The presynaptic Ca2+ homeostasis conditioning ACh release is modified by autoreceptors such as M1-type muscarinic AChR and A2A adenosine receptors. The post-synaptic structure is stabilized by: (i) laminin-network including the muscle-derived agrin; (ii) the extracellular matrix proteins (including collagen Q/perlecan and biglycan which link to MuSK Ig1 domain and CRD); and (iii) the dystrophin-associated glycoprotein complex. The study on MuSK ectodomains (Ig1/2 domains and CRD) recognized by antibodies suggested that the MuSK antibodies were pathologically heterogeneous due to their binding to multiple functional domains. Focussing one of the matrix proteins, biglycan which functions in the manner similar to collagen Q, our antibody assay showed the negative result in MG patients. However, the synaptic stability may be impaired by antibodies against MuSK ectodomains because of the linkage of biglycan with MuSK Ig1 domain and CRD. The pathogenic diversity of MG is discussed based on NMJ signaling molecules.Myasthenia gravis (MG) is a disease of the postsynaptic neuromuscular junction (NMJ) where nicotinic acetylcholine (ACh) receptors (AChRs) are targeted by autoantibodies. Search for other pathogenic antigens has detected the antibodies against muscle-specific tyrosine kinase (MuSK) and low-density lipoprotein-related protein 4 (Lrp4), both causing pre- and post-synaptic impairments. Agrin is also suspected as a fourth pathogen. In a complex NMJ organization centering on MuSK: (1) the Wnt non-canonical pathway through the Wnt-Lrp4-MuSK cysteine-rich domain (CRD)-Dishevelled  signaling acts to form AChR prepatterning with axonal guidance; (2) the neural agrin-Lrp4-MuSK (Ig1/2 domains) signaling acts to form rapsyn-anchored AChR clusters at the innervated stage of muscle; (3) adaptor protein Dok-7 acts on MuSK activation for AChR clustering from “inside” and also on cytoskeleton to stabilize AChR clusters by the downstream effector Sorbs1/2; (4) the trans-synaptic retrograde signaling contributes to the presynaptic organization  These have paved the way to search for the mechanisms underlying myasthenia gravis (MG) weakness  functions as acetylcholine (ACh)-mediated synapse  and titin , anti-programmed death ligand-1 (PD-L1) and anti-cytotoxic T lymphocyte associated-4) to manage various cancers are occasionally associated with immune-related adverse events , <red- limit in pink-MuSK, via Dishevelled .Agrin, a proteoglycan expressed in both motor neuron  are membrane-anchored extracellular proteinases capable of degrading a variety of proteins including agrin; the function of agrin is regulated by the site-specific cleavage by MMPs  effector p21-activated kinase 1  signaling plays a principal role in AChR cluster formation, the cyclin-dependent kinase 5 (Cdk5) concentrated at the postsynaptic NMJ is activated and required for ACh-induced removal of prepatterning AChR clusters which are not stabilized by agrin; this is mediated through two molecular mechanisms  for neuregulin 1  of MuSK ectodomains, we showed that the MuSK Ig1/2 antibodies Y2 in  were posΔCRD/− embryos present defective pre- and post-synaptic differentiation , and a drastic deficit in AChR clustering  superfamily of signaling proteins, have been focused in highlighting insight into the possibility that BMPs work in vertebrate organisms as diffusible signals to ensure proper communication between motor neurons and skeletal muscles  to the active zone proteins  and A2A adenosine receptor along with the interaction of brain-derived neurotrophic factor (BDNF) with receptor tyrosine kinase B (TrkB); these biological mechanisms lead to P/Q-type VGCC activation by the signaling mediated by phospholipase C (PLC)-phosphatidylinositol 4,5-bisphosphate (PIP2)-diacylglycerol (DAG)-protein kinase C  may underlie to compensatory postsynaptic dysfunction.From the standpoint of clinical electrophysiology, the AChR antibody-positive MG and animal models showed that the compensatory ACh release upregulation cannot be sustained at the high frequency of nerve stimulation  triggered by G protein-coupled receptor, it is worthy to note that BDNF in the synapse is activated by β2-adrenoceptor agonists and thereby contributes to the maintenance of the structural and functional integrity of motor end-plate; this signaling pathway may include the cyclic adenosine monophosphate (cAMP)/protein kinase A/cAMP-responsive element-binding protein; Bartus et al., 2-adrenoceptor agonists  have been reported in MuSK MG animal model  caveolin 3  and Wnt-signaling  presynaptic Ca2+ homeostasis by autoreceptors; and (4) the synaptic stability based on the laminin-network including the extracellular matrix and the cytoskeleton. The insight into the NMJ structural mechanisms discussed in this review will foster further immunological approaches to search for new antigenic targets  the AChR cluster formation based on the Wnts-Lrp4-MuSK (CRD)-Dvl signaling (pre-innervated stage) which converges upon the agrin-Lrp4-MuSK (Ig1/2 domains) pathway (innervated stage); (2) the retrograde signaling (from muscle to the nerve) for presynaptic differentiation The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Obstructive sleep apnea‐hypopnea syndrome (OSAHS) is a common breathing disorder during sleep with potential lethality and multi‐complications. Polysomnography (PSG) is now the golden standard for the diagnosis obstructive sleep apnea‐hypopnea syndrome. However, PSG is expensive and time‐consuming. Therefore, it is important to find inexpensive and convenient biomarkers for the diagnosis of OSAHS.The present study aimed to explore the potential diagnostic value of HIF‐1α for OSAHS and its clinical significance.This study consisted of 368 patients admitted to the sleep laboratory. The patients were classified according to their apnea‐hypopnea index (AHI) scores as OSA negative (AHI < 5), mild‐moderate (AHI:5‐30), and severe OSA (AHI > 30), and severe OSA treated with continuous positive airway pressure (CPAP). qRT‐PCR was used to detect mRNA levels in the plasma; Pearson's correlation analysis was performed to analyze the correlation of HIF‐1α mRNA level and the clinicopathological factors of OSAHS; ROC curve was constructed to evaluate the diagnostic value of HIF‐1α mRNA.HIF‐1α mRNA was significantly up‐regulated in the plasma of OSAHS patients, especially patients with severe OSAHS. HIF‐1α mRNA was positively correlated with the AHI and ODI but negatively correlated with the mean oxygen saturation in patients with OSAHS. Results of ROC curve showed that HIF‐1α is a sensitive biomarker for the diagnosis of OSAHS, especially severe OSAHS.HIF‐1α mRNA might be used as s a convenient and inexpensive method for triaging OSAHS patients PSG assessment in the hospital and evaluate the curative effect. HIF‐1α mRNA expression levels in the plasma OSAHS patients. Patients were classified according to their apnea‐hypopnea index (AHI) scores as OSA negative (AHI < 5), mild‐moderate (AHI:5‐30), and severe OSA (AHI > 30). Moreover, 96 patients with severe OSA and treated with continuous positive airway pressure (CPAP) were also included. The exclusion criteria were as follows: (a) psychiatric or neuropathic causes of sleep disorder; (b) diagnosed with metabolic disorders, endocrine, autoimmune disorders; (c) respiratory diseases including asthma and acute respiratory tract infection; (d) diagnosed with liver or kidney disease; (e) hematologic disorders including anemia and leukemia; and (f) age < 18 years. The demographic characteristics such as age, gender, and body mass index (BMI) were collected. BMI was calculated by dividing weight by height squared. Cardiovascular diseases (CVD) were patients who suffered from heart failure, arrhythmia, or coronary artery disease. Diabetes mellitus (DM), hypertension (HT), and smoking history was defined as the risk factor of CVD. This study has been approved by the ethical committee of Nanjing First Hospital, and every patient has signed the inform consent form.2.2Sleep recording was performed by using overnight polysomnography (PSG), comprising continuous recording electroencephalography (EEG), electrooculography (EOG), submental and bilateral anterior tibial electromyography (EMG), electrocardiography (ECG), nasal airflow, and pulse oximetry. A physical examination including weight and height was recorded before PSG. Sleep stages and respiratory events during sleep were scored based on the guideline of American Academy of Sleep Medicine (AASM).2.3Total RNAs were extracted from the plasma by TRIzol reagent (Invitrogen) following the manufacturer's instructions. Next, the purity and quantity of the total RNA were detected with DeNovix DS‐11 and a Spectrophotometer (DeNovix). RNA was reverse‐transcribed to cDNA from 1 µg of total RNA by using AMV reverse transcriptase (Takara) and a RT primer according to the manufacturer's recommendation. The reaction conditions were as follows: 16°C for 30 minutes, 42°C for 30 minutes, and 85°C for 5 minutes. Real‐time PCR (RT‐PCR) was carried out by using a Taqman PCR kit on an Applied Biosystems 7300 sequence detection system (Applied Biosystems), with U6 as the internal control. The reactions were performed in a 96‐well plate at 95°C for 10 minutes, followed by 40 cycles of 95°C for 10 seconds and 60°C for 1 minutes.2.4t test was performed in the multiple comparison. Receiver operating characteristic (ROC) analysis was performed, and areas under curve were calculated to evaluate the diagnostic value of HIF‐1 mRNA. All tests of statistical analysis are two sides, with P < .05 taken to statistical significance.The software of SPSS 20.0 was used for statistical analyses, and all of counting data were expressed in the form of mean ± standard deviation(x ± SD). One‐way ANOVA was used for analyses of interclass variance, and LSD 33.1P < .01). The results of the polysomnographic study showed no significance on sleep stage I of the patients (P = .8353), but there have been significant differences in sleep stage II time/TST and sleep stage III time/TST and sleep efficiency among different groups. Moreover, the AHI and ODI increased while the mean and minimum oxygen saturation decreased with the severity of OSAHS, which was statistical differences between severe OSAHS and other groups. SE showed no significant differences among the groups (P = .0747).A total of 368 patients were included in the study, among which 48 individuals were diagnosed as simple snore, 122 patients were mild‐moderate OSAHS, 102 patients were severe OSAHS, and 96 patients with severe OSA and treated with continuous positive airway pressure (CPAP). Table 3.2P < .01); moreover, the level of HIF‐1α was significantly increased in severe OSAHS group compared with the mild‐moderate group .First, the mRNA expression of HIF‐1αin plasma samples of the patients and healthy controls in each group was measured by qRT‐PCR methods. As shown in Figure 3.3Moreover, we performed ROC curve analysis to identify whether HIF‐1α mRNA can serve as a biomarker in OSAHS. The analysis revealed that the area under the ROC curve (AUC) for distinguishing the OSAHS patients and controls was 0.8438  + OSAHS could also be investigated in future works.None.


Gout is a chronic inflammatory disease caused by precipitation of urate crystals in the joints, kidneys, and urinary tract. Independent of urate deposition disorders, recent studies have shown a positive association between circulating uric acid (UA) levels and cardiovascular (CV) diseases. These results indicate that UA is a precipitating factor of both gout and the progression of CV diseases, including hypertension and/or chronic kidney disease (CKD). A large body of evidence has shown that UA-lowering therapies are effective in preventing the progression of hypertension/CKD and that a causal relationship exists between serum UA level and CV diseases. Despite the urgent need for effective UA-lowering drugs that can be used to obtain better therapeutic outcomes and prognosis, only few drugs have been developed in the past decades. Recently, febuxostat and topiroxostat, which are xanthine oxidoreductase inhibitors, were developed and used in clinical practice. Of note, after the approval of lesinurad, which is a urate transporter-1 (URAT-1) inhibitor, in the United States in 2015, dotinurad (Fig.  The number of patients with hyperuricemia has continuously increased in recent years due to the increased occurrence of metabolic syndrome. Hyperuricemia causes gouty arthritis, gouty kidney, and urolithiasis. Evidence has shown that hyperuricemia is also associated with hypertension –5, chronRegarding the pathogenesis of CKD, UA-induced renal arteriopathy can explain the primary mechanism by which hyperuricemia causes impairment in renal function. UA results in a higher risk of hyalinosis and higher-grade wall thickening in patients with biopsy-proven CKD . In addiIn most individuals, UA levels are genetically determined. Clinical studies of patients with hyperuricemia indicate that the contribution of heritability to hyperuricemia is approximately 70% or higher , 21. ThiAllopurinol, a xanthine oxidoreductase (XOR) inhibitor developed in the 1960s, and benzbromarone (BZB), a urate transporter 1 (URAT-1) inhibitor developed in the 1970s, are long-term drugs widely utilized for hyperuricemia/gout. Since then, only few drugs with UA-lowering potency have been available until quite recently. Febuxostat and topiroxostat, which are XOR inhibitors and alternative drugs to allopurinol, have been developed and widely used within the last 10 years.Regarding drugs with URAT-1 inhibitory property, the Food and Drug Administration (FDA) approved the use of lesinurad in the United States in 2015. Subsequently, dotinurad, a novel promising drug with selective urate reabsorption inhibitor (SURI) property, was recently developed in Japan in 2018.Generally, hyperuricemia is classified into the overproduction type and underexcretion type based on the amount of renal UA excretion. Because the prevalence of the underexcretion type is evidently higher (approximately 60% or higher) than that of the overproduction type in patients with Japanese ethnic extraction , the impAssuming that dotinurad will soon be available in the market, the present review summarized the basic and clinical information obtained from the 11 preliminary clinical trials that assessed the following: (1) efficacy and safety, (2) pharmacokinetics (PK), (3) evaluation results about the non-inferiority of dotinurad to other UA-lowering agents, (4) effects of age, gender, and liver and kidney diseases, (5) factors associated with overproduction- and underexcretion-type hyperuricemia, and (6) drug interaction with NSAIDs.Early- and late-phase clinical trials (phase 2a and 2b studies) were carried out to determine the optimal dose of dotinurad, and phase 3 study was performed to assess the long-term effect of such drug –25 Tabl. Table 1Phase 2a and 2b trials were randomized, multicenter, double-blind, placebo-controlled, parallel-group, and dose-escalation studies. A total of 80 patients were recruited in the phase 2a trial, which revealed that serum UA levels substantially decreased when dotinurad was administered at a dose of 1, 2, and 4 mg for 8 weeks in a dose-dependent manner . SimilarIn addition, the phase 3 trial, a long-term observation study, was independently planned and carried out. More than 300 patients were recruited in this multicenter, open-label, dose-escalation study performed at 26 medical institutions to evaluate the long-term efficacy and safety of dotinurad. The results indicated that the decrease in UA levels was significant even at an early time (2 weeks), and the efficacy was continuously observed throughout an observation period of 58 weeks . The serBased on the findings obtained in the trial, dotinurad is effective in reducing serum UA levels during the long-term observation period.T1/2 was approximately 10 h, regardless of the dosage, age, and gender [In general, PK should be analyzed in various clinical conditions as UA metabolism might be influenced by age and gender, and renal and liver clearances are the two major factors that determine PK parameters. Thus, preliminary PK trials about dotinurad were performed in multiple clinical settings –28 Tabl. A seried gender , and thad gender , 28. BasIn general, clinical trials that assess whether dotinurad is not inferior to other currently available UA-lowering agents may be essential. Non-inferiority clinical trials have been recommended to establish the efficacy of newly developed drugs. For instance, in the CARES trial, the FDA required that febuxostat (FBX) should be compared with placebo and allopurinol to assess the inferiority of FBX to other UA-lowering drugs .In phase 3 trial, Hosoya et al. have investigated the non-inferiority of dotinurad to either BZB or FBX in two separate trials , 31  of dotinurad increased slightly when dotinurad was co-administered with oxaprozin compared to dotinurad alone, the results were still within the acceptable clinical range [The pharmacokinetic interactions of the combined dosing of dotinurad and NSAIDs will be clinically crucial as NSAIDs are frequently administered in combination with UA-lowering agents in patients who present with intractable pain due to gouty arthropathy. In vitro experiments indicated that oxaprozin, a non-steroidal anti-inflammatory drug, may be the most apprehensive candidate among the potential co-administered drugs , which can interact with dotinurad by interfering the glucuronidation conjugate process and decreasing the plasma protein binding rate of dotinurad. In a study that recruited healthy participants, the interaction between oxaprozin and dotinurad was investigated by measuring the blood concentration of dotinurad and UA levels. Although the Tal range  (Table 1As previously mentioned, the predominance of the underexcretion type over the overproduction type highlights the emerging attention for drugs with SURI property. In daily clinical practice, physicians are not always meticulous about making a differential diagnosis of the subtype of hyperuricemia because the measurement of urinary UA excretion is time- and effort-consuming. Thus, even if dotinurad was arbitrarily chosen for a patient whose clinical subtype was unknown, one might still expect a secure UA-lowering effect. To address such issue, the effects of the clinical subtypes of hyperuricemia were the focus of two separate trials , 34 Tab. Namely,However, one must be alert to the potential fear of enhancing UA stone formation induced by uricosuric agents. Although the prevalence of urinary stones of UA origin is approximately 1–3%  among alFinally, the side effects of lesinurad, which is an URAT-1 inhibitor that was recently made available in the market, has gained clinical attention. In terms of the side effects of such drug, the main concern is the increase in serum creatinine concentration . BecauseI feel that dotinurad may practically be given to both subtypes of hyperuricemia/gout patients assuming that the PK parameters are almost comparable between the two subtypes and that the adherence rate of measuring urinary UA excretion rate in the outpatient clinic is quite low. Certainly, it is too early to evaluate the intrinsic advantages of dotinurad. However, drugs with SURI property can be used to achieve the purpose of effectively lowering UA levels, thereby leading to better treatment outcomes in patients with hyperuricemia/gout.Dotinurad is effective in lowering serum UA levels, and its efficacy and safety are similar to those of other UA-lowering drugs in patients with hyperuricemia/gout.


To evaluate fertility knowledge among current Obstetrics and Gynecology (OB-GYN) residents using a recently published validated instrument, the Fertility and Infertility Treatment Knowledge Score (FIT-KS).OB-GYN residents in the United States were recruited through an email to all residency coordinators nationwide. They were asked to voluntarily respond to a short questionnaire including demographic information and the FIT-KS instrument, through an online survey platform. Of approximately 5000 OB-GYN residents in the country, 177 responded.The sample was 91% female, with 69% between the ages of 26 and 30. Participants evenly represented all 4 years of training. Mean FIT-KS score was 21.2 . No statistically significant differences were noted across the level of training. Several knowledge gaps were noted. Residents could define the common assisted reproductive technologies; however overestimated their success rates per cycle.Substantial gaps exist in fertility knowledge among OB-GYN residents, with understanding of male fertility and success rates of Assisted Reproductive Technologies (ART) being particularly limited. Knowledge of fertility does not change throughout residency training, demonstrating consistent gaps in fertility knowledge. Knowledge during post graduate year (PGY)-1 year is consistent with mean scores found in prior research in Internal Medicine residents (65%), as well as a cohort of female medical students and obstetrics and gynecology residents and fellows (64.9%) .The online version contains supplementary material available at 10.1186/s40738-020-00091-2. Age related fertility decline has been highlighted in recent years in the popular media, however misconceptions still exist among the general public. Approximately 48.5 million couples worldwide experience infertility but the majority of the population does not understand natural fertility and age related fertility decline . Prior rIn the general population, criteria for infertility diagnosis are met by approximately 12.5% of women . On averIn one recent study, 24% of female physicians who tried to conceive were formally diagnosed with infertility, of whom 21.7% were ultimately unable to conceive . A substIf, as these data suggest, female physicians have knowledge gaps about their own fertility, it also stands to reason that they may be unable to adequately counsel their patients on this topic. As such, this study aims to evaluate whether obstetrics and gynecology residents are appropriately knowledgeable about natural fertility and age related fertility decline.Permission was received to use the Fertility and Infertility Treatment Knowledge Score (FIT-KS). The FIT-KS is a web-based survey, with 29 multiple-choice items. It assesses knowledge of natural fertility (21 questions) as well as infertility treatment (8 questions). It has previously been validated in both reproductive-aged women in the US as well as in female medical trainees. The correct answers were based on the latest Society for Assisted Reproductive Technology (SART) data from the year of creation of the survey (2014).Institutional review board deemed the study exempt through Temple University College of Medicine (#25003). All participants gave written informed consent through the survey, and risk was deemed to be minimal.In April of 2018, the research team emailed all Obstetrics and Gynecology residency directors with the request to forward the web-based survey to their residents through email. Two follow-up reminder emails were sent over April and May 2018. The survey was hosted by Survey Monkey, and at the end of the survey respondents were able to click on a separate url not linked to their original survey to be entered into a lottery for 1 of 4 $25 Amazon gift certificates. Our response rate calculation is imprecise as the exact number of residents who actually received the recruitment email from their residency directors is unknown, however in number of responses it was consistent with other surveys performed of this population. Of approximately 5000 active OB-GYN residents in the country, 177 responded.Statistical analysis was performed using either an Analysis of Variance (ANOVA) or two sample t tests as appropriate for the data set. Bartlett’s test was used to verify that variances were equal across the samples, in cases where ANOVA was used, such as whether knowledge differed by year of residency training. All statistical analysis was performed in Stata 14.0 . Of the residents who opened the survey, 11 failed to complete all questions and were discarded from the analysis.The FIT-KS score was calculated by dividing the number of questions answered correctly but the total number of questions – 29. The higher the score, the more answers were correct.In designing this project, an attempt was made to avoid bias by several methods. To avoid bias in our question design, we used a previously validated survey instrument, the FIT-KS. For further information on this validation process, please refer to Dr. Kudesia’s previously published paper in Fertility and Sterility .Of approximately 5000 OB-GYN residents in 255 programs in the United States, there were 177 residents who responded to the survey. Of these, 166 completed all questions in the survey. The other 11 residents opened the study, but did not complete all questions. The demographics of those who opened but did not complete all questions were consistent with the other responses from the survey per analysis by our statistician, and they were excluded from the final analysis. We do not know what proportion of respondents came from which residency programs.n = 166).Ninety-one percent of respondents were female. Sixty-nine percent were between the ages of 26–30. They represented an equal distribution between all four levels of training, with 40, 47, 39 and 40 representing each year of study (p = 0.23).The difference in mean score of each year of study was not statistically significant. In total, the highest score available was 29. Post-graduate year (PGY)-1 residents received a mean score of 20.73 , PGY-2 a mean score of 21.64 , PGY-3 a mean score of 20.95 , and PGY-4 a mean score of 21.23 . Gender was also not a significant predictor of score: females’ average score was 21.20  and males’ average scores was 20.57 . Age category also failed to predict score, with 26–30 scoring 21.08 , 31–35 scoring 21.32 , and 36–40 scoring 21.50  (p = 0.57).Thirty-three percent stated they were not concerned about their own future fertility, and level of concern did not predict average FIT-KS score; those unconcerned about future fertility had a mean score of 21.36 , while those with concerns scored an average of 21.15  (p = 0.69. One hundred and thirty-three (82%) stated that they felt comfortable discussing fertility with their patients, with a mean score of 21.29, compared to 20.9  for those who did not feel comfortable discussing fertility with their patients, p = 0.41. Seventeen respondents currently have children, with a mean score of 21.59 , not significantly higher than those who did not at 21.18 , p = 0.45.The majority of residents  stated that they had ever discussed fertility with their patients. Mean score of those who did discuss fertility was 21.21 , compared to 21.60  for those who did not, Significant gaps in knowledge were noted Table . Many anSubstantial gaps exist in fertility knowledge among OB-GYN residents. Without a strong understanding of this topic, they may not be prepared to properly counsel patients and have family planning discussions during routine visits, an essential part of well woman care. Our findings should be viewed by residency program directors as a starting place to encourage more exploration of this gap in knowledge in their own programs.Knowledge of fertility does not change throughout residency training, with this study demonstrating consistent gaps in fertility knowledge. Knowledge during residency is only slightly higher than mean scores found in prior research in Internal Medicine residents (65%), as well as a cohort of female medical students and obstetrics and gynecology residents and fellows (64.9%) , 15. In One area that the survey may not be reflective of current practice is twinning rate. Correct answer in the FIT-KS survey was coded as 21–35% twinning rate, but most recent SART data is closer to 12%, making the majority of resident respondents correct according to the most recent data.As women choose to delay childbearing, they will increasingly rely on ART, and should be sufficiently counseled on success rates that also decrease with aging . In thisN = 255).This study has a number of strengths, including using a newly validated survey, the FIT-KS, which was developed for use in physician populations. We also avoided negative reporting bias by including several analysis that were performed which upheld the null hypothesis, that is differences between the groups did not have an effect on their FIT-KS scores. To avoid selection bias, we sent the FIT-KS survey instrument to all OB-GYN residency directors in the country  rotations occur, and total time on REI rotation. Additional information should be gathered about the different REI curriculums at residency programs as they vary greatly throughout the country.Moreover, interventions, such as an online didactics curriculum on natural fertility and age related fertility decline should be developed for use in residency programs and as continuing medical education to increase knowledge in this area. Data should be collected from these endeavors such as CREOG scores prior to intervention, a pre-test, a post-test several months after intervention to gauge retention of the subject matter, and the next year’s CREOG score.Knowledge of age related fertility decline among obstetrics and gynecology residents is limited. Misconceptions about natural fertility, risk factors, and success of treatments may significantly affect the lives of both physicians and the patients they treat. The FIT-KS instrument is a valuable tool that should be continued to be used in the future in both physician and general populations, however going forward, alternative recruitment methods will assist in generating more useful data for analysis.Additional file 1. SURVEY + FIT-KS Instrument.


The Hippo pathway plays a critical role in tissue and organ growth under normal physiological conditions, and its dysregulation in malignant growth has made it an attractive target for therapeutic intervention in the fight against cancer. To date, its complex signaling mechanisms have made it difficult to identify strong therapeutic candidates. Hippo signaling is largely carried out by two main activated signaling pathways involving receptor tyrosine kinases (RTKs)—the RTK/RAS/PI3K and the RTK-RAS-MAPK pathways. However, several RTKs have also been shown to regulate this pathway to engage downstream Hippo effectors and ultimately influence cell proliferation. In this text, we attempt to review the diverse RTK signaling pathways that influence Hippo signaling in the context of oncogenesis. The uncontrolled growth of cells, due to the occurrence of a variety of biological events, leads to the formation of a neoplastic tissue or a tumor. The malignant form of a tumor, a cancerous tumor, is one of the principal causes of death and has a significant effect on longevity and life expectancy . DespiteThe continually evolving nature and the endogenous complexity of cancer cells provide them with the ability to circumvent conventional treatment strategies. As such, the next generation of therapies are focused on a personalized approach, which may involve the simultaneous targeting of several major biological pathways contributing to the aforementioned hallmarks . These nThere are numerous reports of external and internal signals capable of modulating the Hippo pathway, including events that influence cell polarity, cell adhesion, cell-extracellular matrix (ECM) contact and energy stress ,7,8. HowThe Hippo pathways connection with RTKs is not linear, and these receptors are also under the influence of the Hippo and its effectors. Due to the broad targets of Hippo effectors and their evident expression in different cellular functions, which also involve RTKs, there seems to be a multi-dimensional control mechanism between these two major cellular signaling networks. The current review aims to elaborate on this reciprocal regulatory effect.The MST1/2 phosphorylates itself and the SAV1 accessory proteinMST1/2 in a dimerized form complexed with SAV1 will be cleaved by caspase-3 to enhance the kinase activity of the complex The MST1/2-SAV1 complex phosphorylates MOB1A/B, which then associates with LATS1/2 . It thenThe phosphorylated LATS1/2-MOB1A/B will act as a kinase to phosphorylate YAP1 and TAZ ,13. The Even though the main components of the Hippo pathway were first discovered and studied in fruit flies, the critical role of this network in mammalian cellular signaling was soon evident. The Hippo pathway is a conserved signaling network in mammals with four main components and two main effector proteins. Except for SAV1, there are two, very closely related, proteins for each one of the pathway base proteins, including MST1 and MST2, LATS1 and LATS2, MOB1A and MOB1B; as well as YAP1 and TAZ . The patPhosphorylated YAP1 and TAZ either bind to 14–3–3 proteins, which sequester them in the cytosol, or will be ubiquitinated by the Skp, Cullin, F-box (SCF) complex. This eventually leads to its degradation. Unphosphorylated YAP1/TAZ interact with TEAD transcription factors, and TAZ also interacts with RUNX2 and TBX5, which activate the expression of their target genes ,16.CTGF, CYRG1, NPPA, AREG, etc.), which are mostly involved in cell proliferation and apoptosis [Therefore, the ultimate function of the Hippo pathway is the downregulation of the YAP1/TAZ target genes , which disrupts YAP function through several mechanisms. This protein phosphorylates YAP in two positions—at one position to interrupt its interaction with TEAD  and at aCyr61 and CTGF, both of which are YAP targets [Even though YAP1 activity on increasing cell proliferation and inhibiting apoptosis justifies its high level of expression and function in several cancer types, this is not the only way that YAP affects the persistence and survival of cancer cells. There are studies describing the involvement of YAP target genes in resistance against chemotherapeutic agents. The mechanism of action of anti-tubulin drugs is mediated through the activation of CDK1, which bypasses the Hippo pathway and inhibits YAP by phosphorylation at five sites. However, resistant cancer cells revive YAP1 activity by increasing mutations at these sites . Resista targets .p53 and RB [Hippo proteins act as tumor suppressors that prevent the function of YAP1 and TAZ proto-oncogenes. There are numerous studies evaluating the effects of Hippo deregulation on tumorigenesis, including reports that link LATS2, and critical tumor suppressor genes, 3 and RB ,35. HypeThe main changes to Hippo signaling in cancer occurs through modifications to gene expression or disorder in upstream regulatory signals. The rate of mutation in Hippo effectors, YAP1/TAZ, is negligible; however, modifications in their expression patterns is a common phenomenon in several cancer types. Some reports recount the upregulation of the YAP1/TAZ in the head, neck, and gynecologic cancers . MoreoveThe main Hippo components are differentially expressed across cancer types, such as in the cases of MST1/2 in soft tissue sarcomas ,39,40, LNF2 is highly susceptible to mutations, and its protein product, Merlin, has a direct activatory effect on Hippo. Hence, Merlin’s activity can lead to the downregulation of the YAP1/TAZ targets. RAS genes are molecular switches with critical regulatory effects on many signaling networks. YAP partly controls the transcription level of these genes. Consequently, the inactivation of Merlin can lead to RAS expression due to the formation of the YAP-TEAD complex. Verteporfin is a drug capable of inhibiting YAP-TEAD, which could reverse the effects of Merlin inactivation [As noted, one of the main ways the Hippo pathway influences tumorigenesis is by altering the activity of other cascades. Tumor suppressor gene tivation .Targeting YAP in regulatory T cells (Treg) can disrupt the activity of these cells to promote antitumor immune responses. Treg cells suppress immune activation, and FOXP3 is a critical transcription factor for the function of these cells. By targeting YAP, which has been demonstrated to enhance FOXP3 expression, we can attempt to control tumor growth .During the process of LATS1/2 activation, kibra (WWC1) binds LATS proteins and promotes their function, which causes YAP1/TAZ cytoplasmic localization . The ACTEven though the primary mechanism of YAP hyper-activation is through the dysregulation of the canonical Hippo components, some proteins bypass the Hippo pathway and directly activate YAP. The gain of function mutation in one set of such proteins is in the Gαq family members. It is significantly frequent in uveal melanoma, occurring in approximately 83% of these melanomas. Gαq and Trio, the guanine exchange factor for Gαq, along with RhoA and Rac1 GTPases, initiate a signaling network that promotes YAP activation and leads to YAP-dependent growth and proliferation of cancer cells independent of other Hippo pathway proteins .The examples above highlight the interplay between Hippo proteins and some regulatory proteins on the same level or downstream of the pathway. However, there are several upstream regulators of this signaling cascade, including receptor tyrosine kinases, which can inhibit the initiation of the pathway and increase proliferation signals. The regulatory effects of these proteins on the Hippo pathway, are poorly understood.Protein phosphorylation is one of the most common ways of altering a protein’s function and protein kinases are major players in signaling networks. Tyrosine kinases are switches that can stimulate or deactivate many cellular functions. Even though there are cytoplasmic tyrosine kinases, the membrane-associated RTKs are crucial in the activation state of signaling pathways due to their direct contact with the external environment . The humThe overall structure of RTKs is composed of an external ligand-binding domain, a transmembrane domain, and intracellular catalytic and adaptor domains. The conformational change after binding a ligand leads to dimerization and autophosphorylation of monomers . The tyrTyrosine phosphorylation by RTKs is important for the recruitment and activation of a variety of signaling proteins. Most RTK tyrosine phosphorylation sites are located in non-catalytic regions of the receptor molecule. RTKs should be considered as a platform for the recognition and recruitment of a specific complement of signaling proteins . SH2 , there is evidence that YAP mRNA and protein levels are increased in human PDAC relative to normal pancreatic tissues . In geneActivation of RTK initiates MAPK pathway signaling through RAS GTPase activity. The best-known effectors of RAS are RAF and phosphatidylinositol-3-kinase (PI3K) . The RASCTGF and BIRC5. However, YAP mRNA levels were not affected, implying that YAP protein stability is regulated by ERK. Several studies show a potential link between the Hippo and MAPK pathways [Immunoprecipitation studies in melanoma cancer cell lines  have shown the interaction between RAF and MST . Knockinpathways ; howeverWhen PI3K is activated by RAS, it phosphorylates phosphatidylinositol-4,5-bisphosphate (PIP2) to phosphatidylinositol-3,4,5-triphosphate (PIP3) which then activates AKT (protein kinase B). This activation plays important roles in cell proliferation, cytoskeletal rearrangement, and tumor progression . The PI3Recently, we performed a systematic gain-of-function screen for kinases involved in mammary tumorigenesis. In this screen, we made a kinase overexpressing library (KOL) and established a heterogeneous population of nontumorigenic mammary epithelial cells infected with this KOL library. Following transformation assays, mass spectrometry was performed on positive colonies and it was found that PI3KCB is a transformation-inducing kinase in breast cells . We showPI3K and the Hippo pathways also interact through MST and AKT. Activated AKT phosphorylates MST on T117 and T384. This phosphorylation limits MST interactions, including MST dimerization, transphosphorylation, and binding to the scaffold protein, RASSF1A . Interestingly, YAP forms a positive feedback loop with the PI3K pathway. Analysis of differential gene expression for YAP gain- or loss-of-function with genome-wide identification of YAP-bound loci showed that PI3KCB is a YAP-TEAD transcription target that is sufficient to activate the PI3K pathway and is necessary for cardiomyocyte proliferation and survival . The rolIn summary, two main downstream signaling pathways of RTKs, the MAPK, and PI3K pathways, have been demonstrated to regulate the Hippo signaling pathway. The detailed mechanisms of Hippo pathway regulation by EGFR was determined through studies of Drosophila. EGFR regulates Yorkie nuclear localization and Yorkie is necessary for EGFR-induced cell proliferation in Drosophila . Ajuba, EGFR is highly expressed in epithelial and fibroblast cells. The EGFR family includes four members, ERBB1-4. All members have a glycosylated extracellular domain, a single hydrophobic transmembrane domain, and an intracellular domain with kinase activity . Among sRAS, or acquired resistance, which depends on new mutations. Investigations of gefitinib-resistant A549 and PC9 cell lines compared with parental cells showed that YAP can play important roles in both primary and acquired resistance [In general, resistance to EGFR kinase inhibitors can be divided into two categories including primary resistance, which relies on other oncogenes such as sistance . Interessistance . Consistsistance . Similarsistance .He et al. showed a positive signaling loop between EGFR and the Hippo signaling pathway . YAP uprErBb2, also known as Human epidermal growth factor receptor 2 (HER2), is highly expressed in 15–20% of breast cancers. As ERBB2 (HER2) does not have any known ligand, it needs to interact with other EGFR family member proteins to be activated by transphosphorylation. Lapatinib is a potent inhibitor for both EGFR and HER2, and good responses are achieved for HER2 positive patients. Unfortunately, tumor regrowth is typically observed due to drug resistance . LapatinYAP has been reported to interacts with the carboxyl-terminal PPXY motif of ERBB4 through its WW domain . ERBB4 iCholangiocarcinoma is an aggressive malignancy with limited treatment options, as chemotherapies only increase survival modestly . PlateleCTGF, Cyr61, and CXCL5 [Insulin/insulin growth factor receptor signaling is one of the main regulators of cell metabolism, cell proliferation, and tissue growth. Insulin signaling activates Yorkie in Drosophila to induce cell proliferation . Insulinnd CXCL5 . The useRecently it has been reported that insulin receptor substrate 1 (IRS1) in breast cancer cells is a transcriptional target of TAZ . YAP-TEAThese findings serve as another example of a positive feedback loop relationship between the Hippo pathway and RTKs.Yang et al. showed that nerve growth factor (NGF) receptor tyrosine kinase (NTRK1) regulates YAP activity through the Hippo pathway . They coWith less than a 7% five-year survival rate, PDAC has emerged as one of the most aggressive cancers . PancreaHGF/MET regulates epithelial to mesenchymal transition (EMT) in normal physiology as well as during cancer progression . HGF indEph receptors are the largest family of RTKs, containing more than 14 different RTKs. Eph receptors are involved in a wide variety of biological functions such as cell attachment, cell shape, cell mobility, angiogenesis, and tumorigenesis ,111. TheSLC1A5 and GLS. Both of these genes possess several TEAD binding sites in their promoter region and interact physically with YAP/TAZ through TEAD. Inhibition studies across various points in the pathway, and confirmation of the results with complementary approaches have shown that receptor EphA2 regulates YAP/TAZ through Rho GTPase and ROCK [Glutamine addiction is frequently observed in many cancers . Glutamiand ROCK . In anotand ROCK . AlthougInterestingly, YAP/TAZ also plays important roles in reverse signaling in ligand presenting cells. For example, EphB2 expressing cells in bone marrow interact physically with TAZ. Upon activation, the cytoplasmic domain of EphB2 and TAZ co-localize into the nucleus and regulate the transcription of several important genes involved in bone marrow stromal cell differentiation and bone formation .Blood vessel formation, or angiogenesis, is a dynamic process involving cell migration, proliferation, and differentiation. The Hippo pathway’s critical role in these physiological processes indicates the importance of this pathway in angiogenesis . VEGF reIn a recent study involving a biosensor monitoring LATS kinase activity , signifiIn a similar study, Wang et al. showed that VEGF/VEGFR2 activate YAP/TAZ by regulating actin cytoskeleton dynamics. They found SFKs and Rho GTPases to be responsible for this effect on YAP/TAZ by decreasing LATS phosphorylation . MoreoveThe second important set of receptors involved in angiogenesis are Tie1-Tie2 receptors and their Angiopoietin1-2 (ANG1-2) ligands. Studies show that Tie2 is the functional receptor, and Tie1 can attenuate Tie2 homodimer signaling by making Tie1-Tie2 heterodimers . MoreoveThe regulation of ANG-2 gene expression by YAP1 has been confirmed in several studies ,121,122.Apart from the principal VEGFRs and Tie-ANG receptors, fibroblast growth factor receptors (FGFRs) are also involved in the process of angiogenesis. These receptors participate in several other physiological phenomena including growth, wound healing, and embryogenesis. Members of the FGFR family of RTKs and their ligands interact with the Hippo pathway both in normal and tumor cells. In one study on osteoblastic MC3T3-E1 cells, FGF2 treatment led to decreased levels of the TAZ protein and this effect was correlated with SAPK/JNK MAP kinase activity . AnotherRizvi et al. explored an autocrine Hippo/FGFR signaling in cholangiocarcinoma and determined that FGFR1, −2, and −4 expression is in part driven by YAP/TBX5 complex, but this is not true for FGFR3 because it doesn’t have a TBX5 binding sequence. Interestingly, YAP activity is triggered largely by FGF5/FGFR2 activation. The FGF5/FGFR2 effect on YAP is mediated by reducing LATS cellular levels to prevent YAP phosphorylation . FGFR3 aEven though most of the interactions between RTKs and Hippo occur through PI3K/Akt and MAPK signaling cascades, there are some reports on the direct interaction between ERBB4 and YAP  Figure . In one The aforementioned examples are not the only instances of interconnecting signaling networks between the Hippo pathway and RTKs, as numerous active studies are investigating this relationship. For instance, a recent study uses a novel biosensor  to invesOriginally identified as a key player in physiological development, we have now come to appreciate the broader influence of the Hippo pathway on numerous cellular processes, including in malignancies such as cancer. The intersection of the Hippo pathway with RTKs, master regulators of cell signaling, is unsurprising as Hippo appears to have a seemingly ubiquitous influence across the cell. While Hippo’s main effects are executed through the RTK/RAS/PI3K and RTK-RAS-MAPK signaling pathways, we have highlighted that diverse molecular interactions contribute to the ultimate downregulation of YAP1/TAZ target genes. Continued investigation to better understand this complex signaling pathway may provide the coveted therapeutic targets that will effectively intervene upstream signaling events in the tumor microenvironment.


Ursolic acid (UA) is an anti-cancer herbal compound. In the present study, we observed the effects of UA on anchorage-dependent and -independent growth of human colorectal cancer (CRC) RKO cells.RKO cells were cultured in conventional and detached condition and treated with UA. Cell viability was evaluated by CCK-8 assay. Cell cycle was analyzed by flow cytometry. Apoptosis was identified by Hoechst 33258 staining and flow cytometry analysis. Activities of caspases were measured by commercial kits. Reactive oxygen species (ROS) was recognized by DCFH-DA fluorescent staining. Anoikis was identified by EthD-1 fluorescent staining and flow cytometry analysis. Expression and phosphorylation of proteins were analyzed by western blot.UA inhibited RKO cell viability in both a dose- and time-dependent manner. UA arrested the cell cycle at the G0/G1 phase, and induced caspase-dependent apoptosis. UA inhibited Bcl-2 expression and increased Bax expression. In addition, UA up-regulated the level of ROS that contributed to UA activated caspase-3, − 8 and − 9, and induced apoptosis. Furthermore, UA inhibited cell growth in a detached condition and induced anoikis in RKO cells that was accompanied by dampened phosphorylation of FAK, PI3K and AKT. UA also inhibited epithelial-mesenchymal transition (EMT) as indicated by the down-regulation of N-Cad expression and up-regulation of E-Cad expression.UA induced caspase-dependent apoptosis, and FAK/PI3K/AKT singling and EMT related anoikis in RKO cells. UA was an effective anti-cancer compound against both anchorage-dependent and -independent growth of RKO cells.The online version contains supplementary material available at 10.1186/s12906-021-03232-2. Colorectal cancer (CRC) has the third highest malignancies incidence , and second highest mortality rate  globally . It is nActinidia chinensis Planch (ACP) (Teng-Li-Gen) is a common anti-cancer traditional Chinese medicine for CRC treatment. It has been confirmed that ACP inhibits CRC cell proliferation  × 100%.A total of 1 × 105 RKO cells were seeded into a 6-well plate. After culturing for 24 h, the cells were treated with different doses of UA or same volume of DMSO for 48 h. The cells were collected, fixed with 75% ethanol, stained with propidium iodide (PI), and analyzed by flow cytometry for cell cycle identification. For apoptosis detection, UA treated RKO cells were sequentially stained with FITC Annexin V and PI, and then detected by flow cytometry.A total of 2.5 × 105 RKO cells were seeded into a 6-well plate and cultured for 24 h, at which time, different concentrations of UA or an equal volume of DMSO was added. After 48 h of culture, cells were fixed, followed by staining with Hoechst33258. Cell morphology was then examined by fluorescence microscopy.A total of 2.5 × 105 RKO cells were seeded into a 6-well plate and cultured for 24 h, after which, the cells were treated with different concentrations of UA or same volume of DMSO for 48 h, and harvested. Activities of caspases were detected by commercial kits in which Ac-DEVD-pNA, Ac-IETD-pNA and Ac-LEHD-pNA were used as the substrates of caspases-3, − 8 and − 9, respectively. To address the role of caspases in UA-induced apoptosis, RKO cells were pre-treated with 100 μM of Z-VAD-FMK (a caspases inhibitor) for 2 h, followed by UA treatment and apoptosis detection.A total of 2.5 × 105 RKO cells were seeded into a 6-well plate, cultured for 24 h, and treated with different doses of UA or an equal volume of DMSO for 48 h. The cells were then stained with 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) (diluted at 1:1000) for 20 min, observed by fluorescence microscopy, and detected by a fluorescent plate reader (excitation wavelength of 488 nm and an emission wavelength of 525 nm). For inhibition of ROS, RKO cells were pre-incubated with NAC (200 μM) for 2 h, followed by treatment with UA, and subsequent detection of caspases and apoptosis.ROS was measured according to the manufacturer’s protocol. Briefly, 2.5 × 105 RKO cells were seeded into a poly-HEMA coated 24-well plate and cultured for 24 h. Next day, the cells were treated with different doses of UA or same volume of DMSO for 48 h, and stained with 1 μl of EthD-1 for 1 h at 37 °C. The fluorescence was observed under a microscope, and measured by a fluorescence microplate reader (set at an excitation wavelength of 525 nm and an emission wavelength of 590 nm). For identification of apoptosis, the UA treated RKO cells that were grown in suspension, were sequentially stained with FITC Annexin V and PI, and then analyzed by flow cytometry.A total of 2.5 × 10Protein expression and phosphorylation was evaluated by Western blotting as described previously , 9. Briep < 0.05.The data were expressed as mean ± standard deviation (SD). The differences between groups were compared by One-way analysis of variance (ANOVA), and statistically differences were considered at p < 0.05), the half maximal inhibitory concentration (IC50) value of UA was 18.54 μM. RKO cells growth was completely inhibited by 40 μM of UA. Thus, 14–17 μM UA was selected for subsequent studies. Further study showed that 14–17 μM UA significantly inhibited RKO cells viability in a dose- and time-dependent manner (p < 0.01)  Fig. b. Meanwh01) Fig. c.Fig. 2p < 0.01), while cells at the S phase decreased correspondingly (p < 0.01) after treating with 14–17 μM UA for 48 h. These observations suggested that UA can arrest RKO cell cycle at the G0/G1 phase.The effects of UA on cell cycle were analyzed by PI labeling and flow cytometry. The results were shown in Fig. p < 0.01)  Fig. b and c.p < 0.05)      Fig. a and b. 01) Fig. c-f. Thesp < 0.01). In addition, the anoikis assay showed that suspension grown RKO cells could absorb EthD-1 and emit red fluorescence following UA treatment (p < 0.01)  – which we believe to be anoikis  Fig. b and c, kis Fig. d.Fig. 7FAK is an important protein regulating anoikis, and its downstream signaling is closely related to the PI3K/AKT pathway . In the Anoikis resistance is closely related to EMT , 14. UA Cancer cells proliferate in an uncontrolled manner, for this reason, inhibiting cell proliferation is the basic principle of cancer therapy . ResultsApoptosis is regulated by a cascade of protein-mediated reactions, which is an important mechanism of cancer treatment . Apoptot2•− and H2O2. High level of ROS induces apoptosis by activating caspase-8 or caspase-9 via the mitochondrial or death receptor pathways. DCFH-DA can freely penetrate the cell membrane, which can be hydrolyzed to form DCFH by the esterase, thus generating 2′,7′-dichlorofluorescein (DCF) with fluorescence intensity that is associated with the effects of ROS. The fluorescence intensity of DCF reflects the relative levels of intracellular ROS. DCFH-DA staining showed that RKO cells emitted green florescence after UA treatment, indicating that UA increased ROS levels. Further study showed that NAC antagonized UA activated caspase-3, − 8 and − 9 and promoted apoptosis. These observations suggested that ROS was involved in the apoptosis-inducing effects of UA in RKO cells.Apoptosis is associated with a variety of factors, including ROS production , 20. ROSWe further observed the effects of UA on suspension growth of RKO cells by using poly-HEMA to reduce cell adhesion. The results showed that UA inhibited the suspension growth and stimulated anoikis in RKO cells. Anoikis is a type of apoptosis occurred in epithelial cells due to the detachment from ECM, including epithelial cancer cells, and thus to be known as detachment-induced apoptosis . FAK is EMT is related to anoikis resistance , 14. EMTIn summary, the present study suggested that UA inhibits cell viability, induces cell cycle arrest at the G0/G1 phase, and regulates Bcl-2/Bax expression, increases ROS leading to activation of caspase-3, − 8 and − 9, as well as inducing apoptosis in RKO cells. UA also inhibits suspension growth of RKO cells, elicits anoikis, and may related to FAK/PI3K/AKT singling and inhibition of EMT. This study provided the fundamental basis for applying UA to treat colorectal cancer.Additional file 1: Supplementary Figure 1. UA regulates Bcl-2 and Bax expression. UA treated RKO cells were subjected to Western blot with antibodies against Bcl-2 (A), Bax (B), and GAPDH (C). Supplementary Figure 2. UA regulates anokis related proteins expression. UA treated RKO cells were subjected to Western blot with antibodies against p-FAK (A), FAK (B), p-PI3K (D), PI3K (E), p-AKT (G), AKT (H), N-Cad (J), E-Cad (K), and GAPDH .


Expression changes of the genes responsible for cytotoxicity and apoptosis processes of HAM/TSP patients and asymptomatic carriers were investigated. Expression of the genes involved in cytotoxicity and apoptosis in HAM/TSP patients was decreased; hence, a model was proposed indicating that the spread of immune responses in HAM/TSP may be due to expression of HTLV-1 virulence factors and the resistance of HTLV-1-infected cells to apoptosis.Although human T-cell lymphotropic virus type-1 (HTLV-1) was the first retrovirus among human pathogens to be identified, insufficient information on the pathogenesis of HTLV-1 infection means that no precise mechanism has yet been provided for HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Based on previous studies, it was found that apoptosis and inflammation stimulation were among the most important mechanisms underlying HAM/TSP. The present study provides an  Retroviridae and was first isolated from a young patient with cutaneous T-cell lymphoma by Robert Gallo et al. in 1980 [Tax and HTLV-1 bZIP factor (HBZ), and host epigenetic and immune changes, in particular expression of cytokines, activate NK cells and T-lymphocytes; the altered cell signalling pathways subsequently promote progress toward HAM/TSP and ATLL [Human T-cell lymphotropic virus type-1 (HTLV-1) is one of the most important members of the family  in 1980 . Based o in 1980 . No precand ATLL .The immune system employs a variety of approaches including apoptosis of infected cells, production of granzymes and perforin enzymes, release of nitric oxide radicals, phagocytosis, and antibody production to eliminate infectious agents, especially HTLV-1 . HoweverFas, FasL, perforin, granzyme, granulysin, bax, Bad, BH3-family, BCL2 and caspase8 gene expression changes between the AC and HAM/TSP groups, and a molecular signalling network is presented for the pathogenesis of HTLV-1 based on the target genes.Previous studies have shown that HTLV-1-infected cells undergo apoptosis, which is an important mechanism for virus elimination . HoweverP-values <0.05, and positive fold change numbers were used to indicate upregulation and the negative fold change values downregulation [www.hiv.lanl.gov based on the default instructions.The gene expression profiles of polymorphonuclear neutrophils were primarily obtained in ACs and HAM/TSP individuals under accession number GSE19080 from the NCBI Gene Expression Omnibus (GEO) database. Microarray data were then obtained individually from the AC and HAM/TSP groups. In addition, the GEO2R software and R.3.2.5 software (MetaQC and Affy packages) were used to calculate the fold change and expression changes of differentially expressed genes (DEGs) between the AC and HAM/TSP groups. Analysis of DEGs was performed using Benjamini–Hochberg false discovery rate (FDR)-adjusted The Protein–Protein Interaction Network (PPIN) was plotted using the online STRING  server, and the KEGG and Enrichr  databases were used for gene ontology. Finally, the molecular signalling network was determined according to the collected information.According to our study aims, the expression changes of the most important genes involved in the apoptosis process, including Fas, FasL, perforin, granzyme, granulysin, Bax, Bad, BH3-family, Caspase8 and BCL2, were investigated in ACs and HAM/TSP patients . In geneAnalyses of the gene network indicated that genes associated with the process of apoptosis and the cell cytoskeleton are surrounded by genes in the immune system, and the differentiation and survival process is dependent on immune system changes. Furthermore, the genes responsible for the PI3K-Akt, JNK, NF_κB, TP53 and MAPK signalling pathways are the most common genes that form the gene network . AccordiAccording to the gene ontology data, the analysed genes regulate various processes including apoptosis, oxidative stress, TP53 signalling, and the production of pro-inflammatory cytokines such as IL-6, 3 and 7 . Based oTax and HBZ. In general, it appears that the decrease in apoptosis and cytotoxicity in HAM/TSP patients is due to the expression of HTLV-1 virulence factors and the continuous stimulation of PMN migration by chemokines produced by astrocytes, neurons and oligodendrocytes.Finally, the molecular signalling network was presented based on the findings . It appeDespite extensive studies of the immunopathogenesis of HTLV-1, no precise mechanism has yet been provided for its links to ATLL and HAM/TSP. On the other hand, as the host epigenetic changes also play an important role in the progression of infection to the disease, extensive studies at the transcriptomic level could provide important information on the pathogenesis of HTLV-1 and the mechanism leading to ATLL and HAM/TSP . In addiTax, increase Th1 production and consequently increase the inflammation and tissue destruction in the CNS [Tax transcription is much higher in HAM/TSP patients than in ACs and ATLL patients [HAM/TSP is a chronic neuro-inflammatory infection, which develops slowly but persistently, eventually leading to paralysis . Multi-o the CNS . Based opatients .Cytolytic enzymes and apoptosis are the most important mechanisms of the immune system against viral infections, and do so by destroying cells infected with the virus . PerforiFollowing the stimulation of FasR, an intermediate molecule termed FADD, which can stimulate cellular apoptosis by stimulating caspase 8, is activated; the BH3-family also induces cytochrome C release from the mitochondria and causes the activation of caspase cascade by affecting Bad and Bax, ultimately leading to cell death .et al., expression of the genes involved in cell cytotoxicity and apoptosis in HAM/TSP patients is lower than in ACs [et al., the increased expression of MSH2 (a DNA repair protein) in HAM/TSP patients can lead to increased apoptosis [In the present study, expression changes of key genes of the cytotoxic and apoptosis pathways between the AC and HAM/TSP groups were compared and evaluated. According to the study results, overall expression of cytolytic enzymes in the HAM/TSP group was partially reduced. In addition, the results showed an apparent reduction in the expression of the genes involved in cell apoptosis in the HAM/TSP group, such that there was a significant difference between the Fas and caspase 8 genes. However, it should be noted that these are preliminary hypotheses and further studies are needed for confirmation. Based on a review of studies, the process of apoptosis is one of the most important approaches for the treatment of HTLV-1 infection, particularly for the elimination of IL-2-dependent CD4+ T cells in ATLL patients . Accordin in ACs . In anotn in ACs . It has n in ACs . Despitepoptosis .et al. has also shown that overexpression of Tax can inhibit Bid and Bim expression and the resistance of HTLV-1-infected cells to apoptosis [et al. found that HTLV-1 HBZ leads to a decrease in apoptosis by inhibiting the IRF-1 signalling pathway [et al. also suggested that overexpression of PSMB8 increases apoptosis during HTLV-1 infection [A study carried out by Mühleisen poptosis . However pathway . Mozhgannfection . Howevernfection .Expression of the genes responsible for cytotoxic activities and apoptosis was reduced in HAM/TSP patients compared to ACs, and it appears that the survival of HTLV-1-infected T cells (HTLV-1-infected CD4+ T cells) stimulates and increases IFN-γ production, increases lymphocyte responses and leads to progression towards HAM/TSP. However, this hypothesis cannot be confirmed due to inconsistent literature results, and further studies in this area are needed. Furthermore, based on previous literature reviews, it can be concluded that the processes of cell differentiation, cell survival and apoptosis are among the most important cellular processes that play important roles in the pathogenesis of HTLV-1 infections. Studying and targeting these pathways may lead to the introduction of anti-HTLV-1 drugs.


Hyperuricemia is associated with insulin resistance, pancreatic β-cell dysfunction and consequently with development of type 2 diabetes. Although a direct relationship between high levels of uric acid (UA) and the development of diabetes is still a controversial issue, there is some evidence that strongly points to pancreatic β-cells damage as a result of high serum UA levels. Here, the mechanisms underlying UA-induced β-cell damage are discussed. Available literature indicates that UA can decrease glucose-stimulated insulin secretion and cause β-cell death. The mechanisms underlying these effects are UA-induced oxidative stress and inflammation within the β-cells. UA also stimulates inducible nitric oxide (NO) synthase (iNOS) gene expression leading to NO-induced β-cell dysfunction. Thus hyperuricemia may potentially cause β-cell dysfunction, leading to diabetes. It may be hypothesized that in hyperuricemic subjects, UA-lowering drugs may be beneficial in preventing diabetes. Worldwide, the prevalence of diabetes is about 8–9 % , 2 and iUric acid (UA) is the end product of exogenous and endogenous purine (adenine and guanine) metabolism , 8. The High serum UA levels is a risk factor for type 2 diabetes mellitus (T2DM) as reported in different population-based studies –18. AccoUrate transporters include, (i) urate transporter 1/solute carrier family 22, member 12 (URAT1/SLC22A12), (ii) ATP-binding cassette subfamily G, member 2/breast cancer resistance protein (ABCG2/BCRP), and (iii) glucose transporter 9 (GLUT9/SLC2A9) . ExpressIn 1948, Griffiths reported that feeding rabbits with a diet that was deficient in methionine and cystin for 6–7 weeks decreased blood glutathione levels by about 40–53 % . IntrapeUA inhibits GSIS in isolated pancreatic rat islets , 38, panThe association between UA and insulin secretion is quite complex. It has been shown that UA increases GSIS in isolated perfused rat pancreas . In addiIn addition to decreased GSIS, other mechanisms are involved in hyperuricemia-induced β-cell dysfunction, development of glucose intolerance, and T2DM. These include, increased inducible nitric oxide (NO) synthase (iNOS)-derived NO production , 40, incIn Min6 cells, UA activates the NF-κB signaling pathway by phosphorylation and degradation of inhibitor of κB (IκB) ; NF-κB iHigh levels of UA inhibit the growth of the pancreatic β-cell lines (INS-1 and RIN-m5F) in a time- and dose-dependent manner via the ROS-AMPK-ERK signaling pathway . High coIt has been shown that the timing of serum UA peak (5:08) and serum NO nadir (5:32) coincide in healthy men, suggesting that their concentrations are physiologically related . In addimito) function from efflux to influx. This calcium overload increases ROS production, which decreases eNOS expression and NO release, causing endothelial dysfunction [mito is involved in insulin secretion from β-cells [UA increases iNOS expression in the β-cells, decreases GSIS, and causes apoptosis . Howeverfunction . Because β-cells , one canConsidering UA as a target for prevention/management of diabetes is still premature and needs to be evaluated in clinical trials. However, several lines of evidence indicate a potential favorable outcome of these drugs in diabetes. Zurapamic, an inhibitor of UA reabsorption in the kidneys, protects INS-1 cells and rat islets against UA-induced damage by decreasing URAT1 expression and oxidative stress . AllopurUA induces oxidative stress, the inflammatory response in the β-cells, and decreases GSIS, causing β-cell apoptosis. The threshold theory for the actions of UA on the β-cells hypothesizes that the detrimental effects of UA occurs above a given concentration. In support of this notion, it has been shown that the inhibitory effect of UA on GSIS in rat pancreatic islets has a sudden occurrence at a threshold of 6.7 mg/dL (0.4 mM) . Other hRegarding the association between UA and β-cell function, the effects of UA on the genes and proteins that are involved in insulin biosynthesis and secretion warrants further investigations. In addition, most mechanistic findings have been drawn from in vitro studies or from animal studies. As always, it should be noted that extending results from animal studies to humans needs abundance of caution, as UA metabolism is different between humans and rodents . Unlike All in all, hyperuricemia may potentially cause β-cell dysfunction and predispose subjects to metabolic disorders such as diabetes. If this holds true, then UA-lowering drugs may be helpful in prevention/management of diabetes, at least in subjects who are at risk for both hyperuricemia and diabetes.