Apart from their demanding synthesis, also their quantum‐chemical description is difficult due to their open shell nature and low‐lying excited electronic states. Distorted tetrahedral NiF4 (D2d) and trigonal planar NiF3 (D3h) molecules were produced by thermal evaporation and laser ablation of nickel atoms in a fluorine/noble gas mixture and spectroscopically identified by a joint matrix‐isolation and quantum‐chemical study. Their vibrational band positions provide detailed insights into their molecular structures.Binary transition metal fluorides are textbook examples combining complex electronic features with most fundamental molecular structures. High‐valent nickel fluorides are among the strongest known fluorinating and oxidizing agents, but there is a lack of experimental structural and spectroscopic investigations on molecular NiF 3 and NiF4 are the missing tri‐ and tetrafluorides of the otherwise known binary first‐row transition‐metal fluorides that are predicted to be stable. These elusive high‐valent molecular nickel fluorides were identified in rare‐gas matrices aided by high‐level quantum chemical electronic structure calculations. They are among the most powerful known fluorination and oxidation agents.Molecular NiF Solid MnF4 releases elemental fluorine at temperatures >170 °C.4 and NiF4, solid FeF4 or solid CoF4 are unknown, while the monomeric FeF4 and CoF4 species have been investigated by high‐temperature vapor‐phase mass spectrometry and cryogenic matrix‐isolation spectroscopy.2− salts in anhydrous HF with strong Lewis acids such as AsF5, SbF5, or BF3.4 releases F2 above −55 °C and forms NiF3, which again releases fluorine above 20 °C to form NiF2.3 has not yet been studied spectroscopically, but it has attracted the interest of a theoretical study because of its low‐lying electronically degenerate excited states, which are prone to Jahn–Teller (JT) effects.Solid NiFi2F5 NiII3NiIVF103 and NiF4 for the first time. Different methods were applied to produce these species which were subsequently isolated in cryogenic rare‐gas matrices. First, nickel atoms were vaporized by heating a nickel wire and allowed to react with elemental fluorine diluted in argon prior to deposition on a matrix support at 10 K. Vaporization of atomic nickel was also achieved by laser ablation using the 1064 nm fundamental of a Nd:YAG laser focused onto a rotating nickel target and trapping the products at 6–15 K. The laser ablation process is associated with a hot plasma plume and a bright broad‐band radiation, where excited nickel atoms can react with elemental fluorine and atomic fluorine radicals seeded in excess of noble gases (neon or argon). This method is particularly useful for the generation of highly fluorinated species.[2 and CoF3 but also CoF4 , which could be detected using its known IR band in solid argon reported in a previous study,4 was obtained at 650 K from solid mixtures of CoF3 and TbF4 as atomic fluorine source in a perfluorinated nickel effusion cell.Co‐F modes is CoF4 > CoF3 > CoF2 ≫ CoF, see the Supporting Information.We carried out spectroscopic investigations on molecular NiF species.13, 192 on deposition  at 634.7 cm−1,−1 at the CCSD(T)/AVT(Q)Z‐DK level (Table S4.2). UV and broadband photolysis, associated with the formation of F radicals, resulted in the dramatic growth of the bands due to NiF2 .2 in F2/Ar matrices (see the Supporting Information).Thermally evaporated Ni atoms react with Fn Figure  to yield2 increased while those close to 800 cm−1 decreased, and weak bands at 735–724 cm−1 appeared. These latter bands are assigned to the asymmetric stretch of molecular NiF3, trapped in different matrix sites. CCSD(T) calculations predict a 4A2′ ground state with D3h structure for molecular NiF3 .On annealing  depletes the NiF3 bands  and forms NiF2, while λ=193 nm laser radiation destroyed both, NiF2 and NiF3 . More interestingly, in these experiments a new band at 749.1 cm−1 with a distinct Δν(58/60Ni) isotope splitting of 5.1 cm−1 occurred . Based on this behavior, the new band is assigned to the strongest IR band of molecular NiF4. For a tetrahedral tetrafluoride only a single infrared‐active Ni−F stretching mode is expected, however, for the d6 electronic configuration of NiF4 a Jahn–Teller distorted D2d structure is predicted at all levels of theory .Subsequent nickel laser ablation experiments fully corroborate the assignment of NiFn Figure . Due to 4. We carried out quantum‐chemical investigations of various energetically low‐lying electronic states of NiF4, especially of the lowest‐energy triplet (3A2) and quintet  states of D2d symmetry (Table S3.4). For the triplet ground state (Scheme 4 about 11 cm−1 blue‐shifted from the E′ band of NiF3 at the CCSD(T)/AVTZ‐DK level  was not detected. All four nickel fluorides are also observed in solid neon matrices ,−1 (Co), and 743.8 cm−1  and NiF3  obtained at the CCSD(T)/AVTZ level. A similar frequency‐bond length correlation (Table S3.5) is expected for the stretching vibrations of the tetrafluorides MF4 of Fe to Ni observed in solid argon, which also appear in a narrow spectral range (757 cm−1 (M = Fe),D2d molecular structures of FeF4 and NiF4 and due to stronger matrix interactions in solid argon.A comparison of experimental M−F stretching bands of high‐valent first‐row metal fluorides is particularly revealing. The molecular trifluorides of M = Fe, Co, and Ni all adopt n, with n=1–4,n, n=2–4, the bond length in NiFn, n=2–4, remains almost unchanged (Table S3.5). This different trend for nickel and its predecessors can be traced back to fundamental aspects of their atomic structures, such as a higher nickel ionization energy. This leads to a considerable covalent character of the Ni−F bonds, associated with a low‐spin triplet electronic ground‐state of NiF4 and a considerable radical character of the fluorine ligands .[Apart from these similarities, the higher nickel fluorides are strikingly different from their third‐row predecessors. The rule of thumb that higher fluorides have shorter and stronger M−F bonds that applies to ionic metal fluorides does not apply to the higher nickel fluorides. While CCSD(T)/AVTZ calculations predict a successive M−F bond shortening for the ionic iron fluorides FeFure S11).7, 82, initially formed from the reaction of nickel atoms and elemental fluorine, can be regarded as chemically inert to elemental fluorine, but reacts rapidly with atomic fluorine radicals to form NiF3 and further to NiF4. It has also been shown that the reaction of Ni atoms with elemental F2 to produce NiF2 requires UV photolysis to yield appreciable quantities of product under the cryogenic conditions applied here. Given the highly exothermic reaction energy of this reaction (Table From these experiments we conclude that NiF4 (D2d) and NiF3 (D3h) were produced, isolated in solid noble‐gas matrices, and spectroscopically identified aided by quantum‐chemical calculations. Their weak Ni−F bonds and a considerable fluorine radical character make these molecular species very powerful fluorination and oxidation agents.To summarize, molecular NiFThe authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


An approximate solution of the Schrӧdinger equation for a molecular attractive potential was obtained using the parametric Nikiforov–Uvarov method. The energy equation and the corresponding radial wave functions were calculated. The effects of the potential parameters on the energy eigenvalues were examined. The thermal properties under the molecular attractive potential were calculated and the behaviour of the thermal properties with the maximum quantum state (λ) and the temperature parameter (β) respectively, were studied. Using the molecular spectroscopic parameters, the Rydberg–Klein–Rees (RKR) of cesium dimer and lithium dimer were both obtained and compared with the experimental values. The RKR values of both cesium dimer and lithium dimer calculated aligned with the observed values. The deviation and average deviation of the RKR for each molecule were also calculated. In Refs.7 and8 respectively, the improved Manning-Rosen potential function also formulated by Jia et al.4 was used to calculate the rotation vibrational transition frequency of hydrogen fluoride (HF). The Tietz-Wei potential function was used to study eigensolutions and thermodynamic properties in Ref.9. In Ref.3, an improved deformed four parameter exponential-type potential model was formulated and studied under the RKR of cesium molecule. The same RKR of cesium molecule was studied under Tietz-Hua oscillator in Ref.10. Desai et al.11, in one of their papers, calculated the RKR of nitrogen molecule and hydrogen molecule under the Morse potential and modified Morse potential. Recently, Horchani et al.12, formulated an improved generalized Pӧschl–Teller oscillator and obtained the RKR of potassium molecule. Jia et al.13, in one of the papers, calculated the thermodynamic properties of lithium dimer under the improved Manning-Rosen potential. Onate and Idiodi14, reported Fisher information and complexity measures of the generalized Morse potential model. The thermodynamic properties of Shifted Deng-Fan potential was also reported by Oyewumi et al.15. The scattering state solutions for Manning-Rosen potential was reported by Qiang et al.16. In Ref.17, Idiodi and Onate obtained Fisher information and variance for Frost Musulin potential. Onate et al.18, also reported some theoretic quantities under Tietz-Hua potential. Edet et al.19, calculated the thermal properties of the Deng–Fan–Eckart potential. The bound state solutions and energy spectra for some molecules under some molecular potentials were also studied and reported by different authors. Motivated by the usefulness and influence of the molecular potentials, this study aims to transform an attractive potential model to a molecular potential model and study the one-dimensional Schrödinger equation with the transform potential. The study also intends to calculate the RKR values of two molecules (cesium dimer and lithium dimer) and thermodynamic properties of the potential. The attractive potential is a four-parameter exponential-type potential formulated in 1993 by Williams and Pouliss20. According to these authors, the four parameters are 23. The potential cannot be used to describe the detail features of a molecule since there are no spectroscopic parameters. Here, the attractive potential will be modified into a molecular potential function to fit the description of a given molecule. The molecular attractive potential model in this study is given byA good understanding of a molecular structure depends on the internuclear and molecular potential models. This creates a larger space of study on the molecular potential functions in the diatomic domain, which geared different authors to carry out a number of researches in this area. The molecular potential functions have spectroscopic parameters such as the dissociation energy The parameters From the three-dimensional Schrödinger equation, the radial Schrödinger equation with a centrifugal term is given as25. The method has been widely reported and as such, the detail of the method will not be presented in this work. To use this method, Tezcan and Sever26, formulated a general form of a second-order differential equation of the formIn this work, the authors decided to use parametric Nikiforov–Uvarov method for the calculation of the energy equation and the wave functions. The parametric Nikiforov–Uvarov method is a popular and accurate method to obtain energy equation and the wave functions. It was derived from the original Nikiforov–Uvarov method29Following the work of these authors, the condition for eigenvalues equation and wave functions respectively, are given by26 and other literatures. When Eq. (The detail of the methodology can be found in Ref.When Eq.  are subsWhen Eq. , the radWhen Eq.  becomes9Defining a variable of the form Comparing Eq.  with EqsSubstituting Eq.  into EqsTo calculate the thermodynamic properties for the molecular attractive potential, the energy equation given in Eq.  is writtHaving written the energy equation in a compact form that is suitable for the calculation of the thermodynamic properties, then, the partition function of the system can be define asNow, defining ing Eqs.  into (19The effects of the three potential parameters on the energy eigenvalues are shown in Fig. entclass1pt{minima25 and26. For cesium dimer, 9Since the potential parameters (mentclass2pt{minimmentclasspt{minimaThe solutions of a one-dimensional Schrödinger equation were obtained under a molecular potential with three different potential parameters. It was noted that for negative values of the potential parameters, the potential parameters have the same effects on the energy eigenvalues but for positive values of the potential parameters, the effect of one differs from the effects of the other two parameters. The effect of λ and β on the thermal properties are the same except that of the mean energy. The RKR of both cesium dimer and lithium dimer calculated aligned with the observed values. However, the average deviation of lithium dimer from the observed value is far smaller than the average deviation of cesium dimer from the observed value.


With increasing number of patients undergoing spine surgery, the spinal epidural hemorrhage (SEH) has become a growing concern. However, current studies on SEH rely on case reports or observations from a single center. Our study attempted to demonstrate the incidence rate and risk factors of SEH using a national dataset.A total of 17,549 spine surgery cases from the Health Insurance Review and Assessment Service National Inpatient Sample of 2014 were analyzed. After evaluating the incidence of SEH based on severe cases requiring reoperation, a univariate comparison was performed. Variables found to be significant were included in a multivariable analysis model to determine the risk factors.The incidence of SEH was found to be 1.15% in Korean population, and there were no severe SEH cases. Our analysis confirmed the previous findings that lumbar surgery, intraoperative blood loss, prolonged surgical time, high blood pressure, use of nonsteroidal anti-inflammatory drugs, and concurrent bleeding factors are the risk factors of SEH. Anterior approach showed a protective effect. The use of anticoagulant demonstrated no statistical significance.Although severe SEH cases were not detected, the incidence of SEH was similar to that reported in literature. Given that SEH is a rare complication of spine surgery and constitutes an important research area that needs to be studied further, our study makes a meaningful contribution based on a rigorous national level sample for the first time and provides the academic circle and health professionals with a reliable evidence of improved clinical outcomes in such cases. Globally, spine surgery rates have drastically increased in the last two decades . There aSpinal epidural hemorrhage (SEH) manifests a bleeding in the epidural space along the spinal canal . StimulaAlthough there is no consensus with regard to the risk factors, postoperative anticoagulant use is known to increase the incidence of SEH , 12. It More issues are to be resolved in Asian population who had rarely developed thromboembolism after operation . Recent Considering this, a study that attempts to analyze a large dataset created by utilizing postoperative hemorrhage treatment codes on Asian population could clarify the epidemiology of epidural hemorrhage in patients undergone spine surgery. Therefore, the two main goals of this study were: to investigate the incidence of SEH by analyzing a national database, the Health Insurance Review and Assessment Service National Inpatient Sample of 2014 (HIRA-NIS-2014) and to determine the risk factors that could clearly indicate the cause of SEH development in Korean population.South Korea has a National Health Insurance System (NHIS) that covers approximately 98% of the overall Korean population, and 99% of National Health Insurance (NHI) claims data have been electronically reported since 2005. The claims data of Health Insurance Review and Assessment Service (HIRA) pertains to patients’ diagnosis, treatment, surgical history, procedures performed, and prescription drug information. However, the extensive volume and complicated structure of the claims data limit research approaches. To improve accessibility for researchers to use the claims data, HIRA has established the patient sample data sets that have been validated by five different validity tests .These patient sample datasets are as follows: adult patient group, pediatric patient group, all patient group, and inpatient group. The adult patient sample represents approximately 1 million patients aged > 65 years annually , whereas the pediatric patient sample takes into account 1.1 million patient aged < 20 years annually . All patient samples represents overall 1.4 million patients annually, which represents 3% of the original population. Lastly, national inpatient sample pertains to 700,000 inpatients (13%), and approximately 400,000 outpatients (1%) annually. All sample dataset contain sampling weights allow obtaining estimated national population. With a 95% concordance, the estimated population and the actual population demonstrated a high level of representativeness .Our study analyzed the customized NHIS dataset of HIRA National Inpatient Sample of 2014 (HIRA-NIS-2014), which contains claims data from January 1, 2014 to December 31, 2014 . This peThe data that support the findings of this study are available from HIRA but restrictions apply to the availability of these data, which were used under license for the current study. Thus, these data are not publicly available. However, the data are available from HIRA upon reasonable request and with permission.In Korea, currently there are no disease/treatment codes that exclusively represent SEH. Based on existing studies, this study converted the diagnostic code for postoperative SEH (998.12) indicated in the International Statistical Classification of Diseases and Related Health Problems, 9th revision (ICD-9) to the most appropriate disease code in the 6th Korea Informative Classification of Disease (KOICD-6) codes . As KOICBased on code selection recommendations from the Centers for Disease Control and Prevention/National Center for Health Statistics and KOICD, we first extracted all cases of postoperative hemorrhage and hematoma T81 and T810), and then narrowed down to severe SEH. Severe SEH cases were defined as the ones that required medical treatment codes for the removal or drainage of hematoma in the central nervous system , 25. T81 and T810Next, all selected cases were individually investigated by three different medical professionals for concurrent diseases, treatment provided, and medications to demonstrate detailed information on severe SEH after spine surgery during patient hospitalizations. Subsequently, all claims records of selected patients during the study period, the disease/treatment codes for the complications of surgical and medical care and postoperative disorders of the nervous system after spine surgery were examined. Detailed information on the disease and treatment codes is listed in Supplementary Table As an illustration, in a patient with spinal disease undergoing spine surgery, the incidence of postoperative hemorrhage and hematoma was first examined. Then, the intervention for postoperative SEH or spinal nerve compression was evaluated throughout the study period. If any of the codes indicated postoperative disorders of the nervous system or complication from surgical care, every detail of the case was flagged for further review. All variables for a pertinent case were relevant to the hospitalization period. Detailed information on the SEH codes is presented in Supplementary Table To construct the conceptual framework of analysis, the extant studies on incidence and risk factors of SEH were extensively reviewed. The terms “ AND (spine surgery) AND (risk factor)” were searched in the MEDLINE database. Simultaneously, the codes for spinal disease were referenced by literature review. In case a patient had a spinal disease code and underwent surgery in relation to the main disease, the patient was considered to have undergone spine surgery. In addition, the risk factors considered in this study were based on the results of the literature review. For example, the definition of extended surgery hours (≥ 2 h) and loss of blood were based on the literature search. The use of nonsteroidal anti-inflammatory drugs (NSAIDs) was defined as the administration of NSAIDs at any time during the hospitalization. The total medical cost was the total expenditure on the hospitalization in a case that was claimed by the medical institution. Insurance types were categorized as the NHI and Medical Aid/Veterans Medical Benefits. Blood loss was calculated by the blood transfusion claims codes of the case. Type of hospitals was classified into two categories: general hospitals  and small hospitals . The location of hospitals was divided into three categories: metropolitan area (Seoul), other metropolitan areas, and rural area . The duration of hospital stay was calculated by subtracting the admission date from the discharge date.In case a patient record included any invasive procedures such as lumbar puncture, myelography, and epidural anesthesia, then it was considered that the patient underwent invasive procedures. If a patient had thrombocytopenia or coagulation factor deficiency, the patient was regarded to have a bleeding factor. Trauma case was determined if the patient was admitted through the emergency department.After establishing the analytical dataset containing the variables identified as risk factors, we conducted a descriptive analysis. A pairwise correlation test was applied to determine the level of correlation among the set of variables included in the study model. A chi-square test and independent two-sample t-test were used accordingly to determine if there was any significant association between a set of independent variables and both categorical and continuous dependent variables. As the final step, a multivariate logistic regression was utilized prior to which multicollinearity was examined by referring to the variance inflation factor. Data with a variance inflation factor of > 5 were excluded from the model. In the multivariate logistic regression model, this study tries to identify factors that are associated with the incidence of SEH. Sampling weight was used to obtain better estimation of the original population. All statistical analyses were performed using Stata 14.0 .Based on the inpatient records, out of a total of 31,964,373 cases, there were 17,549 cases of spinal surgery in 2014. As shown in Fig. Based on the extensive review of recent studies, Table N = 25). Most spine surgery procedures wherein SEH occurred (N = 139) were performed at small hospitals, whereas only few such procedures (n = 63) were performed in general hospitals. The results of the detailed descriptive analyses are shown in Table Patient characteristics were obtained from the 17,549 spinal surgery cases (201 patients with SEH and others). The values for age, total medical cost, and duration of hospital stay were greater for SEH cases than for non-SEH cases. Lumbar spine surgery type and posterior approach showed significance in the univariate analysis. Of all SEH cases, 199 involved treatment with NSAIDs. Among all patients with SEH, 14 underwent surgery for > 2 h in contrast to 187 patients who underwent surgery for ≤2 h. Regarding blood loss, 135 patients were reported to have no blood loss in the claims dataset, and 41 had < 0.5 L of blood loss in the SEH group. Others experienced a blood loss of > 0.5 L (p = 0.03), total medical cost (p < 0.001), lumbar spine surgery (p < 0.001), anterior-approach surgery (p < 0.001), hypertension (p = 0.01), use of NSAIDs (p = 0.01), bleeding factors (p < 0.001), surgical time >  2 h (p < 0.001), blood loss > 0.5 L (p < 0.001), number of hospital beds > 200 (p = 0.03), and hospitals located in the rural areas. The variables with marginal significance (0.05 ≤ p < 0.20), such as sex, infections, and small hospital types, were also included in the multivariable logistic regression model. The use of anticoagulant was not found to be a significant risk factor as per the univariate analysis, however, we included the variable in the multivariate analysis since other extant studies demonstrated it as a strong risk factor of SEH. Table A pairwise correlation analysis that was performed before the univariate analysis revealed that there was no significant correlation among the independent variables. In the univariate analysis, we found that most independent variables that have been included in the study model were associated with the incidence rate of SEH. The following variables were found to have a statistically significant relationship with SEH. Age p = 0.0, total mThis is the first study to utilize a national database of Korean population to investigate the incidence and risk factors of SEH. Of total 17,549 cases of spine surgery, 201 (1.15%) were found to be the case of symptomatic SEH, and there was no case of severe SEH detected leading to continuous nerve damage and reoperation.N = 31,380) and reported SEH incidence rate of 0.9%, whereas Anno et al. reported an incidence rate of 0.4% based on data from a single-center ).In our univariate analysis, anticoagulant use was an insignificant risk factor of SEH .


Mmp12 knock-out (KO) mice at 10 (acute) and 60 days (late) after MWCNT instillation.Sarcoidosis is a chronic inflammatory disease of unknown cause characterized by granuloma formation. Mechanisms for chronic persistence of granulomas are unknown. Matrix Metalloproteinase-12 (MMP12) degrades extracellular matrix elastin and enables infiltration of immune cells responsible for inflammation and granuloma formation. Previous studies report increased MMP12 in sarcoidosis patients and association between MMP12 expression and disease severity. We also observed elevated MMP12 in our multiwall carbon nanotube (MWCNT) murine model of granulomatous inflammation. Here we hypothesized that MMP12 is important to acute and late phases of granuloma pathogenesis. To test this hypothesis, we analyzed granulomatous and inflammatory responses of Mmp12 KO mice underwent oropharyngeal instillation of MWCNT. Lungs were harvested at 3, 10, 20, and 60 days post instillation for evaluation of MMP12 expression and granulomatous changes. Bronchoalveolar lavage (BAL) cells were analyzed 60 days after MWCNT instillation for expression of mediators thought to play a role in sarcoid granulomatosis: peroxisome proliferator-activated receptor-gamma (PPARγ), interferon-gamma (IFN-γ), and CCL2 (MCP-1).C57BL/6 (wildtype) and Mmp12 KO mice. In contrast, by 60 days after MWCNT instillation, Mmp12 KO mice revealed markedly attenuated granuloma formation together with elevated PPARγ and reduced IFNγ expression in BAL cells compared to wildtype. Unexpectedly, Mmp12 KO mice further demonstrated increased alveolar macrophages with increased CCL2 at 60 days.Pulmonary granuloma appearance at 10 days after MWCNT instillation showed no differences between wildtype and Mmp12 KO mice suggests that MMP12 is required to maintain chronic granuloma pathophysiology. The increased PPARγ and decreased IFNγ findings suggest that these mediators also may be involved since previous studies have shown that PPARγ suppresses IFNγ and PPARγ deficiency amplifies granuloma formation. Interestingly, a role of MMP12 in granuloma resolution is also suggested by increases in both macrophage influx and CCL2. Overall, our results strongly implicate MMP12 as a key factor in granuloma persistence and as a possible therapeutic target in chronic pulmonary sarcoidosis.The striking reduction of granuloma formation at day 60 in  Pparγ knock out (KO) mice revealed enhanced granulomatous disease as evidenced by increased granuloma size and incidence  correlated with disease severity. These findings make MMP12 biology an area of acute interest in sarcoidosis pathogenesis.MMP12, also known as macrophage metalloelastase is a member of a family of extracellular endopeptidases , 10. MMPituation . As Giand unique . MMP12 w1 . StudieThis study demonstrates that MMP12 deficiency reduces pulmonary granuloma progression and highlights a critical role for MMP12 in the chronicity of granulomatous inflammation. Potential mechanisms involved in granuloma resolution require further exploration, including identifying how the down-regulation of IFN-γ results in elevated PPARγ, and a better understanding of how CCL2 promotes macrophage recruitment. Deciphering MMP12–orchestrated mechanisms in granuloma formation can lead to novel approaches for treating sarcoidosis.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The animal study was reviewed and approved by IACUC of East Carolina University.NN, ES, MM, NL, DO, and DT: acquisition of data. AMo, AMa, WK, BB, SG, LS, and MJT: concept and design. BB, AMa, and MJT: analysis, interpretation, and drafting of manuscript for important intellectual content. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In contrast, the GH10 xylanases; Xyn10 and XT6, and a GH11 xylanase, Xyn2A, showed more than two-fold increased activity on xylan substrates with low sidechain substitutions; BWX and OX, compared to the highly branched substrate, WAX. Interestingly, the FAE-1 and GH10 xylanases (Xyn10D and XT6) displayed a degree of synergy (DS) that was higher than 1 in all enzyme loading combinations during WAX hydrolysis. The 75%XT6:25%FAE-1 synergistic enzyme combination increased the release of reducing sugars by 1.34-fold from WAX compared to the control, while 25%Xyn10D:75%FAE-1 synergistic combination released about 2.1-fold of reducing sugars from WAX compared to controls. These findings suggest that FAE-1 can be used in concert with xylanases, particularly those from GH10, to efficiently degrade arabinoxylans contained in cereal feedstocks for various industrial settings such as in animal feeds and baking.Cereal feedstocks have high arabinoxylan content as their main hemicellulose, which is linked to lignin by hydroxycinnamic acids such as ferulic acid. The ferulic acid is linked to arabinoxylan by ester bonds, and generally, the high substitution of ferulic acid leads to a loss of activity of xylanases targeting the arabinoxylan. In the current study, a feruloyl esterase (FAE-1) from a termite hindgut bacteria was functionally characterised and used in synergy with xylanases during xylan hydrolysis. The FAE-1 displayed temperature and pH optima of 60 ℃ and 7.0, respectively. FAE-1 did not release reducing sugars from beechwood xylan (BWX), wheat arabinoxylan (WAX) and oat spelt xylan (OX), however, displayed high activity of  164.74 U/mg protein on  Agricultural feedstock sources such as maize, wheat, oat, rice and barley consist of arabinoxylans (AX) and glucuronoarabinoxylans (GAX) that are also linked to ferulic acid by ester bonds via the branching arabinose residues (Boz O)5-hydroxy group) of α-L-arabinosyl residues -linked xylose units substituted with acetyl, D-glucuronic acid (GlcA) and/or 4-dues Boz . Accordidues Boz , ferulic−1 dry matter of ferulic acid from oat hull. The cell walls extracted from maize cell suspensions contained about 18 µg mg−1 of dry matter of ferulic acid  from agricultural feedstocks using chemical or enzymatic means , while the GH11 referred to as Xyn2A from Trichoderma viride was purchased from Sigma Aldrich . Insoluble wheat arabinoxylan (WAX) and beechwood xylan (BWX) substrates were purchased from Megazyme  and p-nitrophenyl acetate (p-NP-acetate) and oat spelt xylan (OX) were purchased from the Sigma Aldrich . The purified feruloyl esterase from family 1 (FAE-1) with the following GenBank accession number KC493563 derived from the termite hindgut (Trinervitermes trinervoides) was provided by Dr. K. Rashamuse, Council for Scientific and Industrial Research (CSIR) Biosciences . FAE-1 was expressed and purified according to the method described by Rashamuse et al.   and FAE-1 were determined under standard assay conditions . For the xylanase activity assays, 400 µl of 1% (w/v) WAX, BWX and OX substrates were dissolved or suspended in 50 mM sodium phosphate buffer and 10 µg of enzyme was used. The reaction time for the xylanase activity was 30 min and then the reaction was terminated by heat at 100 ℃ for 5 min. The total reducing sugars released by the enzymes from xylan substrates were determined spectrophotometrically at 540 nm using the modified dinitrosalicylic acid (DNS) method as described by Miller . Xylose Binary enzyme mixtures were formulated using the individual xylanases (Xyn10D or XT6 and/or Xyn2A) and FAE-1 to evaluate their synergistic associations during the hydrolysis of 1% (w/v) BWX, WAX and OX. The enzyme combination ratios of 100:0%, 75:25%, 50:50%, 25:75% and 0:100% on protein mass basis at 10 mg/g of xylan were used to investigate the synergism between enzymes. The reaction was initiated by mixing 1% (w/v) xylan  with the binary enzyme mixtures in 50 mM sodium phosphate buffer (pH 6.0) at 37 °C and run for 12 h. The reducing sugar release was then analysed according to DNS method described in the “The degree of synergy (DS) was defined as the total reducing sugars produced by synergistic actions of the two or more enzymes divided by the theoretical sum of total reducing sugars produced by the individual enzymes  was used to elucidate significant differences in the reducing sugar released by different binary enzyme mixtures referred to in “The focus of the current study was to (1) demonstrate that the FAE-1 derived from a termite hindgut bacteria is active; (2) biochemically characterise the FAE-1; and (3) formulate binary FAE-1 to xylanase synergistic enzyme mixtures to improve the release of reducing sugars during xylan hydrolysis.p-NP-acetate , however, it displayed a high specific activity of about 164.74 U/mg protein on the O-methyl-α-d-glucopyranosyl uronate residues in BWX; Fig. The xylanases  were tested on the xylan substrates; BWX, WAX and OX. Xyn10D and Xyn2A showed the highest specific activity on OX reaching about 156.19 and 212.11 U/mg protein, respectively. XT6 showed the highest specific activity of 128.84 U/mg protein on BWX Table  revealedhttp://www.cazy.org/Glycoside-Hydrolases) can be used in value-added chemical production, baking and feed industries. A few CAZymes (xylanases) that degrade the non-starch polysaccharides (NSPs) were generally used in the baking and feed industry. We propose that understanding the synergy between CAZymes during the hydrolysis of NSPs could lead to improved degradation of feedstocks containing NSPs. Hence, in the current study, binary synergy between (1) Xyn10D and FAE-1; (2) XT6 and FAE-1; and (3) Xyn2A and FAE-1 was investigated for improved hydrolysis of xylans.It was established in the past decade that some CAZymes  is one of the most powerful tools used in establishing synergism between enzymes  hindgut bacteria. In addition, the A. gonensis A4 secreted FAE that displayed a pH optimum between pH 5.5 and 6, and temperature optima at 60 and 80 ℃  than the A. niger BE-2 FAE  family 1 was able to release ferulic acid from wheat bran treated with dilute phosphoric acid. In addition, the EPFAE-1 and a xylanase from GH10 (referred to as EPXYN1) released about 60% of ferulic acid from the wheat bran. The purified pgFAE from Panus giganteus and a GH10 xylanase synergized and resulted in a higher release of ferulic acid of about 112.2 µg/mg substrate compared to 17.6 µg/mg substrate released by pgFAE alone from wheat bran  displayed synergy on WAX, untreated corn cobs, hydrothermal pretreated and acid pretreated corn cobs, resulting in approximately two-fold higher reducing sugars  and FAE-1 did not display effective synergy during the hydrolysis of BWX, WAX and OX samples. XynA derived from p-NP-acetate, suggesting that FAE-1 could hydrolyse the ester bonds between ferulic acids and arabinoxylan. In addition, we demonstrated that xylanases were highly active on the xylan substrate with lesser substitutions like BWX and OX, but their activity dropped significantly during the hydrolysis of a highly substituted xylan substrate, WAX. The synergy of the GH 10 xylanases (XT6 and Xyn10D) and FAE-1 resulted in higher degradation of WAX, no improvement on BWX and OX degradation, but FAE-1did does not exhibit synergy with GH11. These findings contributed to our understanding toward the use of FEA-1 in synergy with GH10 xylanases for hydrolysis of NSPs in the animal feed and baking industries.The FAE-1 does not possess activity on sugars linked by glycosidic bonds, but it displayed a high esterase activity on the model substrate


N = 30) were females, were white (N = 25) and stay at home parents (N = 12). Results: Key barriers included lack of appropriate knowledge and sources of information, problematic skills development, social influences, cognitive overload, and lack of motivation for change. Facilitators included social role, access to resources, positive intentions, beliefs about consequences and reinforcement. In particular, optimal bedtime routines were less likely to be enacted when parents were tired/fatigued and there was a strong effect of habit, with suboptimal routines maintained over time due to past experiences and a lack of awareness about the importance of a good bedtime routine. Conclusions: Several theory-based, and potentially modifiable, determinants of optimal bedtime routines were identified in this study, providing important information for future interventions. Several of the key determinants identified were transient (tiredness) and/or non-conscious (habit), suggesting that future interventions may need to be deployed in real time, and should extend beyond conventional techniques.Objectives: Bedtime routines are a highly recurrent family activity with important health, social and behavioural implications. This study examined perceived barriers to, and facilitators of, formulating, establishing, and maintaining optimal bedtime routines in families with young children. Design: Participants completed a semi-structured interview based on the Theoretical Domains Framework (TDF). Analysis followed a deductive approach. Participants: A total of 32 parents participated in the study. Most participants ( Bedtime routines are one of the most frequently performed family activities encompassing a series of actions undertaken by families with young children in the hour before bed  Most famBedtime routines are essentially a series of behaviours and therefore in order to change them we need to draw from the wealth of behaviour change literature. Existing studies and interventions on bedtime routines have utilised a wide range of theories and approaches (2). A widely used approach within behavioural science and behaviour change interventions is the Behaviour Change Wheel (BCW), a theory-informed and evidence-based guide to designing behaviour change interventions . The BCWIn order to gain a better understanding of our target behaviour (bedtime routines) and enhance our ability to develop evidence-based interventions, the present study utilised a theoretical framework . This evidence-based framework was used to explore the barriers and facilitators that parents from economically deprived households face when developing and maintaining bedtime routines for their children. The TDF is a framework which summarises 84 possible determinants of behaviour into overarching “theoretical domains”, allowing for a comprehensive exploration of all possible determinants of bedtime routines in families with young children . The TDFThis qualitative study uses the TDF to explore perceived barriers and facilitators of the formation, establishment and maintenance of bedtime routines in families with young children.A total of 32 parents with young children between the ages of 3–7 years old were recruited for this study. Parents were recruited in two distinct periods in 2018 and 2019. Parents were recruited from nurseries where a member of the research team approached parents directly providing details about the study and taking informed consent. A total of 40 parents were approached with 32 recruited (80% success rate). Inclusion criteria included: (a) ability to speak and comprehend English and (b) having a child between the ages of 3 and 7 years old. Participants were compensated for their time in the form of £10 vouchers for online shopping. All participants completed the interviews with no dropouts for this study. All elements of this study were approved by the University of Manchester Research Ethics Committee. The study in its entirety including consent forms and all study materials was previously approved by the Health Research Authority (Integrated Research Application System (IRAS) ID: 238552). All participants accepted anonymized use of their data for further analyses and subsequent publication during consent. Written consent was taken during recruitment.Interviews were completed either in person or via telephone depending on participants’ preference. In total, 25 participants completed an in-person interview with 7 opting for a telephone interview. In each interview, a detailed semi-structured interview schedule based on the 14 TDF domains was used . Each TDEach audio recording was transcribed verbatim using a transcription service. Two independent coders with experience in using the TDF framework used a deductive approach to map each statement to one of the TDF domains (or code as outside of the TDF). Any disagreements in coding were resolved through discussion. Remaining disagreements were resolved by a third independent, experienced coder. Barriers and facilitators were identified based on participants’ responses. Overarching themes were also identified and summarized while frequency counts were used to determine the most commonly endorsed domains and specific component constructs.N = 25), three participants of Asian/British-Asian ethnic background and four participants of Black/Black-British/Caribbean ethnic background. In terms of educational level, N = 12 had University education, N = 20 were college graduates (either having completed their A-levels (N = 10) or high school graduates (N = 10)). The majority of participants had either one (N = 15) or two children (N = 15) with only two participants having three children. Most participants were stay at home parents (N = 12), with N = 10 participants working part-time, N = 8 participant working full-time and N = 2 participants studying at University. The majority of participants (N = 27) were in two parent households, a further three were in single parent households and two participants came from households with more than two adults . Finally, in terms of deprivation as calculated by the Index of Multiple Deprivation (IMD) most participants (N = 20) were on the fifth quintile (most deprived). N = 6 were on the fourth quintile with only six participants on the third quintile. Average IMD score was 36.4 (SD = 4.1) classifying as “most deprived”. The IMD is a frequently used metric of social deprivation in England  and it provides data based on participants’ postcodes.In total, 32 individuals  participated in the study. The majority of the participants were white .Cohen’s kappa (κ) was calculated in order to examine inter-rater reliability between the two independent coders. A total of 289 statements were examined and mapped into relevant TDF domains. Based on the results of the analysis and following guideline outlined by Landis and Koch (1977) there was substantial agreement between the two coders (Example quotations are presented below per each TDF domain to showcase participants’ replies.N = 25) of parents reported that they had never been offered advice on bedtime routines when their children were born.In general, all parents reported awareness of the importance of bedtime routines. Most parents were able to describe what a good bedtime routine should look like. Use of electronics before bed was the most common activity that parents did not mention when describing a good bedtime routine. “Reading that the school asks us to do. Spellings and settling them down in a relaxed environment before bedtime and teaching them that it’s healthy to look after their teeth and that is one of the bedtime routines that, as they get older, that they should be doing.”(QI021)“No. It would have been good to get some advice, but no one really said anything about routines when the children were born.”(QI012)Six participants knew about official recommendations or were given some advice when their children were younger, but they could not recall exactly what they were told or who provided them with that information. A total of 15 participants expressed a positive view about how useful an official system or point of contact where they could seek advice on bedtime routines would have been.“If somebody could have told me how to get my kids to sleep that probably would have been really, really helpful.” (QI005)In terms of skills development, most parents reported using the same sort of routines with their own children as they had when they were children. While some parents mentioned external factors that influenced the development of their bedtime routines and most parents were able to identify a variety of skills and techniques they use as part of their bedtime routines.“So when they’re doing their teeth, we have, like, one of their favourite songs will play … so they’ll find a song that’s three and a half minutes long, so they’ve got to brush their teeth while that song is playing, so they’ll dance while they’re brushing their teeth and then once that songs finished, their teeth are done.”(QI004)Parents viewed themselves as an important role model for their children and felt a huge level of responsibility for the overall wellbeing and development of their children. Some parents brought their overall, non-parental, roles and identities as professionals in the context of their responsibility towards their children.“I suppose what I’m doing as a parent is trying to set them up in good habits for the rest of their lives, because the stuff that they do before they go to bed is the stuff that I do before I go to bed.” (QI025)A total of seven parents stated their bedtime routines were generally not perceived to be difficult or challenging. However, parents identified some occasions when routines were perceived as more challenging, for example over the weekend. Ten parents felt that their bedtime routines are difficult and challenging in general.“Difficult but it’s something that we’re all used to, and they’ve done since they were younger and it’s something, like I say, that I’ve always been consistent with but yes, it is difficult.” (QI031)The majority of parents appeared confident and optimistic about how things will unfold in the future regarding their bedtime routines.“I don’t feel anxious about it really, I think... I haven’t really thought about it really. I don’t know.” (QI005)The majority of parents stated clear intentions to try actively to achieve and maintain good bedtime routines for their children in the short and long-term future.“Yes, I mean 100 percent, 101 percent really and in terms of that, it’s maintaining and being consistent and that can get tiring but that’s the length that I personally am happy to go to for them.” (QI011)Most parents mentioned specific outcome expectations associated with problematic bedtime routines. While others reported their overall beliefs about the future of their children and the importance of having a good bedtime routine.“If you’re brushing your teeth so this will give you a future with nothing a problem with your teeth and everything. But if you do brush correctly, in the correct way.” (QI023)Reinforcement was analyzed in two contexts: (a) reinforcement used towards the children as part of the bedtime routine or general parenting and (b) reinforcement experienced by the parents at the end of the night and after the children were off to bed. In terms of reinforcement techniques used with the children, most parents were able to list several techniques covering both positive (reward) and negative (punishment) reinforcement. In terms of reinforcement experienced by the parents, this included immediate benefits of having an optimal routine in place such as free time at the end of a busy day.“It is a nice feeling, if it’s all smooth and everybody goes to bed happy and so on and you don’t feel like you’re on your last nerve, then, yes, of course it’s a nice feeling, because then you look at these two sleeping angels and think that’s lovely.” (QI005)For many of parents, bedtime routines are more than just getting the children to bed, it is about spending good, quality time together and building long-lasting memories. Also, parents gave examples of goal priorities shifting when dealing with changing circumstances in their houses during their bedtime routines.“You’re all busy during the day, the children are at school, you’re at work, so that is a really nice time to talk to the children and find out what’s been going on in their day and yes, they play with each other, it’s their time as well to have a bit of fun with each other.” (QI011)Parents reported a mixed emotional reaction to bedtime routines with some reporting negative emotional reactions towards them.“Calm, quite fine, like I say because we’ve stuck to the same routine. It’s not a chore; it’s a pleasurable thing to do.” (QI008)All parents reported a high level of automation (memory) when it comes to their bedtime routines with little to no thought on what to do and how to do it. However, when tired (cognitive overload), parents reported difficulties in complying with their normal routine as well as issues around forgetting what they need to do.“God, yes, it’s hard, well it can be just because I work full time and by the end of the day I’m shattered so, yeah, because they’re busy and they’re five and three.” (QI023)Houses and the immediate environmental context did not present as an issue. All parents reported adequate access to all required resources  for achieving a good bedtime routine.“Well the children have to share a room which makes things more difficult. It would have been nice to be able to have separate rooms for them but that’s not a possibility unfortunately.” (QI012)Peer support  was important for 15 parents in this study especially due to lack of any other available source of information. Meanwhile, 15 parents reported comparing their routines to their peers  with some of them expressing beliefs on whose routine is better and why.“Yes, they always resist, every night they resist at bedtime and obviously at the weekends, I’m a little bit more lenient but no I think they enjoy the bedtime routine.” (QI011)In terms of self-monitoring, a total of 14 parents reported not using any type of self-monitoring with regards to their bedtime routines reflecting the automated, habitual nature of the routines. However, 15 parents reported using specific self-monitoring techniques. Some parents reported specific habit breaking events that led to a significant change of behaviour in the past.“Just in my head and keeping track of how it works well for the children and varying it upon that.”(QI007)Across the whole dataset, overarching themes, or factors that emerged as most important in relation to bedtime routines included: (a) lack of provision of information from respected sources, especially when children were younger and routines were being developed; (b) skills development and social support through peers; (c) parents’ beliefs that looking after their children’s bedtime routines is part of their parental role, their responsibility; (d) parents’ self-confidence and the emotional reactions associated with bedtime routines; (e) optimism about the future with clearly defined intentions to achieve and maintain good routines for their children; (f) positive reinforcement from good bedtime routines and negative reinforcement from bad bedtime routines; and (g) the level of automation and self-monitoring during bedtime routines.The key barriers and facilitators identified regarding formation, establishment and maintenance of optimal bedtime routines are summarised in This study examined perceived barriers to, and facilitators of, the formation, establishment, and maintenance of bedtime routines using the TDF. The examination of barriers and facilitators is a vital step for the development of theory-informed and evidence-based interventions to support and assist parents with their bedtime routines.In line with recent studies in this area  it is evPast actions are a strong predictor of future behaviours and, people tend to stick with their behaviours unless they prove to be problematic early on ,14,15,16Parental tiredness/fatigue and cognitive overload acted as additional barriers to systematically and consistently achieving optimal bedtime routines even when parents knew what they needed to do and how to do it. In general, parental tiredness is a nearly universal experience . MultiplFinally, lack of motivation, negative emotions and automation of routines are another set of barriers identified in this study. These barriers can be grouped under the term “behavioural inertia” . BehavioBehavioural inertia and status quo bias in the right context can be useful in maintaining optimal behaviours however, in cases where change would be beneficial they transform to detrimental factors perpetuating problematic behaviours . For bedThe development of the interview schedule to reflect and capture all TDF domains might have resulted in a more rigid and structured discussion. However, this particular possible limitation was managed through the establishment of prior good rapport that allowed for participants to feel more comfortable and express themselves in their own way. Participants were also given the freedom to discuss anything outside of the topic guide that they felt was relevant to any aspect of bedtime routines. Also, the disproportionate number of white and mainly female participants, while problematic for generalization of results, it is not surprising given this particular area of research and the context of recruitment. Also, the household composition of our participants, mainly two parent households, limits generalizability in more diverse household types that might be more relevant and prevalent in other countries and/or specific areas. Finally, another possible limitation can be found in the selection of participations using a convenience sampling approach that could have resulted in biases around demographic and socio-economic characteristics.Using an evidence-based approach there is a possibility to map identified barriers and facilitators into existing literature and evidence around behaviour change and techniques. Those techniques could either maintain and promote facilitators  while removing barriers . With some crucial barriers clearly identified, future work will focus on how to address them using a theory-informed and evidence-based approach that can maximise the effectiveness of subsequent intervention.Parents of young children face a series of barriers to achieving optimal bedtime routines ranging from lack of appropriate knowledge to lack of motivation and tiredness. These barriers can prove detrimental for bedtime routines with possible health, behavioural, and social consequences for parents and children. Gaining a better understanding of the determinants of optimal and suboptimal bedtime routines, is an important first step for future more in-depth examinations and potentially intervention studies.


In particular, we focus on correlations manifested by Kochen–Specker–Klyachko box (KS box), scenarios involving n-cycle non-contextuality inequalities and Popescu–Rohlrich boxes (PR box). We provide the criteria for optimal classical simulation of a KS box of arbitrary n dimension. The non-contextuality inequalities are analysed for n-cycle setting, and the condition for the quantum violation for odd as well as even n-cycle is discussed. We offer a simple extension of even cycle non-contextuality inequalities to the phase space case. Furthermore, we simulate a generalised PR box using KS box and provide some interesting insights. Towards the end, we discuss a few possible interesting open problems for future research. Our work connects generalised PR boxes, arbitrary dimensional KS boxes, and n-cycle non-contextuality inequalities and thus provides the pathway for the study of these contextual and nonlocal resources at their junction.Quantum communication and quantum computation form the two crucial facets of quantum information theory. While entanglement and its manifestation as Bell non-locality have been proved to be vital for communication tasks, contextuality  has shown to be the crucial resource behind various models of quantum computation. The practical and fundamental aspects of these non-classical resources are still poorly understood despite decades of research. We explore non-classical correlations exhibited by some of these quantum as well as super-quantum resources in the  The quantum mechanical description of nature is incompatible with any local hidden variable theory and consequently is said to exhibit Bell non-locality . This con-cycle graph. In particular, we explore the correlations manifested by Kochen–Specker–Klyachko box (KS box), Popescu–Rohlrich boxes (PR box) and scenarios involving n-cycle non-contextuality inequalities. The KS box was first introduced by Bub et al. in 2009 [p), which determines the nature of box namely classical, quantum and post-quantum. Bub et al. showed that it is impossible to simulate the KS box statistics for p, the best classical strategy has a success probability of approximately Our focus revolves around the correlations manifested by three different objects from quantum and post-quantum theories with underlying structure governed by the  in 2009  who anal 0.94667 . The aut 0.94667 . It is i 0.94667 ,19,20. Fe PR box .Now, we turn to the last object of our study. The Bell nonlocal nature of theories can be witnessed via the violation of certain inequalities, referred to as Bell inequalities and non-contextuality inequalities in the general case of contextuality . In theiThe KS boxes were motivated by KCBS non-contextuality inequality .The KCBS non-contextuality inequality belongs to the same family of inequalities as the CHSH inequality .The maximum value of CHSH inequality for no-signalling theories is provided by PR boxes .The PR box can be simulated by KS box for p=12 .It is important to observe the connections among KS box, non-contextuality inequalities and PR boxes.n-cycle non-contextuality inequalities. Our work provides the pathway for the study of these generalised contextual and nonlocal resources at their junction.In this paper, we further explore the interconnections among the generalised versions of the aforementioned objects, namely generalised PR boxes, arbitrary dimensional KS boxes and n-dimensional case and provide the optimal classical strategy as well as corresponding success probability for simulating the box using classical resources. Our results provide the minimum gap between the optimal classical strategy and the KS box based strategies for arbitrary p and n. We observe that the optimal success probability for classical simulation decreases monotonically with the dimension of the KS box.In n-cycle contextuality scenario and the corresponding non-contextuality inequalities. We explore the odd cycle generalisation of the well-known Klyachko–Can–Binicioğlu–Shumovsky (KCBS) inequality [In equality ,23 and eequality . Followiequality , we discequality .p (tunable parameter) for the KS box required to saturate classical, quantum and no-signalling bounds.Within no-signalling theories, the maximum violation of CHSH inequality is obtained by Popescu–Rohlrich box, also known as PR box . The PR Finally, we conclude in A Kochen–Specker–Klyachko box or KS box is a bipartite no-signalling box with two inputs and two outputs  [A KS box with marginal probability domness) . We wantN-gon with a M “1s” for a given N-gon corresponding to an N-dimensional KS box is referred to as a chart of degree M, in short To capture the essence of classical strategies, we use the language from graph theory. Consider an Simulating a Proposition 1.Given the chart  ⊥ condition is given byProof. N-dimensional KS box, the total number of possible input pairs for Alice and Bob are For an This completes the proof. □N-dimensional p.For Theorem 1.For a given Proof. Assume that Alice and Bob play the charts M, with Now, observe that the objective function isThis completes the proof. □Let us have a look at the simulation efficiency in a bit detail. Numerical evidence refer to  suggestsp, the nonlocal nature of KS box increases with dimension of the box and hence the simulation efficiency for the optimal classical strategy decreases. Moreover, for a KS box with fix dimension, its nonlocal nature increases with increase in p.For a particular value of n-cycle non-contextuality inequalities.Having studied the KS box, we move next to the n-cycle generalisation of KCBS and CHSH inequalities, we would like to discuss the prior art briefly required to understand our work.Before we analyse the A, B and C such that A lies in two contexts defined by the sets K. Now, it is possible to have a theory which violates the bound in Equation  theories is given by independence number of the underlying exclusivity graph, denoted by α(G) . The uppG) [ϑ(G) . Formallm theory ,22.CHSH inequality is a special case of non-contextuality inequality where the context is provided by space-like separation of parties involved, e.g., Alice and Bob. The scenario corresponding to inequality corresponds to four measurements, two for each party. Each of Alice’s measurements are compatible with Bob’s measurements and vice versa. Suppose Alice’s measurements are given by X and Z are Pauli matrices, The local hidden variable theories respect the bound in , howevernd state . These oThe inequality in Equation  has beenThe odd cycle generalisation of KCBS inequality has been studied extensively in literature ,21,23,27cle case ,23,28. Tn-cycle generalisation of KCBS inequality, the projectors corresponding to the optimal quantum violation are given byWe represent the density matrices in the standard basis Proposition 2.A qutrit violates the odd n-cycle generalisation of KCBS non-contextuality inequality if and only if Proof. n-cycle scenario can be defined asThe generalised KCBS operator for the odd Adding all the projectors  and and23) aFor a two qubit density matrix n-cycle chained Bell inequality becomesThe condition for violation of The eigenvalues of n-cycle chained Bell inequality turns out to beUsing Equations  and 25)25), the This completes the proof. □The set of quantum states form a convex set. Since the non-contextuality inequality in Equation  is a linSince the system under consideration is a two qubit density matrix. We have the following constraints on the eigenvalues:The constraints in Equations  and 29)29) implyUsing Equations  and 30)30), we gThe Equation  providesRemark 2.It is easy to see that set of two-qubit quantum states that can violate chained Bell inequality shrinks as we increase n See . In the The even cycle non-contextuality inequalities can be extended to phase space case quite easily following the work of Arora et al. , where tEquation . The ineWe know thatFor z-axis with angle L. This operator is not Hermitian and hence we introduce the following symmetric combination to make it Hermitian,Let us look back at the operator in Equation  more cloLet L. and Let Let The appropriate entangled state which shows the violation isN. The experimental implementation of the phase space extension is quite simple and follows directly from the work of Arora et al. [The quantum violation for the state in Equation  correspoa et al. .The KS box is a powerful resource which can be used to efficiently simulate the most non-local no-signalling box, i.e., PR box . The PR Definition 2.A PR box is a no-signalling resource with input pair which means that the outputs are different if and only if the inputs are n-dimensional Now, suppose Alice and Bob are equipped with an arbitrary dimensional KS box. KS box is more powerful than PR box and can be used to simulate the same . We ask Proposition 4.A PR box of dimension (number of inputs for each party) n can be simulated efficiently using a KS box of dimension Proof. To prove our claim, we provide the following strategy: Alice relabels her inputs for PR box as follows:Similarly, Bob relabels his inputs as follows:The relabelled inputs are used as fresh input for the p in the Given the even cycle generalisation of CHSH inequality, the marginal probabilities Remark 3.For a large value of n, all the above probability expressions tend to one half. However, the quantum probability approaches the PR box limit of h 12 see .n-cycle non-contextuality inequalities in this work. We provided the optimal classical strategy and the corresponding success probability for classically simulating the KS box. For future work, it is worth exploring the optimal quantum strategy for this purpose. We provided the sufficient condition for the violation of the generalised KCBS inequality and necessary condition for the violation of even-cycle generalisation of CHSH inequality. We also discussed the phase space extension of even-cycle generalisation of CHSH inequality. We leave the phase space extension of KCBS and generalised KCBS inequality for future work. We also studied the strategy for simulating a generalised PR box using KS box. It is also interesting to explore further how the generalised PR box, arbitrary dimensional KS box and n-cycle non-contextuality inequalities are related to each other and their implications.We studied arbitrary dimensional KS box, generalised PR box and n-dimensional case. We also provided the sufficient condition for violation of odd n-cycle non-contextuality inequalities. Since contextuality is the chief resource behind various models of quantum computation, our result can help select the resources required to get necessary quantum speed-up. Moreover, our phase space extension of chained Bell inequalities make them suitable for experimental purposes and also harness the underlying Bell non-locality for various quantum communication tasks such as secure key distribution for example. Our work helps quantify the Bell non-locality of KS box in terms of impossibility of classical simulation for general


Ucp1 or Dio2 mRNA expression in BAT explants. Taken together, the investigated agonists do not seem to exert lasting and physiologically relevant effects on BAT thermogenesis after peripheral administration, demonstrating that D1- and D2-receptors in iBAT are unlikely to constitute targets for obesity treatment via BAT activation.Brown adipose tissue (BAT) thermogenesis is considered a potential target for treatment of obesity and diabetes. In vitro data suggest dopamine receptor signaling as a promising approach; however, the biological relevance of dopamine receptors in the direct activation of BAT thermogenesis in vivo remains unclear. We investigated BAT thermogenesis in vivo in mice using peripheral administration of D1-agonist SKF38393 or D2-agonist Sumanirole, infrared thermography, and in-depth molecular analyses of potential target tissues; and ex vivo in BAT explants to identify direct effects on key thermogenic markers. Acute in vivo treatment with the D1- or D2-agonist caused a short spike or brief decrease in BAT temperature, respectively. However, repeated daily administration did not induce lasting effects on BAT thermogenesis. Likewise, neither agonist directly affected  It is therefore highly important to elucidate the complex pathways of energy homeostasis in order to develop approaches to counteract these dysregulations of metabolism. In addition to advocating individual responsibility regarding energy intake and expenditure, which may present particularly difficult in highly obese individuals4, an endogenous increase of energy expenditure proposes a potential strategy. Regarding this, activation and recruitment of brown adipose tissue (BAT) thermogenesis have been discussed recently8.Obesity and its metabolic consequences like type 2 diabetes have spread in recent years to become a worldwide pandemic10, making this tissue a promising strategic target to treat metabolic disorders. Therefore, a safe and long-lasting way of BAT thermogenesis activation would be required, and consequently, knowledge of the underlying modes of activation is crucial to assess its true potential for safe exploitation.Brown adipocytes have the unique ability to combust energy from metabolic substrates directly into heat11. NE binds to the adrenergic receptors α1, α2, and β, which all exist in BAT12. Nevertheless, the interaction between NE and β3-adrenoceptors (ADRB3) is the most relevant to induce thermogenesis in mature brown adipocytes14. Binding of NE to the Gs-coupled ADRB3 induces an adenosine cyclase (AC)- and cyclic adenosine monophosphate (cAMP)-mediated cascade that increases expression of uncoupling protein 1 (UCP1), which in turn bypasses oxidative phosphorylation in the respiratory chain and causes heat dissipation16. Interestingly, NE simultaneously binds to Gi-coupled α2-adrenergic receptors and inhibits AC activity, counteracting ADRB3-mediated activation of BAT thermogenesis17. The purpose of this counter regulation is not understood to date, but underlines that endogenous neurotransmitter-receptor relationships are complex and not always specific20. To dissect the significance of distinct receptor-mediated pathways on BAT thermogenesis, they need to be studied separately, e.g., by using specific receptor agonists instead of the endogenous ligand.The catecholamine neurotransmitter norepinephrine (NE) is a key player in BAT thermogenesis activation22. Dopamine receptors are categorized into D1-like and D2-like dopamine receptors23. D1-like dopamine receptors (D1 and D5) are Gs-coupled receptors and stimulate cAMP synthesis, whereas D2-like dopamine receptors  are Gi-coupled and inhibit cAMP synthesis upon ligand-binding25. Interestingly, it was suggested that dopamine—structurally highly similar to NE—also modulates BAT thermogenesis in rodents, due to comparable mode of action27. Additional studies have shown that dopamine and the D1-agonist SKF38393 increase thermogenesis and mitochondrial mass of brown adipocytes in vitro28. However, whether dopamine receptor signaling directly affects BAT thermogenesis in vivo has not been studied. Here, using primary mouse BAT explants and mouse models, we characterize the direct effects of the D1-agonist SKF3839329 and D2-agonist Sumanirole31 on BAT thermogenesis in detail. Our results show, that neither the D1-agonist, nor the D2-agonist have a sustained BAT-specific effect on thermogenesis upon direct application ex vivo or peripheral administration in vivo.The catecholamine neurotransmitter dopamine is the direct metabolic precursor of NE, also binding to G-protein-coupled receptorsUcp1 and Dio2 mRNA expression and decreased mRNA expression of Adrb3 itself  mRNA expression  tissue explants. We validated this ex vivo experimental model using the ADRB3-agonist CL316,243 as a positive control (PC). As expected, the PC increased To capture rapid effects of the D1- and D2-agonist on iBAT thermogenesis, we continuously and non-invasively measured iBAT temperature in wild type mice up until 1 h after i.p. injection using infrared video thermography. While the D1-agonist significantly increased iBAT temperature for a total of 15 min between minutes 20 and 45 compared to baseline  indicative of no WAT browning, while Dio2 mRNA expression in iBAT, iWAT and gWAT was also not altered by the D1-agonist .In addition to the physiological parameters, we investigated adipose tissue homeostasis. Repetitive treatment with the D1-agonist resulted in significantly decreased weight of the collected fat depots, namely iBAT, inguinal and gonadal white adipose tissue (iWAT and gWAT) Fig. A; while ist Fig. D. Howeveist Fig. E, indicaoup Fig. F, but hioup Fig. G. Treatmoup Fig. E,F, Dio2ion Fig. K, cAMP pion Fig. L, or lipion Fig. M. Furtheion Fig. N. Intereice Fig. I. Howeveice Fig. B. We alsice Fig. A–D, indiPyrk  may yield only a minor transient effect, which does not occur when stimulating both receptors simultaneously with the endogenous ligand dopamine.We observed rapid, yet very brief, acute effects of the D1- and D2-agonist on iBAT temperature in mice. According to their respective nature of being GUcp1 nor iBAT temperature were increased. This is contrary to the well-known effect of an NE-induced cAMP increase36 and suggests differences in the downstream signaling cascade that, in case of the D1-agonist, do not further promote thermogenesis. The lack of downstream signaling could be caused by the biased agonism of SKF38393, namely G-protein signaling in absence of β-arrestin recruitment37, which can prevent late signaling and therefore avert sustained thermogenesis39. Moreover, the brief, acute changes in iBAT temperature could be indirect consequences of the D1- and D2-agonist affecting the vascular system40, either via peripheral action or via centrally mediated effects41.After 1 week of repeated D1-agonist administration—in spite of elevated cAMP in iBAT—neither Ucp1 and Dio2 ex vivo in isolated mouse iBAT explants. This was surprising, as it seems to contradict the recent suggestion of dopamine receptors D1 and D2 being present, and D1 being involved in brown adipocyte thermogenesis regulation in a murine in vitro model28. There are, however, several differences between the model system of iBAT explants, as used in our study, and immortalized brown adipocytes from mice28, e.g., in their degree of proliferation, multi-cellular interaction, and overall thermogenic capacity . Our ex vivo model, which showed the expected BAT activation response upon stimulation with an ADRB3-agonist44, interestingly also showed a decrease of Ardb3 mRNA upon stimulation with the D1-agonist. However, as this was not accompanied by activation of thermogenesis markers Ucp1 or Dio2, it suggests only a negligible biological relevance for dopamine receptor D1 in the recruitment of BAT thermogenesis. While dopamine itself exerted similar effects on thermogenic markers Ucp1 and Adrb3 in iBAT explants, it additionally increased Dio2 mRNA expression, suggesting a potential direct role of dopamine in tissular iBAT thyroid hormone production. Nevertheless, as we did not include data on UCP1 protein or DIO2 activity, we cannot fully exclude any additional non-transcriptional effects at present. Most importantly, however, dopamine—just like NE—does not exclusively bind to its designated receptors19, which allows the possibility that other receptors may contribute to this effect. Unfortunately, due to its extremely short half-life and rapid oxidation in vivo45, longterm studies as conducted with the individual agonists cannot be performed with dopamine in a similar setup.The possibility of indirect action is supported by the lack of significant direct effects of the D1- or D2-agonist on mRNA expression of key thermogenic markers 46, thus questioning previous findings of D1 or D2 receptor protein in the tissue. Hence, it remains to be investigated whether dopamine receptors are truly present on brown adipocytes using more elaborate quantification techniques, e.g., on a single cell level.Furthermore, to fully understand the direct role of dopamine receptors in BAT function, it has yet to be elucidated whether dopamine receptors are at all present on brown adipocytes. Our own dopamine receptor expression analyses in mice exhibited mRNA transcript levels of dopamine receptors D1–D5 in iBAT close to the detection limit (data not shown), which could also be caused by the remaining vasculature in the samples. Moreover, several commercially available antibodies for dopamine receptors D1 and D2 have recently been demonstrated to be unspecific48. This is supported by our finding of an increased tail base temperature, while core body temperature remained unaltered. The elevated tail base temperature could also result from D1-agonist-induced vasodilation51; however, if vasomotor-mediated tail heat loss were the primary effect, iBAT thermogenesis would be stimulated in order to compensate the heat loss and prevent a consequent decrease of core body temperature48—two effects not observed in our animal model.Despite the lack of BAT thermogenesis activation, repeated D1-agonist administration still increased metabolic turnover in vivo, as evidenced by the reduced fat depot weights and lower hepatic glycogen levels. Ironically, this metabolic activation by the D1-agonist may even prevent activation of BAT thermogenesis in vivo, as it elevates obligatory thermogenesis, thus attenuating facultative BAT thermogenesis. Accordingly, emission of surplus heat would be probable, e.g., via tail vasodilation, which constitutes a major thermoregulatory mechanism in rodents53 or by increased excitation of skeletal motoneurons54. This would subsequently lead to an elevated metabolic demand, which may be reflected by the reduced hepatic glycogen stores in our D1-agonist treatment group. Further studies, dissecting the role of dopamine receptors in muscle metabolism, are therefore warranted.Another possible mechanism could involve increased skeletal muscle activity, e.g., by enhanced locomotion as observed previously in D1-agonist-treated mice55. However, in this study the effects of the D2-agonist were demonstrated to be mediated via centrally initiated activation of the sympathetic nervous system. Together with the low transport of the agonist across the blood brain barrier56, these results complement our findings suggesting that D2 receptors play a role in the central control of BAT thermogenesis, but not directly in BAT.The lack of effects of the D2-agonist on iBAT thermogenesis shown in our study seemingly contradicts a recent study, where hypothalamic D2-agonist administration decreased body weight and stimulated iBAT activity in rodents57, as dopamine can also activate adrenoceptors—albeit less efficiently than NE58. Due to the extremely short half-life and rapid oxidation of dopamine after injection, this would, however, be difficult to test directly in a similar in vivo experiment and therefore needs to be studied separately. Taken together, our findings suggest that dopamine receptor D1- and D2-agonists might not constitute a promising therapeutic approach for peripherally inducing BAT thermogenesis for treatment of obesity or associated metabolic disorders.Overall, based on the lack of pronounced effects observed upon treatment with the D1- and D2-agonist in our study, we conclude that dopamine receptors D1 and D2 are unlikely to play a major physiological role in the peripheral regulation of BAT thermogenesis in mice. However, this does not fully exclude a potential role for dopamine itself in BAT thermogenesis activationad libitum access to food  and water. All animal procedures were approved by the Ministerium für Energiewende, Landwirtschaft, Umwelt, Natur und Digitalisierung (MELUND) Schleswig–Holstein, Germany. All methods were performed in accordance with relevant guidelines and regulations.All studies were carried out with 10–12 weeks old male wild type C57BL/6NCrl mice, purchased directly from Charles River, Germany. Upon arrival, the animals were housed in groups , had one week to acclimate to the facility with 23 ± 1 °C, a constant 12 h light–dark cycle, and D1-agonist ( ±)-SKF-38393 hydrochloride , D2-agonist Sumanirole maleate , dopamine hydrochloride , ADRB3-agonist CL316,243 hydrate , and NE compound “Arterenol”  were solubilized to make identical stock concentrations in water or saline for ex vivo or in vivo studies, respectively. Final dilutions were freshly made in complete culture medium or saline for ex vivo or in vivo studies, respectively.60. Briefly, iBAT was excised from 8 mice (12 weeks of age) per experiment, cut into four pieces and transferred into basal differentiation medium . Pooled iBAT was minced into pieces of 1–2 mm in diameter, rinsed with DPBS, and distributed evenly into 12-well culture plates (~ 10 mg tissue/well) filled with 1 ml complete differentiation medium , 2,5 nM dexamethasone, and 150 µM L-ascorbic acid). Explants were synchronized in 37 °C and 5% CO2 for 24 h and then rinsed with DPBS, which was subsequently replaced by culture medium with added treatment substances . Finally, explants were incubated in 37 °C and 5% CO2 for 24 h, rinsed with DPBS, collected, snap frozen, and stored at − 80 °C until further processing.The protocol for iBAT explant organ culture and medium composition was adapted from previous studiesTo study acute effects of the D1- and D2-agonist on iBAT thermogenesis, mice (n = 6) received saline, the D1-agonist SKF38393 (10 mg/kg), D2-agonist Sumanirole (3.2 mg/kg), or dopamine (100 µg/kg) via intraperitoneal (i.p.) injection. One animal was injected with 1 mg/kg NE (s.c.) as a positive control reference. Infrared videos (1 picture/s) were shot using a VarioCAM hr head (InfraTec). First, 10 min were filmed for baseline measurements. Subsequently, animals were briefly restrained for i.p. injection, released back into the cage, and filmed for another 60 min. iBAT temperature of one representative picture/min was scored using the IRBIS3 software (InfraTec). Changes in iBAT temperature were calculated by subtracting the 10 min baseline average score from 5 min interval average scores.62); each treatment had its own independent saline control group.To study chronic effects of the D1- and D2-agonist on iBAT thermogenesis, mice were monitored for two weeks. Measurements of the first week were collected for baseline data, while measurements of the second week were collected under treatment. During the second week, animals received saline (n = 6) or one of the agonists (n = 6) via daily i.p. injection  infrared photo camera, on the last two days of both, the first (baseline) and second (treatment) week. iBAT and tail base temperature was scored using the FLIR Tools Software (FLIR Systems). The scored temperature was normalized to rectally measured core body temperature .Simultaneous measurements of heart rate (pulse), systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP) were recorded using the non-invasive SC1000 Single Chanel System (Hatteras Instruments). An automated sequence of 15 measurements was conducted in each session. The mean of at least 12 values for each parameter from sessions on two consecutive days was used for statistical analyses.Isolation of total RNA and transcription into cDNA were done according to manufacturer’s instructions for the RNeasy Mini/RNeasy Lipid Tissue Mini Kit  and RevertAid First Strand cDNA Synthesis Kit , respectively. Genes of interest were analyzed by qRT PCR. Fast Start Universal SYBR-Green Master (ROX)  was used for all qRT PCR experiments, according to the manufacturer’s instructions. Primers were used in final concentrations of 50 nM. Primer sequences (5′-3′) are listed below.63, OXPHOS Rodent WB Antibody Cocktail , DAT (dopamine transporter) , MAO-A (monoamine oxidase A) , or SERCA2 (sarco/endoplasmic reticulum Ca2+-ATPase)  overnight at 4 °C. Incubation with a secondary antibody  was performed for 1 h at room temperature, before bands were detected with Clarity Max Western ECL substrate  and a ChemiDoc Touch Imaging System (Bio-Rad). Semi-quantitative analysis was performed in Image-Lab (Bio-Rad). Target protein abundance was normalized to total protein.Separation of isolated proteins was achieved by SDS-PAGE with 12% gels  and 20 µg total protein per lane. Total protein was detected for semi-quantification using Stain-Free imaging according to the manufacturer’s instructions (Bio-Rad). Wet transfer was conducted to a PVDF membrane. Membranes were incubated with primary antibodies UCP164, with minor modifications. Specifically, after 2 × volume ethanol (> 95%) was added to 50 µl supernatant, samples were incubated at 4 °C overnight. Furthermore, after re-suspension in 80 µl Lugol Reaction Mix, sample reaction developed for 10 min before OD600 was measured (75 µl/well in a 96-well plate). Values were normalized to the amount of tissue used.Hepatic glycogen was determined as published by Vujovic et al.cAMP concentration and lipase activity in fat depots of mice were measured by following the manufacturer’s instructions for the Non-Acetylation EIA Procedure of the Amersham cAMP Biotrak Enzymeimmunoassay (EIA) System  and Lipase Activity Kit colorimetric , respectively.Hematoxylin and eosin (H&E) staining was performed on 5 µm histological sections of paraffin embedded tissue. Sections were dewaxed in xylene (2 × 15 min), hydrated in descending ethanol concentrations , and washed in de-ionized water (6 min). Nuclear staining was attained in a hematoxylin bath (4 min), followed by flowing tab water (15 min) and a bath in de-ionized water (2 min). Next, non-nuclear staining was achieved in an eosin bath (2 min) and de-ionized water (10 s). Lastly, sections were dehydrated in ascending ethanol concentrations  and cleared in xylene (2 × 15 min). Specimens were mounted with Pertex  and covered with a glass cover slip.Values are presented as mean ± SEM. Statistical analyses were performed using Prism6 (Graphpad Software) and considered significant with *P < 0,05; **P < 0,01; ***P < 0,001; and ****P < 0,0001. Statistical tests were applied as indicated in figure legends. Grubb’s outlier test was performed with α = 0.01 for ex vivo and α = 0.05 for in vivo experiments. Statistically significant outliers with significant impact on results were excluded from graphs and calculations.Supplementary Information


Enterocytozoon hepatopenaei (EHP) is an obligate, intracellular, spore-forming parasite, which mainly infects the gastrointestinal tract of shrimp. It significantly hinders the growth of shrimp, which causes substantial economic losses in farming. In this study, we established and optimized a SYBR Green I fluorescent quantitative PCR (qPCR) assay based on the polar tube protein 2 (PTP2) gene for the quantitative analysis of EHP-infected shrimp. The result showed that the optimum annealing temperature was 60 °C for the corresponding relation between the amplification quantitative (Cq) and the logarithmic of the initial template quantity (x), conformed to Cq = −3.2751x + 31.269 with a correlation coefficient R2 = 0.993. The amplification efficiency was 102%. This qPCR method also showed high sensitivity, specificity, and repeatability. Moreover, a microscopy method was developed to observe and count EHP spores in hepatopancreas tissue of EHP-infected shrimp using Fluorescent Brightener 28 staining. By comparing the PTP2-qPCR and microscopy method, the microscopic examination was easier to operate whereas PTP2-qPCR was more sensitive for analysis. And we found that there was a correspondence between the results of these two methods. In summary, the PTP2-qPCR method integrated microscopy could serve for EHP detection during the whole period of shrimp farming and satisfy different requirements for detecting EHP in shrimp farming. Enterocytozoon hepatopenaei (EHP), first discovered in Thailand Penaeus monodon [Microsporidia are obligate, intracellular, spore-forming parasites, and diverse species infect almost all invertebrates and vertebrates, as well as some protists, with different species exhibiting various degrees of host specificity ,2. It is monodon ,7, mainl monodon . Althoug monodon . Nowaday monodon ,11,12,13Ent. bieneusi, which is known to infect immune-suppressed and immunodeficient humans, such as patients with AIDS [SSU rRNA) gene, a housekeeping gene, is a universal diagnostic target in EHP molecular detection methods. But it is well known that the SSU rRNA gene is highly conserved among microsporidia, which may give false-positive test results [SSU rRNA, a more specific diagnosis target needs to be chosen.EHP is closely related to ith AIDS . Most miith AIDS . Althougith AIDS ,16. Therith AIDS ,18. Howeith AIDS ,20,21. T results . Hence, PTP2 gene encoding a 35-kDa protein was first identified from microsporidium Encephalitozoon cuniculi [Enc. intestinalis, Enc. hellem, Paranosema grylli, Nosema ceranae, N. bombycis, and so on [PTP2 gene was also reported to be a single copy in the EHP genome [PTP2 gene was selected as the EHP detection target for recombinase polymerase amplification (RPA) and CRISPR-Cas 12a fluorescence assay [PTP2 gene sequence in this study. Moreover, to provide real-time monitoring of EHP in the field, we attempted to quantify the EHP spores using microscopy. The integrated method of qPCR and microscopy to quantify EHP spores is first reported in our study and will provide a reference for the detection of EHP in shrimp farming.The polar tube, a highly specialized invasion organ, is one of the important taxonomic indexes of microsporidia . Up to ncuniculi . This gend so on ,28,29. TP genome . Due to ce assay . HoweverWe collected shrimp from Chongqing Province, China. Thirty mg of hepatopancreas tissue was used for genomic DNA extraction as follows: add 500 µL CTAB  20 mL, 0.5 M EDTA 8 mL, sterilized water up to 200 mL) and 20 µL Proteinase K (20 mg/mL) before incubation at 56 °C for an hour. Total DNA was purified using a standard phenol-chloroform method .EHP-PTP2 gene (GenBank No. MT249228), SSU rRNA gene (GenBank No. FJ496359.1) and β-Tubulin gene (GenBank No. KY593130) of EHP were amplified via PCR to confirm the EHP-infected shrimp sample. All PCR primers were designed using Primer Premier 5.0 and are listed in The EHP-PTP2 sequence (238 bp) was inserted into the pMD19-T vector and transformed into Escherichia coli DH5α. Positive colonies were selected to extract the plasmid and verified via sequencing . The recombinant plasmid was extracted by the Mini Plasmid Extraction Kit  and determined with a spectrophotometer  to be 54.6 ng/µL, which was equal to 1.7 × 1010 copies/µL. The recombinant plasmid was used as the quantitative standard and stored at −80 °C.The amplified DNA fragment of the partial ® qPCR SYBR Green Master Mix kit . The final concentration of EHP-PTP2-F and EHP-PTP2-R primers was 0.2 μM in the optimized reaction system  1 μL, and nuclease-free water were added to make a total volume of 10 μL. The amplification reaction was performed in the LightCycler® 96  and the optimized reaction procedure was 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. The data analysis was performed using the LightCycler® 96 Software 1.1 (Roche).The reaction mixture of PTP2-qPCR was formulated on ice according to the description of Hieffn system . The oth7 copies/μL and made a 10-fold series of 7 gradients (1.0 × 107–1.0 × 101 copies/μL). Three parallels of each dilution were used as the template of the qPCR assays. A standard curve corresponding to the Cq value of the standard plasmid copy number was constructed. The correlation coefficient and amplification efficiency were also analyzed.The standard plasmid was diluted to 1.0 × 10L. vannamei infected with different shrimp pathogens such as white spot syndrome virus (WSSV), shrimp hemocyte iridescent virus (SHIV), as well as Vibrio parahaemolyticus (VPAHPND) causing acute hepatopancreatic necrosis disease were used as templates to conduct qPCR amplification. The total DNA of healthy L. vannamei was used as the template of the negative control, the total DNA of EHP-infected L. vannamei was used as the template of the positive control, and the blank control used water as the template, respectively.To analyze the specificity of PTP2-qPCR, the total DNA of 5–1.0 × 101 copies/μL) were used as qPCR templates, and water was used as the negative control. The highest dilution which could be detected while still showing an S-shaped amplification of the curves was considered the lowest template copy concentration of the qPCR. The same test was performed by conventional PCR, and the highest dilution that could provide a visible band on the agarose gel was equivalent to the lowest template copy concentration of the PCR.In order to determine the sensitivity of PTP2-qPCR, serially diluted positive plasmids DNA  at room temperature for 25 min, followed by washing with PBS (pH 7.0) three times. Fifty µL of Fluorescent Brightener 28  was added and incubated for 5 min, then washed with PBS (pH 7.0) three times. EHP spores were observed by the fluorescent microscope , and the spore number in twenty random fields was recorded.Regarding the microsporidian chitin-staining method , 0.8 mg Cq value of the standard template copy number. The corresponding relation between the amplification quantitative (Cq) and the logarithmic of the initial template quantity (x) showed a good linear correlation when x was within the range of 1.0 × 101 to 1.0 × 107 copies/μL: Cq = −3.2751x + 31.269, correlation coefficient R2 = 0.993, and the amplification efficiency was 102%. From the amplification curve shown in The optimized reaction system was used to establish the standard curve corresponding to the AHPND. Only the EHP positive template showed a significant amplification curve, while no fluorescent signal existed in other templates, indicating that this quantitative method had good specificity  and coefficient of variation (CV) were calculated by reliable .PTP2 gene  shared 93% identity with the SSU rRNA gene of Enterospora nucleophile (GenBank No. KF135641.1), and the identity shared with the other five microsporidia was higher than 85%. According to the latest report, the PTP2 gene exhibited a good detection target in recombinase polymerase amplification (RPA) and CRISPR-Cas 12a fluorescence assay [PTP2 gene of fluorescence SYBR Green I using real-time quantitative PCR was established to detect EHP, and the minimum copy concentration was up to 10 copies/μL EHP, which suggested this diagnosis had a high sensitivity. Additionally, there was no interference reaction with other shrimp pathogens verified in our study (All microsporidia possess a unique, highly specialized structure: the polar tube. The polar tube is an important organ of the unique infection mechanism of microsporidia which can transport cytoplasm to host cells upon appropriate environmental stimulation . Many kiur study , implyinNosema spores in field measurements [6 copies/mg EHP could be detected by PTP2-qPCR, 7 to 20 spores vs. 105 copies/mg, 2 to 6 spores vs. 104 copies/mg, and 1 to 2 spores vs. 103 copies/mg. When examining the number of spores via microscope, in this case, the EHP concentration would be easily predicted.One of our aims is to provide a more convenient EHP detection method for shrimp culture. Microscopy can be more accessible to detect pathogens in the field, as it does not require professional technicians and instruments. A field-portable and cost-effective smartphone-based platform was presented for the detection and quantification of chitin-positive urements . In thisAbove all, the use of the PTP2-qPCR method was recommended as early detection for EHP-infection, and staining microscopy was more suitable for real-time monitoring of EHP in the field. This integrated methodology could serve for EHP detection during the whole period of shrimp farming and provide a reference for the epidemiological study of EHP.To our knowledge, this study is the first integrated qPCR and staining microscopy method for EHP detection. In shrimp culture, when EHP infection is serious, it can be directly detected by a microscope, and when EHP infection is mild, it can be detected by qPCR. The combination of these two methods not only makes the test results more accurate, but also prevents and controls EHP timely and effectively. We recommend that the integrated method be used to study EHP transmission routes in the shrimp–human food chain to monitor food chain safety.