23FXXLF27 and 178LXXIL182 motifs (Del 2–360) on AR foci formation. Interfering with both, DNA binding or N/C interactions, significantly decreased AR's ability to form foci in LNCaP cells . Taken together, cellular and in vitro data suggest that interactions mediated by the LBD and NTD are required for phase separation while the DNA-binding interface of AR may be necessary for the recruitment to androgen response element (ARE) in the genome and the formation of foci at these elements. To evaluate the effect of 14449 further, we tested whether the ARE from the promotor of KLK3 gene is recruited to AR droplets and whether 14449 can impede this recruitment. As seen in Figure KLK3 gene indeed colocalized with AR-mEGFP-MBP droplets. Importantly, the addition of ARE significantly increased the size of AR droplets  interactions, we also tested the effect of these AR antagonists on droplets formation o Figure , E usings Figure , G and us Figure  and H. Ts Figure .in vitro and in PCa cells that have liquid-like properties. However, whether such dynamic assembles could have functional relevance is not clear. Therefore, we aimed to determine if foci formation is associated with AR transcriptional activity. Therefore, we first confirmed using a DNA-FISH probe the colocalization of AR-rich foci with FKBP5, an AR target gene associated with SE programs  and the initiation of transcription ,47. Andrin vitro, we expressed and purified the IDR of MED1 with an N-terminus OFPSpark red tag and mixed it with AR-mEGFP-MBP  upon dihydrotestosterone (DHT) stimulation compared to enhancer regions . We obseKLK3 and TMPRSS2 SEs  of AR in presence and absence of 1,6-HD. We observed a strong enrichment of AR binding following androgen stimulation at the s Figure . Interesn Figure . Howeverin vitro. Droplet formation in vitro is inhibited by high salt concentration and exposure to 1,6-HD, which suggests that both electrostatic and hydrophobic interactions are key for their formation. Importantly, FRAP experiments reveal similar diffusive exchange properties for in vitro droplets and foci formed by AR in PCa cells upon androgen stimulation. Moreover, recovery half-lives calculated from these FRAP experiments are similar to the ones measured for MED1 foci  and oncogenic drivers (e.g. ERG and MYC) (7). The direct interaction between the AR and ERG oncogene has recently been confirmed by a report of the cryo-EM structure elucidating this interaction . Consistent with this current understanding of PCa biology, we found an increase of SEs and AR-occupied SEs with the advancement of PCa in patients. Given that MED1 and AR interact not only in androgen-dependent PCa cells at SEs  and LBD inhibitors , which impede homotypic (N/C) and heterotypic (coactivator) interactions, reduce foci formation in PCa cells and condensate formation in vitro (Under physiological conditions, SE programs control genes involved in cell identity. In cancer cells, by contrast, oncogenes acquire SE in order to assemble the transcriptional apparatus at high density and enable enhanced transcription of target genes ,14,53. M and MYC . Consists at SEs , it is l and MYC . Consistin vitro ,40. AddiIn summary, we reveal that dynamically formed foci are important to the transcriptional activity of AR in PCa cells. This transcriptional activity can be modulated by changing the foci content genetically or chemically. A better understanding of foci assembly mechanism and their mode of action may open novel therapeutic venues to treat advanced forms of PCa, specifically those that are addicted to androgen-driven SE transcriptional programs.The data that support the findings of this study are available from the corresponding author, upon reasonable request.gkac1158_Supplemental_FilesClick here for additional data file.


In this paper, four pyramided lines, including gn1a-gs3, gn1a-ipa1, gs3-ipa1, and gn1a-gs3-ipa1, were further generated by conventional cross-breeding to be tested. Agronomic traits analysis showed that: (1) the stacking lines carried large panicles with an increased spikelet number in the main panicle or panicle; (2) the grain weight of the stacking lines, especially gs3-ipa1 and gn1a-gs3-ipa1, were heavier than those in single mutants; (3) both gn1a-gs3 and gs3-ipa1 produced more grain yield per plant than single mutant lines; (4) pyramided lines were higher than single mutants and transcriptome analysis found improved expression levels of genes related to lipid, amino acid, and carbohydrate transport and metabolism in lines pyramiding three mutant alleles, possibly as a result of complementary and additive effects. Accordingly, the alteration of gene-expression patterns relating to hormone signaling, plant growth, and seed size control was characterized in pyramided lines. The present study not only investigates the effects of pyramiding genes, but also may provide an efficient strategy for breeding super-high-yield rice by reducing the time cost of developing pyramided lines.Pyramiding of quantitative trait loci (QTLs) is a powerful approach in breeding super-high-yield varieties. However, the performance of QTLs in improving rice yield varies with specific genetic backgrounds. In a previous study, we employed the CRISPR/Cas9 system to target three yield-related genes,  Oryza sativa L.) is one of the major staple crops around the world. As the population continues to rise and less arable land becomes available in agricultural production, improvement of agricultural productivity is in an upsurge to satisfy global food demand [IDEAL PLANT ARCHITECTURE1 (IPA1)/WEALTHY FARMER’S PANICLE (WFP)/SQUAMOSA PROMOTER-BINDING PROTEIN LIKE 16 (OsSPL 14), containing a mutation in miR156 cleavage site was found in both Taichung Native 1 (indica cultivar) and Aikawa and Shaoniejing (japonica cultivar), which were high-yielding varieties characterized with ideal plant architecture with a small number of unproductive tillers, more grains per panicle, and thick and sturdy stems [Grain number 1a (gn1a) encodes a CK oxidase/dehydrogenase (CKX) that catalyzes active cytokinin (CK) degradation. A mutation or reduced expression of gn1a causes the accumulation of CK in inflorescence meristems and an increased number of reproductive organs, thus, promoting grain production in Habataki (indica cultivar) [Gn1a was also detected in the restorer line R1128, a parental line of hybrid rice featured giant panicles [DENSE AND ERECT PANICLE (DEP1) and Grain Size 3 (gs3) encode atypical G-protein γ subunits. The mutation of the DEP1 allele in high-yield varieties of Shennong 265 and Jiahua 1 (japonica cultivar) resulted in dense and erect panicles [gs3 (indica cultivar) produced long grains and an increase in grain weight [DROUGHT AND SALT TOLERANCE (DST), grain number, plant height, and heading date7 (ghd7) involved in regulating rice grain number [Rice , a typical heavy panicle restorer line, was pyramided with gn1a and gs3 alleles to produce a heavy panicle with an increased grain number and large grains [Sd1 for plant height, Hd1 and Ehd1 for heading date, gn1a for spikelet number, and ipa1 for ideal plant architecture [gs3 and gs3gn1a in five different japonica varieties and found that gs3gn1a mutants were not only characterized with increased grain length, as in gs3, but also with a larger number of grains on main panicles. IPA1 regulates the crosstalk between growth and defense to have impacts on both disease resistance and grain yield [gn1a-type 3 and OsSPL14 produced higher grain yield than two single-gene introgressed lines, respectively. A recent report from Reyes et al. [gn1a and ipa1 introgression lines developed by marker-assisted backcross breeding resulted in significantly higher spikelet numbers per panicle and primary branching compared with recurrent parents in selected NERICA cultivars. The modification of gs3 or the introgressed segment carrying the gs3 allele was found to promote rice grain length and weight [ipa1 led to increased grain weight and number per panicle [Over the past decades, several rice breeding strategies contributing to the sustainable increase in rice yield have been successfully practiced. Breeding methods, such as conventional hybridization, heterosis breeding, and wide hybridization, are effective in combining yield-promoting traits from different cultivars to breed high-yielding varieties ,12. Wangitecture . In anotitecture  investigin yield . Miura es et al.  indicated weight ,18,19,20 panicle . These sHabatakign1a (type 3) was observed to significantly increase grain number per panicle (GNPP) in japonica cultivars Koshihikari, Sasanishiki, and Kongyu131 [gn1a-type 3 allele was not found to be effective in promoting GNPP. Since QTLs could have opposing effects on grain yield [The key goal of crop breeding is to incorporate multiple desirable traits, such as high yield, superior quality, pest resistance, and environmental stress tolerance, into a single variety ,17,21,22ongyu131 ,23. Howeongyu131 , the gn1in yield , the genGn1a, DEP1, GS3, and IPA1) using the CRISPR/Cas9 system in “Zhonghua11” and obtained phenotypes similar to those previously described [gn1a-gs3, gn1a-ipa1, gs3-ipa1, and gn1a-gs3-ipa1 by conventional crossing. The objectives of this study include: (1) to stack multiple single elite genes contributing to a large panicle and big grain into japonica ‘Zhonghua 11’, and to produce a high-yielding plant with thicker culm; (2) to study the interplay of gn1a, gs3, and ipa1 in regulating rice yield-related traits; (3) to generate a series of rice core germplasms with different desirable traits, and to provide a theoretical basis for molecular-designed breeding of super-high-yield japonica rice cultivars in tropical area.In our previous study, we successfully edited four yield-related genes . However, when counting the spikelet number per panicle level, ipa1 (163.75) is significantly more than that in gn1a (144.85)  and more primary branching (17.05) than gn1a , and more spikelet per the main panicle (240.3) than gn1a (209.65)   , these r(209.65) , suggestghua 11’ .ipa1 was heavier (25.60g) compared with WT (23.27g) and gn1a (23.20g) (gn1a-ipa1 (25.72g) was more than those of gn1a and WT, but close to ipa1 grain  . The 100a1 grain . Overallgn1a, seed setting rates in ipa1 and gn1a-ipa1 dropped by about 10%, suggesting the low-seed setting rate of gn1a-ipa1 was caused by ipa1. The grain yield of gn1a-ipa1 fell between those of gn1a and ipa1  and longer main panicle (24.92 cm) than in the single mutants gs3  and ipa1 . The results demonstrated the superimposed effect of gs3 and ipa1 on increasing the spikelet number and panicle size, and the dominant role of ipa1 in controlling the number of primary branching and spikelet numbers per plant in gs3-ipa1.In the current study, as it was shown in gs3-ipa1 and gs3; however, the values of 1000-grain weight increased when comparing gs3-ipa1 (27.69 g) with the single mutant line ipa1 (25.60 g)  . The resgs3-ipa1 significantly increased (32.63g/plant) compared with single mutant gs3 (24.31 g/plant ) and ipa1 (16.10 g/plant)  . It was gn1a, gs3, and ipa1 together, we obtained the homozygous line of gn1a-gs3-ipa1. The main panicle length of gn1a-gs3-ipa1 (25.5 cm) was significantly longer than in gn1a-gs3 and gn1a-ipa1, indicating a larger superimposed effect on the length of the main panicle. The number of the primary branching of gn1a-gs3-ipa1 significantly increased by 34%, compared with gn1a-gs3, but was not significantly different to that of ipa1, gn1a-ipa1, and gs3-ipa1  and ipa1 (78.23%), but lower than that of gn1a-gs3 (85.34%) and gs3-ipa1 (88.46%), indicating the dominant role of ipa1 in decreasing seed setting rate. Overall, there was a greater improvement of grain yield per plant of gn1a-gs3-ipa1 (26.12 g/plant) than that of gn1a-ipa1 (22.40 g/plant), but lower than that of gn1a-gs3 (31.03 g/plant) and gs3-ipa1 (32.63 g/plant) (The lines pyramiding n weight . The seeg/plant) .gs3, gn1a, and ipa1 plants were taller than WT. After gs3 was stacked to gn1a and ipa1, respectively, both gn1a-gs3 (99.57 cm) and gs3-ipa1 (112.84 cm) were on average much higher than single mutant gs3 (91.65 cm), gn1a (93.9 cm), and ipa1 (101.62 cm). The gn1a-ipa1 plant was higher than gn1a but not significantly different to the ipa1 plant. Triple mutant gn1a-gs3-ipa1 (115.59 cm) was even higher than gn1a-gs3, gn1a-ipa1, and gs3-ipa1 (112.84 cm). These results indicated that gs3, gn1a, and ipa1 might have a superimposed effect on increasing plant height, while gn1a might only promote gs3’s height instead of gs3-ipa1’s.As it was shown in ipa1 plant was characterized by fewer sterile tillers (three to five tillers per plant) than WT, while it had a minor impact on the number of effective tillers in gn1a and gs3  compared with gn1a-gs3 (31.03 g) and gs3-ipa1 (32.63 g) due to less effective tiller and seed setting rate, possibly caused by the ‘ipa1 effect’ . Ten items based on their function correlated with energy and yield were selected . Compare and gs3 A. It stred yield . In gs3-gs3-ipa1 B, DEGs fgs3-ipa1 . We alson1a-ipa1 C, more Dn weight . Finallygs3-ipa1 D, we fougs3-ipa1 . That isgs3-ipa1 .Hormones play important roles in controlling plant growth and development and pyramided lines were found to alter the expression pattern of many hormone-related genes . AccordiFinally, we examined transcriptome data from young panicle materials  and founBRASSINOSTEROID UPREGULATED 1-LIKE1; OsBUL1/POSITIVE REGULATOR OF GRAIN LENGTH 2; PGL2) and Os09g0510500 were upregulated in gs3-ipa1 rather than in a single mutant (osbul1) produced erect leaves and smaller grains, whereas overexpression of OsBUL1 increased lamina inclination and grain size [gs3-ipa1  is a drought-inducible receptor-like cytoplasmic kinase and secures grain yield under drought and well-watered conditions. The gudk mutants feature a significant grain-yield reduction under normal well-watered conditions or when facing drought stress at the reproductive stage [gs3-gn1a, gs3-ipa1 and gn1a-gs3-ipa1 influences grain chalkiness, rice yield, and many other quality traits. It encodes a vacuolar H+-translocating pyrophosphatase (V-PPase) with inorganic pyrophosphate (PPi) hydrolysis and H+-translocation activity. Elevated expression of Chalk5 increases the chalkiness of endosperm and produced chalky grain [gs3, gn1a-gs3, and gn1a-gs3-ipa1  encodes a new-type GNAT-like protein with intrinsic histone acetyltransferase activity. Elevated OsglHAT1 expression enhances grain weight and yield by enlarging spikelet hulls via increasing cell number and accelerating grain filling, and increases global acetylation levels of histone H4 [gn1a-gs3, gn1a-ipa1, and gn1a-gs3-ipa1(dwarf 18) encodes a gibberellins 3β-hydroxylase, and its knockout mutant exhibits extremely dwarf phenotypes suitable for large-scale indoor planting [ipa1, gn1a-gs3, and gn1a-gs3-ipa1 , introgression of the ipa1 allele showed its potential in breeding novel high-yielding lines [ipa1 phenotype by targeting the region containing the OsmiR156 target site [ipa1 exhibited ‘Ideal Plant Architecture’ and featured a reduced effective tiller number, increased grain number and weight per panicle, and a strong stem  that catalyzes active cytokinin (CK) degradation, it has a dominant effect on stimulating meristem cell division, and thus, increases grain number, the role of this gene is in accordance with previous studies [Cytokinin plays important roles in regulating plant growth and development, for example, in shoot and root growth, seed development, and apical dominance . As earld allele ,23,24, silencing , or the PR/Cas 9 . CRISPR/ studies .gs3 contributed specifically to energy conversion, lipid and carbohydrate transport and metabolism, gn1a in transcription and signal transduction, and ipa1 in cell cycle, cell motility, and signal transduction, which correlated well with the traits in a single mutant , gn1a (with a 1-bp deletion), and gs3 (with a 47-bp deletion and 23-bp insertion) were selected to produce gene-stacking lines by conventional crossing. Homozygous double mutants, gn1a-gs3, gn1a-ip1a, and gs3-ipa1, and triple mutant gn1a-gs3-ipa1 were examined by PCR amplification of the target sites, further confirmed by sequencing  and wild type (WT) plants, were planted in experimental fields at South China Normal University in Guangzhou, P. R. China. Their phenotypes were investigated in three successive years from 2019 to 2021. The lines were sowed on 10th March each year and harvested on 30th June. At the mature stage, major agronomic traits were recorded from 20 plants of each line, which were randomly chosen from 50 tested plants. These traits include plant height, effective tiller number, main panicle length, spikelet number per the main panicle, spikelet number per panicle, number of primary per the main panicle, 1000-grain weight, and grain yield per plant. Since results generated from each of the three years provide similar implications, only the representative data from 2021 was presented in the current study.T3 generation homozygous lines  according to the manufacturer’s protocol. RNA-Seq was performed by Novogene Bioinformatics Technology Co., Ltd.  with Hiseq-PE150  and the raw data was analyzed using the software package of Transcriptome Analysis with Reference Genomes in the BMKCloud cloud server2. Gene expression level quantification was estimated by fragments per kilobase of transcript per million fragments mapped (FPKM). A value of |log2(foldchange)| ≥ 1 (FDR < 0.01) was set as the threshold for differentially expressed genes (DEGs). Data from two biological replicates were used for the analysis.For RNA-Seq analysis, total RNA was extracted from 10 young panicles (about 1 cm) from each line using TRIzolLOC_Os03g50885) was used as internal control and three technical replicates were designed for each sample. Primers used for qPCR are listed in Briefly, 10 young panicles (0.5–1 cm length) from one mutation line were collected for RNA extraction, using Trizol reagent . RNA samples were treated with DNaseI following the manufacturer’s protocol, and to be quantified under Nanodrop Spectrophotometer . First-strand cDNA synthesis and qRT-PCR (quantitative reverse transcriptase polymerase chain reaction) were performed according to the instructions of commercial kits . PCR amplification was performed following the procedure: 30 s 94 °C preincubations, 45 cycles of 5 s denaturation at 94 °C, and 30 s annealing and extension at 55 °C. The relative expression levels were calculated by the delta-delta Ct method . The Ric


Background: Most therapeutics targeting cardiac voltage-gated sodium channels (Nav1.5) attenuate the sodium current (INa) conducted through the pore of the protein. Whereas these drugs may be beneficial for disease states associated with gain-of-function (GoF) in Nav1.5, few attempts have been made to therapeutically treat loss-of-function (LoF) conditions. The primary impediment to designing efficacious therapies for LoF is a tendency for drugs to occlude the Nav1.5 central pore. We hypothesized that molecular candidates with a high affinity for the fenestrations would potentially reduce pore block.Methods and Results: Virtual docking was performed on 21 compounds, selected based on their affinity for the fenestrations in Nav1.5, which included a class of sulfonamides and carboxamides we identify as ARumenamide (AR). Six ARs, AR-051, AR-189, AR-674, AR-802, AR-807 and AR-811, were further docked against Nav1.5 built on NavAb and rNav1.5. Based on the virtual docking results, these particular ARs have a high affinity for Domain III-IV and Domain VI-I fenestrations. Upon functional characterization, a trend was observed in the effects of the six ARs on INa. An inverse correlation was established between the aromaticity of the AR’s functional moieties and compound block. Due to its aromaticity, AR-811 blocked INa the least compared with other aromatic ARs, which also decelerated fast inactivation onset. AR-674, with its aliphatic functional group, significantly suppresses INa and enhances use-dependence in Nav1.5. AR-802 and AR-811, in particular, decelerated fast inactivation kinetics in the most common Brugada Syndrome Type 1 and Long-QT Syndrome Type 3 mutant, E1784K, without affecting peak or persistent INa.Conclusion: Our hypothesis that LoF in Nav1.5 may be therapeutically treated was supported by the discovery of ARs, which appear to preferentially block the fenestrations. ARs with aromatic functional groups as opposed to aliphatic groups efficaciously maintained Nav1.5 availability. We predict that these bulkier side groups may have a higher affinity for the hydrophobic milieu of the fenestrations, remaining there rather than in the central pore of the channel. Future refinements of AR compound structures and additional validation by molecular dynamic simulations and screening against more Brugada variants will further support their potential benefits in treating certain LoF cardiac arrhythmias. Na). Mutations in the SCN5a gene, encoding Nav1.5, result in loss-of-function (LoF) or gain-of-function (GoF) abnormalities in the protein, leading to an array of cardiac disorders (The cardiac voltage-gated sodium channel (Nav1.5) is responsible for electrical depolarization in cardiac tissue through the conduction of sodium current  and Magnetococcus marinus (NavMs) confirmed that at rest, drugs, especially lipophilic ones, have a high access into the channel through the dilated fenestrations and await the dilation of the central pore to elicit their pharmacological action  at 3.2–3.5 Å resolution was captured in a state in which all four voltage sensors were partially activated; hence, the channel was also partially inactivated. The four fenestrations were identified: Domain II-III fenestration was the largest compared to the others . FlecainA major caveat of the cyro-EM structure of rNav1.5 is that it does not fully depict fenestration size at rest, nor in activated or inactivated states. Crystal structures obtained of NavAb inactivated states are compatible with slow inactivation in eukaryotic Nav channels, since NavAb lacks the fast inactivation particle , 2012. UV1.5, DI-II and DIII-IV fenestrations are dilated, and DII-III and DI-IV fenestrations are constricted  and their radial size is determined mainly by isoleucine and phenylalanine residues in S5s . The crystricted .Na conduction. In this study, we used virtual docking and whole-cell patch clamp to assess the efficacy of a new class of compounds, ARumenamide (AR), that dock within the fenestrations, in an effort to target LoF. We screen high profile ARumenamides against E1784K to validate their effect in attenuating LoF.Our hypothesis is based on the premise that compounds dock in the dilated fenestrations during activation, and the fenestrations are prevented from constricting during inactivation, thereby maintaining a dilated and unblocked central pore, thus maintaining Iswissmodel.expasy.org)  . We geneThe pre-activated bacterial crystal structure of NavAb (2.7 Å resolution) was used as a template against the Nav1.5 sequence. Modeling was done according to the protocol established by blaster.docking.org)  . The higing.org) , selecteing.org) . PyMOL-pARs for functional characterization using patch clamp electrophysiology. We based this decision on each AR compound’s affinity and probability of adhering to the fenestrations  in powder form. They were dissolved in 100% DMSO to create a stock solution of 50 mM and diluted with external solution to the desired final concentration.The six chosen ompounds  can be gAR-051 (MolPort-002-662-051/ZINC38767171/STK615542)AR-189 (MolPort-004-279-189/ZINC40014265/Z26997243)AR-674 (MolPort-001-911-674/ZINC39699427/STK742976)AR-802 (MolPort-010-669-802/ZINC64470729/G801-0062)AR-807 (MolPort-010-669-807/ZINC64470737/G801-0091)AR-811 (MolPort-010-669-811/ZINC64470745/G801-0104)V1.5 is TTX-resistant, we used lidocaine to confirm the ARumenamide effects observed in E1784K.Vehicle (0.05% DMSO) controls were often performed to enable correction for any compound-independent decrease of currents. Since Na2. The α-subunits (WT or E1784K) were transiently co-transfected with the β1 subunit and green fluorescent protein, eGFP . The cDNA mixture was then allowed to incubate with the HEK293 cells before plating on coverslips.HEK293 cells were grown at pH 7.4 in a DMEM (1x) nutrient medium , supplemented with 10% FBS and maintained in a humidified environment at 37°C with 5% CO2, 1 MgCl2, and 10 HEPES (pH 7.4 adjusted with CsOH). Solutions were titrated with CsOH to pH 7.4. Pipettes were fabricated with a P-1000 puller using borosilicate glass , dipped in dental wax to reduce capacitance, then thermally polished to a resistance of 1.0–1.5 MΩ. Low resistance electrodes were used to minimize series resistance between pipette and intracellular solution resulting in typical access resistances of 3.5 MΩ or less, thereby minimizing voltage measurement error. Pipettes were filled with intracellular solution (in mM): 130 CsF, 10 NaCl, 10 HEPES, and 10 EGTA titrated to pH 7.4.Whole-cell patch clamp recordings were performed in extracellular solution containing (mM): 140 NaCl, 4 KCl, 2 CaClAll recordings were made using an EPC-9 patch-clamp amplifier  digitized at 20 kHz using an ITC-16 interface . Data were acquired and low-pass-filtered (5 kHz) using PatchMaster/FitMaster software  running on an Apple iMac . Leak subtraction was performed online using a P/4 procedure. Bath solution temperature was controlled using a Peltier device driven by a TC-10 Temperature Controller , and maintained at 37°C. When screening the effects of AR-802 and AR-811 on E1784K, bath solution temperature was maintained at 22°C since it was difficult to record at higher temperatures due to the inherent instability of cells.ARumenamide compounds chosen for biophysical characterization were screened against wild type (WT) and E1784K Nav1.5 by bath perfusion following an initial series of voltage clamp protocols (below).The six Conductance was measured by a protocol that depolarized the membrane from -100 mV to +80 mV in increments of +5 mV for 19 ms. Prior to the test pulse, channels were allowed to recover from fast inactivation at −130 mV for 197 ms. Channel conductance was calculated from peak INaNa is sodium channel conductance, INa is peak sodium current in response to the command potential V, and ERev is the reversal potential. Calculated values for conductance were fitted with the Boltzmann function:max is the normalized conductance amplitude, VM is the command potential, z is the apparent valency, e0 is the elementary charge, V1/2 is the midpoint voltage, k is the Boltzmann constant, and T is temperature in °K.Fast Inactivation Onset: At voltages greater than −50 mV, the fast inactivation τ values were calculated from the mono-exponential fit to the decay of sodium current.ss is the plateau amplitude, α is the amplitude at time 0 for time constant τ, and t is time.Persistent INacurrent was measured by holding the potential at -110 mV, −90 mV, or −70 mV for 1 s followed by a depolarization to 0 mV for 200 ms and repolarization to −110 mV for 50 ms. The fast inactivation τ values were also calculated from the mono-exponential fit to the decay of sodium current measured in this protocol.Use-Dependence was used to indirectly measure slow inactivation (SI), which is a physiologically relevant protocol. The protocol includes a series of 110 ms depolarizing pulses to 0 mV followed by a –90 mV recovery pulse for 55 ms at a frequency 6 Hz. With repetitive depolarizations, Nav1.5 channels accumulate into slow inactivation. Normalized current amplitude as a function of time was fit with a double exponential.ss is the plateau amplitude, α1 and α2 are the amplitudes at time 0 for time constants τ1 and τ2, and t is time.p < 0.05.Statistical analysis was performed using a one-factor completely randomized design ANOVA followed by a post hoc Tukey test. Our statistical model was a full factorial in which all the factors were allowed to interact together. Data are shown as mean ± SEM. Statistical significance was considered at ARumenamides described here are under IP protection .ARumenamide hits, AR-051, AR-189, AR-674, AR-802, AR-807, and AR-811, are shown in ARumenamides displayed the same binding affinity  whether they were docked against the individual fenestrations or the entire channel.Six ARumenamide hits screened against rNav1.5 interacted with Domain I–II and Domain II–III fenestrations  at 10 µM and progressively more with higher compound concentrations. AR-051, AR-189, AR-802, and AR-807 all significantly blocked peak INa at −20 mV at concentrations of 50 µM and higher. AR-811 was the only compound that did not block peak INa within the concentrations tested (1–100 µM) (Na block at −20 mV in Na compared to the rest of the compounds.All six t −20 mV . AR-674 –100 µM) . A dose-ARumenamide compounds affected the midpoint potential value of conductance (p < 0.05) the conductance slope at 50 µM and higher. While AR-802 and AR-807 significantly decreased (p < 0.05) the conductance slope at 100 µM (The voltage-dependence of conductance was assessed using a series of depolarizing test pulses. None of the ductance . AR-674 ductance . Other ct 100 µM .p > 0.05) at −50 mV. However, AR-802 and AR-807 slowed the onset of fast inactivation (higher τ - Open-state fast inactivation was measured at voltages greater than -50 mV . The comARumenamide compounds displayed similar trends in their effect on use-dependent decrease of current amplitude as they did with peak INa block (p < 0.05) use-dependence in Nav1.5 at 10 µM and higher concentrations .To confirm their effects in potentiating I control . Neither control . Fast in control . However for 1 s . AR-811  1 s  Ihythmias . Most Gohythmias . The undhythmias . SuppresOther cardiac conditions, including progressive cardiac conduction disorder (PCCD), sick sinus syndrome, progressive familial heart block, atrial fibrillation, sudden infant death syndrome, and dilated cardiomyopathy, also degenerate into lethal arrhythmias . HoweverARumenamide compounds we screened were selected from a cohort of other hits based on their affinity for the fenestrations. Although docking ARumenamides against the Nav1.5 model based on the NavAb template is temperamental, this approach allowed us to shortlist compounds for electrophysiological testing. We tested our hypothesis that ARumenamides block the fenestrations and restrict the motion of Nav1.5 from naturally progressing into inactivation, thereby maintaining INa conduction. Although we are the first to treat LoF by targeting the fenestrations, previous attempts have been made to attenuate hyperexcitability in Nav through “non-blocking” modulation as with riluzole, which indirectly blocks peak INa in Nav1.4 by binding to one of the fenestrations  that lipophilic drugs access their binding sites via an alternative route, slowly slithering through the fenestrations into the central pore . Structutrations . Our resAR candidates in its effects on Nav1.5. Although virtual docking suggested its high binding affinity for the fenestrations, AR-674 blocks INa and suppresses Nav1.5 availability with time via use-dependence. Independent of any particular theory, the biophysical shifts differentiating AR-674 from the other compounds, or at least its counter sulfonamides that share the same backbone structure, seems to be that its functional group is aliphatic and not aromatic. We predict that an inverse relationship exists between AR’s aromaticity and INa block. The aliphaticity of AR-674 appears to lower the affinity to bind within the fenestrations. Lower affinity within the fenestrations would be predicted to allow greater access of AR-674 into the central pore, which may help explain the 34% reduction in peak INa at -20 mV and the significant use-dependence at concentrations as low as 10 µM. AR-674 may also interact with the voltage sensors during activation, suppressing their movement and accounting for the decreased slope in conductance.Our results suggest AR-674 differs from the other ARs seem to increase binding affinity within Nav1.5 fenestrations. AR-802, AR-807, and AR-811 have aromatic benzene in their side chains compared to the aliphatic side chain of AR-674. Compound binding within the fenestrations may impede changes in fenestration size during fast inactivation. These three compounds slow the onset of fast inactivation at different concentrations. The fluorine atom attached to the benzene ring of AR-811 seems to mediate the potentiator effects in Nav1.5; however, this conclusion is based only on comparing AR-811 effects with other relatable sulfonamides. The presence of the fluorine atom may account for the high resistance to peak INa block and the unaltered use-dependence. These results suggest that AR-811 mediates its apparent “potentiating” effects (compared to AR-674) at elevated depolarization rates through an open/inactivated compound-block mechanism. The observed deceleration in fast inactivation kinetics caused by AR-802 and AR-811 is seen only after E1784K channels are pre-conditioned at −110 mV, −90 mV, and −70 mV for 1 s. With prolonged depolarizing pre-conditioning pulses, it seems reasonable to hypothesize that AR-811 mediates its “potentiating” effects by targeting fast and potentially slow inactivation. The benefit of attenuating LoF in E1784K via the aromatic compounds, AR-802 and AR-811, in particular, is that their speculated adherence to the fenestrations reduces their likelihood of blocking peak or persistent INa, a result that we clearly observed.Aromatic functional groups in Na as much at 10 µM compared to AR-051. The branched alkyl side chain adjacent to the furan may increase the hydrophobicity of AR-189, increasing its binding affinity within the fenestrations.Compared to the carboxamides, the sulfonamides, AR-051 and AR-189, also inform us about the structural features required to generate Nav1.5 potentiators. Although not statistically significant, AR-189 seems to be slightly better in not suppressing IAR compounds have preference for a particular fenestration based on the virtual docking alone; however, our modeling clearly showed an affinity of AR compounds screened to the Domain III-IV fenestration in Nav1.5-NavAb and Domain VI-I fenestration in rNav1.5. Molecular dynamics simulation could be used to verify AR interaction with the fenestrations in Nav1.5. Additional screening of AR compounds against other Brugada variants of Nav1.5 will help confirm our initial experiments that suggest ARs may have therapeutic potential to treat LoF.Virtual docking and the Nav1.5 homology models have limitations. Not all the fenestrations were targetable, especially in rNav1.5. Crystallography and recent cryo-EM structures do not fully depict the fenestrations in their various voltage-dependent conformations . Thus, oNa and attenuate GoF, drugs like GS-967, eleclazine, ranolazine, and mexiletine analogs block peak INa at varying concentrations  attenuate LoF in the most common Brugada Syndrome Type 1 and Long-QT Syndrome Type 3 mutant, E1784K, by decelerating fast inactivation onset kinetics. Neither AR-802 nor AR-811 block peak INa or enhance persistent INa in E1784K, two highly sensitive parameters that must be controlled to avoid exacerbation of phenotype. Successfully optimizing AR compounds and testing them in LoF models related to cardiac disorders may ultimately lead to lowering the economic burden and health costs associated with untreated arrhythmias.Our results suggest that


Allergic rhinitis (AR) is a noninfectious inflammatory disease of the nasal mucosa mediated by IgE after atopic individuals are exposed to inhaled allergens and involving a variety of immune cells and cytokines . AR is cTLR1, TLR10, GATA3, IL33, NFKB1, etc., which are involved in both innate and adaptive immune pathways and bridge the environmental insults and cell responses [With regards to the epidemiology of AR, factors such as prevalence, disease classification, allergen sensitization, co-morbidities, risk factors, genetic susceptibility, and costs, etc., are widely discussed . Globallesponses . Epidemiesponses . Two majesponses . Future esponses .With respect to prevention, graded prevention of AR has been proposed . PrimaryAllergen avoidance, drug therapy and allergy immunotherapy (AIT) are the main clinical intervention modalities for AR . The firAvailable asthma biologics targeting type 2 response, such as omalizumab, an anti-IgE antibody and dupilumab targeting the IL-4/IL-13 pathway, have been attempted in patients with AR. Two meta-analyses revealed that omalizumab was effective and safe to treat uncontrolled AR patients ,15. AlthAIT is considered as the only etiological and most effective therapy for AR . HoweverA major outstanding question is, when the above therapies fail, what can we do next? For those patients, surgical operations may be considered. Surgical intervention may aim at reducing nasal obstruction as a means of improving quality of life of AR patients . Other sThere are several items that should be addressed in future research efforts for AR. First, to integrate genomics, transcriptomics, proteomics and metabolomics into AR pathogenesis investigation will promote the understanding of the interactions between environmental factors, allergen sensitization and disease development. Second, biomarkers are urgently needed to predict treatment response and tailor the management of AR patients. Third, research into novel vaccines and improvement of treatment schemes in AIT is needed to increase safety while maintaining or even enhancing the efficiency of AIT. Vaccines targeting B or T cells have been tried, and some of them have demonstrated promising results ,32. FourJournal of Clinical Medicine, which extends our knowledge of AR and gives us the confidence to fight against this highly prevalent disorder. The present Special Issue invites authors to contribute original research articles as well as review articles related to all aspects of AR in epidemiology, prevention and clinical treatment. We hope that our readers will enjoy this issue of the