Understanding the molecular mechanisms underlying prostate cancer (PCa) progression towards its most aggressive, castration-resistant (CRPC) stage is urgently needed to improve the therapeutic options for this almost incurable pathology. Interestingly, CRPC is known to be characterized by a peculiar hormonal landscape. It is now well established that the androgen/androgen receptor (AR) axis is still active in CRPC cells. The persistent activity of this axis in PCa progression has been shown to be related to different mechanisms, such as intratumoral androgen synthesis, AR amplification and mutations, AR mRNA alternative splicing, increased expression/activity of AR-related transcription factors and coregulators. The hypothalamic gonadotropin-releasing hormone (GnRH), by binding to its specific receptors (GnRH-Rs) at the pituitary level, plays a pivotal role in the regulation of the reproductive functions. GnRH and GnRH-R are also expressed in different types of tumors, including PCa. Specifically, it has been demonstrated that, in CRPC cells, the activation of GnRH-Rs is associated with a significant antiproliferative/proapoptotic, antimetastatic and antiangiogenic activity. This antitumor activity is mainly mediated by the GnRH-R-associated Gαi/cAMP signaling pathway. In this review, we dissect the molecular mechanisms underlying the role of the androgen/AR and GnRH/GnRH-R axes in CRPC progression and the possible therapeutic implications. Prostate cancer (PCa) still remains the second leading cause of cancer-related deaths in Western countries, although a higher survival rate and a long-term decline in mortality have been recently reported . Most PCIt is now well established that the androgen/androgen receptor (AR) axis remains a key player in the growth of CRPC ,11,12,13Second-line antiandrogen therapy is indicated for the treatment of CRPC patients, supporting the idea that the androgen/AR axis is still active in this progression phase of the tumor ,39,40,41About two decades ago, it was reported that, after ADT therapy, intratumoral levels of androgens remain high in spite of serum castration levels of testosterone . This obIt has also been suggested that, in CRPC tissues, androgens can be synthesized from cholesterol; however, this issue is still a matter of debate ,50. CYP1AR gene copy number [CRPC tissues (about 80%) from patients who progressed after ADT express high levels of AR; 30–50% of these tissues were reported to carry AR amplification, due to the presence of a high y number ,55,56. Ay number . The ovey number ,59.AR gene, belonging to the steroid hormone receptor superfamily, composed of eight exons, is mapped on chromosome Xp11-12 and encodes a 110 kDa (920 amino acids) protein. The full-length AR protein consists of three domains: the NH2-terminal transactivation domain , the central and conserved DNA-binding domain , a flexible hinge region containing a nuclear localization signal and the COOH-terminal ligand-binding domain . The NTD domain contains two trinucleotide repeats that encode polyglutamine and polyglycine tracts. The interaction between NTD and LBD is necessary for the receptor transcriptional activity. In the absence of androgens, the AR is located in the cytoplasm, where it is present in an inactive conformation associated with heat shock proteins. Once bound by androgens , the receptor translocates into the nucleus, where it is activated through dimerization, recruitments of coregulatory/epigenetic factors and stimulation of specific target genes [The et genes ,60,61,62Gain-of-function mutations of the AR are quite frequent (about 50%) in CRPC patients after antiandrogen therapy; these are usually single point mutations occurring mostly in the LBD of the receptor ,64. The AR receptor genes were reported to be easily detectable in cell-free DNA (cfDNA) obtained from CRPC patients. These data strongly support that the presence of specific circulating mutant ARs can represent a useful biomarker in terms of personalized therapy in CRPC [Recently, mutant  in CRPC ,75,76,77The most frequent mutations of the AR in CRPC patient tissues and plasma (cfDNA) are reported in AR gene can undergo alternative splicing, giving rise to different splice variants (AR-Vs) [During the last two decades, it has become increasingly clear that, in PCa cells, the  (AR-Vs) ,80,81,82 (AR-Vs) . The exp (AR-Vs) . In nude (AR-Vs) ,86; in l (AR-Vs) . In huma (AR-Vs) ,90,91,92 (AR-Vs) ,95,96,97AR gene in CRPC [Additional transcript variants are generated by alternative splicing of the  in CRPC ,98,99. I in CRPC ,100,101.Mechanistically, the AR-Vs can form homodimers or heterodimers (by combining with other variants), or they can dimerize with the full-length AR. At the nuclear level, the dimers bind to response elements in the promoter region of specific downstream genes , thus modulating their expression and promoting the development of CRPC ,91,102.Based on these observations, targeting the AR-Vs and their signaling pathways might represent a novel and effective therapeutic strategy for the treatment of CRPC patients.AR-mediated gene transcription requires the interaction of the receptor with different coregulators  and transcription factors (such as GATA2 and FOXA1) ,105,106.The transcriptional activity of AR requires the recruitment and cooperation of transcription factors. Among these, the GATA family of transcription factors, consisting of six members, was reported to be involved in the AR-mediated signaling in CRPC cells ,43,106. FOXA-1 (forkhead box A1) is another pioneer transcription factor involved in AR-promoted gene transcription. It was demonstrated to play a key role in AR-mediated tumor growth and progression in CRPC cells . MechaniCoregulators modulate the activity of several proteins in the transcription complex through chemical modifications and are also involved in the recruitment of general transcription factors associated with RNA polymerase II to the constitutive promoter of target genes ,111. SpeTaken together, these observations support the notion that the interaction of AR with specific transcription factors and coregulators plays a key role in promoting PCa growth and progression. These mechanisms are presently considered a possible molecular target for novel therapeutic approaches for CRPC.The most relevant molecular mechanisms underlying the persistent activity of the androgen/AR axis in CRPC cells are summarized in GnRH agonists  bind to pituitary GnRH-Rs and, after an initial stimulation , induce their down-regulation and desensitization leading to the suppression of gonadotropins (LH and FSH), and subsequently, of testosterone secretion ,120. To In the last three decades, it has been widely demonstrated that GnRH and GnRH-Rs are expressed also in tumor tissues, including PCa and, specifically, CRPC ,126,127.1-His2-Trp3-Ser4-Tyr5-Gly6-Leu7-Arg8-Pro9-Gly10-NH2) is a decapeptide synthesized in a small number of neurons and released in a pulsatile way into the portal blood vessels, through which it reaches the gonadotrope cells at the pituitary level. Here, it binds to its specific receptors, GnRH-Rs, to stimulate gonadotropin secretion and, consequently, gonadal androgen production [2) domains of the decapeptide are essential for its binding to GnRH-Rs. Hypothalamic GnRH , known to be involved in the biological functions of the neurohormone. On the other hand, GnRH-II conserves the amino acid sequence of GnRH in both the N- and C-terminal domains, supporting that it may recognize, bind and activate the same receptors. The presence of a receptor specific for GnRH-II in vertebrates is still a controversial issue [Petromyzon marinus); its structure differs from that of GnRH in amino acids 5–8 ; this isoform was shown to possess a very low gonadotropin-releasing effect in vertebrates [In addition to the classical form of GnRH , other forms of the peptide have been identified. The isoform II (GnRH-II) has been observed in most vertebrates . It is aal issue ,133,134.tebrates ,135,136.GnRH was reported to be expressed in different types of cancer cells. Specifically, GnRH mRNA expression and immunoreactivity were identified in breast, ovarian, endometrial as well as in PCa cells, both androgen-dependent and castration-resistant. In tumor cells, including CRPC cells, this peptide was shown to be endowed with a significant antiproliferative/proapoptotic biological activity ,140,141.The molecular structure of the pituitary GnRH-R, belonging to the GPCR (G protein coupled receptor) receptor family, was identified by Kakar et al., in 1992 . Its gen2) into the two second messengers, inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DG), leading to protein kinase C (PKC) activation and increased cytoplasmic levels of Ca2+, respectively. Both PKC and Ca2+ trigger specific downstream signaling pathways involving proteins of the MAPK cascade , finally mediating the biological activity of the receptor ligands, i.e., expression and secretion of the two gonadotropins [Endogenous GnRH, as well as its synthetic agonists, by binding to this receptor, triggers the recruitment/activation of the Gαq/11 subunit of the G protein complex, thus activating the downstream effector phospholipase Cβ (PLCβ). PLCβ, in turn, catalyzes the hydrolysis of phosphatidylinositol 4,5-biphosphate  in human tissues has been widely investigated; however, this receptor still must be cloned and sequenced. It has been proposed that the GnRH-II-R might correspond to a five transmembrane domain protein, which lacks the transmembrane regions 1 and 2 . AnotherThe presence of the GnRH-II-R was also investigated in human cancers. Emons and coworkers reported the presence of a functional GnRH-II-R in tumors of the female reproductive system  ,33,166. The activation of locally expressed GnRH-Rs, by means of GnRH agonists, was widely reported to induce antitumor/proapoptotic effects in PCa cells. Specifically, we demonstrated that GnRH agonists, significantly and dose-dependently, reduce the proliferation of both androgen-dependent (LNCaP) and castration-resistant  human PCa cells, both in vitro and in vivo ,169,170.GnRH-R activation was further reported to be associated with proapoptotic effects ,176. In It is now well known that GnRH-R activation interferes with the protumor activity of growth factors and their locally expressed receptors in cancer, and, specifically, in PCa cells ,183,184.The insulin-like growth factor (IGF) signaling axis, composed of two receptors (IGF-IR and IGF-IIR) and their specific ligands (IGF-I and IGF-II), was widely shown to be up-regulated in PCa cells and tissues ,186,187 The EGF/EGF-R  signaling pathway is also deeply involved in PCa growth and progression ,194,195.Based on the undesired initial flare effect triggered by GnRH agonists, GnRH antagonists, able to compete with the binding of the endogenous decapeptide to its pituitary receptors, were subsequently developed, and it was expected that these compounds might act as GnRH-R antagonists also in tumors. Surprisingly, it was widely demonstrated that GnRH antagonists act as agonists in cancer cells, including PCa cells, exerting a significant antiproliferative/proapoptotic activity both in vitro and in vivo ,201,202.To explain the agonistic behavior of GnRH antagonists at the level of tumor cells, Millar and coworkers proposed that these receptors may adopt different conformations according to the cell context in which they are expressed, thus selectively binding to the different GnRH analogs  . In addition to the classical form of GnRH, its GnRH-II isoform has also been reported to be expressed in tumors, including PCa ,31,212. CRPC is frequently associated with the development of metastasis, specifically at the bone, lymph node and visceral level; a better understanding of the molecular mechanisms involved in the metastatic events associated with PCa progression might help identify novel biomarkers and possible targets to increase the therapeutic approaches against this almost untreatable disease ,216,217.A remodeling of the intracellular cytoskeleton as well as of the extracellular matrix is deeply involved in the motility and invasive behavior of cancer cells associated with the tumor metastatic spread. We demonstrated that, in CRPC cells, GnRH-R activation significantly reduces both cell migratory and invasive behavior. Moreover, GnRH agonists interfere with the prometastatic activity of IGF-I by affecting cell morphology, cytoskeleton organization and the expression of the αvβ3 integrin, involved in the cell to extracellular matrix adhesion in PCa tissues . In line with these data, it was demonstrated that not only GnRH but also the GnRH-II isoform inhibits CRPC cell motility through the remodeling of actin cytoskeleton .Dondi and coworkers reported that, in DU145 and PC3 CRPC cells, GnRH analogs inhibit the activity of the plasminogen activator (PA) system, implicated in the local degradation of the extracellular matrix. Specifically, they showed that both the GnRH agonist leuprolide and the antagonist cetrorelix decrease cell motility and invasiveness by reducing the enzymatic activity and the secretion of uPA (urokinase-type PA), while increasing the expression of the PA inhibitor PAI-1 . In lineIt is well known that metastasis is a complex process that involves the cooperative actions of different cancer cell subpopulations, in which cancer stem cells would be responsible for the final step of colonizing premetastatic niches. Cancer stem cells are also deeply involved in the mechanisms of drug resistance, being able to avoid the effects of standard antitumor therapies . RecentlAngiogenesis is the process by which new vascular vessels form from the pre-existing vasculature. In tumors, the formation of new blood vessels is necessary to provide an appropriate blood supply to support cell viability and proliferation. Hence, this process plays a key role in tumor progression and is now recognized as one of the hallmarks of cancer ,225. VasIn our laboratory, we observed that GnRH-Rs are expressed in HUVEC  cells; GnRH agonists reduce HUVEC cell proliferation and counteract the effects of VEGF-A on their ability to form capillary-like tubes . RecentlThe inhibitory effects of GnRH-R activation on tumor growth and progression suggested that, in cancer cells, these receptors might be coupled with intracellular signaling mechanisms different from those found at the pituitary level. It is now well accepted that, while GnRH-Rs expressed on the gonadotrope cell membranes are associated with the Gαq/PLC/PKC signaling pathway, in cancer cells, these receptors are mainly coupled with the Gαi/cAMP/PKA cascade of transduction mechanisms ,239,240.c-fos) and, consequently, of their protumoral effects [We demonstrated that, in PCa and, specifically, CRPC cells, GnRH-R activation significantly interferes with the forskolin-induced increase in cytoplasmic cAMP levels while pertussis toxin counteracts the antiproliferative effects of GnRH analogs , suggest effects ,218,239. effects . The JNK effects ,177. Sim effects ,241,242.Naor’s laboratory widely demonstrated that the Gαq/PLC/PKC pathway is also involved in the antiproliferative/proapoptotic activity of GnRH analogs in cancer cells . It was Taken together, these data strongly support the notion that different GnRH-R-associated signaling pathways are involved in the opposite effects of GnRH analogs in pituitary vs. CRPC cells.The main signaling pathways associated with the GnRH/GnRH-R axis in CRPC cells are summarized in ADT, based on GnRH agonists or antagonists, still remains the mainstay treatment of hormone-dependent PCa. Results from different clinical trials also support the use of AR antagonists, such as enzalutamide and apalutamide, as well as of androgen synthesis inhibitors, such as abiraterone, for the treatment of hormone-sensitive PCa ,250,251.Taxane-based cytotoxic chemotherapy, i.e., docetaxel, is considered a therapy of choice for CRPC patients; cabazitaxel was also introduced in the clinical settings; however, its efficacy was found to be lower than that of docetaxel in a phase III clinical trial (FIRSTANA) . ImmunotGiven the persisting role of the AR in the progression of PCa to the CRPC stage, inhibitors of the androgen pathways are commonly used for the treatment of CRPC patients ,257,258.The presence of a GnRH/GnRH-R axis associated with antitumor activities in PCa and specifically in CRPC cells supports the notion that it might be considered an additional direct target of GnRH analog-based (agonists and antagonists) ADT. Gnanapragasam and coworkers reported that a high expression of GnRH-Rs correlates with a better clinical outcome in CRPC patients treated with GnRH agonists . MoreoveAs discussed above, by binding to GnRH receptors expressed in PCa cells, as specifically in CRPC cells, GnRH antagonists behave as agonists triggering marked antitumor effects. To this purpose, it must be underlined that these compounds elicit a faster suppression of testosterone, as well as of FSH and PSA levels, in PCa patients ,274. AbuRecently, cytotoxic GnRH bioconjugates were developed as a new therapeutical approach for tumors expressing GnRH-Rs; these compounds consist of a GnRH-derivative covalently linked to a cytotoxic drug. It is expected that, by binding to its receptor, the GnRH derivative may specifically carry the cytotoxic drug to cancer cells without affecting normal cells. AEZS-108 , a bioconjugate consisting of a GnRH derivative covalently linked to doxorubicin via an ester bond, was developed, and its anticancer activity was investigated in different types of tumors ,283,284.It is now well established that the progression of PCa towards its most aggressive CRPC phase is characterized by an active hormonal landscape.Specifically, a persistent activity of the androgen/AR axis has been demonstrated to be present in CRPC cells. This is correlated to different molecular mechanisms, including: intratumoral synthesis of androgens; AR amplification; AR mutations; alternative splicing of the AR mRNA leading to the expression of AR splice variants ; increased expression/activity of transcription factors and coregulators of the AR. Based on these data, second-generation non-steroidal antiandrogens  as well as inhibitors of enzymes involved in the androgen biosynthesis  are commonly used for the treatment of CRPC patients.In addition, a GnRH/GnRH-R axis, widely reported to be associated with a substantial antiproliferative/proapoptotic, antimetastatic and antiangiogenic activity, is also expressed in CRPC cells. In line with these data, a high expression of GnRH-R in PCa tissues correlates with a better clinical outcome in CRPC patients. Interestingly, in cancer cells expressing the GnRH receptor, GnRH antagonists trigger the same antitumor activity of GnRH agonists, suggesting that differences in the molecular structure/mechanisms of these receptors might exist at the tumor vs. pituitary level. In accordance with these observations, ADT interventions, based on a gonadotropin-releasing hormone agonist, with or without an antiandrogen drug (AR antagonist or inhibitor of androgen synthesis), represent a mainstay treatment for PCa, even in the CRPC stage ,257,258.In spite of these promising results, the incidence of PCa is still increasing. Underlying the molecular pathways of PCa development and progression towards the most aggressive, castration-resistant stage is mandatory for the identification of novel molecular markers/targets and therapeutic approaches for CRPC patients.AR gene copy number, in plasma DNA is a minimally invasive tool for the identification of the development of resistance in PCa patients escaping enzalutamide- or abiraterone-based therapies. Recently, Beltran and coworkers reported that CRPC patients with high plasma AR copy number do not have a worse response to taxane-based chemotherapy compared with patients with normal plasma AR. These authors speculate that the analysis of plasma levels of AR copy number in the different stages of PCa might improve therapy selection for CRPC patients [Assessing the AR status, and, specifically, the patients .ARs were reported to be easily detectable in cfDNA from CRPC patients [Several studies are presently ongoing with the aim to solve the issue of the role and subsequent potential targeting of AR mutations in PCa. As discussed in this review, specific AR mutations not only confer resistance to antiandrogens  but can also convert these compounds in AR agonists, indicating a rapid development of drug resistance. In recent papers, by means of molecular docking and molecular dynamics simulations, novel AR antagonists were developed and found to exert a significant antitumor effect in CRPC cells as well as in PCa cells engineered to overexpressed the F876L mutant AR ,299. Morpatients ,75,76,77patients ,300. HowAR-V7 is the most frequent AR splice variant detected in PCa, and, specifically, in CRPC tissues and its levels of expression, appears to be strictly correlated to resistance to enzalutamide and abiraterone. CTCs are now considered a clinically relevant biomarker of disease progression in different pathologies, including PCa. Recently, it has been reported that AR-V7 mRNA expression, assessed via the Adna test platform and the EPIC sciences CTC-based platform , can be detected in the plasma from patients with mCRPC treated with enzalutamide. Compared with AR-V7 negative patients, AR-V7 positive patients treated with enzalutamide showed a shorter PFS and OS ,302. ThuExtracellular vesicles  carry different types of bioactive molecules to recipient cells in the tumor microenvironment. EVs mediate intercellular communications, including re-education of stromal cells, modulation of cancer metabolism, and also development drug resistance. The bioactive molecule profiles/signatures of tumor-derived Es change over time, reflecting the real-time status of cancer cells. Specifically, different bioactive molecules have been identified in PCa-derived EVs, such as AR and AR-V7, caveolin-1 (involved in cancer cell stemness), has-miRNA-940 , integrin αvβ3 and αvβ6 (involved in PCa cell metastatic behavior), miR-409 (involved in prostate tumorigenesis), TGFβ  and MDR-1  ,303. BasAs discussed in this review, GnRH receptors, associated with a significant antiproliferative activity, are expressed in PCa, and, specifically, in CRPC cells and tumors. Moreover, GnRH-R expression was also demonstrated in PCa stem cells isolated from explants of human prostate tumors. PCa patients expressing high levels of GnRH receptors, have a better clinical response to the GnRH-analog based therapy. GnRH analogs, either alone or together with AR inhibitors or taxane derivatives, still represent the mainstay therapy for CRPC patients.Based on this observation, GnRH analog -based cytotoxic bioconjugates have been recently developed with the aim to specifically carry the cytotoxic drug  to cancer cells expressing the GnRH-R, while sparing normal cells. In particular, the bioconjugate AEZS-108 (AN-152) consists of a GnRH derivative linked to the cytotoxic drug doxorubicin. This compound was found to significantly inhibit the growth of CRPC cells, both in vitro and in vivo. Phase I and II clinical trials showed that AEZS-108 efficiently reduces PSA levels and increases PFS in CRPC patients. Further studies are needed to confirm these as promising. In conclusion, a deeper clarification of the expression and activities of both the androgen/AR and GnRH/GnRH-R axes in the CRPC stage will likely lead to the identification of novel predictive biomarkers as well as to the improvement of the therapeutical options for this almost untreatable disease, in terms of precision medicine.


The retarding performance and acid–rock reaction rates of AR-1 and AR-2 were evaluated, and the experimental results indicated that AR-2 had a better retarding performance than AR-1, and the acid–rock reaction rate of AR-1 was higher than AR-2. This could be because AR-2 with its hydrophobic chains forms a thicker adsorbed film. This was confirmed by X-ray photoelectron spectroscopy (XPS) and adsorption behavior studies. The scanning electron microscope (SEM) images, contact angle, and XPS results showed that both retarders form adsorption films on the surface of rocks, indicating that the introduction of AM and APEG-1000 makes the retarder adsorb on rock surfaces. The introduction of hydrophobic chains for AR-2 enhanced the thickness of adsorbed film, indicating that adding hydrophobic chains to the acidizing retarder significantly improves its retarding performance.Acidizing retarders are used to reduce the reaction rate between hydrochloric acid (HCl) and carbonates to increase the efficiency of acidification of oil and gas wells and increase oil and gas recovery. We synthesized two polymeric acidizing retarders (AR-1 and AR-2) to explore ways to improve the efficiency of this process. Retarder AR-1 is based on acrylamide (AM) and allyl polyethylene glycol (APEG-1000), and retarder AR-2 is composed of AM, APEG-1000, and octadecyl dimethyl allyl ammonium chloride (C-18). The molecular structures of AR-1 and AR-2 were characterized by Fourier transform infrared (FT-IR) and  Acidizing retarders are used to reduce the reaction rate between hydrochloric acid (HCl) and carbonates to increase the efficiency of acidification of oil and gas wells and increase oil and gas recovery. Therefore, there has been a search for chemicals that could be added to the HCl to reduce the reaction rate between the HCl and carbonates and allow the HCl to penetrate more deeply into the reservoir.Because of the world's continuing reliance on oil and natural gas, there has been concern about the decrease in the recovery rates of oil fields.8 foam acid,9 diverting acid,10–13 and others have been used to reduce the acid–rock reaction rate.14–16 The high viscosity of thickening acid effectively reduces the diffusion rate of hydrogen ions to the rock surface, thus reducing the reaction rate of acid–rock. However, the secondary damage caused by the thickening acid is a problem, and it is difficult to pump it in and out, so its application is limited. Foaming acid is a system with a mixture of acids, such as hydrochloric acid, hydrofluoric acid, and formic acid, introduced with bubbles created by adding a water-soluble polymer as a foaming agent and a foam stabilizer to the acid solution. Foaming acid with its high apparent viscosity and small filtration loss can effectively slow down the acid–rock reaction, but its foam stability is poor under high temperature conditions.17 The mixture of a chemical diverting agent and acid is called diverting acid. The existing diversion methods include in situ cross-linked acid and self-diverting acid.18,19 The diverting acid blocks some of the channels produced by dissolution, changing the flow profile and forcing the acid solution to flow into a relatively low permeability formation, which improves the acidification effect, but it is expensive and has poor temperature resistance and a complex construction technology.In the past, thickening acid,+ ions by increasing the viscosity of acids, the other separates the acid solution from the rock by forming a film on the surface of the rock, thus reducing the reaction rate between the acid and rock.At present, the retarding mechanisms are divided into two types. The first, reduces the diffusion rate of the H20–22 research group proposed that a retarding acid with low viscosity could be adsorbed on the surface of carbonate rock and form a hydrated film that would delay the acid–rock reaction, but no published research has described a study of which factors could influence the film-forming properties.The adsorptive acidizing retarders include small molecular surfactants and macromolecular polymers synthesized using acrylamide. However, the former has the shortcomings of large dosage and high price, the latter mainly reduces the acid–rock reaction through the method of adsorbed film. Quan'sIn this paper, we describe our development of two low viscosity acidizing retarders (AR-1 and AR-2) that differ with respect to the presence of hydrophobic functional groups. AR-1 was synthesized using AM and APEG-1000, and AR-2 was synthesized using AM, APEG-1000, and C-18. The addition of C-18 adds hydrophobic groups to AR-2. We compared the retarding performance of the two acidizing retarders to better understand the key role of hydrophobic chains in retarding performance.2.2.1Acrylamide , allyl polyethylene glycol , hydrochloric acid , anhydrous ethanol (>99.5%), and potassium bromide  were purchased from the Chengdu Kelong Chemical Factory. Octadecyl dimethyl allyl ammonium chloride  was bought from Jiangsu Fumiao chemical reagent plant. 2,2′-Azobis(2-methyl-propionamidine)dihydrochloride  was purchased from Aladdin chemicals.2.2The two reactions were completed in 250 mL three-necked flasks. We synthesized AR-1 by combining AM and APEG-1000 in a mole ratio of 24 : 0.25, and we synthesized AR-2 by combining AM, APEG-1000, and C-18 in a mole ratio of 22 : 1 : 1. We added V50 as an initiator. The two reactions were conducted at 50 °C for 5 h, and the two products were purified with ethanol and dried at 40 °C for 24 h.2.32.3.11H-NMR spectra of AR-1 and AR-2 in D2O using a Bruker ASCENT-400 NMR (Switzerland) at room temperature.We generated FT-IR spectra of AR-1 and AR-2 using a WQF-520 Fourier transform infrared spectrophotometer, and we generated 2.3.2We determined the molecular weights of AR-1 and AR-2 using gel permeation chromatography .2.3.3We allowed the HCl in AR-1 and AR-2 to react with carbonate rock for 45 min at 50 °C, and we observed the morphology of the rock surface using scanning electron microscopy .2.3.4We prepared AR-1 and AR-2 with different mass concentrations and reacted them with carbonate rock for 10 minutes at 50 °C. We then put water droplets on the rock surface and determined the contact angle of the rock surface using an interface parameter measuring instrument .2.3.52. We calculated the retarding rate using m is the dissolution mass of the rock sample (g), m1 is the mass of the rock sample before the reaction (g), and m2 is the mass of the rock sample after the reaction (g). s is the surface area of rock (cm2), Δt is the reaction time (s), K is the retarding rate (%), V0 is the reaction rate of the AR-1 or AR-2 acid and rock (mg (cm2 s−1)−1), and V1 is the reaction rate of the hydrochloric acid without AR-1 and AR-2 and rock, (mg (cm2 s−1)−1).We added AR-1 and AR-2 to HCl solutions to yield mass concentrations of 20%. The reactions between HCl solutions with the AR-1 and AR-2 added and carbonate rock were carried out at 30 °C, 50 °C, and 70 °C, with a rock surface area of 5 cm2.3.62 gas produced by the reaction of the retarding acid and carbonate rock with a gas-collecting device. The acid–rock reaction rate was calculated using the volume of the collected CO2,23,24 and we calculated the reaction rate using u is the reaction rate of the retarding acid and carbonate rock (g cm−2 min−1), n3)(CaCO is the amount of substance of CaCO3 (mol), M3), n2)(CO is the volume of CO2 gas per unit of reaction time (mL), V1 is the molar volume at 25 °C.We added different amounts of AR-1 and AR-2 to 20 wt% HCl to obtain different concentrations of AR-1 and AR-2 retarding acid solutions. We combined these solutions with carbonate rock at 50 °C and collected the CO3.3.1−1 for AR-1 corresponds to its O–H stretching vibration, and the peak at 3423 cm−1 for AR-2 corresponds to its O–H stretching vibration. The peaks at 2933 and 2877 cm−1 for AR-1 correspond to the C–H stretching vibrations of its –CH2 and –CH3 groups, and the peaks at 2942 cm−1 and 2867 cm−1 for AR-2 correspond to the C–H stretching vibrations of its –CH2 and –CH3 groups. Characteristic absorption bands at 1656 cm−1 (AR-1) and 1673 cm−1 (AR-2) correspond to C–O stretching vibrations of polyacrylamide, and the peak at 647 cm−1 for AR-2 correspond to the absorption peak of the C–H bonds of C-18. These results indicate that we successfully synthesized AR-1 and AR-2.1H-NMR spectra of AR-1 and AR-2 that we used to provide further evidence of the successful synthesis of AR-1 and AR-2.2–CH– and –CH– scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O)–) appear at 1.52 (marked as 3) and 2.20 ppm (marked as 6), respectively. The peak for –CH–CH2–O– is at 2.11 ppm (marked as 5). The peak at 3.59 ppm (marked as 9) is for –CH2–CH2–O–. The spectrum for AR-2 is similar to that of AR-1. The chemical shift peak for –CH–CH2–N– is at 2.11 ppm (marked as 5), and the characteristic chemical shift peaks for –CH2–N– and –N–CH3 appear at 3.28 (marked as 7) and 3.32 (marked as 8), respectively.For AR-1, the chemical shift peaks of –CH1H-NMR analysis, we successfully synthesized AR-1 and AR-2, and their structures were consistent with our expected structures.According to the results of our FT-IR and Mn) and the weight-average molecular weight (Mw) of the two retarders, and We measured the number-average molecular weight  with the XRD diffractogram recorded between 10° and 60° with Cu Kα X-rays at 85 W. 3.25The interplanar spacing and the intensity of the diffraction peaks shown in 3.3We added AR-1 and AR-2 to 20 wt% HCl to obtain different concentrations of samples, and we measured the viscosities of the mixtures. The viscosities were measured by a rotational viscometer  at 30 °C. 26,27The results in 3.4−1, their viscosities were the same, but when the concentrations were greater than 6000 mg L−1, the viscosities of AR-1 were higher than for AR-2. Because AR-2 has higher retarding rates than AR-1 despite its lower viscosity, the retarding mechanism must be different from the traditional method for which higher viscosity leads to decreased acid–rock reaction. The results also suggested that the introduction of hydrophobic chains on AR-2 molecules played a significant role in retarding performance.3.52 production for the reaction of acid and carbonate for different concentrations of AR-1 and AR-2.2 produced during the acid–rock reaction decreased with increasing polymer concentration. Comparison of the acid–rock reaction rate of AR-1 and AR-2 shows that the reaction rate of AR-1 was higher than for AR-2, indicating that the introduction of hydrophobic chains on AR-2 cause it to do a better job of blocking the reaction of HCl with the carbonate rocks. This will be explained below.3.6The microstructure of carbonate rocks treated in different ways was obtained by SEM. + ions from coming into contact with the rock surface, thus reducing the reaction rate between the acid and rock. The smoother and more obvious film shown in the SEM images for AR-2 helps to explain why AR-2 acts as a more efficient retarder.3.728Q is the adsorption capacity, mg g−1; C0 is the concentration before adsorption, mg L−1; C1 is the concentration after adsorption, mg L−1; w is the concentration of adsorbent, g L−1.Adsorption isotherms describe the relationship between the adsorption capacity of adsorbent and the equilibrium concentration of adsorbate in the liquid when the adsorption achieves equilibrium at a given temperature.29 The two models are most commonly used to determine isotherms for different adsorbent/adsorbate systems, to interpret solid–liquid adsorption, and predict their equilibrium parameters.C is the equilibrium concentration (mg L−1), qe (mg g−1) and qm (mg g−1) are the equilibrium and maximum adsorption capacity, respectively. The KL (L mg−1) is the Langmuir coefficient and the Kf (mg g−1) is the Freundlich coefficient. The “n” parameter indicates the adsorption intensity.We further explored the adsorbing behavior and mechanism by fitting the data using Langmuir and Freundlich models. The Langmuir and Freundlich isotherm equations were expressed by R2) for AR-1 was close to 1 for the Langmuir model, indicating that the Langmuir model was appropriate for generating the isotherm adsorption data of AR-1. Because the polyoxyethylene ether chain at AR-1 helps AR-1 to be attracted to a carbonate surface by hydrogen bonds, the adsorption tended to be monolayer adsorption.The Langmuir model yields the homogeneous adsorptive energies on the adsorbent surface. 30R2) for AR-2 was close to 1, indicating that the Freundlich model was suitable for generating the isotherm adsorption data of AR-2. Because its hydrophobic chain is pointed away from the carbonate rock when AR-2 is adsorbed on the rock surface, and the association between the hydrophobic chains leads to multi-layer adsorption.The Freundlich isotherm model yields the heterogeneous adsorptive energies on the adsorbent surface.The difference of adsorption isotherm models between AR-1 and AR-2 demonstrates that the introduction of hydrophobic chains enhanced the thickness of adsorption film, which can further explain why the retarding effect of AR-2 is superior to AR-1.3.831,32We used contact angle to test the wettability of the rock surface.−1, the contact angle of the rock surface gradually increased with increasing concentration, but still was less than 90 degrees. This was because during the adsorption process of the AR-2, the hydrophobic chains of AR-2 were outward, which gradually changed the rock surface from hydrophilic to more hydrophobic. When the concentration of AR-2 was beyond 8000 mg L−1, the contact angle on the rock surface was greater than 90°, which could be due to the intermolecular association of polymers, and with the number of hydrophobic chains decreased, the rock surface was identified as water wetness gradually.−1 AR-2 for different acid–rock reaction times, and To further understand the wettability of the rock surface after AR-2 acid interacted with it, we measured the contact angles of 8000 mg L33–36 When the reaction time was longer than 40 min, the wettability gradually changed from increasing hydrophilicity to increasing hydrophobicity. This could be due to desorption of the AR-2 from the rock surface, exposing the hydrophobic end of AR-2 on the rock surface. These trends provide evidence that there was multilayer adsorption of AR-2 on the rock surface.In summary, the contact angle experimental results showed that the rock surface itself was hydrophilicity. Although the addition of the retarder AR-1 decreased the hydrophilicity of the rock, the rock surface remained hydrophilic. The adsorption of the retarder AR-2 on the rock surface led to a more significant decrease in the hydrophilicity of the rock surface with increasing AR-2 concentration and acid–rock reaction time, indicating that the introduction of a hydrophobic chain improved the film-forming ability of the AR-2 on the rock surface, making AR-2 a better retarder.3.937,38 on the rock surface after retarding acid interacted with rock. We did X-ray photoelectron spectroscopy  analysis to determine the elemental composition of the substancesI0 and Id are the initial and the thickness of the photoelectron intensity, d is the thickness of the adsorption layer, EK is the optoelectronic kinetic energy, λ(EK) is the average escape depth of photoelectrons.We used 4.We synthesized two retarders, AR-1 and AR-2, and studied their retarding properties. The results showed that the retarding rate of AR-2 with its hydrophobic chain was higher than that of AR-1, and this was further verified by acid–rock reaction rate and SEM experiments. SEM images showed that there was an adsorption film on the rock surfaces treated by both retarding acids, and the thickness of the adsorption film was measured by XPS showed that the adsorption film thicknesses of AR-1 and AR-2 on the rock surface were 1.5371 nm and 2.2864 nm, respectively. The XPS and contact angle analysis of the rock surfaces after the acid–rock reactions verified that the two retarders were adsorbed on the rock surface. We found that the Langmuir model (monolayer adsorption) was a better fit for AR-1, and the Freundlich model (multi-layer adsorption) was a better fit for AR-2. This shows that the introduction of a hydrophobic chain on AR-2 increases the thickness of the adsorption films on rock surfaces, thereby improving the retarding rate of the retarding acid. This provides a direction for further research on retarding acids.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported on this paper.


The treatment of Lumbar Disc Herniation (LDH) in elite athletes is a debated topic that lacks consensus in the literature due to varying outcome reporting methods. The objective of this study was to quantify the overall performance of a sample of professional athletes before and after receiving a lumbar discectomy or microdiscectomy in a cohort of players in the National Football League (NFL), National Basketball Association (NBA), National Hockey League (NHL) and Major League Baseball (MLB).The authors identified publicly accessible data from a cohort of different types of professional players who received either a lumbar discectomy or a microdiscectomy. These records were identified through newspaper archives, injury reports, player profiles and press releases between 1993 through 2015. Fantasy and Wins Above Replacement (WAR) scores were calculated for each player.A total of 38 professional players met study inclusion criteria. NFL players had the lowest return-to-play (RTP) at nine of 14 (64%). The RTP for NBA, NHL and MLB players were comparable with 6/7 (86%) vs 8/9 (89%) vs 7/8 (88%). NFL players had the lowest average career length after surgery at 34.8 months, while NBA players had the longest average career length at 48 months. MLB players on average required the longest time to return to presurgical level of performance (24 months) and required the longest average recovery time at 12 months.Based on these results, the average performance of most elite athletes are likely to decrease after undergoing a lumbar discectomy. Although it appears that performance peaks in the initial years after the operation for some players, there was an overall long-term decline in this sample of elite athletes. Study limitations included small sample size, lack of controlling for possible confounding variables  and use of variable reporting sources. Additional studies with larger sample sizes and age-matched controls are needed to examine the effects of lumbar discectomy more comprehensively in elite athletes. Symptomatic lumbar disc herniation (LDH) is common in the general population.The success of such an operation is generally evaluated by relief of symptoms and the ability of the patient to return to his/her daily functioning.In 2011, Hsu et al. investigated outcomes after the diagnosis of LDH in a sample of professional athletes of American football, baseball, hockey, and basketball.The clinical outcomes of LDH after a discectomy have been well studied in the general population. However, the treatment of LDH in elite athletes is a debated topic that lacks consensus in the literature.Fantasy sports games have been used to provide real-life information about a player’s performance known as “fantasy scores”. Such “fantasy scores” have been observed as a validated form of performance in professional athletes after injury.The aim of this study was to quantify the performance of professional athletes before and after a lumbar discectomy or a microdiscectomy using assigned fantasy scores in a cohort of NFL, NBA, NHL and MLB players. Prior studies have assessed clinical success based on the return to play rate (RTP), recovery time, and career length.In addition, the authors examined the RTP, average career length, average length of time to return to presurgical performance and recovery time. The authors’ primary hypothesis was that elite athletes would exhibit a decrease in performance after undergoing a lumbar discectomy or a microdiscectomy because of the unusual physical demands.http://www.nba.com), NBA (http://www.nba.com), NHL (http://www.nhl.com) and MLB (http://www.mlb.com).Using a previously published methodology,http://www.rotoworld.com). Player information for MLB players were obtained from FANGRAPHS (http://www.fangraphs.com). Both data sources contained in-depth sports reporting and sponsor fantasy leagues on their respective platforms.Inclusion criteria included professional athletes who missed at least one game during their regular or postseason season after receiving a lumbar discectomy operation. Player information including primary position, dates of operations, number of games played, fantasy scores and WAR were compiled. For NFL, NBA and NHL players, this information was obtained from ROTOWORLD  points scored by the player, (2) field goal percentage, (3) free throw percentage, (4) three-point field goals, (5) assists, (6) steals, (7) blocks, (8) rebounds, and (9) turnovers.To track the performance of MLB players, WAR was used instead of fantasy points. WAR values for each MLB player were obtained from FANTASYGRAPHS. WAR is one statistic that assesses a player’s contribution to the team. WAR utilizes various inputs, which that illustrated in the following formula for position players:Essentially, the formula calls for the sum of batting runs, base running runs, fielding runs, positional adjustment and league adjustment. Replacement runs are added to compare the player’s performance to the replacement level rather than the average player. This sum amounts to runs above replacement (RAR). The RAR is subsequently divided by runs per win to yield WAR. WAR is all-inclusive and provides a useful reference point for comparing two different players or comparing players against themselves from season to season.The formula for WAR for pitchers was different from positional players. Pitching WAR is based on the number of innings the pitcher threw, park pitched in and fielding independent pitching (FIP). FIP is a measure of a pitcher’s run prevention independent of the performance of their defense. The formula for FIP is illustrated below:Where HR is number of home runs, BB is walks, HBP is hit by pitch, K is strikeouts and IP is innings pitched. The FIP constant is calculated via the following formula:Where lgERA, is the league earned run average, lgHR is league home runs, lgBB is league’s walks, lgHBP is league hit by pitch, lgK is league strikeouts and lgIP is league innings pitched.For review of data findings, the player’s name, position, date of operation, season played, games played and fantasy score  or WAR (for MLB players) were tabulated in a Microsoft Excel 2016 spreadsheet. Next, the average number of games played and fantasy score or WAR prior to and after the date of the index operation were calculated. This enabled the authors to compare a player’s performance against themselves before and after the surgery.Finally, the average number of games played and the fantasy score or WAR were graphed with respect to each season the player played in as a visual representation of a player’s performance and to discern the pattern of postoperative performance. The average of the number of games played and average fantasy score prior and after surgery were compared to assess how the surgery affected a player’s performance.Other parameters that were examined included: RTP, career length after the index operation, length of time to return to presurgical level of performance and recovery time. For this study, the RTP was defined as the percentage of athletes that returned to professional play after undergoing the procedure. Career length after the index operation was defined as the number of years played after undergoing surgery.Length of time to return to presurgical level of performance was defined as the time it took for the player to reach a fantasy value after surgery that was equivalent to his fantasy score the year before surgery. Recovery time was defined as the length of time between surgery and return to competitive play in the patients’ sports.A total of 38 (92.7%) professional athlete players met study inclusion criteria of approximately 41 first-identified athletes. Sample players were split into four cohorts . For each player, the number of games played and fantasy score for each season was tabulated and graphed as illustrated in The authors also examined the performance of 14 NFL players. Ten players (71%) exhibited a decrease in the number of games played and fantasy score after undergoing a lumbar discectomy. Next, the authors examined performance outcomes of seven NBA players. Four players (57%) showed a decrease in the number of games played and fantasy score after undergoing the index operation, while one player did not return to play. Notably, there was one (14.3%) NBA player who demonstrated an increase in fantasy value and decrease in the number of games played. In addition, there was one (14.3%) other NBA player who exhibited a decrease in fantasy value, but increased games played.The authors also examined the statistics of nine NHL players. Four (44.4%) NHL players showed a decrease in the number of games played and fantasy value status post a lumbar discectomy. Two (22.2%) NHL players saw an increase in games played, but they still exhibited decrease in fantasy value. Therefore, six (67%) players were found to have a decrease in fantasy value. Interestingly, three (33%) players exhibited an increase in games played and fantasy value.Finally, the authors reviewed the statistics of eight MLB players. Seven MLB players (88%) exhibited a decrease in games played and WAR. However, one player (a pitcher) exhibited an increase in games played and a decrease in WAR.In addition, the performance graphs for all players (available by request from the corresponding author) were generated. In summary, 12 (31%) of all sample players demonstrated an increase in games played and fantasy score or WAR within the first two post-procedure years. However, after the first couple of years a steep decline in performance occurred. Interestingly, this spike in performance after surgery was seen in NFL, NBA and NHL players, but not in MLB players.Lumbar discectomy and microdiscectomy for LDH herniation are very successful operations in the general population, with clinical success rates ranging from 75 to 90%.A total of 38 players with a confirmed lumbar discectomy or microdiscectomy and missed time were included in our review. The average RTP across the four leagues was 31 of 38 (82%). This was a rate that was consistent with previously published data.In terms of post-operative performance, NBA players exhibited the longest career length at an average of 48 months, and MLB players had the longest length of time required to return to presurgical level of performance at 24 months. These patterns may be a reflection of the relative physical demands of each sport and differences in medical adherence protocol or differences in medical clearance protocols.The average recovery time of 2.8 to 12 months after lumbar discectomy in this sample of elite athletes was greater than that in the general population .Sample players who returned to play after undergoing a discectomy had lower average games played [14.63 (pre-op) vs 13.5 (post-op)], lower fantasy scores and WAR values [73.94 vs 43.63] compared to prior the operation. This phenomenon could be attributed to athletes not allowing enough time to recover from their operation because there is so much emphasis about “getting back to the game” in addition to the unusual physiologic demands.Notably, there was one (14.3%) NBA player who demonstrated an increase in fantasy value and decrease in the number of games played as well as one (14.3%) other NBA player who exhibited a decrease in fantasy value, but increased games played. Both of these scenarios appear to be anomalies, although previous studies have shown that physical performance in the NBA tends to decline with age even though postoperative playing time and technical performance remains stable or increased.The summary performance graphs in the appendix reveals a trend in which some athletes display an increase in performance in the first few years after the operation, followed by a sharp decline. This increase in performance could be attributed to the player now playing pain free. Without pain, the player is better at playing the game. However, our data shows that their performance is expected to decline as time passes.Our study results are subject to several limitations. First, our small sample of male-only professional athletes may not comprise a true representation of all types of elite athletes. Another limitation concerns the inherent variations of reporting sources of performance-based outcomes and complications that may have skewed our outcome measures. Our study lacked age-matched controls which may have provided a more rigorous longitudinal performance representation of aging athletes over time.A final limitation concerns the use of statistics websites and reliance on publicly accessible news information sources prone to reporting bias. The detailed medical information of each athlete was limited to medical personnel documentation and data regarding some complications or patient specific factors that could have confounded our interpretation of available data was not always available.Based on these study results, the average performance of an elite athlete tends to decrease after undergoing a lumbar discectomy. Although it appears that performance peaks in the initial years after the operation for some players, there is an overall long-term performance decline. More studies are needed to better understand the effects of lumbar discectomy in elite athletes. Future larger-scale prospective studies could also be conducted to define the best treatment and recovery regimens for LDH in different types of elite athletes.NoneNone


Background:Dermatophagoides pteronyssinus 1/2 (Der p 1/Der p 2) are regarded as important allergens of house dust mite (HDM). However, the effect of both products on the epithelial barrier and immune response of patients with and without HDM allergic rhinitis (AR) remains unclear.Methods: Air–liquid interface (ALI) cultured human nasal epithelial cells (HNECs) derived from control subjects (non-AR) (n = 9) and HDM-AR patients (n = 9) were treated with Der P 1 and Der P 2, followed by testing the transepithelial electrical resistance (TEER), paracellular permeability of fluorescein isothiocyanate (FITC)-dextrans and immunofluorescence of claudin-1 and ZO-1. Interleukin-6 (IL-6) production was evaluated by ELISA.Results: Der p 1 reduced TEER significantly in a transient and dose-dependent manner in HNEC-ALI cultures from HDM-AR and non-AR patients, whilst the paracellular permeability was not affected. TEER was significantly reduced by Der p 1 at the 10-min time point in HDM-AR patients compared to non-AR patients (p = 0.0259). Compared to no-treatment control, in HNECs derived from HDM-AR patients, Der p 1 significantly cleaved claudin-1 after 30 min exposure  and induced IL-6 secretion (p = 0.0271).Conclusions: Our results suggest that patients with HDM-AR are more sensitive to Der p 1 than non-AR patients with increased effects of Der p1 on the mucosal barrier and induction of inflammation, indicating an important role for Der p1 in sensitization and HDM-AR development. Allergic rhinitis (AR) is a common disorder involving immunoglobulin E (IgE)-mediated type I allergic inflammation of the nasal mucosa following allergen exposure . TypicalDermatophagoides pteronyssinus 1/2 (Der p 1/Der p 2) are major components of HDM extracts and are regarded as the most important HDM allergens. Der p 1 is a 25-kDa protein and has enzymatic activity as a cysteine protease. It is classified as a digestive tract enzyme as Der p 1 is found mainly in the excrement of mites. Previous studies reported that Der p 1 increased the permeability of airway epithelial cells via its protease activity , formed between the apical surface of adjacent cells, are considered to be vital components in maintaining the epithelial cell and mucosal membrane integrity. TJs limit the diffusion of solutes through the intercellular space and provide cellular polarity separating apical and basolateral domains , 9. In aIn order to develop effective treatments for AR it is vital to understand the mechanisms of how allergens affect the nasal mucosa. The aim of this study was to evaluate the effect of Der p 1 and Der p 2 on the barrier of air-liquid interface cultured primary human nasal epithelial cells derived from patients with and without HDM-AR.This study was performed with approval of The Central Adelaide Local Health Network Human Research Ethics Committee (reference HREC/15/TQEH/132 and 13604). All participants provided informed written consent in accordance with the Declaration of Helsinki. Primary human nasal epithelial cells (HNECs) were obtained from the inferior turbinate surface with sterile nasal brushes during paranasal sinus, nasal septum or pituitary tumor surgeries. The diagnosis of HDM allergy was based on the International Consensus Statement on Allergy and Rhinology  and incubated with 0.05% trypsin . After 5 min the trypsin reaction was neutralized with 10% fetal bovine serum in PBS, the cells were centrifuged  and resuspended in Ex medium. Cell suspensions  were seeded in 100 μL Ex medium onto collagen coated apical chambers of 6.5-mm-diameter polyester membranes with a pore size of 0.4 μm . 500 μL Ex medium was added in the basolateral chamber. After 2 days, the medium was removed from the apical chamber and the basolateral chamber medium was changed to ALI medium to allow differentiation of cells. ALI medium consisted of PneumaCult™-ALI Basal Medium  along with PneumaCult™-ALI 10X Supplement, PneumaCult™-ALI Maintenance Supplement  and penicillin-streptomycin/amphotericin B . Medium was changed every 48–72 h. The cells were cultured for at least 21 days before conducting any experiment , then HNECs were collected after centrifugation (500 g for 7 min) and resuspended in Ex-medium consisting of PneumaCult™-Ex Plus Basal Medium  along with PneumaCult™-Ex Plus 50X Supplement, Hydrocortisone Stock Solution  and penicillin-streptomycin/amphotericin B . The resuspended cells were incubated in culture plates coated with anti-CD68  to remove macrophages for 20 min at 37°C. Supernatants containing HNECs were seeded in collagen-coated T75 cell culture flasks  and grown in Ex-medium at 37°C, 5% COALI cultured HNECs were treated with purified protein Der P 1 or Der P 2 (purity of 98%)  at 0.4, 2, and 4 mM concentrations (diluted in PBS) and TEER was measured with a voltohmmeter at various time points up to 6 h  whilst putting cell cultures on a heating plate . PBS was added as a negative control and 5% Triton was added as a positive control. TEER measurements at each time point were normalized with the values at time = 0 before treatments exposure .2, 95% humidity for 2 h. Samples were then taken from the basolateral compartment and transferred to a clear bottom black 96-well plate. The amount of passaged FITC-dextran was measured by a FLUOstar Optima 96-well fluorescence microplate reader  at excitation and emission wavelengths of 485 and 520 nm, respectively.To measure the paracellular flux, 4-kDa fluorescein isothiocyanate (FITC)-dextran  was used as a tracer. FITC-dextran was diluted in PBS at a concentration of 3 mg/ml. The apical chamber of HNEC-ALI cultures was washed two times with PBS after the last (6 h) TEER measurement followed by incubation with 100 μL FITC-dextran at 37°C, 5% COHNEC-ALI were fixed in PBS containing 2.5 % formalin for 10 min and preserved at −20°C. HNEC-ALI were permeabilized with 1% sodium dodecyl sulfate on ice for 15 min. Permeabilized cells were then blocked with serum free blocker  at room temperature for 1 h, followed by overnight incubation with primary antibodies diluted in PBS: 1:50 rabbit anti-claudin-1; 1:100 mouse anti-zonula occludens (ZO)-1 . This was followed by incubation of donkey anti-rabbit Cy3 (1:200 in PBS) and donkey anti-mouse IgG Alexa Fluor 488  and incubated for 1 h at room temperature. Then nuclei were stained by incubation with 200 ng/ml of 4′,6-diamidino-2-phenylindole  for 10 min at room temperature. Finally, HNEC-ALI cultures were mounted with a drop of fluorescence anti-fade mounting medium  before cover slipping. The samples were washed with PBS four times between each step. Images were recorded using a confocal laser-scanning microscope LSM700  with × 20 magnification. Image processing was performed with ZEN Imaging Software . Three different areas were randomly selected for quantification in individual samples. Digital image stacks were created as 20 μm thickness with 21 sequential slices. The mean fluorescence intensity of claudin-1 and ZO-1 was normalized against DAPI.Interleukin-6 (IL-6) levels produced by HNEC-ALI cultures were measured with enzyme-linked immunosorbent assay (ELISA) kit  after application of 4 mM Der p 1, 4 mM Der p 2 for 6 h. PBS was used as negative control and 10 μg/ml poly (I:C) LMW  was used as positive control. The medium in the basolateral chamber was harvested after completion of the TEER experiment and stored at −20°C. Amounts of IL-6 in the medium were determined according to the manufacturers' protocols. All samples were measured in duplicate. The optical density (OD) was measured at 450 nm and IL-6 concentration was determined using the standard curve prepared for individual assay , 23, 24.t test or Fisher exact test. P < 0.05 were considered statistically significant.All results are presented as the mean ± standard error of the mean (SEM). The collected data were analyzed with statistical software . The comparisons in multiple treatment conditions were conducted by one-way analysis of variance (ANOVA), followed by a Dunnett's test. Statistical significance between two groups was determined by using a 2-tailed unpaired n = 9; non-AR, n = 9). Demographic factors and comorbidities of patients are detailed in p = 0.30)  and 1-h time point  (p < 0.0001 in both groups), followed by a gradual restoration of TEER back to baseline from the 2-h time point onwards. A significant reduction in TEER was observed in HNEC-ALI cultures treated with 4 mM Der p 1 at the 10-min time point in cells derived from HDM-AR patients (p = 0.0321) but not in cells derived from non-AR patients (p = 0.1136). TEER values were significantly reduced upon challenge with 4 mM Der p 1 at the 10-min time point in HDM-AR patients compared to non-AR patients .HNEC-ALI cultures were established from 18 subjects  . In HNECR group) . The maxn = 4, HDM-AR; n = 4) (p = 0.0286). Similar to the transient TEER change, the claudin-1 localization normalized at the 4 h time point in both groups. The localization of ZO-1 was not altered by challenge with Der p 1 (p > 0.05)   and trea; n = 4) . Moreove > 0.05) .p = 0.0271) but not in cells derived from non-AR patients. Der p 2 did not significantly induce IL-6 production in cells derived from HDM-AR or non-AR patients  and 10 μg/ml poly (I:C) LMW were used as negative and positive control respectively. Compared to negative control, Der p 1 induced IL-6 secretion in cells derived from HDM-AR patients (patients .Our study demonstrates a dose-dependent transient Der p 1-induced detrimental effect on the nasal epithelial barrier in ALI cultured HNECs associated with a cleavage of claudin-1. The effect was significantly higher in patients with HDM-AR compared to non-AR patients and was accompanied by a higher IL-6 production in those patients. These results suggest that the epithelium of patients with HDM-AR is more sensitive to Der p 1 than that from patients without AR. This may possibly create a higher chance of sensitization to Der p 1 in these patients with mucosal barrier disruption and induction of inflammation.Previous research has shown an impaired nasal epithelial barrier function in patients with HDM-induced AR, indicated by a low TEER and low mRNA expression of TJ genes compared to control . Whilst in vivo (Interestingly, our data showed that the Der p 1-dependent effect on TEER was accompanied by a reduced expression of claudin-1 but not ZO-1. Together with the finding that paracellular permeability of larger molecules was not affected, these findings suggest that Der p1 affects mainly the pore pathway and does not promote an increased paracellular flux of larger non-charged solutes . Given tin vivo . It is tin vivo –30. Intein vivo . FurtherOur study is the first to show recovery of TEER and claudin-1 protein expression of HNEC-ALI cultured cells after Der p-1 exposure. These results are in line with other studies showing a reversible effect of Der p1 on the mucosal barrier . ConversClaudin-1 is expressed in the paracellular space, while ZO-1 is an intracellular molecule , 35. TheWe studied the IL-6 production because previous studies indicated the relationship between the dysregulation of TJ protein expression and levels of inflammatory cytokines such as IL-6 and tumor necrosis factor , 44. DerIn summary, Der p 1 induces a reversible degradation of claudin-1 in HNEC-ALI cultures derived from HDM-AR patients with a reduced mucosal barrier structure and function and accompanied by an induction of IL-6 secretion. Our data therefore support an important role for Der p1 in sensitization and AR development.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by The Central Adelaide Local Health Network Human Research Ethics Committee. The patients/participants provided their written informed consent to participate in this study.KO, JF, and MR conducted the biological experiments. CB, AP, and P-JW collected the clinical samples. SL, AP, P-JW, and SV designed the study. MS, MR, and SV analyzed the data. SF, KO, and SV drafted the manuscript. All authors critically reviewed the manuscript and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


Whistleblowers who expose wrongdoing often face several concerns, pressures, and threats of retaliation before reaching a final decision. Specifically, this study examines the effects of perceived seriousness of wrongdoing (PSW) and perceived threat of retaliation (PTR), as well as the impact of rationalization (RNL), comparing perceived seriousness of wrongdoing, perceived threat of retaliation and whistleblowing intention. Furthermore, this study aims to determine the mediating effect of anticipated regret (AR) on the relationship between perceived seriousness of wrongdoing and whistleblowing intention. We validated our model by analyzing data gathered across three stages from employees in the telecom sector in Pakistan. The key findings of our research may be summarized as follows: (i) individuals’ willingness to ‘blow the whistle’ increases as a result of perceived seriousness of wrongdoing; (ii) whistleblowers are more likely to opt to remain silent if they anticipate a greater threat of retaliation, and (iii) our study establishes a positive connection between perceived seriousness of wrongdoing and whistleblowing intention, indicating that perceived seriousness of wrongdoing enhances people’s willingness to blow the whistle, and whistleblowers are more likely to choose to emerge if the behaviour is more serious in nature; (iv) the data we have uncovered indicates a moderating role of rationalization in regulating the connections between perceived seriousness of wrongdoing, perceived threat of retaliation, and whistleblowing intention; and (v) the findings demonstrate that anticipated regret mediates the connection between perceived seriousness of wrongdoing and the intention to report wrongdoing. Additionally, the results are discussed in terms of their significance for corporate ethics researchers and managers, as well as for end-users who are interested in whistleblowing. Strong financial incentives for whistleblowersAnonymous reportingMost individuals are eligible to receive awardsSEC rejects “gag clauses”Protections for SEC whistleblowersIn July 2010, Congress approved the Dodd-Frank Act, which established the US Securities and Exchange Commission (SEC) Whistleblower Initiative in response to the global financial crisis of 2008. For over a decade, the program has produced high-quality recommendations that have helped the SEC to uncover and stop fraud schemes while protecting investors. Approximately $700 million in compensation for whistleblowers has been given out by the SEC Office of the Whistleblower since 2012. Penalties against wrongdoers resulting from SEC efforts relating to the disclosures totaled more than $2.7 billion, which was paid out in the form of fines and penalties. According to the American Council on Fraud and Ethics’s 2020 Report to the Nations, there has been a considerable increase in the number of complaints received via business hotlines or reporting systems since 2010. Additionally, individuals should consider the SEC Whistleblower Program as another essential weapon in their collection of tools for exposing fraud and wrongdoing. Whistleblowers who met the criteria for the ACFE’s award received around $368 million in compensation, including the five highest awards in the program’s history—two awards of over $50 million and one each of $39 and $37 million. In 2020, hard work took place to process claims faster than ever before, even with the impact of COVID-19. This has allowed the distribution additional rewards [The Association of Certified Fraud Examiners (ACFE), the world’s premier anti-fraud organization, published a report on professional fraud and abuse for the tenth time in 2018, marking the organization’s establishment as a global leader in the field of fraud prevention. According to the ACFE’s most recent study, which examined 2690 real cases from 125 countries, whistleblowers were responsible for detecting or exposing 40% of fraud situations ,4,5,6,7.Whistleblowing is a vital tool to prevent and uncover corporate misconduct, whether in the public or private sector ,9,10. ToCuliberg and Mihelič  stated tAccording to ,51, the Whistleblowing is defined as “the disclosure by organizational members (former or current) of illegal, immoral, or illegitimate practices under the control of their employers, to persons or organizations that may be able to effect action” . The degThe whistleblower first evaluates the depth of the wrongdoing before whistleblowing. This activity is performed to judge the seriousness and course of action to be completed before it causes harm to the organization or employee ,38,57,58Hypothesis 1 (H1).The perceived seriousness of wrongdoing has a positive effect on whistleblowing intention.According to researchers, perceived seriousness of wrongdoing is a significant positive predictor of whistleblowing intention ,63,64,65Hypothesis 2 (H2).Rationalization moderates the relationship between perceived seriousness of wrongdoing and whistleblowing intention.Whistleblowers face several threats that have negative implications and even mentioning wrongdoing might result in serious repercussions. There are several threats related to blowing the whistle that influence whether someone will go forward with their information  ,22,43. RHypothesis 3 (H3).Perceived threat of retaliation has a negative effect on whistleblowing intention.The threat level in various situations of whistleblowing has been observed in previous research, at varied percentages. For example,  demonstrHypothesis 4a (H4a).Rationalization moderates the relationship between the perceived threat of retaliation and whistleblowing intention.The seriousness of the wrongdoing has an influence on the possibility of having regrets about not reporting it sooner rather than later. According to the definition, the seriousness of wrongdoing relates to the extent to which a certain unlawful behaviour may cause harm to others after they show an intention to blow the whistle ,73. In tHypothesis 4b (H4b).Anticipated regret mediates the relationship between perceived seriousness of wrongdoing and Whistleblowing Intention.Gender differences in whistleblower behaviour have been reported in the past . When deThe data were collected from 450 employees of telecommunications companies in Islamabad, Pakistan, through emails and also through physical visits to office premises, by adopting a convenience sampling technique at the start of 2020. Several procedural techniques were investigated in this study to reduce the common method variance , e.g., bAll aspects of this study were measured using a 5-point Likert scale, ranging from 1 = “strongly disagrre” to 5 = “strongly agree”.To measure whistleblowing intentions, the scales in  have beeTo measure PSW, a scale developed by  was usedTo measure PTR, a five-item scale adopted from the study of  were useTo measure RNL, we adapted the five-item scale developed by ,25,85, wAnticipated regret was assessed with a seven-item scale, developed by .2/df = 2.03, p < 0.001; comparative fit index (CFI) = 0.91; root mean square error of approximation (RMSEA) = 0.03; GFI = 0.89; root mean square residual (RMR) = 0.03. To analyse the factor structure of our survey measures, we utilised IBM SPSS Amos 22 to run a series of confirmatory factor analyses ,88. In oOn the recommendation of , a singlp 0.01), and PSW has a significant correlation with whistleblowing intention . Additionally, the more significant correlation is between rationalization and anticipated regret ; , which supports Hypothesis 1 (Model 3). As for Model 4, perceived threat of retaliation negatively affects perceived seriousness of wrongdoing , thus supporting Hypothesis 3. As displayed in p < 0.001, Model 1), and anticipated regret had a significant influence on whistleblowing intention . To further confirm the mediation effect of anticipated regret (perceived seriousness of wrongdoing → anticipated regret → whistleblowing intention), we adapted bootstrapping of 5000 cases’ analyses with a 95% bias-corrected confidence interval. We found that anticipated regret partially mediated the path between perceived seriousness of wrongdoing and whistleblowing intention , and that the indirect effect exists as a consequence of the confidence interval excluding zero between the upper and lower boundaries.  for employees with low rationalization, and a significant positive association  for employees with high rationalization (see p < 0.001) for employees with low rationalization, and a significant positive association  for employees with high rationalization. Thus, hypothesis Hypotheis 4a is confirmed.By examining the link between perceived seriousness of wrongdoing and perceived threat of retaliation, we determined the influence of rationalization on whistleblowing intention. To quantify the interaction effects, we used the methods developed by Hayes (2018). To begin, we evaluated the model without taking into account the interaction effects. Following that, we retested the model by including interaction effects and determining the degree of significance. Finally, we used the Hayes PROCESS macro to create a visual graph. tion see . ExamineWhistleblowing may be used as a preventative approach to reduce the likelihood of misconduct and discrepancies occurring. It is more likely that managers will identify inconsistencies early on if employees and other key stakeholders are given the authority to raise the alarm when anything is wrong. The implementation of steps to enable whistleblowing in companies that take their Code of Conduct seriously will include the establishment of a secure corporate whistleblowing system or hotline, and the development of policies and procedures for reporting misbehaviour.The whistleblowing approach have been investigated and recognized as a mechanism for exposing misconduct in organizations by academics from a broad variety of fields. Employees from Pakistan’s telecom industry are used as a sample in this study, which is based on the theory of planned behavior. We examine the impacts of perceived seriousness of wrongdoing and perceived threat of retaliation on whistleblowing intention, as well as the mediating roles of anticipated regret and the moderating roles of rationalization . In partOur hypothesis was put to the test, and we found five key results. First, we tested the link between perceived seriousness of wrongdoing and whistleblowing intentions. We found evidence of a positive relationship between perceived seriousness of wrongdoing and whistleblowing intention. Whistleblowers are more inclined to blow the whistle when they notice major wrongdoing in the workplace. Our results support data from prior research on the seriousness of wrongdoing ,26,28,29The findings of our study, which we discuss in more detail below, have a wide range of theoretical and practical implications. This study makes one of the most significant contributions to the literature on whistleblowing because it is the first to examine anticipated regret as a mediator in the relationship between perceived seriousness of wrongdoing and whistleblowing intention, and RNT as a moderator in the relationship between perceived seriousness of wrongdoing, perceived threat of retaliation, and whistleblowing intention. Despite earlier study focusing on the individual, situational, and contextual aspects that impact whistleblowing intention, the process of determining whether to blow the whistle still remains unclear ,19,35. RWe believe that managers and other stakeholders in organisations will be able to use our results to reduce the potential of retribution against whistleblowers, while also assisting them by offering freely available reporting channels to the general public . IncreasCertain limitations apply to this study. Our research does not take into account elements that might alter the association between relevant factors, such as demographics . For exa


Physical rehabilitation is often prescribed immediately following a neurological event or a neurological diagnosis. However, many individuals require physical rehabilitation after hospital discharge. The purpose of this scoping review was to determine the amount of physical rehabilitation that individuals living in the community with neurological conditions receive to understand current global practices and assess gaps in research and service use.This scoping review included observational studies that 1) involved adults living with a neurological condition, and 2) quantified the amount of rehabilitation being received in the community or outpatient hospital setting. Only literature published in English was considered. MEDLINE, EMBASE, AMED, CINAHL, Cochrane Library, and PEDro databases were searched from inception. Two independent reviewers screened titles and abstracts, followed by full texts, and data extraction. Mean annual hours of rehabilitation was estimated based on the amount of rehabilitation reported in the included studies.Overall, 18 studies were included after screen 14,698 articles. The estimated mean annual hours of rehabilitation varied greatly (4.9 to 155.1 h), with individuals with spinal cord injury and stroke receiving the greatest number of hours. Participants typically received more physical therapy than occupational therapy (difference range: 1 to 22 h/year). Lastly, only one study included individuals with progressive neurological conditions, highlighting a research gap.The amount of rehabilitation received by individuals with neurological conditions living in the community varies greatly. With such a wide range of time spent in rehabilitation, it is likely that the amount of rehabilitation being received by most individuals in the community is insufficient to improve function and quality of life. Future work should identify the barriers to accessing rehabilitation resources in the community and how much rehabilitation is needed to observe functional improvements.The online version contains supplementary material available at 10.1186/s12913-022-07754-4. Physical rehabilitation, such as physical and occupation therapy, is commonly prescribed following the diagnosis of a neurological condition or the occurrence of a neurological event. The goal of physical rehabilitation is to optimize physical functioning so people can continue to complete tasks that are important to them, as independently and safety as possible. More specifically, physical therapy typically views movement on a continuum while considering the physical, pathological, social, and psychological aspects . Common Quality of life – which may be one of the most important self-perceived measure of function – has been shown to improve with access to physical rehabilitation in the community setting . The comIndividuals with long-term neurological conditions have qualitatively reported that their physical rehabilitative needs are not met . Howeverth, 2020.This scoping review was conducted according to the guidelines presented in the PRISMA Extension for Scoping Reviews . A protoArticles were uploaded to Covidence  where they were initially deduplicated. Articles were then screened for eligibility. Articles were included if they met the following criteria: 1) adults 18 years of age or older; 2) living in the community with a neurological condition defined as traumatic brain injury, multiple sclerosis, spinal cord injury, stroke, and Parkinson’s disease , 20; 3) Two reviewers independently reviewed all deduplicated titles and abstracts to determine which articles to include for full text screening. If consensus could not be achieved between the two reviewers, the other team members were consulted to determine the article’s eligibility. During full text screening, two reviewers independently assessed the potential articles for their eligibility. If the article was not included, a reason was provided based on the inclusion criteria. The same procedures as title and abstract screening were used in the event of reviewer disagreement. Once full texts were selected for study inclusion, backwards citation tracking was conducted to consult the references of included studies for additional articles.Following full text screening, data extraction was performed based on a standardized, piloted extraction form developed by the research team. Data extracted included: year and country published in; study design and objectives; population and patient demographics included in the article; method of data collection; time spent in rehabilitation; and type of rehabilitation. The two primary reviewers independently extracted the data for each study and an additional team member cross-referenced the two data extraction forms with the included journal article. The collected data were then summarized using descriptive statistics  and presented in tabular and graphical forms. Annual time spent in rehabilitation was estimated in hours for each study based on the information provided. One hour was allocated for each session in the event that number of sessions was reported based on previous research, which has shown that 1 session is approximately 1h on average .A PRISMA diagram outlining the screening process can be found in Fig. n = 6) or the United Kingdom (n = 4). No research was found prior to 1990 in the literature, with an incremental increase in the presence of these studies over the last three decades ; medical charts or electronic patient record (n = 5); health claims data (n = 3). Some studies used a variety of methods to gather this information (n = 2), or custom data collection sheets (n = 2) were also implemented in some studies.The most commonly used data collection tools to evaluate the amount of time in physical rehabilitation were questionnaires, interviews, and surveys (n = 13), with SCI (n = 2), TBI (n = 2), and multiple long-term neurological conditions (n = 1) also being evaluated. Chronicity of the condition varied from immediately following hospital discharge until 40 years post-injury. Half of the studies distinguished between occupational and physical therapy (n = 9), and the rest of the studies grouped all types of physical rehabilitation (n = 9).Stroke was by far the most common condition studied  reported that the amount of rehabilitation received by those with mild traumatic brain injury was greater than those with moderate/severe injury beyond 10 years post-injury . These fA second main finding of this review was that the vast majority of studies reported increased hours spent in physical therapy compared to occupational therapy . There are a few potential reasons for the finding. Firstly, there are approximately 37% more registered physical therapists than occupational therapists . The avaWith respect to the location of services, results were split with half of the studies finding patients utilized more in-home services compared to outpatient services , 25, wheDespite our findings demonstrating that people with neurological diagnoses commonly do receive some physical rehabilitation after discharge from hospital inpatient units, the amount of rehabilitation received is likely insufficient. Research indicates many people continue to have disability long-term following diagnosis, and evidence shows that failure to access occupational and physical therapy is associated with continued issues following a brain injury , 32. FurThe present scoping review highlighted various gaps in the literature pertaining to physical rehabilitation use in those with neurological conditions living in the community. Firstly, while we sought out to study individuals with discrete and progressive neurological conditions, we did not identify any studies on those with a degenerative condition with the exception of Jackson et al., (2014) which included various neurological conditions including 21 subjects (5%) with progressive long-term neurological conditions. It is important to note that the focus of rehabilitation differs between these two disease types. Rehabilitation for degenerative neurological disorders aims to manage the condition and slow the decline of physical function by developing compensatory strategies and increasing support over time . ConversFurthermore, while studies did not track service use over time within the same cohort of subjects, it will be important for researchers to track service use longitudinally in order to further understand these trends and factors that affect them. As most of the studies included in this review focused on the first year following injury, there remains a large gap in services utilized over the long term in these populations. Determining the optimal timing and length of treatment is important in both progressive and non-progressive conditions as individuals have long-term needs related to improving or maintaining independence and slowing the rate of functional decline –37. In tThis review is not without its limitations. Firstly, the calculation of time spent in rehabilitation was estimated based on the number of sessions when time in minutes or hours was not provided. Many factors may influence the length of therapy sessions, and these may vary over time. However, the purpose of these calculations was to provide a broad picture of the landscape of community rehabilitation for those with neurological impairments. In addition, due to the small number of studies and only three studies including subjects more than one year following injury , 22, 38,This scoping review found that the amount of physical rehabilitation received by individuals with neurological conditions living in the community varied greatly, with individuals who experienced a spinal cord injury or stroke receiving the greatest amount of care. In addition, more time is spent on average in physical therapy compared to occupational therapy in the community. These findings highlight the heterogeneity of physical rehabilitation received by individuals with a neurological condition and point to various avenues for future research including studying service use over time, the impact of community rehabilitation on functional outcomes and quality of life, and rehabilitation use in individuals with progressive neurological conditions.Additional file 1: Medline (OVID) Search Strategy.