Characterized by an excessively increased uric acid (UA) level in serum, hyperuricemia induces gout and also poses a great threat to renal and cardiovascular systems. It is urgent and meaningful to perform early warning by noninvasive diagnosis, thus conducing to blockage of disease aggravation. Here, guanidinocalix[5]arene (GC5A) is successfully identified from the self‐built macrocyclic library to specifically monitor UA from urine by the indicator displacement assay. UA is strongly bound to GC5A at micromolar‐level, while simultaneously excluding fluorescein (Fl) from the GC5A·Fl complex in the “switch‐on” mode. This method successfully differentiates patients with hyperuricemia from volunteers except for those with kidney dysfunction and targets a volunteer at high risk of hyperuricemia. In order to meet the trend from hospital‐centered to individual‐centered testing, visual detection of UA is studied through a smartphone equipped with a color‐scanning feature, whose adaptability and feasibility are demonstrated in sensing UA from authentic urine, leading to a promising method in family‐centered healthcare style. A high‐throughput and visual detection method is provided here for alarming hyperuricemic by noninvasive diagnosis. G value of images acquired with a portable smartphone.A noninvasive and individual‐centered method is accomplished for monitoring uric acid (UA) in urine by executing indicator displacement assay, which successfully differentiates hyperuricemia patients from volunteers and targets a volunteer at high risk of hyperuricemia. To fit the family‐centered healthcare style, optical supramolecular sensing of UA is achieved by reading  Hyperuricemia is the most crucial risk factor for the occurrence of gout triggered by the deposition of monosodium urate crystals around the joints derived from the supersaturation of urate in the blood. Also, hyperuricemia greatly increases the patient's susceptibility to chronic renal insufficiency, cardiovascular diseases, and diabetes, resulting in reduced quality of life. Therefore, it is of great significance to prevent and interrupt the pathophysiologic process of hyperuricemia by monitoring UA.Following hypertension, hyperlipidemia, and hyperglycemia, hyperuricemia has become one of the most common metabolic diseases,1 whic−6m for women and >416.5 × 10−6m for men) by enzyme‐coupling method, which requires invasive blood collection, specific instruments, and well‐trained hospital operators. Moreover, it can be a considerable challenge for the elderly and handicapped patients to accomplish the diagnosis. Nowadays, preventive medicine has entered the spotlight and attracted global attention, which will contribute to the economic utilization of medical resources and a great improvement in the quality of life for people. Accordingly, in order to realize the shift from hospital‐centered to family‐centered testing, there is an imperative need to develop a noninvasive, portable, accessible, and environment‐friendly method for daily monitoring based on the obvious fact that invasive blood collecting is not an ideal choice in family‐centered healthcare style.Generally, the clinical diagnosis of hyperuricemia is determined according to the blood UA level , and HPLC‐mass spectrometry, all of which can be employed to monitor UA in urine with the merits of high sensitivity and excellent specificity to facilitate the diagnosis of hyperuricemia. Noticeably, the use of harmful reagents as mobile phase or solvent poses a threat to the environment and human health, which deviates from the concept of green development. What is important is that the reported methods exhibit obvious limitations which hindered the individual‐centered monitoring of UA, as evidenced by the high cost of employing such professional instruments together with the laborious and time‐consuming operation for sample pretreatment.As the most optimal research object, urine can be noninvasively collected and carries important physical and chemical information related to many metabolic diseases, such as diabetes, gouty arthritis, and renal disease diacrisis.17 Man enabling the translation of the microcosmic behavior of molecular recognition into the macroscopic signal of fluorescence. Recently, IDA has become prevalent, which utilizes specific cavity microenvironments to perform intermolecular interactions with analytes to facilitate the determination of diagnostic biomarkers. In order to satisfy the diversified needs for monitoring analytes, supramolecular macrocycles, such as cyclodextrins (CDs), cucurbit[n]urils (CBs), and calixarenes (CAs), were designed to produce different cavities, charged head bases, and hydrophobic alkyl chains. By executing IDA, the ideal biomarkers of thrombotic diseases (trimethylamine N‐oxide) and renal dysfunction (creatinine) were successfully detected in body fluids via supramolecular sensing with macrocycles. Supramolecular sensing shows a promising prospect for application in family‐centered healthcare for disease diagnosis.As a rapidly evolving frontier cross‐discipline, supramolecular chemistry sheds the light on an approach to monitor the analytes which surpasses the classic spectral and chromatographic methods. As a facile, environmentally friendly, and high‐throughput supramolecular strategy, indicator displacement assay (IDA) offers an alternative noncovalent approach for determination by molecular sensing in aqueous solution with advantages of simplicity, modularity, and tunability,24 enaIn our study, a noninvasive and individual‐centered method to monitor UA in urine was successfully performed by a supramolecular optical strategy. For accomplishing the detection of UA through implementing IDA, the key challenge is to design artificial receptors that are capable of strongly and selectively binding UA in the assay. From ourself‐built macrocyclic library, including CAs, CDs, and CBs, guanidinocalix[5]arene (GC5A), was screened based on the association ability between macrocycles and UA by employing fluorescein (Fl) as a fluorescent indicator. A “switch‐on” sensing of fluorescence was successfully undertaken for UA, from which a synchronous increase in fluorescence signal was witnessed along with increasing UA level. Excitingly, we successfully applied the established method to distinguish hyperuricemia patients from volunteers except for those with renal impairment. Meaningfully, one volunteer was identified as a high‐risk person. With accessible smartphones equipped with color‐scanning function, the improved method enabled visual sensing of UA in urine from volunteer and hyperuricemia patient, which offers a prospective access to an early warning of dangerous UA level for family‐centered healthcare.22.1Ka) of the indicator‐host and analyte‐host complexes, a competitive analyte can reversibly displace the indicator from the artificial receptor in an IDA, causing the “switch‐on” or “switch‐off” of the fluorescence signal. Therefore, it is the prerequisite issue to screen UA artificial receptors from ourself‐established macrocyclic library for performing a successful assay of supramolecular sensing.In the light of association constant Ka of the Scheme Tableα‐cyclodextrin, α‐CD; β‐cyclodextrin, β‐CD; (2‐hydroxypropyl)‐β‐cyclodextrin, HP‐β‐CD; sulfobutylether‐β‐cyclodextrin, SBE‐β‐CD; methyl‐β‐cyclodextrin, Me‐β‐CD; γ‐cyclodextrin, γ‐CD), CBs , and CAs ‐31,32,33,34,35‐penta (4‐methylpentloxy)calix[5]arene, QAC5A; GC5A).In our macrocyclic library, seventeen macrocyclic receptors Scheme 1 were oα‐1,4‐glycosidic bonds with a hydrophilic external surface and a hydrophobic hollow cavity, conducing to the design of stimuli‐responsive supramolecular systems for the varied analytes. As for the indicators always combined with CDs, six reporter pairs of CDs were usually employed, including α‐CD·p‐dimethylaminobenzonitrile (DMABN),β‐CD·methylene blue (MB), HP‐β‐CD·MB, SBE‐β‐CD·neutral red (NR), Me‐β‐CD·NR, and γ‐CD·8‐hydroxypyrene‐1,3,6‐trisulfonate (HPTS), each of which was tested in monitoring UA. Unfortunately, for the reporter pair of α‐CD·DMABN, it was proved to be infeasible because of the apparent interference with the fluorescence signal of DMABN by UA styryl]‐1‐methylpyridinium iodide (DSMI), CB[7]·acridine orange (AO), and CB[8]·N,N’‐dimethyl‐2,7‐diazapyrenium (Me2DAP). By competitive fluorescence titrations, there was no binding toward UA obtained by the CBs hosts  modified with multiple sulfonate head groups were employed to monitor UA with the lucigenin (LCG) as an indicator. In the competitive fluorescence titration, the fluorescence signals of the complexes did not exhibit remarkable changes as UA level increased, suggesting that SCnAs are undesirable candidates for sensing UA  as the reporter pair . However, GC4A‐4C, GC4AOEG, and QAC5A were incapable of affording UA, leading to the unsatisfactory association constant detected by executing IDA with Fl, eosin Y (EY), and EY as the indicators, respectively  unlimited possibilities”.51 CAs2.2Ka = (5.0 ± 1.0) × 106m−1) by the fluorescence titration and UV–vis titration. Moreover, UA did not interfere with the fluorescence intensity of Fl in the range of 0–447.08 × 10−6m  between UA and GC5A  was mapped on the molecular van der Waals surfaces of GC5A and UA. A complementary manner of the MEP was witnessed between the positively charged guanidine group (blue areas) of GC5A and the negatively charged oxygen atoms (red areas) of UA in the GC5A·UA complex. Collectively, there are electrostatic, hydrogen bonding, π−cation interactions, and MEP that promoted the stable binding between GC5A and UA, contributing to the satisfactory performance for UA sensing by the GC5A·Fl reporter pair.The complexation of UA with GC5A was further confirmed by a 100 ns molecular dynamics (MD) simulation Figure . From th2.3R2 = 0.988) excited by the extruded Fl from the GC5A·Fl complex (0.80 × 10−6/1.00 × 10−6m) accompanied by the increasing UA level in HEPES buffer solution. The low detection limit (LOD) of UA was calculated to be 1.53 × 10−6m using the 3σ/slope method. In order to investigate the sensing specificity for UA by GC5A, typical endogenous substances in urine were added into the GC5A·Fl complex (0.80 × 10−6/1.00 × 10−6m), including xanthine (Xan), hypoxanthine (HX), allopurinol (All), guanine (Gua), creatinine (Cre), adenine (Ade), urea, ascorbic acid (AA), glucose (Glc), Cl–, K+, aspartic acid (Asp), glutamic acid (Glu), tyrosine (Tyr), arginine (Arg), human serum albumin (HSA), bovine serum albumin (BSA), and immunoglobulin G (IgG), respectively. The fluorescent interference caused by endogenous substances was negligible compared to the fluorescence response triggered by UA , including UA+HSA, UA+IgG, UA+Xan, UA+HX, UA+Arg, UA+K+, and UA+Glc, respectively. The addition of endogenous substances did not lead to a significant variation on the fluorescence response caused by UA  between fluorescence response and UA level (0–47.64 × 10−6m), as displayed in Figure −6m)  was revealed between fluorescence and the HPLC method. Evidently, according to the fluorescence response  between the volunteers (330.89 ± 44.71) and hyperuricemia patients. Also, it is a substantial difference (p < 0.001) in fluorescence signals from hyperuricemia patients (410.20 ± 19.37) and hyperuricemia patients with kidney dysfunction (307.11 ± 31.96). However, no discrepancy in fluorescence signals (p > 0.05) was witnessed between hyperuricemia patients with kidney dysfunction and volunteers, revealing that the kidney, an important excretion organ, would suffer from incapacitation to remove harmful metabolites from the body through urine with dysfunction. Obviously, UA level in the blood mostly depends on the balance between its production and excretion. Overproduction and/or underexcretion will lead to hyperuricemia. Prolonged hyperuricemia induces progressive gout with joint damage and chronic synovitis, which also threatens kidney function severely. This is how an unfortunate vicious circle starts. Therefore, as an early‐warning method, there exists an urgent demand for UA family‐centered and daily detection, which will facilitate the precaution of hyperuricemia.As shown in Figure uricemia.58 Pro2.5 we succeeded in performing the optical supramolecular sensing of UA with a smartphone equipped with a color‐scanning feature.Recently, a widespread outbreak of chronic diseases has further constrained limited medical resources, triggering increased overcrowding in the hospital and uncomfortable experiences of patients. Fortunately, with the popularization of smartphones and various applications, individual‐centered healthcare has come true. Inspired by the successful application of real‐time/on‐site monitoring of environmental pollutants and biological molecular,62 we Figure−6/5.00 × 10−6m), no marked difference is visually observed under daylight in a series of HEPES buffer solution with varying concentrations of UA (0–0.8 × 10−3m) added. The phenomenon that intrigued us was that as soon as the samples were excited by ultraviolet (366 nm), the response of green fluorescence  increased linearly in a UA concentration‐dependent pattern. Alternatively, same phenomena were witnessed in 200‐fold diluted urine from volunteer and hyperuricemia patient, which was further titrated using different concentrations of UA (0–0.8 × 10−3m). The additional UA was added into urine samples from volunteer and hyperuricemia patient to demonstrate the linear relationship between the UA level and the G value even in practical urine samples, which is informative for quantitative detection. This portable, noninvasive strategy enables the implementation of home‐based UA optical supramolecular sensing.As shown in 3G value of images with a portable smartphone. Collectively, this work offers a rapid and sensitive strategy for sensing UA in urine through noninvasive diagnosis, providing a promising access to home‐based UA optical supramolecular sensing in our daily life.In conclusion, from our self‐built macrocyclic library, the artificial receptor, GC5A, was identified to selectively and sensitively sense UA by employing Fl as an indicator in fluorescence “switch‐on” mode. Successful UA sensing was accomplished in artificial urine and authentic urine from human. Taking advantage of the specificity and reliability, we applied the established IDA method to distinguish volunteers from hyperuricemia patients with the exception of those suffering from hyperuricemia with kidney dysfunction, implicating that the excretory function of kidney plays an indispensable role in balancing the UA level in the blood. To accommodate the family‐centered healthcare style, optical supramolecular sensing of UA was exploratively carried out by reading the 4α‐CD, β‐CD, Me‐β‐CD, HP‐β‐CD, SBE‐β‐CD, γ‐CD, CB[6], CB[7], CB[8], Xan, HX, Cre, HSA, IgG, and methanol were purchased from Sigma‐Aldrich Co., Ltd. . HEPES and NR were provided by Tianjin Heowns Biochemical Technology Co., Ltd. . Glu, Asp, Tyr, Arg, Glc, and AA were manufactured by Yuanye Bio‐Technology Co., Ltd. . BSA was obtained from Beijing solarbio science and technology Co., Ltd. . MB was offered by Merck KGaA Co., Ltd. . Urea, potassium chloride, sodium chloride, potassium chloride, and sodium phosphate (monobasic) were bought from Tianjin Fengchuan Chemical Reagent Technology Co., Ltd. . Triethylamine was procured from Aladdin Biochemical Technology Co., Ltd. . Phosphoric acid was acquired from Tianjin Jinke Fine Chemical Industry Research Institute . Me2DAP was given as a gift from Prof. Frank Biedermann from Institute of Nanotechnology, Karlsruhe Institute of Technology . GC5A, QAC5A, SAC4A, GC4A‐4C, GC4AOEG, SC4A, SC5A, and SC6A were synthesized as previously reported.UA, Fl, EY, DSMI, HPTS, LCG, AO, RhB, reported.65−3m, pH = 7.4) was obtained by dissolving HEPES (2.38 g) in ultrapure water (1 L) at 25 °C. The HEPES buffer solution was further verified to pH 7.4 with sodium hydroxide (0.1 m). Artificial urine solution was prepared as previously reported.HEPES buffer solution (10 × 10Written informed consent was signed by 47 volunteers (25 ± 1 years old) and 13 patients (55 ± 18 years old). The acquired urine samples were stored at −80 °C before analysis. All urine specimens were collected from the midstream of morning urine with the corresponding supernatant collected after 15 min centrifugation (12 700 rpm) prior to analysis.−3m, pH = 7.4) at 25 °C under atmospheric pressure. From a 40.0 µL syringe, a constant volume (2.0 µL per injection) of the UA solution (500 × 10−6m) was injected into the reaction cell (280 µL) filled with α‐CD solution (50 × 10−6m) within 4 s under stirring (750 rpm). Eighteen successive injections were conducted to record the microcalorimetric titration curve of the complexation of host·guest.The ITC measurements were undertaken by a fully computer‐operated isothermal calorimetry instrument . All titrations were performed in HEPES buffer solution (10 × 103 quartz cell (light path = 10 mm) by Varian Cary Eclipse spectrometer  equipped with a single‐cell peltier accessory. In order to obtain the association constant (Ka), data were fitted by a host–guest binding stoichiometry (1:1) in the direct host–guest titrations. From the competitive titrations, the data fitting was nonlinearly conducted by a 1:1 competitive binding model. All experiments were carried out in HEPES buffer solution  at 25 °C. The calibration lines were established at 25 °C in the light of the fluorescence responses of GC5A·Fl reporter pair upon gradual titration of UA in HEPES buffer solution, artificial urine solution, and urine from volunteers, respectively.The steady‐state fluorescence spectra were recorded in a conventional 10 × 10 × 45 mmonstant Ka, data wonstant Ka, data wx¯±SD . Data were fitted in a nonlinear manner for direct host·guest titrations and competitive titrations, by using the fitting modules downloaded from the website of Prof. Nau's group (http://www.jacobs‐university.de/ses/wnau) under the column of “Fitting Functions”.The experiments were repeated three times in this study with the results expressed astrations,67 by t‐test. Significance was indicated by asterisks: ***p < 0.001. Pearson correlation analysis was conducted to test the correlation.Data obtained from this study were used without preprocessing unless otherwise stated. Data were expressed as the mean ± SD for three independent experiments. The difference between two groups was analyzed using Student's The Originpro 2016 software  and SPSS 21.0 software  were used for the statistical analysis. All figures were drawn by Originpro 2016 software  and Adobe Illustrator CC2020 . with the CHARMM36 force field and the TIP3P water model were employed to perform MD simulation. With the visual molecular dynamics (VMD) program , visualization and analysis of all the MD trajectories were carried out. By adding four Cl– ions, the GC5A·UA complex was overall charge neutrality. Before simulation, the system  underwent 5000 steps minimization and 100 ps MD simulation with host·guest restrained, and 2 ns water equilibration without restrained. Then, the interaction between UA and GC5A was systematically investigated by performing a 100 ns MD simulation.The scalable program nanoscale molecular dynamics (NAMD) 2.1469 witQuantification of UA in urine from volunteers and hyperuricemia patients was executed on an LC‐20AT HPLC system . After deproteinization with methanol (1:1), all urine samples were diluted fivefold by 50% methanol aqueous solution (v/v), which were centrifuged for 15 min at 12 700 rpm. The assay was performed in triplicate for all samples. A series of UA standard solutions with different concentrations were employed for the construction of the calibration curve.–1 and 10 µL, respectively.0.1% Phosphoric acid aqueous solution with 0.05% triethylamine (v/v) (A) and methanol (B) were applied for excellent separation of UA on a Diamonsil C18 column  at 30 °C. The gradient program was undertaken as follows: 5% B in 0–4 min and 5–30% B in 4–13 min. The flow rate and injection volume were 1 mL min−6/5.0 × 10−6m) reporter pair was employed to sense UA (0–0.8 × 10−3m) in HEPES buffer solution , and the 200‐fold diluted urine from volunteer and hyperuricemia patient, respectively. Sample solutions in 2.0 mL centrifuge tubes  were excited with ultraviolet (366 nm) in thin layer chromatography scanner . Then, the excited fluorescence from samples was scanned by an iPhone XR , which was processed by a color scanning application from Apple Store .The GC5A·Fl (10.0 × 10The authors declare no conflict of interest.Y.Z., H.Y., and S.C. contributed equally to this work. Y.Z., H.Y., D.‐S.G., and Y.W. conceived the experiment and designed the study. S.C. collected urine samples from patients in the hospital. W.‐C.G. and J.‐J.L synthesized host macrocycles. W.‐C.G. performed the theoretical calculation. Y.Z., H.Y., S.C., and Y.‐X.Y. performed all the other experiments. S.C., X.C., and L.W. investigated the project and analyzed data. Y.Z., H.Y., W.‐C.G., D.‐S.G., and Y.W. contributed to writing of this paper and all authors commented on it.Supporting InformationClick here for additional data file.


When small proteins such as cytokines bind to their associated receptors on the plasma membrane, they can activate multiple internal signaling cascades allowing information from one cell to affect another. Frequently the signaling cascade leads to a change in gene expression that can affect cell functions such as proliferation, differentiation and homeostasis. The Janus kinase-signal transducer and activator of transcription (JAK-STAT) and the tumor necrosis factor receptor (TNFR) are the pivotal mechanisms employed for such communication. When deregulated, the JAK-STAT and the TNF receptor signaling pathways can induce chronic inflammatory phenotypes by promoting more cytokine production. Furthermore, these signaling pathways can promote replication, survival and metastasis of cancer cells. This review will summarize the essentials of the JAK/STAT and TNF signaling pathways and their regulation and the molecular mechanisms that lead to the dysregulation of the JAK-STAT pathway. The consequences of dysregulation, as ascertained from founding work in haematopoietic malignancies to more recent research in solid oral-gastrointestinal cancers, will also be discussed. Finally, this review will highlight the development and future of therapeutic applications which modulate the JAK-STAT or the TNF signaling pathways in cancers. Cells can communicate with each other and this is frequently accomplished by molecules, that are produced by one cell, then detected and interpreted by another. One class of these signaling molecules are secreted proteins, including cytokines, interferons and growth factors, otherwise known as ligands. These are detected by discrete receptor molecules which then initiate a cascade of signaling responses in the receptor cell that may result in, for example, small or large, acute or chronic (days to months) and reversible or irreversible changes (live or die) in the receptor cell. It is rare for particular receptor ligand combinations to work in isolation or to signal only one specific outcome, i.e. one signaling event may initiate multiple signaling branches that may in turn intersect, overlap or be affected by other signaling branches occurring concurrently. This makes analysis of signaling challenging but permits cells to respond with a degree of finesse to environmental cues.The term cytokine encompasses interleukins, chemokines, interferons and tumour necrosis factors and all bind to corresponding receptors to activate cellular signaling pathways, including two related and evolutionary conserved signature signaling entities, the Janus Kinases (JAK: a family of tyrosine kinases) and the Signal Transducer and Activator of Transcription (STAT) protein family, which together constitute the JAK-STAT signaling pathway and has been studied in great detail –5. Anoth  , a kinase (JAK kinase) and finally a transcription factor (STAT proteins) , 19, cou, 21, 22 , 5, 23. (NLS)  . Within (NLS)  , 25, 26]via . In addition, the idea of hierarchical role for JAKs, with one JAK family member functioning as the upstream activator of a neighbouring heterotypic JAK family member within a cytokine receptor complex is another factor contributing to their functional complexity  resultind IGF1R   are the main cells that express STAT1 and p-STAT1 , an inhibitor of T-cell-mediated tumor cytotoxicity , are major cellular components of the OSCC stroma and communicate with tumor cells to stimulate cancer cell growth, survival, and invasion and associated with poorer survival outcomes. Epiregulin (EREG) a member of the Epithelial Growth Factor (EGF) family promotes tumor development, migration and invasion . We receJAK2, as well as 38 other genes  or Epstein-Barr virus (EBV) infection or prolonged exposure to gastric irritants, such as a diet high in salt and nitrated foods   . A smallpe (CIN) . Many cype (CIN) .CDH1) resulting in an autosomal dominant GC predisposition  and PDCD1LG2 (PD-L2) were also described in the EBV+ GC subtype (CD274 (PD-L1) and PDCD1LG2 (PD-L2) are involved in immunosuppression by tumor cells to evade cytotoxic T cell mediated killing and are associated predominantly with the EBV+ sub-type . Signalling through Gp130, a shared receptor element of many of these transducers, is often dysregulated in GC due to elevated levels of IL-6 and IL-11  mouse, which carries a homozygous mutation (Y757 residue) in the gp130 receptor chain , a member of the canonical NF-κB signaling pathway develop inflammation-driven invasive gastric adenocarcinoma  are the mainstays for advanced OSCC/HNSCC, because effective and targeted therapies which are still wanting , 217. SiTherapeutic targeting of STAT transcription factors is not without challenges. Transcription factors have been deemed “undruggable” due to their lack of catalytic function , 222. WhSTAT1 inhibitors are still under development using comparative virtual screening and docking validation . An impoBecause of its strong pro-oncogenic function, most STAT inhibition studies have focused on this transcription factor with the aim of blocking phosphorylation and/or STAT dimerization . However). STAT3 signaling activity can also be attenuated by Stattic, a small molecule STAT inhibitor, which targets the SH2 domain, resulting in the modulation of invasion and migration of OSCC cell lines  cancer showed no improvement in overall or progression free survival , which have been documented in many phase I trials elected; Table 1lected;  and is clected; . Phase Ilected;  and locaThe small-molecule competitive STAT3 inhibitor TTI-101 (formerly C188-9) developed by Tvardi, targets the pY-peptide binding site within the SH2 domain . TTI-101-SOCS3) or recombinant, cell-permeable proteins has been shown to efficiently control aberrant STAT signaling in hepatocellular carcinoma . A SOCS1, 2A) and TNF, which signals through the TNF family .Another approach to enlist TNF signaling are the smac-mimetics, a drug class that antagonises Inhibitor of APoptosis proteins . Several small molecule smac-mimetic compounds have been developed and many have been or are currently in clinical trials, including Debio 1143/AT-406/SM-406, LCL161, APG1387, BI 891065 and ASTX660 , 282. Th+ also a subtype which might benefit)  boosts the cytotoxic activity of tumour antigen-specific T cells and has revolutionized treatment and increased survival for a subset of cancers , 293. Bobenefit) . These sbenefit) . Howeverbenefit) . These cbenefit) , 298.+ GC, which exhibits amplified JAK2, ErbB2, PD-L1 and PD-L2   for recurrent GC based on TCGA sub-type and trialling immunotherapy  plus ipatasertib (AKT inhibitor) for EBVnd PD-L2 Table 1.2646748) –301.Cytokine therapy is challenging because of their high degree of pleiotropism, resulting in many adverse effects. However, their real value in cancer therapy, particularly for oral and gastric cancers may be in enhancing the safety of immunotherapies. Thus, ICI or CAR-T cell therapy can result in inflammatory toxicities, such as immune-related adverse events (irAEs) or cytokine release syndrome (CRS). These are life threatening systemic diseases affecting multiple organs , 303. ByImprovements in selectivity and prevention of drug resistance in cancer treatment may be achievable with a novel approach using Proteolysis Targeting Chimeric (PROTAC) technology developed by the Crews and Deshaies labs , 307. InL  using an alternative E3 ligase, VHL (von Hippel-Lindau) for both hematologic and solid tumours  as the E3 ligase . These P tumours . The suc tumours . Pertine tumours , 319 and tumours  and JAK- tumours  signalinin vivo was also achieved with SD-36 and was well tolerated, bringing this PROTAC into the realm of possible therapeutic utility , of proximal membrane bound proteins , 325. Soytokines Figure 2In vitro studies have shown that IRX-2, enhances DC maturation, T cell activation, and NK cell stimulation, to overcome tumor-mediated immunosuppression by activating the tumor environment , the yellow pigment found in turmeric. This natural compound targets many signaling pathways and has been shown to have multiple anti-inflammatory and anti-tumor qualities including anti-tumor metastatic activities through a variety of mechanisms . High doJAK-STAT/cytokine signaling pathways have been comprehensively studied due to their pivotal roles in both inflammation and many disease conditions including cancer. The core JAK unit activating the transcription factor STAT element is triggered by a variety of receptors and activates a wide range of downstream targets. Deregulated JAK-STAT signalling and particularly dysregulation of JAK2, STAT3, Il-6 and TNF are commonly associated with driving inflammation and carcinogenesis, including both OSCC and GC. Therefore, the targeting of these signaling pathways with inhibitors holds potential as clinical interventions for these cancers. There are a number of specific inhibitors of the JAK-STAT/cytokine cascade that have already been approved by the FDA for inflammatory conditions and which are being investigated for efficacy in clinical trials for haematopoietic malignancies and a few select solid tumors. However, despite promising clinical outcomes in inflammatory immune diseases and in tumor animal studies, toxicity issues have hindered adoption of these therapeutic approaches in the clinic. Investigating other avenues, such as the addition of cytokine blockade or smac-mimetics may provide synergy for more efficacious inhibition of OSCC and GC tumorigenic progression. More recent advances such as blocking JAK-STAT signaling with small molecules such as PROTACs may provide even better therapeutic precision in conjunction with other anti-cancer agents already in the clinic such as immunotherapy.LO’R wrote the original review and YN, JL and JS reviewed edited and assisted with figures. All authors have read and agreed to the published version of the manuscript.This review was in part funded by a Cancer Council Victoria Grant-in-Aid project, grant number 1161400 (to LO’R), a Cancer Australia and a Cancer Council New South Wales project grant number G20-13 (to LO’R), the Victorian State Government (OIS grant), NHMRC fellowships to (to JS) NHMRC 1107149 & 1195038 and National Natural Science Foundation of China Grant Nos. 81772880, 82173159 (to YN), a Key Research and Development Project (Jiangsu Province) No.BE2020628 (to YN).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Human T lymphotropic virus 1 (HTLV-1) may be transmitted from mother to child and affects at least 5–10 million individuals worldwide, with severe consequences on health. Strategies to prevent transmission are important, as there is no treatment or vaccine. This systematic review aimed to identify interventions to prevent HTLV-1 mother-to-child transmission and to determine their effectiveness. Exclusive formula feeding, short-term breastfeeding, use of freeze–thaw milk, milk pasteurization, maternal and infant antiretroviral drugs, caesarean section, early clamping of umbilical cord, screening of milk donors and avoidance of cross-breastfeeding were identified as possible strategies. Avoidance of breastfeeding is an intervention that prevents 85% of transmissions. This strategy is recommended in Japan, Brazil, Colombia, Canada, Chile, Uruguay, the USA and some regions of French Guyana. Whilst breastfeeding for <3 months does not increase the risk of transmission compared to exclusive formula-feeding, concerns remain regarding the limited number of studies outside Japan, and the lack of information on women having higher risk of HTLV-1 transmission and on the ability of women to discontinue breastfeeding. Additional interventions are plausible, but data on their effectiveness are limited. The acceptance of interventions is high. These findings may guide healthcare professionals and support policymakers in implementing policies to avoid HTLV-1 mother-to-child transmission. Human T lymphotropic virus type 1 (HTLV-1) is a retrovirus that may be transmitted from seropositive mothers to their babies. It is estimated that 20% of babies breastfed from women living with HTLV-1 will become infected. Mother-to-child transmission (MTCT) also occurs in approximately 2.5–5% of exclusively formula-fed babies born from seropositive mothers, indicating that there are other transmission routes, although less common .Remark 10.Training of healthcare workers is essential to guarantee that they can support women to make an informed decision regarding strategies to prevent HTLV-1 mother-to-transmission.Arguments on the psychological distress of HTLV-1 antenatal screening on women, as well as the impact of interventions on women’s wellbeing and health, have been raised. Evidence regarding the negative impact of such interventions is lacking. In addition, the impact that of the lack of interventions has on families in which transmissions have occurred is not documented. In Brazil, although women living with HTLV-1/2 may experience anxiety, sadness, fear and guilt  about reAs familiar aggregation is common in HTLV-1 infection ,91,92,93An integral approach is important to ensure that healthcare professionals are properly trained and that pregnant women have the support needed before screening and during notification of test results, and advice on strategies to reduce risk of transmission. A good practice was described in Bahia, Brazil, where a multidisciplinary centre organizes workshops for women living with HTLV-1 regarding strategies to increase the mother/child bond during bottle feeding and provides psychological support during and after pregnancy . This integral support is important as many women have questions regarding preparation of formula and have difficulties following medical advice. In Kagoshima, 1/8 of women who choose short-term breastfeeding failed to stop at the recommended time. A trial with regular visits of public health nurse has started in the area , but theRemark 11.Mothers living with HTLV-1 may persist breastfeeding for longer than planned. Integral approach is essential to support those women living with this virus.Refraining from breastfeeding and provision of formula is the intervention with the largest body of evidence and is considered the most effective strategy currently available preventing 87% of early-life infections. This intervention has been recommended in the prefecture of Nagasaki since the establishment of antenatal screening, in the late 1980s. This is also recommended in Brazil, Chile, Uruguay, Canada, Colombia, the USA and some regions of French Guyana. When exclusive formula feed is not AFASS, short-term breastfeeding (up to 3 months) should be considered. Acceptance of HTLV-1 antenatal screening and interventions to limit or avoid breastfeeding is high (>90%). New interventions that will enable seropositive women to breastfeed their babies without imposing risk of HTLV-1 transmission are welcome, but in the meantime, there are interventions, such as exclusive formula-fed, that are highly effective to prevent HTLV-1 mother-to-child transmission and the implementation of such policies should be encouraged in all regions where HTLV-1 infections occur.


This study aims at exploring the effect of obstructive sleep apnea–hypopnea syndrome (OSAHS) on the liver and kidney function indexes of patients and analyze the changes in these indexes after minimally invasive surgery. n = 51) who were diagnosed via polysomnography (PSG) and received minimally invasive surgery in the sleep disorders diagnosis and treatment center of the West China Fourth Hospital of Sichuan University from January 2017 to January 2019 were selected as test subjects and placed in the OSAHS group. At the same time, 79 healthy people with no snoring or breathing difficulties were selected from the medical examination center of the hospital as the control group . These two groups were used to compare the differences in the related indexes of serum liver and kidney function and evaluate the changes in sleep monitoring and related liver and kidney function indexes in patients with OSAHS after minimally invasive surgery.  Patients with OSAHS . Of the patients with OSAHS, 17 were followed up for one year. After minimally invasive surgery, ALT decreased from 44.29 ± 20.61 to 26.47 ± 9.91 (t = 4.395), AST decreased from 27.71 ± 8.32 to 21.82 ± 4.81 (t = 3.673), and UA decreased from 397.35 ± 92.14 umol/L to 362.94 ± 106.76 umol/L (t = 2.580), and these differences were statistically significant (p < 0.05).The changes in ALT (r = −0.635) and AST (r = −0.504) were related to the difference in the lowest blood oxygen saturation (p < 0.05), and the change in UA was related to the difference in the apnea–hypopnea index .  The alanine aminotransferase (ALT), aspartate aminotransferase (AST), and uric acid (UA) levels were higher in the OSAHS group  than in the control group (21.91 ± 11.61, 22.18 ± 6.19, and 330.49 ± 64.45 and  There are some abnormalities in liver- and kidney-function-related indexes in patients with OSAHS, and minimally invasive surgery can help to improve liver and kidney function in these patients. Obstructive sleep apnea–hypopnea syndrome (OSAHS) refers to an apnea–hypopnea index (AHI) ≥ 5 times/hour in a night consisting of at least seven hours of sleep, and it is a clinically common sleep and respiratory disease with obstructive respiratory events . PatientOSAHS group: a total of 51 patients who came to the Department of Otorhinolaryngology of the West China Fourth Hospital of Sichuan University for snoring and sleep apnea, were diagnosed by polysomnography (PSG) as having OSAHS, and who received minimally invasive surgery from January 2017 to January 2019 were selected as the OSAHS group.Control group: a total of 79 healthy people with no snoring or sleep apnea symptoms were selected from the physical examination center of the Fourth Hospital of West China as the control group which was diagnosed by PSG (AHI<5).Exclusion criteria were as follows: (1) having received OSAHS-related treatment before the current treatment, such as surgical, drug, or continuous positive pressure ventilation treatment; (2) suffering from serious cardiovascular, cardiopulmonary, or cerebrovascular diseases; malignant tumors and other organics; sexual diseases; insomnia; or anxiety, depression, or other neuropsychiatric diseases; and (3) having incomplete clinical case data.This study was conducted with approval from the Ethics Committee of West China School of Public Health and West China Forth Hospital (HXSY-EC-2021022). This study was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants. All subjects were informed of the purpose of the study and agreed to their clinical data being used for the study.2), and the microarousal index.In this study, the German SOMNO screen™ plus PSG monitor was used for sleep monitoring for at least seven hours. All study subjects were told in advance not to overwork; take drugs that affect sleep; or drink alcohol, coffee, strong tea, or other beverages on the day of sleep monitoring to ensure normal sleep at night. After the sleep monitoring, data were automatically analysed by the computer and checked by a sleep technician. The data collected were the apnea–hypopnea index (AHI), lowest arterial oxygen saturation , aspartate aminotransferase (AST), urea, creatinine, uric acid (UA), and other indicators. All the above hematological examinations were conducted by the laboratory department of the West China Fourth Hospital of Sichuan University.Inferior turbinate: by using the low-temperature plasma system (Reflex45 cutter head), surgical electrodes are inserted into the middle and lower portions of the front edge of the inferior turbinate in multiple levels, and advanced from front to back until they are close to the back of the inferior turbinate, and they ablate for about 10–15 seconds.Oropharyngeal plane: ablate the soft palate, uvula, and tonsils, and make an outward 45° “V”-shaped incision along the junction of the soft palate and the uvula on both sides with a radio frequency electric knife (U-shaped blade) to the tongue and palate arch and remove the surrounding hypertrophic palate tissue. For the thick uvula, part of the uvula can be cut off.Tongue plane: A low-temperature plasma system (Reflex55 cutter head) was used, which was generally inserted in and around the midline of the tongue base. 4 or 6 treatment points (1.0∼1.5 cm apart) were selected to insert electrodes into the mucosa, and then they were moved backward and downward for about 1.0 cm, lasting for about 10 s.The diagnostic and treatment center for sleep and respiratory diseases at the Fourth Hospital of West China developed a minimally invasive surgery system by combining the American Ellman radiofrequency knife and the low-temperature plasma system , which hThe patients were observed in the postoperative ward for 5–7 days, and intravenous antibiotics were used to prevent infection and were supplemented with local semiconductor laser irradiation treatment for inflammation and swelling.t-test was used for comparison between groups, and the paired t-test was used for comparison before and after minimally invasive surgery. The correlation analysis for continuous variables was done using Pearson correlation analysis. p < 0.05 was considered to be statistically significant.The EpiData 3.3 software was used to input the data, and SPSS 22.0 was used for statistical analysis. Quantitative data were expressed as mean ± standard deviation, and count and grade data were expressed as a frequency or composition ratio. The independent group p > 0.05).As shown in t = 6.514, 3.549, and 6.373, respectively; p < 0.05). This suggests that patients with OSAHS may have abnormal liver and kidney metabolism.As shown in 2 increased from 71.78 ± 11.87% to 78.70 ± 11.58%, ALT decreased from 44.29 ± 20.61 U/L to 26.47 ± 9.91 U/L, AST decreased from 27.71 ± 8.32 U/L to 21.82 ± 4.81 U/L, and UA decreased from 397.35 ± 92.14 umol/L to 362.94 ± 106.76 umol/L (p < 0.05).In this study, a total of 17 patients with OSAHS were followed up after minimally invasive surgery. As shown in 2 , AST and LsaO2 , and UA and AHI .As shown in Previous studies have found that even if patients with OSAHS did not have obesity or metabolic syndrome, the prevalence of NAFLD would still increase significantly, and the severity of NAFLD was related to the increase in the severity of OSA . In addip < 0.05). Qu Yinfei [r = 0.293, p < 0.05 and r = 0.275, p < 0.05), and ALT levels were negatively correlated with LSaO2 , suggesting that as the severity of OSAHS increases, ALT and AST levels increase. ALT and AST are the most sensitive test indicators of liver function damage. Many studies have shown that the severity of OSAHS is a risk factor for increased serum aminotransferase [The levels of ALT and AST in the OSAHS group were higher than in the control group, which is consistent with previous studies. Atan Doğan's  study ofu Yinfei  found thnsferase . Patient2 , suggesting that patients with OSAHS are at risk of renal impairment.In this study, the OSAHS group had higher levels of UA than the control group. Increased blood UA is primarily seen in patients with gout, and it is closely related to renal dysfunction. Studies have shown that with the continuous progression of chronic kidney injury, the glomerular filtration rate gradually decreases, and the ability of the kidney to excrete metabolic waste also decreases, which is manifested by rising continuous blood UA levels. UA is an index that is used clinically to assess the impairment of renal function. Asymptomatic patients with hyperuricemia are 1.6 times more likely to have chronic kidney injury than the general population . This st2 increased, which objectively indicated that minimally invasive surgery improved the nighttime breathlessness and hypoxia of the patients, reduced the number of nighttime awakenings, and improved sleep quality. These findings are consistent with previous studies [After minimally invasive surgery in patients with OSAHS, AHI decreased and LsaO studies , 12, and2. It shows that minimally invasive surgery can reduce serum transaminases in patients with OSAHS, which may be related to the reduction of postoperative dyspnea, improvement of nocturnal hypoxia, and reduction of oxidative stress.There is no report of improvement in liver- and kidney-function-related indexes in patients with OSAHS who receive minimally invasive surgery. This study found that after minimally invasive surgery, the ALT levels decreased from 44.29 ± 20.61 to 26.47 ± 9.91 and the AST levels decreased from 27.71 ± 8.32 to 21.82 ± 4.81. Using correlation analysis, these decreases were found to be statistically significantly correlated with the change in LsaOThis study also found that after minimally invasive surgery, the UA levels in patients with OSAHS decreased, but no changes in urea or creatinine levels were found. There were no significant differences in the urea and creatinine levels between the OSAHS and the control group. Urea and creatinine are common indicators used in the clinical assessment of renal impairment, but the results are affected by many factors, such as age, gender, and muscle metabolism, and abnormalities often appear in the late stage of renal injury. However, the reduction in UA in patients with OSAHS after minimally invasive surgery was statistically significant, suggesting that minimally invasive surgery can improve uric acid metabolism in OSAHS patients.However, our study still has limitations. The sample size we included is small, and the follow-up time is only 1 year. We will continue to expand the sample size and extend the follow-up time in future studies, looking forward to more accurate data.In summary, this study showed that there are certain abnormalities in the levels of liver and kidney function indicators in patients with OSAHS, and minimally invasive surgery treatment can help to improve liver and kidney function damage in these patients. The research conclusions will be further confirmed by future follow ups , 16, 17.


The hippo signaling pathway is a highly conserved evolutionary signaling pathway that plays an important role in regulating cell proliferation, organ size, tissue development, and regeneration. Increasing evidences consider that the hippo signaling pathway is involved in the process of respiratory diseases. Hippo signaling pathway is mainly composed of mammalian STE20-like kinase 1/2 (MST1/2), large tumor suppressor 1/2 (LATS1/2), WW domain of the Sav family containing protein 1 (SAV1), MOB kinase activator 1 (MOB1), Yes-associated protein (YAP) or transcriptional coactivator with PDZ-binding motif (TAZ), and members of the TEA domain (TEAD) family. YAP is the cascade effector of the hippo signaling pathway. The activation of YAP promotes pulmonary arterial vascular smooth muscle cells (PAVSMCs) proliferation, which leads to pulmonary vascular remodeling; thereby the pulmonary arterial hypertension (PAH) is aggravated. While the loss of YAP leads to high expression of inflammatory genes and the accumulation of inflammatory cells, the pneumonia is consequently exacerbated. In addition, overexpressed YAP promotes the proliferation of lung fibroblasts and collagen deposition; thereby the idiopathic pulmonary fibrosis (IPF) is promoted. Moreover, YAP knockout reduces collagen deposition and the senescence of adult alveolar epithelial cells (AECs); hence the IPF is slowed. In addition, hippo signaling pathway may be involved in the repair of acute lung injury (ALI) by promoting the proliferation and differentiation of lung epithelial progenitor cells and intervening in the repair of pulmonary capillary endothelium. Moreover, the hippo signaling pathway is involved in asthma. In conclusion, the hippo signaling pathway is involved in respiratory diseases. More researches are needed to focus on the molecular mechanisms by which the hippo signaling pathway participates in respiratory diseases. Hippo signaling pathway, as a highly conserved evolutionary pathway, plays an important role in regulating cell proliferation, organ size, tissue development, and regeneration.Accumulative evidences suggest that the hippo signaling pathway is involved in respiratory diseases.YAP promotes PAVSMCs proliferation and pulmonary vascular remodeling via PI3K/AKT pathway; thereby PAH is aggravated.When the stiffness of the extracellular matrix increases, YAP enters the nucleus and binds to TEAD to promote Twist1 gene transcription, cell proliferation, and collagen deposition, which cause the pulmonary fibrosis.What are the core components of the hippo signaling pathway in cells?How the hippo signaling pathway plays the functions in cells?What are the mechanisms that the hippo signaling pathway regulates respiratory diseases?The hippo signaling pathway is first identified in Drosophila melanogaster during screening for genes that negatively regulate tissue growth and is a highly conserved evolutionary signaling pathway that plays a central role in controlling tissue homeostasis, development, regeneration, and organ size through the regulation of cell proliferation and apoptosis –3. IncreThe core components of the hippo signaling pathway consist of a kinase cascade transcription of the upstream and effector factors of the downstream , 13. TheYes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) is the downstream effector of hippo signaling pathway and regulates the expression of target genes , 12. WheThe hippo signaling pathway is regulated by G-protein-coupled receptors, mechanical cues, cell adhesion, cell polarity link-related proteins, and cell energy state , 28, 29.G-protein-coupled receptor (GPCR) and its related ligands regulate YAP activity and the expression of YAP by regulating LATS1/2 kinase , 31. StuIn addition, the hippo signaling pathway is regulated by mechanical cues such as cell density, mechanical tension, and extracellular matrix stiffness . In diffAdditionally, it is reported that cell adhesion inhibits LATS1/2 by stimulating the FAK-SRC-PI3K-PDK1 pathway and then induces YAP transferring into the nucleus . AccordiMoreover, proteins involved in cell polarity and cell connection may play an important role in regulating the hippo signaling pathway –45. By tAdditionally, when cells suffer from energy starvation, AMPK activates and directly phosphorylates the amino acid residues of Ser94 in YAP, which inhibits YAP binding to TEAD, and then the YAP is inhibited . MoreoveALI is characterized by the damage of alveolar epithelial cells and pulmonary capillary endothelial caused by noncardiogenic factor and results in acute hypoxic respiratory insufficiency , 53. SevThe respiratory epithelial cells of mature lung are stationary under normal physiological conditions, but a variety of epithelial cells, such as type II alveolar epithelial cells (AECIIs) and basal cells, significantly regenerate when lung is injured –60. It iThe basal stem/progenitor cells (BSCs) are activated and recruited to the site of injury in lung, where they help to regenerate the lung epithelium . DiffereMoreover, alveolar epithelial cells are repaired by exogenous bone marrow-derived mesenchymal stem cells (BMSCs) and down-expression of LATS2 improves BMSCs repairing the tissue in ALI, thereby alleviates the pathological damage of lung tissue . In addiIn addition, the repair of pulmonary capillary endothelial is important for ALI. YAP promotes the germination and remodeling of neovascules through various angiogenesis factors such as Ang2, MMP2, VE-cadherin, α‐SMA, and PGC1α , 69. KnoPAH is a chronic, progressive pulmonary vascular disease with abnormally elevated pulmonary arterial pressure , 77.Pathogenesis of PAH is associated with pulmonary artery cell proliferation, vascular remodeling, increased anti-apoptosis, and orthotopic thrombosis , 79. VasIn addition, S1P promotes the activation of signal transduction and transcriptional activator 3 (STAT3) through S1PR2 or autocrine loop signaling, which further leads to STAT3 translocating to the nucleus and leads to reduced ubiquitination degradation of YAP in PAVSMCs , 83. MeaPneumonia refers to inflammation of the terminal airway, alveoli, and interstitium of the lung and is caused by pathogenic microorganisms, immune damage, physical and chemical factors, and allergies –90. BactThe expression and nuclear localization of YAP in AECIIs were significantly increased in mice with bacterial pneumonia, which causes the production of inflammatory cytokines and affects the activation of YAP-IκB . IκB is IPF is a chronic, progressive, age-related, irreversible, fibrotic interstitial lung disease characterized by excessive deposition of extracellular matrix proteins and destruction of alveolar structures –99. MostLung fibroblasts are the main effector cells of IPF, and the YAP mediates the proliferation, migration, and collagen deposition of lung fibroblasts induced by mechanical signals , 101. WhMoreover, IPF development is a SMAD-3-dependent process, which increases collagen deposition in AECs. It is reported that YAP promotes the expression of SMAD-3 in pulmonary fibrosis mice . In addiTransforming growth factor-β (TGF-β) promotes the expression of plasminogen activator inhibitor-1 (PAI-1), and then PAI-1 promotes fibrosis and regulates degradation of fibrin and stromal adhesion of lung fibroblasts by affecting pericellular plasminase activity, which results in IPF . SerpineAsthma affects >300 million people around the world and its prevalence is increasing –110. TheThe immune tolerance disorder of Notch4-mediated is a main mechanism that induces chronic inflammation in asthma. It is suggested that the Notch4 causes Treg cell dysfunction through the hippo signaling pathway, which promotes allergic airway inflammation . When alAbnormal proliferation and migration of ASMCs play an important role in airway hyperresponsiveness and airway remodeling in asthma , 117. S1The hippo signaling pathway is mainly composed of MST1/2, LATS1/2, SAV1, MOB1, YAP, or TAZ, and TEAD and is involved in ALI, PAH, pneumonia, IPF, and asthma. YAP/TAZ, as the cascade effector of the hippo signaling pathway, plays an important role in these respiratory diseases. When YAP is activated, YAP transfers from the cytoplasm to the nucleus and accumulates in the nucleus, thereby acting on target genes such as Twist1 and Serpine1. Subsequently, the activation of target genes affects the proliferation and migration of cells such as AECs, PAVSMCs, lung fibroblasts, and AECIIs. However, the specific mechanisms of the hippo signaling pathway regulating respiratory diseases remain unclear and more researches are needed in further.


Hepatozoon spp. have been mainly studied in domestic animals and peri-urban areas. The epidemiology of Hepatozoon spp. is poorly investigated in natural systems and wild hosts because of their scarce veterinary and economic relevance. For most habitats, the occurrence of these parasites is unknown, despite their high ecosystemic role. To fill this gap for alpine small mammals, we applied molecular PCR-based methods and sequencing to determine the Hepatozoon spp. in 830 ear samples from 11 small mammal species  live-trapped during a cross-sectional study along an altitudinal gradient in the North-Eastern Italian Alps. We detected Hepatozoon spp. with an overall prevalence of 35.9%. Two species ranging from 500 m a.s.l. to 2500 m a.s.l. were the most infected: My. glareolus, followed by Apodemus spp. Additionally, we detected the parasite for the first time in another alpine species: C. nivalis at 2000–2500 m a.s.l. Our findings suggest that several rodent species maintain Hepatozoon spp. along the alpine altitudinal gradient of habitats. The transmission pathway of this group of parasites and their role within the alpine mammal community need further investigation, especially in consideration of the rapidly occurring environmental and climatic changes.Among the Apicomplexa parasites,  Plasmodium spp. or the Toxoplasma spp. transmitted in urban cycles that include domestic species and humans. Other protozoan parasites, such as the Hepatozoon genus represented by 340 species, have also been commonly recorded in peri-urban cycles sustained by domestic species  [Hepatozoon spp. have been detected in amphibians, reptiles, birds that act as intermediate hosts, and mammals, which can be both paratenic and reservoir hosts [Protozoa parasites are a highly diverse group of successful organisms globally distributed via a wide range of hosts  and whosnd dogs) ,6,7,8, and dogs) ,10. Furtir hosts .Hepatozoon spp. can occur through different modalities that most often involve the ingestion of infected vectors or hosts. In mammals, for example, animals can become infected through grooming behavior [The transmission of behavior  by eatinbehavior , fleas [behavior , and mosbehavior . Furtherbehavior . Additiobehavior .Hepatozoon spp. have been found in a diversity of habitats across the globe, such as temperate broad-leaved forests [Hepatozoon spp. in some crucial ecosystems remain unexplored, for example, across altitudinal gradients where the rapid succession of habitats often corresponds to the high biodiversity of partially sympatric hosts. Under global and climate changes, such mountain habitats are particularly exposed to abiotic and biotic variations that favor upward distributional shifts of host mammal species, with local modifications of the communities [Hepatozoon spp., is therefore of crucial importance in these habitats undergoing intense dynamics. This is the case for Alpine ecosystems, where Hepatozoon spp. were identified in wild carnivores [Hepatozoon spp. to rodents are blood ectoparasites, such as ticks, fleas, mites, and mosquitoes [ forests ,19, coni forests , Mediter forests , subtrop forests , and tai forests , likely munities ,25. Thismunities ,27. The rnivores ,20 but hrnivores . The occsquitoes , the occsquitoes .Hepatozoon spp. life cycles involve schizogony, which occurs in various organs, such as muscles, lymph nodes, the spleen, and generally in the liver, while gametogony occurs in lymphocytes, monocytes, or occasionally, in granulocytes [Hepatozoon genus were detected in European rodents, e.g., H. lavieri [H. erhardovae [H. sylvatici [H. griseisciuri [H. muris (formerly known as H. perniciosum) [Hepatozoon spp. may be pathogenic [Hepatozoon spp. may compromise the immune status, thus predisposing it toward coinfection by other pathogens.In small mammals, ulocytes . Furtherulocytes . Several lavieri , H. erhahardovae ,32, H. sylvatici ,34, H. geisciuri , and spoiciosum) ,35. In rthogenic ,38,39,40thogenic . Conversthogenic , felids thogenic , mustelithogenic , and in thogenic , HepatozHepatozoon spp. across the altitudinal gradient of the Alpine habitat also in consideration of the anthropic pressure on such habitat. To the best of our knowledge, this is the first study investigating Hepatozoon spp. occurrence in small mammals, and more generally in rodents in Italy, with a special focus on the Alpine habitat.In this work, we conducted a cross-sectional study, i.e., we assessed the parasite prevalence without specifically considering the temporal pattern from 2019 to 2021 in the Italian Alps by intensively live-trapping small mammals from forested habitats up to above the tree line. Our main aim was to evaluate the occurrence of The study was carried out in two sites located in the Province of Trento : Cembra Valley  and Calamento Valley  . CEV com® Ltd., Douglas, UK). At each capture event, we recorded information on individuals, such as species, PIT tag code, sex, body mass, breeding status, and ectoparasites load. Additionally, at first capture, an ear biopsy  was collected for each animal and individually placed in tubes stored at −80 °C.Small mammals were captured using standard Ugglan Multiple Live Traps  set in a grid array, following a protocol that aimed at capturing especially rodents to compare assemblages across an altitudinal gradient. Capture–mark–recapture (CMR) techniques were adopted, and each animal was individually tagged with a subcutaneous Passive Integrated Transponder (PIT) tag .For DNA extraction, the ear samples (506 from CEV and 326 from CAV) were incubated overnight at 56 °C and isolated using the DNeasy 96 Blood & Tissue Kit  according to the manufacturer’s instructions.Hepatozoon spp. was a bycatch while assessing the protocol for Babesia spp. detection on ear tissue samples. We applied a conventional polymerase chain reaction (PCR) method using the Babesia BJ1 5′–GTC TTG TAA TTG GAA TGA TGG–3′ and BN2 5′–TAG TTT ATG GTT AGG ACT ACG–3′ primers [Babesia spp., BJ1 and BN2 primers can amplify the 18S RNA gene of some other protozoans [Hepatozoon spp. [Babesia spp. not described) by specifically adapting the PCR reactions and temperature cycling profiles for each protozoan [This study is part of a wider project investigating tick-borne pathogen circulation in small mammals. Therefore, the identification of  primers . Indeed,otozoans , such asoon spp. ,49,50. Trotozoan .Hepatozoon spp. detection, the PCR reactions were composed of 3-mM MgCl2, 0.1-mg/mL BSA, 0.2-mM each dNTP, 0.3-μM of each primer, 5× Flexi buffer, and 1.25-U/μL Go Taq Hot Start Polymerase (Promega) in 50 μL of total volume, including 1 μL of extracted DNA. After the initial denaturation at 95 °C for 2 min, 40 cycles of 95 °C for 20 s, 46 °C for 15 s, and 72 °C for 50 s were performed before a final incubation at 72 °C for 5 min. Negative controls, i.e., samples that did not contain DNA but only reagents, were included in each molecular essay to cross-check the quality for DNA extraction and PCR amplification. The success of amplification was confirmed using the Qiagen QIAxcel® capillary electrophoresis system. Positive PCR products were purified using ExoSAP-IT  according to the manufacturer’s instructions and then sequenced using Sanger sequencing. The DNA sequences were analyzed and aligned using Sequencher software version 5.4.6  and Clustal X software version 2.0 [In particular, for sion 2.0  and subsTo verify the robustness of the PCR method, we also analyzed and sequenced double ear samples on a subset of individuals .Hepatozoon spp. was calculated for each rodent species with a 95% confidence interval (CI) using the EpiR package [p-values were <0.05.The prevalence of  package  in the R package . We applHepatozoon spp., controlling for the life history traits. Specifically, we applied two analytical designs: (i) we evaluated the effect of anthropogenic pressure on rodent infection by comparing the study areas , only for a homogeneous altitude; (ii) we assessed how the probability of infection would vary across species along the alpine altitudinal gradient in a particularly wild context . To this end, we fitted Generalized Linear Mixed Models  with bi .Apodemus flavicollis), 10 wood mice (Apodemus sylvaticus), 24 snow voles , 2 common voles , 2 pine voles (Microtus subterraneus), 1 short-tailed field vole (Microtus agrestis), 199 bank voles (Myodes glareolus), 25 common shrews (Sorex araneus), 3 alpine shrews , 1 pygmy shrew (Sorex minutus), and 1 bicolored-shrew (Crocidura leucodon) : 562 yedon) see . AdditioHepatozoon spp. and were therefore not included in the following analysis , A. sylvaticus (30.0%), C. nivalis (33.3%), M. arvalis (50%), and My. glareolus (58.8%)  , captured . Hepatoz × 10−6) a.My. glareolus showed a statistically significant higher prevalence compared to A. flavicollis and C. nivalis  , four rodent species tested positive . My. glactively) b. Among Hepatozoon spp. Conversely, both human pressure and the altitudinal gradient were not selected in the best models, indicating their scarce relevance in influencing the probability of becoming infected by Hepatozoon spp. . In the second analysis, the infection of Hepatozoon spp. depended on the additive effect of the species and breeding status, confirming that My. glareolus was more prone to becoming infected if compared to A. flavicollis , while no significant difference emerged between A. flavicollis and C. nivalis , while we did not detect any significant difference between adults and subadults  previously assigned to Hepatozoon spp. were instead confirmed as Babesia microti .Hepatozoon spp. in wild rodents have been generally based on blood [Hepatozoon spp. can be identified only through blood sampling, to the best of our knowledge [Hepatozoon spp. prevalence, especially with low intensity of infection [C. nivalis [Hepatozoon spp., e.g., by comparing ear tissue samples with skin ones as those collected in studies [European studies on on blood ,47,61,62on blood ,22,23, aon blood ,63 samplnowledge ,32. This nivalis , limitin studies ,63, coulHepatozoon spp. was detected, its first identification in C. nivalis (33.3%), and its occurrence at 2000 and 2500 m a.s.l., the highest elevation at which these parasites have ever been recorded, represent key findings to speculate on the transmission pathways of Hepatozoon spp. in the Alpine range. Additionally, this is the first record of Hepatozoon spp. in A. sylvaticus (30.0%) using molecular approaches [Hepatozoon spp. mainly though the ingestion of infected vectors, it is reasonable to consider Hepatozoon spp. as a vector-borne infectious agent for small mammal hosts and specifically in our case, although we did not assess the infective status and transmission dynamics of arthropod vectors. Our results suggest that Hepatozoon spp. are common in alpine environments, showing a high prevalence in a broad spectrum of alpine small mammal species. In particular, we did not detect any relevant difference in Hepatozoon spp. prevalence between anthropic and wild systems, implying a stable and ubiquitous presence of Hepatozoon spp. in the analyzed small mammal species. This is interesting, as Hepatozoon spp. have been previously studied mainly in domestic species [Hepatozoon spp. [A. sylvaticus generally shares food resources, part of the habitats and vectors, with other rodent competitor species, especially in case of high rodent and vector density [My. glareolus and Apodemus spp.) and, in turn, the survival and development of arthropod vectors  [Hepatozoon spp. in C. nivalis and high altitudes that we recorded is compatible with these dynamics, although there is no track record of previous investigations at the same altitudes in small mammals  ,71,72. Td ticks) ,74,75,76eference ).Hepatozoon spp., a broad spectrum of both domestic . This aspect of the transmission pathway of Hepatozoon spp. needs further investigation.The high rate of infection that we observed in different rodent species across a diversity of Alpine habitats may induce relevant cascading effects across the food web. In particular, being carnivore-competent hosts for erd dogs ) and wilerd dogs , meso- aerd dogs ,79, and erd dogs ) may bece stages  infective stages . The pare stages . Howevere stages , predatoHepatozoon spp. that we recorded in the most common rodent hosts, i.e., My. glareolus and A. flavicollis, is in partial accordance with other studies in Europe. In particular, we found that My. glareolus showed the highest prevalence of Hepatozoon spp. among all captured rodent species (58.8%), as was also observed in other studies [A. flavicollis (28.1%) is higher than all previously published estimates that were below 10% [Hepatozoon spp. infection at lower elevations. The small sample size of Microtus spp. voles does not allow conclusive indications on the rate of infection of this genus , as was the case also for other studies [The prevalence of  studies ,32,47,63 studies ,62,82). elow 10% ,47,48,63 studies ,31,32,47Hepatozoon spp. in new wild hosts and habitats importantly contributes to the understanding of the ecological role of these parasites, especially under the current global change. Collectively, by detecting Hepatozoon spp. in a broad range of rodent hosts throughout the altitudinal gradient of Alpine habitats, we indicated the widespread occurrence of this parasite as likely supported by an expanding availability of arthropod vectors, due to variations in the temperature and moisture linked to climate change [Hepatozoon spp. across the food chain in habitats particularly exposed to climate change, such as the Alpine range. In particular, studies investigating vector competence and distribution, as well as host ecology and their role in maintaining and transmitting Hepatozoon spp., are needed to clarify the function of this group of parasites in such changing ecosystems.In conclusion, Apicomplexa remains one of the most poorly investigated groups among protozoa, so that accurate identification (via PCR-based methods coupled with sequencing) of e change . Therefo


We discuss a simplified form of Stokes operators for quantum optical fields that involve the known concept of binning. Behind polarization analyzer photon numbers  are measured. We have two outputs, say, for horizontal and vertical polarization. If the value obtained in horizontal output is greater than in vertical one we put 1. Otherwise, we put − 1. For equal photon numbers, we put 0. Such observables do not have all properties of the Stokes operators, but can be employed in Bell type measurements, involving polarization analyzers. They are especially handy for states of undefined number of photons, e.g. squeezed vacuum and their realisation is intuitive. We show that our observables can lead to quite robust violations of associated Bell inequalities. We formulate a strongly supported numerically conjecture that one can observe with this approach violations of local realism for the four mode squeezed vacuum for all pumping powers . This started with the response by Bohr2. Many years later, after the paper of Bell3 the challenge of revealing non-classicality, in terms of violation of local realism, has entered the core of contemporary research. All that in the meantime gained in importance with the emergence of quantum information and communication.The discussion about what is the essence of quantumness started with the first attempts of formulating quantum mechanics. With the emblematic paper of Einstein et al.7. However, if one moves to situations with undefined numbers of particles, still the situation is quite open. This is of course e.g. the case of general quantum optical fields. A lot of approaches are tested.The ultimate test of non-classicality is the violation of Bell inequalities. This is now also the essence of testing of device-independent quantum communication protocols. Formulations of Bell’s theorem for situations of fixed numbers of particles have already a vast literature, and well established methods, see e.g. reviews9. A deceptively obvious step in the direction towards optical fields of undefined photon numbers is to use quantum Stokes observables. The usual definition of these runs as follows. If one assumes that the intensity of light is proportional to the photon number, then (standard) quantum Stokes observables are given by i and i’s to represent horizontal-vertical, 10.Polarization entanglement experiments are classic examples of experimental tests of Bell’s inequalities. The two photon experiments are a realization of two qubit-entanglementA and B in the form of 11. This is because such attempts involve additional assumptions, beyond the usual ones for Bell inequalities, which limit the range of local hidden variable theories for with such Bell inequalities must hold.If we are interested in the degree of polarisation of light we use 14:Bell inequalities for Stokes parameters can be formulated if one introduces normalized Stokes observables16 the discussion about normalized Stokes parameters stops to be only theoretical and its use in experiments is becoming feasible.It has been shown that such operators allow for the construction of stronger entanglement criteria, and they are a handy tool for formulation of Bell inequalities. One of their properties, crucial in this case, is the fact that these operators have a spectrum bounded by z component of pseudo-spin is 18. The spectrum of pseudo-spin operators is the same as the spectrum of Pauli matrices, but their use introduces great difficulties from the experimental point of view. Even a loss of one photon (due to e.g. detector inefficiency) or a single dark count reverses the result of a measurement.Note that what makes Pauli operators so straightforwardly applicable to Bell inequalities is their dichotomic nature. One of the attempts to construct field operators of a similar property was the formulation of pseudo-spin operators. For example, the 19. The approach to binning presented here is concurrent with the method used in20 in the context of correlation in Bose-Einstein condensates. With the observables, we construct Bell inequalities. We test their resilience under losses and noise for Here we analyze a simpler approach, which leads to proper Bell inequalities for polarization measurements of quantum optical fields. Our aim is to construct a family of operators that would have the usual spectrum for Bell experiments and would be robust with respect to experimental noise. We present polarization quantum field observables that have spectrum limited to 14 and its GHZ-like generalization (BGHZ)21. However, for a higher mean number of photons, the violation of Bell inequalities by these states is quickly damped. This results in lowering of the threshold values for pumping strength after which violation cannot be observed.It was shown that Bell inequalities constructed with normalized Stokes operators can be violated by macroscopic states of light such as s denotes the chosen setting related with the corresponding polarization basis with the eingenstates given by s and H and V refer to horizontal and vertical polarizations, and the operator H, V into another orthogonal pair of, in general, elliptic polarization modes G(s) are We address those problems by another normalization scheme, based on the so-called binning, which we call Sign approach normalization. To obtain new operators, we use the sign function and apply it to Stokes operators:25.The action of the We shall call the new operators sign Stokes operators. Following the usual approach, we shall define a triad of sign Stokes operators, related to the three maximally complementary settings of a polarization analyzer. For the usual triad of such settings, we denote by i: standard, normalized, and sign ones, as they depend solely on the measured photon numbers s to The sign operators share some properties of Stokes and normalized Stokes operators. Importantly, once one has a photon-number-resolving detection setup, the data collected in each run allows one to compute the obtained values of each of Stokes operators for the given basis 13. These norms remain invariant under any unitary transformation between two triads of mutually maximally complementary polarization analysers. This transformation can be put as f is a polynomial of both variables. We put this relation as Standard Stokes operators form a Stokes vector. We will discuss this property for pure states. However, it works also for mixed ones. We have For such mode transformations we have 13, which work via a simple replacement of Pauli operators in entanglement conditions for qubits, by Stokes operators, standard or normalized. Still, as we shall see, there is no obstacle to using this method in the case of construction of Bell inequalities.Such properties are not shared by sign operators. Let us construct covariance of polarization variables is not a necessary feature required to derive Bell inequalities . This allows one to construct CHSH and CH inequalities for fields with sign Stokes observables.Rotational s, X. We define the local hidden values for sign operators as To derive Bell inequalities satisfied by any local realistic description, we start by defining local hidden values that predetermine the output of the measurement of sign Stokes operators . We deno14. Following ideas of14 we modify sign Stokes operators as follows:As this modification does not change local hidden values However, this inequality cannot be violated by states with a significant vacuum component, e.g. the (polarization) four-mode squeezed vacuum state, which will be our working example, see next sections. This situation is analogous to the case of normalized Stokes operators, seeA, and B. The amplification gain determines the intensity of the pumping field and thus We are going to analyze how the use of sign Stokes operators in CHSH inequality helps to reveal the non-classicality of quantum states. Our working example is 14.Assume that both observers choose to measure only linear polarizations. thus, the angles by which the measurement polarization basis is rotated with respect to 14 as a function of the amplification gain Figure In Fig. relative angle of the orientation of the polarization analyzers at the two spatially separated observation stations.Because of the rotational invariance of vacuum term, that is CHSH inequality averaged over the vacuum component of BSV and non-vacuum term. The vacuum and non-vacuum terms in . No additional “reasonable” assumptions are used. As, according to our numerical estimates, one can conjecture that the associated inequalities are violated for an arbitrary Supplementary Information.


A full-scale model for predicting low-velocity impact (LVI) damage and compression after impact (CAI) strength was established based on a subroutine of the material constitutive relationship and the cohesive elements. The dynamic responses of the laminate under impact load and damage propagation under a compressive load were presented. The influences of impact energy and ply thickness on the impact damage and the CAI strength were predicted. The predicted results were compared with the experimental ones. It is shown that the predicted value of the CAI strength is in good agreement with the experimental result. As the impact energy reaches a certain value, the CAI strength no longer decreases with the increase in the impact energy. Decreasing the ply thickness can effectively improve the damage resistance and CAI strength. One of the advantages of composite laminates is that different fiber-reinforced materials and design angles can be selected according to the load requirements of various structures, which provides greater freedom for the structural design and ply scheme optimization. However, the obvious anisotropy and low inter-laminar strength characteristics of the composites can also lead to multiple failure modes of structures, including fiber breakage, resin cracking ,2 and deThe impact damage and residual strength of composite laminates have attracted lots of attention. A lot of related works have been published, and experimental testing is one of the most direct and effective research methods. Using an experimental method, Sergii  investig4s sub-laminate scaled laminates can be predicted accurately, and the maximum error is 10%. Yang An impact damage prediction model of the composite laminate was established to calculate the response under the impact load. According to the ASTM D 7136 standard, the kinetic energy of the impactor was implemented using the initial velocity, and the impact energy was set at 30 J. The damage states of typical interfaces are shown in The laminate state at the end of impact was used as the initial condition of the compression simulation. The failure state of the fiber, matrix and delamination at the beginning of compression was defined by field variables. Considering the storage space and computing efficiency of the computer, it was impossible to simulate the compression loading rate under real experimental conditions. The loading rate needs to be increased to ensure that the calculation time is within an acceptable range, and the enforced displacement loading rate used in this research was 50 mm/s.The selection of the damage criterion affects the prediction of the in-plane properties of the composite plies, and then, it affects the prediction of structural failure process and final damage state. The influences of the damage criteria on the prediction results of the impact damage and the CAI strength were studied through the FEM that was established in this paper. The 3D Hashin criterion is shown in The composite structures may be subjected to different impact energies, which will cause the bearing capacity of the composite structures to decline by different degrees. To study the influence of impact energy on CAI strength of the composite materials, the impact damage and CAI strengths under impact energies of 10, 20, 40 and 50 J were compared with the result of the test using 30 J. The impact damage of the composite laminate was calculated, and According to To further explain the above phenomenon, Raman  shows thTo further analyze the mechanism of the effect of the single ply thickness on the CAI strengths, The comparison of contact forces and energy absorption with different single ply thicknesses are shown in An FEM for predicting the impact damage and CAI strength of composite laminates was established. The whole analysis process of the impact damage and CAI strength was conducted using the FEM. The predicted CAI strength of the composite laminate was compared with the experimental one. The influences of impact energy, damage criterion and single ply thickness on the impact damage area and CAI strength were investigated. Four important results emerging from the research are as follows:The damage zone of the fiber breakage and matrix cracking caused by the impact is approximately circular.The predicted CAI value is in good agreement with the experimental one, and the error is 4.7%.The CAI strength decreases as impact energy increases from 10 to 30 J. After the impact energy exceeds 30 J, the CAI strength remains basically unchanged.Decreasing the single ply thickness can effectively improve the damage resistance and CAI strength of the composite laminates. When the single ply thickness decreases to 0.04 mm, the CAI strength increases by 22.4%.


In the 1940s, Alfred Jost demonstrated the necessity of testicular secretions, particularly androgens, for male internal and external genitalia differentiation. Since then, our knowledge of androgen impacts on differentiation of the male internal (Wolffian duct) and external genitalia (penis) has been drastically expanded upon. Between these two morphologically and functionally distinct organs, divergent signals facilitate the establishment of tissue-specific identities. Conversely, conserved actions of androgen signaling are present in both tissues and are largely responsible for the growth and expansion of the organs. In this review we synthesize the existing knowledge of the cell type-specific, organ specific, and conserved signaling mechanisms of androgens. Mechanistic studies on androgen signaling in the Wolffian duct and male external genitalia have largely been conducted in mouse model organisms. Therefore, the majority of the review is focused on mouse model studies. Androgen signaling pathway is essential for development of male organs across vertebrate animals . This paDue to the dependency on androgen signaling, male reproductive structures are particularly sensitive to disrupted fetal development and adulthood dysfunction , 5. The In this review, we discuss the diverse and conserved androgen actions in two critical reproductive organs, the Wolffian duct-derived tissues and the male external genitalia, specifically the penis, during embryonic development. We review the literature with the goal of understanding the pleiotropic nature of AR. To do this, we tease apart the cell populations of each organ. Then we investigate the androgen signaling programs that are specific to each cell population. By reviewing the literature in this way, we can begin to discern the molecular determinants that specify the distinct androgen signaling programs in each cell population. To further understand the general functions of AR, we discuss common androgen signaling events in both the Wolffian duct and male external genitalia. Because our knowledge about androgen signaling in male sexual differentiation derive largely from mouse studies, the majority of the following sections focus on mouse models. Reviewing both the internal and external reproductive tract provides a roadmap for future studies and allows us to investigate the many diverse and conserved action of androgen signaling.Androgen signaling pathway is mediated by the androgen receptor (AR), which mainly acts as a ligand-activated transcriptional factor . Loss ofCyp17a1) and 3β-Hydroxysteroid (3β-HSD)  . These t(3β-HSD) , 12. The(3β-HSD) . BindingAr with only genomic or non-genomic actions in the absence of endogenous AR  exposure during sexual differentiation decreased Egf in male mice embryos , which regulates a wide range of cellular events including cell survival and proliferation . Egf exp embryos . Recepto embryos . Global  embryos . Egf knohenotype , suggestCoup-tfII increased expression of two mesenchymal growth factors Fgf7 and Fgf10, which activated their receptor FGFR2 in Wolffian duct epithelium to promote Wolffian duct survival in XX embryos in the absence of AR actions   double males  to caudal (i.e. posterior) regions Figure 1Hoxa9 , Hoxd9 , and th Hoxd13)    and is highly expressed in the mesenchyme of the anterior Wolffian duct before the initiation of epididymal morphogenesis  region, AR action promotes epididymal coiling which relies on the action of inhibin β A (Inhba) . Inhba iogenesis Figure 1ogenesis . Androge studies . Male ems intact .Lgr4-6, the leucine-rich repeat-containing G-protein-coupled receptors) and preventing the destabilization of the Wnt receptors. As a result, RSPOs act in concert with WNTs to promote the activation of the β-catenin-mediated intracellular signaling (Pkd1 (a receptor for mediating WNTs actions) is deleted in the epithelium  is a member in a gene family of generally extracellular signaling peptides, which are key regulators in organ development. The mesenchyme of the caudal Wolffian ducts expressed high Fgf10, which is increased by androgen treatment , suggesting that Fgf7 is dispensable in AR actions in promoting Wolffian duct development in vivo. These observations demonstrate that mesenchymal AR actions regulate region-specific morphogenesis via paracrine growth factors.In the caudal region, another secreted ligand of TGFβ superfamily GDF7 is required for seminal vesicle growth, morphogenesis, and epithelial differentiation Figure 1reported . Gdf7-/-al cells . In addiignaling , 55. Fgfreatment . Global elopment . Wolffiaan ducts . FGF7 waondition , 58. How-/-Wnt9b male embryos fail to form Wolffian duct derived organs at birth despite of normal testis development , Myh9, and F-actins  and Myl6 and is enriched with actin-cytoskeleton signaling pathway components, suggesting that it may exert similar mechanical forces on the distal urethra  to distal (tip) fashion along the ventral aspect of the penis . At E16. urethra . The expEsr1 knockout mice develop mild hypospadias in adulthood, similar to estrogen exposed mice  compared to the rest of the penis . Estrogesed mice . These dFgf10 causes severe hypospadias with no urethra closure occurring throughout the penis  and myosin light chain 9 (Myl9), which are both expressed in the developing prepuce  , which ied penis . The rolThe action of AR must be imposed within a specific fetal programming window. For the Wolffian ducts, the masculinization programming window is E15.5–E17.5 in rat , presumain vivo, classic tissue recombinants studies provide evidence that the mesenchymal AR actions governs androgen-induced epithelial morphogenesis .Extensive studies have demonstrated the essential role of AR action in the mesenchyme of Wolffian ducts and penis Figure 4ogenesis   that mediate mesenchymal AR actions in controlling survival and differentiation of Wolffian ducts and penis , 74, 109nization . These rAlthough AR is a singular transcription factor, it can fill many roles throughout Wolffian duct and male external genitalia development. In both organs, plenty of research has identified androgen responsive programs, but there many questions about the molecular drivers of AR pleiotropy. Future studies that conduct cell type-specific investigations of AR co-regulator interactions, chromatin accessibility of AREs, and multi-organ androgen signaling conservation will bring us drastically closer to understanding the prevalence of androgen-related human birth defects.CA, FZ, and H-CY were responsible for creating figures, writing content, and editing the manuscript. All authors contributed to the article and approved the submitted version.Intramural Research Program of National Institute of Environmental Health Sciences Z01-ES102965 (H-CY). National Institute of Child Health and Development R00-HD096051 (FZ).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.