ZZ interaction with transmon qubits. We then use this interaction to engineer efficient, high-fidelity qutrit CZ† and CZ gates, with estimated process fidelities of 97.3(1)% and 95.2(3)% respectively, a significant step forward for operating qutrits on a multi-transmon device.Ternary quantum information processing in superconducting devices poses a promising alternative to its more popular binary counterpart through larger, more connected computational spaces and proposed advantages in quantum simulation and error correction. Although generally operated as qubits, transmons have readily addressable higher levels, making them natural candidates for operation as quantum three-level systems (qutrits). Recent works in transmon devices have realized high fidelity single qutrit operation. Nonetheless, effectively engineering a high-fidelity two-qutrit entanglement remains a central challenge for realizing qutrit processing in a transmon device. In this work, we apply the differential AC Stark shift to implement a flexible, microwave-activated, and dynamic cross-Kerr entanglement between two fixed-frequency transmon qutrits, expanding on work performed for the  Qutrits, or quantum three-level systems, can provide advantages over qubits in certain quantum information applications, and high-fidelity single-qutrit gates have been demonstrated. Goss et al. realize high-fidelity entangling gates between two superconducting qutrits that are universal for ternary computation. The surface code4 using qubits is considered the main route to fault tolerance8, though its technical challenges have led the community to explore other approaches that could have more favorable QEC schemes, such as storing a two level system in the large Hilbert space of quantum oscillators10. Another alternative is to use d-dimensional quantum objects, or qudits, which mobilize a larger and more connected computational space than their qubit counterparts. Qutrits, the simplest form of qudits, can provide advantages in QEC for magic state distillation12, compactly encoding qubits13, and can be used to encode logical qutrits themselves16. Additionally, there are several proposals utilizing qutrits to improve quantum applications such as factoring with Shor’s algorithm17, performing the quantum Fourier transformation18, providing speedups for oracle based quantum algorithms19, improving quantum simulation20, and asymptotically improving algorithms such as Grover’s search22. Realizing multi-qudit systems, however, is challenging due to the complexities of the larger Hilbert space. Nonetheless, coherent control of qudits has been performed in several physical platforms28. While state of the art experiments have demonstrated high-fidelity qudit entangling gates with trapped ions24 and photonic circuits26, generating high-fidelity, maximally entangling two-qudit gates remains a major challenge in superconducting circuits.Quantum error correction (QEC)29, the transmon30, is well suited to be operated as a qutrit due to its weak anharmonicity. Technical advancements in microwave engineering and improved fabrication techniques have increased transmon coherence times31, enabling coherent control of the full qutrit Hilbert space. Furthermore, dispersive readout can be used for high-fidelity single shot qutrit readout27. In addition, high-fidelity single qutrit operations33, quantum information scrambling28, compact decompositions of multi-qubit gates37, and improved qubit readout38 have all been demonstrated using transmons as qutrits. Nonetheless, past qutrit entangling gates in transmons have been limited by relying on either a slow, static interaction which can only be sped up at the expense of increased quantum crosstalk on the qutrit processor or an interaction that restricts the entanglement to only a subspace of the qutrit.The most commonly used qubit in superconducting circuits42 on two fixed frequency transmon qutrits with static coupling and leverage it to generate dynamic qutrit entangling phases. The tunable nature of this entangling interaction enables a large on/off ratio, allowing for future high-fidelity, simultaneous single-qutrit and two-qutrit operations in transmon qutrit processors. With this interaction, we engineer the ternary controlled-Z gate (CZ) and its inverse (CZ†). Both gates performed in our work are universal for ternary computation, maximally entangling, and Clifford gates needed for QEC in qutrits. We achieve an estimated process fidelity of 97.3(1)% and 95.2(3)% for the CZ† and CZ respectively, measured using cycle benchmarking43 and our generalization of the cross-entropy benchmarking routine44. The fidelity of the CZ† represents a factor of 4 reduction in infidelity over previous two qutrit transmon gates32. Finally, we numerically demonstrate that our gate scheme is efficient for generating additional two-qutrit Clifford gates.In this work, we characterize the differential AC Stark shiftZZ errors from the always-on dispersive coupling between the transmons. Secondly, unlike in the CR gate, the frequency of the microwave drive can take a range of values. This flexibility in drive frequency affords significant advantages in avoiding frequency collisions with other transmons or spurious two-levels systems45. The generalization of conditional Stark shifts to qutrits is straightforward. Working in the energy eigenbasis of our two qutrit Hilbert space, up to single-qutrit phases, one’s system evolves under the cross-Kerr Hamiltonian:αij (ZZ-like) terms are given by:i and ϕi are respectively the amplitude and phsae of the drive on transmon i. The coefficients Aij are functions of the proximity of the microwave drive frequency (ωd) to nearby transitions. We note that this Hamiltonian generates entanglement between the entire two-qutrit Hilbert space, contrary to the CR case, where the entanglement is mostly restrained to a subspace of the qutrit46. This dynamic, driven cross-Kerr interaction is depicted schematically in Fig. Recent works by refs. ZZ interaction in the qubit case can be performed by a conditional Ramsey experiment or through a dynamically decoupled JAZZ (Joint-Amplification-of-ZZ) sequence that removes the low frequency drift48. In the larger Hilbert space of two qutrits, we need to measure four of these entangling phases with a rate of accumulation set by αij in Eq.  in our device. In an experimental setting, the flexibility of this entanglement allows us the freedom to set the drive frequency far from any of these features.We demonstrate in Fig. l in Eq. . In Fig.l in Eq. . We also† gate are both maximally entangling and members of the two-qutrit Clifford group making them particularly useful gates for ternary computation. The CZ gate is defined as:ω = eiπ/32, the third root of unity; the CZ unitary follows directly from generalizing the qubit Pauli group to qutrits and is explained in further detail in Supplementary Note α11 = α22 = 2α21 = 2α12}, a task that is not broadly feasible. Practically speaking, we desire a compromise between the most general and robust gate scheme and this “fine-tuned” approach, while still taking advantage of the dynamical nature of our cross-Kerr interaction. By employing the pulse scheme in Fig. α11 + α22) ≈ − (α12 + α21) for performing the CZ gate and (α11 + α22) ≈ 2(α12 + α21) for performing the CZ† gate.The Stark drive parameters that meet these conditions are provided in Supplementary Note τ, we will have approximately acquired the desired entangling phases found in Eq. % and 4.8(3)% respectively. By contrast, the highest fidelity, two-qutrit gate performed previously with transmons had an error rate of 11.1%28. CB also allows us to construct the Weyl-twirled error per channel32 of the unitary in Fig. We first benchmark our two-qutrit gates with cycle benchmarking (CB)† gate, we generalize the cross-entropy benchmarking (XEB) routine53 to tailor all gate errors into a depolarizing channel, for work with qutrit unitaries. In the qutrit case, we find sufficient tailoring of our noise can be performed by interleaving random SU(3) gates around our target gate. The circuit diagram for qutrit XEB is in Fig. † dressed with random SU(3) gates agrees with the estimate of the process fidelity from our Weyl twirled CB results within a standard error. Additional discussion of the qutrit XEB method is provided in Supplementary Note As an added confirmation of the fidelity of the CZ55. The corresponding estimates are shown in Fig. † gate. This disagreement is likely due to the fact that the CB data reveals that the noise is dominated by single-qutrit phase errors. As these errors are likely to fluctuate around a mean, they will add dephasing errors that are not captured by the depolarizing unitary model used to analyze XEB. Another possible contributing factor is that the noise fluctuated between the XEB and CB experiments, which were performed in separate batches.Finally, we would like to be able to characterize what fraction of the errors present in our two qutrit unitaries are coherent on the time scale of multiple experiments, and thus could be removed by improved calibration. As we show in supplementary note †  to synthesize other two-qutrit gates, and compare them to two-qutrit entangling gates that only entangle a subspace of the qutrit, such as the controlled-exchange (Cex) and controlled-increment (Cinc) gates performed on a trapped ion system in ref. m, which we use to synthesize target circuits belonging to either the two qutrit Clifford group or set of Haar random gates. The gate synthesis is performed by optimizing the ansatz parameters to minimize the distance between V and U, i.e. the infidelity 1-To study the expressibility of the two-qutrit gates in this work, we numerically explore the ability of the ternary CZ/CZ†, 7 for Cinc, and 9 for Cex. The synthesis success rate for all 3 unitaries in terms of target Clifford circuits are shown in Fig. †, with 100% success at depth 3. By contrast, Cex and Cinc did not demonstrate as much improvement at synthesizing Clifford gates over Haar random gates, achieving 100% success for target Clifford gates at depth 6 for Cinc and 9 for Cex. Additionally, unique amongst these gates, the CZ/CZ† can generate maximally entangled two qutrit states with a single iteration of the gate. We demonstrate the power of this feature in the experimentally reconstructed qutrit Bell-state density matrix in Fig. † gates have low intrinsic errors and can also can synthesize a very important family of gates for QEC (the Cliffords)1 at much lower depths than the two-qutrit gates which only entangle a subspace of the qutrit.We perform this numerical investigation on 1000 Haar random gates and 1000 Clifford gates. We find that all 1000 Haar random gates can be synthesized at depth 6 for CZ/CZWe realized a microwave-activated, dynamic cross-Kerr entangling interaction that can be employed to engineer qutrit entangling phases with high precision. Leveraging this interaction, we generated two maximally entangling and high-fidelity two-qutrit gates on two separate pairs of fixed-frequency transmon qutrits. We demonstrated numerically that these two qutrit gates are efficient for producing additional two-qutrit unitaries, especially other Clifford gates. Future work developing a systematic gate tune up procedure may prove essential in improving the fidelity and scalability of our approach. Additionally, a study of the effects of this gate scheme on spectator qutrits will also be necessary for determining its scalability. We expect that by being maximally entangling and a member of the two-qutrit Clifford group, the gates performed in this work will prove especially powerful in future efforts to employ qutrits for QEC, quantum simulation, and quantum computation. Perhaps most importantly, all of this work was performed on multi-transmon quantum processors which are normally used for qubit experiments; the untapped potential of transmons as qutrits is only beginning to be explored. As a final note, the two-qutrit Hilbert space is larger than even the three qubit Hilbert space; as the community continues to explore qudits, we propose that metrics and benchmarks should be developed to reasonably compare qudit vs. qubit gates.Supplementary InformationPeer Review File


Low back disorders were the most frequent cause for SL, while lower limb disorders tended to cause the longest average SL. The SL durations were similar across divisions within the sector, while the incidence rate tended to be higher in the “accommodation” than the “food and beverage services” division. Conclusions: Special attention needs to be paid to reducing the risk of low back disorders, which are by far the most common cause of SL, and lower limb disorders, which cause the longest SL. We recommend the implementation of countermeasures that focus on early detection and rapid treatment/recovery of MSDs in older workers.Objectives: The aim of this study was to analyze Slovenian data on sick leave (SL) due to the most common work-related musculoskeletal disorders (MSDs) among workers in sector I “accommodation and food services activities” from the NACE Rev2 classification. Methods: We investigated both SL incidence  and disease severity  by body site, gender, age and divisions within the sector. In addition, trends in SL data (difference between 2015 and 2019) were analyzed. The effect of age group, gender and division was also assessed with relative risk (RR). RESULTS: Female gender was associated with higher risk of MSDs in young (RR = 1.91 (1.53–2.43)) and older (RR = 2.24 (1.90–2.65)) subgroups. Older age was associated with greater SL incidence and longer SL duration, regardless of gender and division within sector I. This was also reflected in relative risk calculations between older and younger groups (females: RR = 4.43; CI = 3.75–5.01;  Musculoskeletal disorders (MSDs) are still the most common work-related health issue in the European Union (EU). MSDs have consequences for workers, businesses and society, including absenteeism from work . In SlovThe accommodation and food services activities sector (NACE Rev2 Sector I) is an important job creator, employing 8.3% of the EU workforce in 2019 . This seOur previous study also showed that workers in the “accommodation and food service activities” sector in Slovenia are at high risk for MSDs. For example, female workers from this sector aged 45.0–64.9 years have on average the fourth highest incidence of work-related low back disorders . TherefoWe analyzed Slovenian national data for SL determinants due to the most common work-related MSDs in the NACE Rev2 sector I “accommodation and food services activities”. The analysis was made for the period 2015 to 2019 by gender, body region and two age groups: 20.0–44.9 years (20–44 y) and 45.0–64.9 years (45–65 y). Results are presented for both sector I as whole and separately for its divisions.Data regarding SL represent an important source of information on the health status of the working population. The data for this study were collected by the Slovenian Institute of Public Health (NIJZ). All data were anonymized at all stages of the study. The study does not contain data that could be linked to an individual person. The NIJZ collects, analyzes and disseminates data on SL of employed and self-employed workers who are enlisted in the compulsory health insurance in Slovenia. Data collection covers the work force in all NACE Rev2 economic activities. Data collection has a legal basis in the Health Care Databases Act  and Personal Data Protection Act (ZVOP-1—Ur. L. RS 94/07). Article 17 in the Personal Data Protection Act considers scientific research for historical or statistical purposes as a lawful processing operation and therefore provides a legal basis for further processing. The source of data is the Certificate of justified abstinence from work due to health conditions (eBOL). The data are obtained from health care providers. The distribution of the data in this paper was also approved by the Ethical Committee of the National Institute of Public Health . The study was conducted in accordance with the guidelines of the Declaration of Helsinki.According to the NACE Rev 2—Statistical classification of economic activities in the European Community—the activities are separated into 21 sectors. The NACE Rev 2 sector I, “accommodation and food services activities”, contains two divisions: “accommodation” (No. 55) and “food and beverage service activities” (No. 56). Accommodation activities include the provision of short-stay accommodation for visitors and other travelers. Food and beverage service activities provide complete meals or drinks fit for immediate consumption .For the analysis, we received anonymous data as numbers representing a body-region-specific SL determinant by gender and age in “accommodation and food services activities” and it’s divisions. The average values for SL determinants from 2015 until 2019 and by single year were used for analysis. First, we analyzed (i) the incidence rate, as the number of SL cases per 100 employees in one year (SL incidence) and (ii) the severity of MSDs, expressed as the average duration of one absence from work due to a health condition (SL duration). The number of SL cases is considered to be a number of completed SL due to MSDs in a calendar year (1 January–31 December) regardless of when the SL started. The most common work-related MSDs (according to the ICD-10-AM classification) by body region  included in the study are listed in The number of employees in the sector “accommodation and food services activities” sector I, has increased from 32,526 in 2015 to 37,928 in 2019. They account for 4.2% of the Slovenian workforce. There are more females than males employed in this sector (approximately 1.5:1 ratio), with the number of females ranging from 19,452 to 22,529 and the number of males ranging from 13,074 to 15,399 in 2015–2019. In addition, there are about 1.8 times more workers in the 20–44 age category  than in the 45–65 category . Most workers (71%) are in the “food and beverage services activities” and 29% are in the “accommodation” division. For more details, see The bottom chart shows the data on the average sick leave duration. The data showed high variability across the body regions . In the back and upper limbs, the older group generally had a longer SL duration (range = 32.7 to 87.7 cases) than the younger group (range = 14.5 to 31.5 cases), with the exception of the wrist, where there was little difference between age groups. SLs due to hip disorders had the highest severity, with average SL duration ranging from 94.8 to 146.5 days. There were no major differences between genders; in the older group, males had more severe upper back disorders , while older group females had longer SLs due to ankle disorders .p < 0.001) in the young subgroup. In the older males, the SL incidence range from 2.52 to 3.39 cases. Among older females, the numbers were even higher, with 5.32 cases in 2015, followed by an increasing trend toward 7.59 cases in 2019. Female gender increased the risk of SLs for 124% compared to males  in the older subgroup. Older groups had noticeably higher incidence rate, which is also reflected in relative risk calculations .Data on SL duration  show consistent values over the years, with males consistently showing slightly shorter duration (20.5–27.8 days) than females (26.9–33.9 days). SL duration, like SL incidence, appears to be increasing in the older female group . Males had even larger values in 2015–2017 (difference ~ 10–11 days), but decreased after 2017 and ended up at lower values than females in 2019 (54.8 days).p = 0.081), but it was statistically significantly higher in “accommodation” than “food and beverage services” for young females , older males  and older females .The analysis of SL incidence by division shows that age has a greater impact than divisions  were by far the most common (SL incidence) work-related MSDs in both gender and age groups . This fiOur study revealed  a genderConsistent with previous publications ,26, oldeIt appears that in sector I, older age contributed more to the incidence and severity of the most common work-related MSDs than occupational exposure in each division . This fiThe major limitation of the study is the lack of data on specific workplaces within sector I and its divisions. At least a small proportion of workers were engaged in administrative tasks, and it may be that this proportion varies between age groups and genders. Rather than dividing sectors into divisions, further research should consider classifying individuals by job type to identify which individuals need special attention in terms of MSD prevention. We also need to be cautious about generalizing the results to other geographic locations.This study provides a nationwide overview of SL incidence and SL duration of the most common work-related MSDs in sector I “accommodation and food service activities” from the classification NACE Rev2. Special attention should be paid to reducing the risk of low back disorders, which are by far the most common cause for MSD-related sick leave, and lower limb disorders, which cause the longest SL. Older age has been associated with higher SL incidence and longer SL duration. Therefore, we recommend countermeasures that focus on early detection and rapid treatment/recovery of MSDs in older workers. Prevention strategies should consider the role of gender and age, while the influence of division on SL risk is likely to be small.


Four treatment groups have been established, named control (0), 2, 20, and 200 μg/L MET. Results showed that at moderate temperatures such as 25°C to 30°C, tilapia's feed consumption increased and body weight improved. SOD, CAT, and GSH in the liver of GIFT indicated the significant increase under MET exposure. MET reduced the growth rate of GIFT, and water spinach reduced part of the water quality indexes in the MET (<200 μg/L) groups. Water spinach altered GIFT's hepatic oxidation system to some extent and effectively absorbed MET in water and transferred it to itself, and the degradation time was lower than the dietary standard time which termed as 15–20 days. Growing water spinach in farmed waters partially decomposes MET and prevents it from causing damage to GIFT's liver.This study was conducted to investigate the effect of methomyl (MET) on the growth and antioxidant system of GIFT (5.28 ± 0.12,  MET is considered to be highly toxic to fish and aquatic invertebrates [50 values for crucian carp ranged from 0.9 to 3.4 mg/L, and the LC50 values for Daphina magna were from 0.022 to 0.026 mg/L [Channa striatus [Oreochromis niloticus [μg/L). As well as in the liver, the results showed significant increases in the activities of GST, GR, and GPx and levels of GSSG accompanied by a decrease in GSH levels.Methomyl (MET) is a broad-spectrum pesticide that is used to get rid of insect pests. The main routes of agricultural pesticide transport to different aquatic ecosystems are through drains from agricultural fields, spray drifts, disposal through wastes, and deliberate use in aquatic environments. MET C5H0N2O2S is2O2/alcohol and O2). GSH reduces H2O2 and lipid hydroperoxides with the GPx enzymes, which is an important part of the integrated antioxidant system and maintains other nonprotein antioxidants in their reduced and biologically significant state.Oxidative stress is an imbalance between the production of reactive oxygen species (ROS) and the cell's ability to reduce ROS, and MET can cause an increase in ROS production in the cells of the exposed organisms . On the Ipomoea aquatica) is one of the effective aquatic plants that is used for heavy metal removal from water. We constructed the determination method for MET in plants [Ipomoea as a part of the biofilter is documented. Thus, reactive oxygen species (ROS) induced in aquatic ecosystems through pollutants can be evaluated via antioxidant enzyme activity measurement and could be excellent biomarkers [Water spinach  were obtained from the fish farm in the Yixing research center of FFRC-CAFS, China. Before the experiments, the fish were acclimated under laboratory conditions. After four weeks, the fish were transferred to the aquarium. The commercial feed  was purchased from Jiangsu Zhe Ya Food Co. Ltd., China. Fish were fed twice daily with commercial feed (extruded pellets), and the feeding rate was 5% of total body weight. This ration size was built on preliminary observations to drive the fish to feed on the roots, stems, and leaves of mint, also to prevent water quality deterioration, as well as for cost-saving. The experiments started when no mortality was observed in the acclimated population. Water spinach was placed at the surface of all twelve tanks at 800 g using plastic floaters.MET was obtained from Shanghai Focus Biological Technology Co., Ltd, China  and dissolved in pure water to form stock solutions, and then the experimental concentration in aquariums was diluted immediately before use. The study applied a static system with a floating bed. A total of 12 glass aquaria  were used. Erstwhile to use, mint height of 10 cm was planted in several floating pots and placed in a tank. Mint was stored in a 1000 cmWt)–initial body weight (W0). Relative body weight growth rate  = (Wt − W0)/W0 × 100. Specific growth rate  = (lnWt–lnW0)/(t2-t1) × 100. Feed conversion rate (FCR) = dry feed fed/body mass gain where t1 represents the initial time of the test and t2 represents the end time of the test. The SGR assumes an exponential growth of fish over the whole life cycle, which is incorrect. Fish growth rate is weight dependent and, therefore, SGR will vary over different life stages of fish . Therefore, SGR may not be a suitable parameter in comparing the growth of fish between different studies. Feed conversion is the total of nutrients accumulated into tissue for weight gain. It covers all of the nutrients. FCR depends on the composition of the diet, quantity of feed, feeding frequency, age, and species of fish.Absolute weight gain (BWG) = final body weight (μg/L (12 tanks for 4 groups in triplicate). Fifteen fish were introduced in each concentration in an open-static system with the total experimental fish of 180 (n = 15 per tank for different groups in triplicate). 800 g of water spinach was added to all the experimental tanks, including the control with the weight of water spinach coming up to 9600 g, and the water did not change until the end of the experiment. The entire experiment lasted for ninety days, including an acclimatization period, but the total exposure was sixty days. The first water samples were collected after 2 (named as w2), 4 (named as w4), 6 (named as w6), and 8 (named as w8) weeks of exposure to the MET concentrated water, while the fish liver samples were collected at w2, w4, and w6. All the liver samples were collected using the same format—fish caught and the weight and length recorded— they were decapitated and the liver was extracted and kept in the refrigerator at −85°C pending analysis.A set of juveniles were randomly distributed into 1500 L plastic tanks containing 750 L of water with different MET sublethal concentrations as follows: 0, 2, 20, and 200 4+-N), nitrite-nitrogen (NO2−-N), nitrate-nitrogen (NO3−-N), and total nitrogen (TN). All samples were filtered with a Whatman filter paper of a pore size of 0.45 μm before laboratory analysis.Monitoring and data collection started one week after the plant and fish were acclimatized to the system. Water quality was measured daily for two weeks in aquariums. To test the mint as part biofilter, triplicate water samples from each of the treatment sets were transferred to a 500 mL polyethylene bottle to test physicochemical parameters of ammonia-nitrogen  0.9 g was dissolved in 100 ml and was used for the extraction of the supernatants. The tissues were homogenized using a portable homogenizer. 25 ml of saline solution was added to the homogenized tissue and centrifuged at 250 nm for 10 min. The supernatants were collected into a tube and kept at 4°C. This supernatant was used to carry out the assay of CAT, SOD, GSH, and protein content. The supernatants were prepared using test assay and taken to the spectrophotometer for results . A spectμm organic membrane filter using a syringe, and the sample was taken to HPLC for analysis.The plant samples  were collected at two-week intervals and dried in an electrical oven at a temperature of 105°C. It was powdered using an electric grinder. The plant samples (1.5 g) were put into a 50 ml centrifuge tube, mixed with 10 mL acetonitrile and homogenized at 2500 rpm for 5 minutes, and centrifuged at 3500 rpm for 5 minutes (this was done twice), and the supernatant was transferred into a 50 mL tube and taken to the solid face extraction for further extraction. Here, 10 mL of acetonitrile-toluene (3 : 1 v/v) was used to activate the cartridge and the supernatant was filtered through the cartridge. 20 mL of acetonitrile-toluene (3 : 1 v/v) was measured, and 2 mL of the solution was used 3 times to wash the tube and added to the cartridge. The cartridge was then eluted with 19 mL of acetonitrile-toluene (3 : 1 v/v). The eluent was then collected and evaporated to dry at 40°C using the rotary evaporator. The dried residue was dissolved in 1 ml of acetonitrile and filtered with a 0.45 P < 0.05 with a lowercase letter.The data collected was analyzed by one-way ANOVA using the Statistical Package for Social Sciences (SPSS 26.0) to determine the level of CAT, SOD, and GSH in GIFT. GSH was expressed as nmol GSH/mg protein. The data are presented as mean ± SE values. The numbers of animals per group were stated in the table or figure legends. The statistical analysis of the data was performed using one-way analysis of variance (ANOVA). The significance of the results was ascertained at P > 0.05) in FCR and SGR at the treatment group 2 and 20 μg/L exposed to MET sublethal concentrated water as compared to control group, but there was a significant increase (FCR) and decrease  within groups (There was no significant difference (n groups .3−-N in the control group were significantly higher than those in other groups (μg/L treatment group showed a significant increase when compared to the controls at w8. TN (at w6 and w8) and NH4+-N  in the 2 μg/L treatment group showed a significant decrease when compared to those in the control group, while NH4+-N at w4 showed the converse tendency. At w2, NO3−-N in the control treatment group showed a significant decrease (P < 0.05) as compared to 200, 20, and 2 μg/L MET groups. At w2, NO2−-N in the 200 μg/L treatment group showed a significant increase (P < 0.05) as compared to 20 and 2 μg/L and the control groups. The NO3−-N concentrations at w4, w6, and w8 showed that there was no significant difference (P > 0.05) (2−-N in the 2 μg/L treatment group and the control groups showed a significant decrease (P < 0.05) as compared to the 20 and 200 μg/L treatment groups at w2  and NOr groups , while T > 0.05) . NO2−-N P < 0.05) in the 2 μg/L treatment group compared to the other treatment group at w4  compared to the control group.The effect of SOD in the liver of GIFT indicates a significant increase (P < 0.05) at w4 and w6 as compared to those at w2  compared to the other treatments. At w6, CAT in the 200 μg/L group was significantly lower (P < 0.05) as compared to the other treatments, whereas CAT in the 2 and 20 μg/L groups significantly decreased and increased (P < 0.05) compared to the control and 200 μg/L treatment groups, respectively.Glutathione was a good antioxidant enzyme and was vigorous against oxidation in living things. The presence of CAT in the liver of GIFT has shown a significant difference (se at w2 . In the P < 0.05) at w2, w4, and w6. GSH in the 200 μg/L treatment group at w2 significantly decreased (P < 0.05) compared to other groups at w2, and there was no significant difference between lower groups at w2. A significant increase of GSH occurred in the 20 μg/L group at w4/6 compared to 0, 2, and 200 μg/L groups, while GSH in the 2 μg/L group at w4/6 was significantly higher than those in the control (including 200 μg/L group at w6) groups.In the above GSH graph , there wIn this section of study, water spinach was exposed to MET solution at various concentrations for 60 d. The samples of the plants were collected from the roots and evaluated to establish the significance of the pesticide residue in the plant.P < 0.05). MET residue in the 2/20/200 μg/L treatment groups at w2 showed a significant increase than the controls, while w1 revealed no significant difference among each group. MET residue in the 20/200 μg/L treatment groups at w4 (with no significant difference in 20/200 μg/L groups) and w6 (in a dose-dependent manner) significantly decreased and increased (P < 0.05) as compared to the 2 μg/L groups, respectively. After w6 MET exposure, the MET residue had significantly increased with the increased concentrations.The MET residue in roots of the control groups in all the weeks significantly increased than those in the other groups (P > 0.05 among three MET groups), w4 (P > 0.05 among three MET groups), and w6 in the treatment groups showed significant increase (P < 0.05) as compared to those in the control group, while MET residue in the 20 μg/L treatment group showed no significant difference as compared to those in the 2 μg/L treatment group. The MET residue in the 200 μg/L groups significantly increased compared to those in the 2/20 μg/L groups. The similar tendency is also seen in the leaves at w2/4, and only MET residue at w6 showed a significant increase (P < 0.05) in leaves with the dose-dependent manner.The residual ability of MET in the stem of the water spinach at w2 (μg/L MET group were without significant differences at w4/6/8 except for NH4+-N (w4/8) and NO2−-N (w6). The antioxidant enzymes at w4/6 also showed the same tendency except for CAT (w6) and GSH (w4) when compared to the short-term exposure duration. The FCR and SGR showed a significant increase and decrease in the 200 μg/L MET when compared to the controls. This experiment indicates that the highest concentration of MET sublethal dose (200 μg/L) in water did not badly affect the fish, but there was a slower pace of growth in the fish with the highest concentration of the pesticide. From observation during feeding, all feed was consumed by the fish, and because of this, parameters like ammonia, nitrite, and nitrate, were at very low concentrations in the fish tanks. Another contributing factor would be natural environmental factors such as the tanks exposure to nature. It was also observed that fish eat the roots of the water spinach that had been placed in the tanks, which has turned out to be one of the health benefits. The MET residue in the root, leaf, and stem showed a significant increase in the MET exposure groups when compared to the controls, and especially revealed a dose-dependent manner at w6. The plants eaten by the fish helped to revamp their immune systems, which induced a higher production and more healthy status [Petroselinum crispum) seed meal alleviated MET-adverse impacts on tilapia growth [Water quality may affect fish's health when under toxicant exposure, and the present study showed the detected parameters in the 200 y status –15. Recey status  and parsa growth . Our prea growth  and intea growth . It was C. punctatus exposed to pyrethrins [μg/L of carbamate carbosulfan for 60 days greatly increased the liver CAT activity when compared to controls [Pesticides have been identified to cause overproduction of ROS, which if not thoroughly neutralized by antioxidant mechanisms, may lead to oxidative stress and potential tissue damage . The increthrins . Gills arethrins . Differecontrols .Channa punctatus tested after 24 h of treatment with endosulfan [C. punctatus. The first line of defense against oxidative stress comprises antioxidant enzymes , and a decrease in their activity alters the redox status of the cells. It is therefore possible that an increase in the activity of the enzymes contributes to the elimination of ROS induced by cells exposed to pesticides. GSH is also widely used as an environmental biomarker [Clarias gariepinus to fenthion formulations resulted in the induction of the liver GSH activity [P < 0.05) occurred in 2/20 μg/L MET groups, accompanied by the subsequent decrease in the form of homeostasis balance. The present study suggested that growing with water spinach may have a positive effect on the prevention of GIFT's liver toxic damage. Whether the eating of water spinach by tilapia may be attributed to the positive effect should be determined.Equally so, oxidative stress levels in fish from waters contaminated by different concentrations of pesticides were totally different, and in some cases a clearly inhibitory effect was experienced. The study observed a decrease in CAT activity in the liver of silver catfish exposed to the herbicide clomazone. There were similar effects found in the liver of the freshwater fish iomarker . In someiomarker . In accoiomarker . Neverthactivity . The preP < 0.05). There was no significant difference at w2 and w4. The leaf, which was one of the most important parts of this economical plant does not have much significant difference, and only MET residues at w6 also showed a significant difference (P < 0.05). MET was revealed in the liver and muscle of fish [Ipomoea aquatic, when exposed to MET-concentrated water, had some MET absorption effects on the plant.Results showed that the roots and the leaves were more affected by the pesticide as compared to the stem. At w4 and w6 of the experiment, MET residual potential in the root was of significant difference as compared to those at w2. Results from the stem indicate that only w6 showed a significant difference ( of fish , and theSupervised residue data from trials in several other countries showed that MET  residues were detected on most above-ground crops at the time of harvest . Higher μg/L groups, while those activities were increased in 2 μg/L groups, especially GSH after long-term MET exposure duration. Water spinach is a good source of nutrients that accumulate toxic residue, especially in the root and the leafy parts. The degradation time of MET was lower than the dietary standard time, which was termed 15–20 days.MET reduced the growth rate of GIFT, and water spinach decreased part of the water quality indexes. SOD, CAT, and GSH were significantly decreased in 200


The storage of antimicrobials at home is frequently in-practice in various developing countries, resulting an irrational use, antibiotic resistance, and toxicities. This condition may worsen more in conflict zones where health facilities are limited. This study aimed to determine the storage and use of leftover antibiotics among households (HHs) along with knowledge and awareness about antibiotics and antibiotic resistance (ABR).A descriptive cross-sectional study design was employed. Members of HHs were invited to participate in the survey while using a convenient sampling technique. The data were obtained using a validated questionnaire and analyzed through SPSS.n = 50, n = 46), with most of the participants being men between the ages of 18 and 28  years. The majority of HHs  had six to eight total family members, with one to two chronic diseases , individual families , and with  LRTIs (lower respiratory tract infections). The HHs were aware of the word “antibiotic”  and gave correct replies to amoxicillin as an antibiotic ; on the other hand, HHs also thought of paracetamol as an antibiotic . They identified the most common brands of antibiotics easily, and a majority of them  had never heard of ABR before and had lower levels of awareness. The most stored antibiotic at home  was azithromycin (J01FA10). In addition, they had multiple needless  and antibiotics in their houses. Age had a strong association  affected the mean knowledge scores related to antibiotic use. Association of education levels  and occupation  with proper antibiotics use were found to be significant. However, family members with more than one chronic illness  showed a significant relationship with their awareness of antibiotics.A total of 96 HHs were randomly selected from two districts (Participants generally stored various antibiotics of different classes in their homes. Lack of knowledge related to the appropriate usage of antibiotics, use of leftover antibiotics, and awareness related to ABR were unknown to the participants. A majority of households (HHs) around the world keep medicines on hand for a variety of reasons, including the treatment of both short-term and long-term illnesses, as well as for emergencies. If the directions for storage are not followed, drug stability may be impacted, which could result in ineffective therapy , 2. The Staphylococcus aureus) is found in 60.5% patients, and resistance types and rates are similar in patients from Yemen, Syria, and Iraq , is a rural district in Malakand Division Khyber Pakhtunkhwa, Pakistan, with a population of 2.3 million people. About 86% of the total population lives in rural areas. District Shangla was once a part of Swat state but was carved out in 1995 as a separate district and adjunct to the Swat district having a population of 0.7 million . The curThis study employed a door-to-door survey method, which is an excellent strategy for gathering meaningful measures of health. In the vicinity of both district rural regions, the HHs were prioritized for data collection. Permanent residents of the area or residents who have lived in the area for the past 5 years and do not have a disability were eligible to answer questions and respond to the research teams. Participants under the age of 18, as well as HHs with no male members and those who refused to consent, were all excluded from the study. Following their visits to each HH, the data collection teams (DCTs) received preliminary training to ensure that the norms and local traditions are considered. For a total of 2 weeks, DCTs received prior training daily throughout their working days.The face and content validity of the questionnaires were evaluated with the help of specialists in the field. The study tool was piloted with a group of 15 HHs to determine its internal consistency. Visits to both research sites were part of the pilot project, which were utilized to validate the questionnaire. This part of the study provided feedback to the research team, confirming that they had a good understanding of the questions .The internal consistency of the amended questionnaire was calculated using Cronbach alpha on 22 HHs. The Cronbach alpha result  was adequate and acceptable, indicating that the questionnaire was internally consistent.Medical records were utilized as data sources, and all the information was self-reported by the participants. The questionnaire requested details on demographic factors , as well as respondents' awareness of antibiotics, ABR, and antibiotic storage procedures at their homes. Each question prompted replies in a variety of ways, including yes/no/don't know, closed-ended questions with numerous answers, and open alternatives. The questionnaire was developed using relevant information from prior studies on the subject , 28–32.The final questionnaire had three sections. Section I sought demographic details and had a total of 10 questions. Section II had questions related to knowledge  of antibiotics and 16 statements to which the respondents were asked to respond. Two false and two true (Amoxicillin is an antibiotic/Paracetamol is an antibiotic) statements were also included in the identification of antibiotics. Three false questions were asked about the use of antibiotics (antibiotics are often needed for cold and flu), and six questions were related to ABR (misuse of antibiotics can lead to ABR) with the correct approach. A total of eight statements were also employed for the assessment of overall HHs' knowledge of antibiotics use, ABR, and infectious diseases. The responses to these statements were evaluated . Section III included 15 statements/questions related to the HH storage of antibiotics and factors related to storage conditions.The questionnaire was translated by two native translators through the forward and backward translation methods. All data was gathered using a structured paper questionnaire with questions written in the local and national languages (Pashto/Urdu). Forward and reverse translation procedures were undertaken by experts due to the questionnaire's cultural adaption.A convenient sampling technique was adopted to collect data. We intended to recruit as many HHs as feasible over the course of the study to increase the sample's prospective representativeness. As a logical consequence, we did not determine the minimum sample size in advance . As a reA significant amount of group orientation was provided to the DCTs, covering areas like guidelines for conducting the interview and HHs survey, advice on proper attire to respect the locale's cultural norms, and other specifications. The DCTs were coached by researchers who were familiar with the clinical situations and the rural community environment. Before administering the survey, the researchers and DCTs visited 22 residences (Swat = 12 and Shangla = 10) in both districts jointly to create familiarity and confidence.Several efforts were made to achieve maximum standardization and preserve consistency in the data gathering. All data were assigned a special code of identification. The research team members entered the data and cross-checked it. All files were stored, and the confidentiality of the data was assured.Statistical Package for the Social Sciences (SPSS version 21) was used for data entry and data compilation. Additional test statistics were employed to compare categorical variables, and descriptive statistical analyses were provided as frequencies (percentages). If p>0.05, differences were judged statistically significant. Due to the non-normal data distribution, continuous data were presented as median and interquartile ranges (IQRs). The comparison of continuous data across various samples was conducted through Kruskal–Wallis, and Mann–Whitney tests, where appropriate. Antibiotic use, identification, and ABR median scores (which measure associated characteristics) were also calculated and compared with demographic data such as age, gender, occupation, education level, the total number of families, average income, and family setup. The median scores and testing statistics scores for the types of infectious diseases and total chronic diseases patients present per HH were also tested and tabulated.n = 45, 46.9%) years. The majority of its members had primary education , were self-employed , and  with an average monthly income of PKR 21000–30000. Most of the families had six to eight members , had family members with one or two chronic diseases , were individual families , and had prevalence of LRTIs . A small  number of healthcare professionals were present at home  participated voluntarily, resulting in an 88.8% response rate response rate was observed. Considering the cultural values in the study locations, only male members of the HHs took part in the study with the mean age group 18–28 ( at home .n = 55). But less than half of the respondents could correctly identify amoxicillin as an antibiotic. Additionally, a majority (61.5%) provided incorrect responses to the correct use of antibiotics and believed that ABs were efficacious in flu and cold. About half of the participants answered the side effects statements and reported that antibiotics kill useful bacteria and ABs can cause (67.7%) of allergic reactions. However, they were unaware of ABR as just 28.1% expressed little knowledge about drug resistance . Knowledge also had a relationship with age group (p < 0.05), occupation (p < 0.05), and family income (p < 0.05), all of which have been proven to be favorable to the knowledge of antibiotics due to their level of education (Table 5). The type of family structure, the number of family members, and the type of infectious disease were not significant indicators of antibiotic use or knowledge of AMR  aware of the term “antibiotic” and that such medicines can fight against infectious diseases. Participants reported correct responses related to  amoxicillin as an antibiotic, and more than half  identified paracetamol as not an antibiotic . The Median (IQR) Knowledge score regarding the identification of antibiotics was 2 (1–3) (The households were (tibiotic . Knowled 2 (1–3) .n = 52, 54.2%), and only half  knew “good bacteria” was present in everybody's bodies  provided accurate information regarding allergic reactions as HHs experienced such allergic reactions. They reported numerous instances of adverse reactions caused by antibiotics.Most of the HHs believed that antibiotics killed the germs  had not heard the term ABR before and they showed less awareness and knowledge about ABR. As evident from their responses, many of them provided incorrect answers about the treatment after bacteria/germs get resistant to the antibiotics; 45.8% (n = 44) responded with “don't know” about such incidents  (see Respondents believed that the storage of unnecessary antibiotics is not a cause for ABR and only 31.3% (n=30), answered correctly. The Median (IQR) Knowledge score regarding ABR was 1(0–3). A majority of the respondents also had misconceptions regarding the misuse of antibiotics and it was common among HHs (.5%) see .n = 33, 34.4%) agreed that antibiotics should be identified alongside other medications, even if they are prescribed separately. The urinary and respiratory tract infections can be treated only with antibiotics  and just a small proportion of HHs had such knowledge. Only 25 HHs strongly agreed with the statement regarding penicillin-triggered allergic reactions and the test dose . The majority of HHs  were unconcerned about the statement that ABR is caused by antibiotics failing to kill or inhibit bacterial growth. Within households, family members shared antibiotics and less than half of HHs  agreed to the prospect of ABR owing to an incomplete antibiotic course. ABR can develop due to incomplete doses (as a result of HHs storing antibiotics for future illnesses), and the majority of HHs remained indifferent , with fewer than half  agreeing with the assertion on ABR  together with any other drugs for multiple purposes. Most of them were willing to show their stored antibiotics , and a significant proportion of the antibiotics  were in the oral dosage form. Antibiotics used for present illness by HHs , were also used by other HH members , and/or retained for future use. The antibiotics used by the majority of the HHs  for their current illness were purchased from the pharmacy/drug store staff . Antibiotics were available without prescription and only a small number  of HHs got their antibiotics through prescription. Nearly half of the HHs  received entire strips of antibiotics, more than the exact number required for the antibiotic course  under different brand names. The second most stored antibiotic was “amoxicillin along with clavulanic acid” (J01CR), a common brand of the multinational pharmaceutical industry , and “amoxicillin” (J01CA04) . The fluoroquinolones group “ciprofloxacin” (J01MA02) was found as the third most reported  antibiotic among HHs. Third-generation cephalosporin “ceftriaxone” (J01DD04) which is common in rural areas was reported the fourth  most stored antibiotic among HHs,  followed by “cefixime” (J01DD08). Few HHs also had “levofloxacin” (J01MA12) and ofloxacin (J01MA01)  respectively. Metronidazole (J01XD01) use was found concurrently  with other antibiotics to treat various infectious diseases. Many HHs stored more than one antibiotic in combinations. They had several unnecessary 1–2  and 3–4  antibiotics at their homes (p < 0.05) and maintained fewer antibiotics at home than the other family members, the cross-tabulations reveal that the number of family members had no bearing on how antibiotics were stored (Azithromycin (J01FA10) was the most stored antibiotic  and affected the mean knowledge scores related to antibiotic use. Education was one of the main factors that determined proper storage and use, and this study found a significant association  between education and HHs' understanding of antibiotics. Similarly, employment had a direct effect on proper antibiotic use and a strong association  was observed between employment and HHs' perspectives. The family setup , the total number of family members , and monthly income  of HHs did not affect the understanding of antibiotics identification, use, and ABR and no relationship was found among the variables. Although, family members having more than one chronic disease  had a near significant relationship in their understanding of antibiotics and their uses  (see Various demographic parameters were checked against antibiotics knowledge and use scores. Different age groups had a strong association (735) see .The overall results show that immediate attention is needed to enhance the rational use of antibiotics in households that do not have adequate knowledge of antibiotics, their identification, proper uses, and ABR-related factors.The current study is the first of its kind in Pakistan's post-conflict districts. The results of this survey provide an up-to-date picture of HHs and medicines storage (antibiotics), which will contribute to the development of community education initiatives to promote sensible antibiotic use. The purpose of this study was to examine the knowledge and habits of HHs on antibiotic use and storage. Instructing HHs on medicines and their storage is one of the most important aspects of antibiotic use after receiving them from the community pharmacy, as the rest of the therapy outcomes are dependent on their proper intake at home. Our study showed a significant lack of awareness regarding the proper use of antibiotics, ABR, and needless medication storage at home, which indicated poor antibiotic practices at HHs.The accurate identification of antibiotics is essential. Knowledge gaps in antibiotics use can lead to ABR, and we observed such a lack of knowledge among HHs in the present study. We found that antibiotic identification was relatively poor 39.6%) and similar results have been reported by Atif and his colleagues, and Tawseef et al., in their studies , 35. Thi.6% and sOne of the most important findings of the current study is that respondents incorrectly believed that they could stop taking antibiotics midway without completing the course; about 43% of the Serbian population also believes likewise . The sitIn the current study, the majority of customers preferred to store unnecessary antibiotics for future usage, and near similar findings have been reported from China (31%) and Jordan (49%) , 54. TheAccording to studies, areas that have experienced continuous conflict are prime locations for bacteria to develop resistance and misuse of antibiotics . ViolentPublic awareness efforts and awareness campaigns are needed to discourage people from using leftover antibiotics at home for themselves or their families and to encourage people to properly dispose of leftover medicines. Our findings show that personalized interventions can reach people with a wide range of demographic and socioeconomic factors. Family members with a history of infectious diseases like respiratory tract infections were a significant risk factor for keeping antibiotics at home, according to our findings , 44.“World Antibiotic Awareness Week” was organized by the National Institute of Health (NIH), Pakistan, on 18–24 November 2021. Such occasions can result in a step-up in Pakistani society's consciousness. The NIH must stress the significance of not keeping antibiotics at home and educate HHs in the affected areas. Therefore, now is an excellent time (spring) to launch a program for HHs and communities, particularly in Pakistan's post-conflict rural areas, to inform the people about the proper use of antibiotics and the adverse effects (ABR) of inappropriate antibiotics consumption. The findings revealed by Khan et al. , are verThere are a few limitations that should be considered while interpreting the results of our study. First, it was a cross-sectional study of Pakistan's post-conflict areas that focused on the most impacted districts from militancy and army operations. It did not cover the rest of the Khyber Pakhtunkhwa, Malakand division districts; thus, the findings cannot be generalized. However, these results can be considered for similar other conflict areas in the country. Second, due to cultural norms, women's participation was not documented, as the men always represent their families during any visit to the HHs in these areas; all these limitations are an integral part of the rural areas of Swat, Khyber Pakhtunkhwa customs, and traditions. However, we believe that female participation will not largely impact the results as these regions are primarily led by men who are decision-makers in their families and a convenient sampling method was employed. Third, the sample was taken from only two sites and the sample size is small. Fourth, we only included 'antibiotics' among the stored medications at home, no additional medications were stated by the HHs. In Pakistan's rural areas, more research on medicines storage among HHs is required.This study showed that storage of antibiotics for future use is quite common among HHs residing in post-conflict regions of Pakistan. Households identified antibiotics among the stored drugs based on packaging and several common brands but were unable to explain why leftover antibiotics should be saved for future use as the storage of antibiotics was observed. The majority of HHs were not aware of ABR and did not explain how antibiotics cause resistance. Furthermore, ABR is the result of irrational antibiotic use, and antimicrobial/antibiotic oversight measures are still weak in Pakistan. The ABR and antibiotic stewardship are still unknown to the rural population. In terms of healthcare, Pakistan's post-conflict areas are the most neglected. More research studies are needed to raise awareness of the ABR problem and to educate HHs on how to use antibiotics properly when they are needed.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and the Ethics Committee for Medical Research at Xi'an Jiaotong University has given its approval to our study (Ref.No: 2020-1342). The patients/participants provided their written informed consent to participate in this study.FaiK and YF: the study concept and design. FaiK: writing of the manuscript. QK: data analysis. TA and FarK: obtaining data. TM and KH: interpretation of results. TM and YK: critical revisions of the manuscript. All authors approved of the version for submission.This work was funded by the National Natural Science Fund (71974156), Young Talent Support Plan, High Achiever Plan of Health Science Center, Xi'an Jiaotong University, and the Central University Basic Research Fund (2015qngz05).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This study aimed to elucidate the changes and associated mechanisms of circulating CD28- cytotoxic T lymphocytes (CTLs) in patients with IgG4-related disease (IgG4-RD).Fifty IgG4-RD patients and 15 healthy controls (HCs) were recruited. Peripheral blood mononuclear cells (PBMCs) were isolated, the levels of circulating CD28- CTLs were detected by flow cytometry, and the proportions of CD127lo or GZMB+CD28- CTL subsets were analyzed in the meantime. Mechanistically, PBMCs isolated from IgG4-RD patients were stimulated with IL-7 in the presence or absence of the JAK inhibitor tofacitinib. Flow cytometry was used to analyze the proliferation of CD28- CTLs and the changes in related subpopulations.ex vivo culture of PBMCs showed that IL-7 could induce the amplification of CD4+CD28- CTLs and CD8+CD28- CTLs in IgG4-RD. Furthermore, IL-7 promotes the proliferation and functional subset changes of these CD28- CTLs in this disease. The selective JAK inhibitor tofacitinib significantly inhibited the effects of IL-7 on CD4+CD28- CTLs and CD8+CD28- CTLs.Circulating CD4+CD28- CTLs and CD8+CD28- CTLs were significantly increased in IgG4-RD patients compared with HCs, accompanied by an elevation of CD127lo or GZMB+ CTL subsets. The IL-7 can affect the immune balance of IgG4-RD patients by promoting the expansion and function of CD4+CD28- and CD8+CD28- CTLs in IgG4-RD through the JAK pathway. Blockade of the IL-7 signaling pathway may be a new therapeutic strategy for IgG4-RD. Most Many studies have focused on T cell subsets to identify the immune mechanisms of this disease, of which cytotoxic T lymphocytes (CTLs) have attracted increasing attention , 7. CD28Interleukin-7 (IL-7), a 25 kDa secreted soluble globular protein, plays an irreplaceable role in T cell development and homeostasis –11. Its In this study, we analyzed the changes of CD4+CD28- CTLs and CD8+CD28- CTLs and their related subsets in the circulation of IgG4-RD patients by flow cytometry. We further investigated the effects of IL-7 on CD28- CTLs in IgG4-RD patients and inquired into the molecular mechanisms.A total of 50 patients with IgG4-RD and 15 healthy controls (HCs) were recruited from outpatient and inpatient sections of Peking University People’s Hospital between March 2021 and Jan 2022. The diagnosis of IgG4-RD was performed according to the 2011 comprehensive diagnostic criteria . Forty-sSerum levels of IgG4 were measured by nephelometry using a Siemens BN II Nephelometer  and Siemens reagents. Serum IgE levels were tested by a Cobas e601 Electrochemiluminescence Immunoassay Analyzer .Peripheral blood mononuclear cells (PBMCs) were separated from fresh blood samples by gradient centrifugation with a human lymphocyte separation medium  and then washed twice with PBS.Enriched PBMCs were resuspended in RPMI 1640 , supplemented with 10% FBS. Cells were stimulated with recombinant human IL-7 (50 ng/mL)  in the presence or absence of 150 nM tofacitinib  and cultured for up to 4 days in 96-well plates.Fresh or cultured PBMCs were stained with antibodies against surface markers, including CD3-APC, CD4-PE/Cy7, CD8-APC/Cy7, CD28-PE, CD127-PerCP/Cy5.5. Then intracellular staining was performed using a transcription factor staining buffer kit , according to the manufacturer’s instructions. After fixation and permeabilization, cells were incubated with antibodies against intracellular markers including GZMB (granzyme B)-FITC and Ki67-PerCP/Cy5.5 for 30 minutes. The mean fluorescence intensity (MFI) of GZMB was calculated. All fluorescent antibodies were purchased from BioLegend . Samples were analyzed on FACSCanto using Diva software .2 test and were shown as percentages. Continuous variables were presented as mean with SD or median with an interquartile range. The unpaired two-tailed Student’s t-test or two-tailed Mann–Whitney test was used for the comparison of two groups as indicated. Cytokine-treated and untreated samples were compared using paired two-tailed Student’s t-test. Statistical significance was determined using GraphPad Prism software V.7.0 . Statistics with P values less than 0.05 were considered to be significant.Categorical variables were analyzed with a χP < 0.0001). Similarly, IgE levels were significantly elevated in IgG4-RD patients compared to HCs .Totally 50 patients and 15 healthy controls were enrolled in this study. The demographics and the levels of serum IgG4 and IgE were listed in We analyzed CD4+CD28- CTLs and CD8+CD28- CTLs in the blood of patients with IgG4-RD and age-matched HCs by flow cytometry. We observed a significant increase in the levels of CD4+CD28- CTLs and CD8+CD28- CTLs in IgG4-RD patients Figure 1We further analyzed the correlations between the levels CD4+CD28- CTLs or CD8+CD28- CTLs and IgG4-RD RI or the concentrations of serum IgG4 and IgE. No significant correlation was found between them 2.We further analyzed the changes of CD28-CD127lo CTLs in peripheral blood of IgG4-RD patients and HCs. Similarly, the levels of CD4+CD28-CD127lo CTLs and CD8+CD28-CD127lo CTLs were significantly higher in patients with IgG4-RD compared with HCs Figure 2GZMB is one of the most important effector molecules of cytotoxic T cells. We used CD28 surface molecules to characterize CTLs, and we need to clarify the expression of GZMB inside them. As shown in We next investigated the effects of IL-7 on CD28- CTLs from IgG4-RD patients and analyzed the role of JAK signaling downstream of IL-7. PBMCs from IgG4-RD patients were treated with IL-7 in the presence or absence of tofacitinib. The percentages of CD4+CD28- CTLs and CD8+CD28- CTLs were significantly increased by the stimulation of IL-7 -derived signals in IgG4-RD. A previous study stated that memory CD4+ T cells were promoted by either IL-7R or TCR signaling, whereas naive CD4 T cells require the cooperative activity of both of these receptor signaling pathways . So the In particular, tofacitinib is a potent inhibitor of JAK1/JAK3 and inhibits JAK2 at high concentrations . Thus itOur study still has many shortcomings, first of all, the sample size is not large enough, but it is enough to explain the effects of IL-7 upregulating JAK to promote the expansion of CD28- CTLs. We did not directly detect the JAK molecule, but only reflected its effect through the inhibitor because it was difficult for us to obtain enough CTLs for western blot detection through the limited blood routine remaining peripheral blood for cell sorting experiments. In addition, the current study did not address the clinical value of circulating CD28- CTL levels in the diagnosis of IgG4-RD patients, because it is difficult to collect a sufficient number of primary patients to elucidate its clinical value, which will be investigated in systematic research in the future.In conclusion, in this study, we demonstrated the critical role of IL-7 in IgG4-RD, finding that administration of exogenous IL-7 could induce the expansion of CD4+CD28- CTLs and CD8+CD28- CTLs in IgG4-RD patients. The effects of IL-7 were inhibited by the administration of the JAK inhibitor tofacitinib. Therefore, IL-7 can affect the immune balance of IgG4-RD patients by promoting the expansion and function of CD4+CD28- CTLs and CD8+CD28- CTLs in IgG4-RD through the JAK pathway. Meanwhile, blocking the IL-7 signaling pathway has a potential therapeutic effect on IgG4-RD.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of Peking University People’s Hospital. The patients/participants provided their written informed consent to participate in this study.C-SX and CL designed the study; YL, AA, and XZ performed the experiments; YYL was responsible for subject recruitment; C-SX and CL wrote the paper. All the authors had approved the final draft submitted.This work was supported by grants from the National Natural Science Foundation of China (81871230), the Doctoral Fund of Ministry of Education of China (20120001120053), and Peking University People’s Hospital Scientific Research Development Funds (RDT 2020-01).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Processing construction waste into aggregate and reusing it in asphalt pavement is beneficial in terms of environmental protection and resource utilization. However, recycled aggregate (RA) possesses some property defects. Therefore, RA usually needs to be strengthened by modification technologies prior to use. In order to promote the convenient and low-cost utilization of construction waste, a new method of preparing construction waste into powder and using recycled powder (RP) as asphalt filler is proposed in this research. The property defects of RA and the applicability of RP used as filler were first analyzed based on their material characteristics. Then, asphalt concrete with RP was designed according to the Superpave method, and the engineering performance of the asphalt mixture was further investigated. According to the results, we recommend the use of acidic RP in combination with other highly alkaline fillers, such as Portland cement (PC), with a suitable blending ratio of RP to PC of 1:1. Preparing asphalt concrete with filler composed of RP and PC can achieve satisfactory engineering performance. Roads are an important part of the transportation network, providing support for national economic development and daily travel of residents. Asphalt concrete pavement is widely used in the surface layer of roads due to its satisfactory driving comfort, low noise, easy maintenance, etc. . It is mNew construction and demolition of infrastructure produce a large amount of construction waste, usually comprising a mixture of steel, rebar, wood, concrete, etc. . In someTherefore, RA usually needs to be strengthened prior to use in asphalt concrete. Many methods can be applied to improve the properties of RA, such as organic and inorganic modifications. Zhu et al. found that sealing the surface of RA with organic silicone resin can effectively eliminate its property defects and improve the engineering performance of asphalt concrete containing RA . However3). Alkaline calcium-based fillers are believed to improve the compatibility of weak acid bitumen and filler particles in asphalt mastic and increase the bonding performance between asphalt mastic and aggregate [Many attempts have been made to use construction waste as asphalt concrete filler, such as in the form of glass powder (GP) , brick pggregate .In this research, we used construction waste resulting from the demolition of concrete pavement. Compared with other types of construction waste, its components are simpler. It consists entirely of crushed stone aggregate and cement mortar. Waste concrete blocks were first crushed and ground into powder; then, the recycled powder (RP) was used as asphalt filler. The acidity or alkalinity of RP is determined by the crushed stone aggregate, although cement mortar is highly alkaline. This is because the amount of crushed stone aggregate in waste concrete is much higher than that of cement mortar. Crushed stone aggregate can be divided into three categories according to composition: acidic, neutral and alkaline aggregates. If RP produced from waste concrete containing acidic crushed aggregate can be used to produce asphalt concrete with satisfactory performance by taking some appropriate technical measures, RP produced from waste concrete containing neutral and alkaline crushed aggregates can also achieve the goal. Accordingly, this study used the RP produced by processing waste concrete containing acidic crushed stone aggregate as the research object. According to previous research, when acidic materials are incorporated into asphalt concrete, highly alkaline fillers, such as hydrated lime  and PortBased on the above information, in the present study, we (1) analyzed the unsuitability of directly using RA in asphalt concrete and the applicability of RP as an asphalt filler according to its material characteristics. (2) Then, asphalt concretes were designed according to the Superpave method, and the appropriate blending ratio of RP and highly alkaline filler was determined based on the test results of the engineering performance of asphalt concretes, including moisture damage resistance, high-temperature deformation resistance and low-temperature crack resistance.The raw materials used in this research included two types of coarse aggregates, namely limestone and recycled aggregate (RA); a limestone fine aggregate; three types of fillers, namely limestone powder (LP), recycled powder (RP) and Portland cement (PC); and an SBS-modified asphalt binder. Aggregates, LP and RP were provided by a local crushed stone factory in Hangzhou, China. RA and RP were obtained by crushing and grinding waste concrete blocks, respectively. The waste concrete blocks mainly consisted of granite crushed stone and cement mortar. PC was provided by the Zhejiang Southern Cement Co., Ltd., Hangzhou, China. The SBS-modified asphalt was from the Zhejiang Transportation Resources Investment Co., Ltd. Asphalt Technology Branch, Hangzhou, China.According to the Chinese technical specification , the basThe basic technical properties of RA and common limestone coarse aggregate, including apparent specific gravity, water absorption, crush value and abrasion value, were first compared to reveal the property defects of RA. Then, a JSM-IT300 scanning electron microscope (SEM) from JEOL, Japan was used to observe the micromorphology of RA, the reasons for the property defects were analyzed based on the characteristics of the SEM images.The mineral phases and chemical compositions of RP and LP were analyzed by a D8 Advance X-ray diffraction (XRD) from Bruker, Germany, and a Zetium X-ray fluorescence (XRF) from PANalytical, Netherlands, respectively. The XRD data were analyzed by means of Jade software with embedded PDF data from the International Centre for Diffraction Data (ICDD). A scheme suitable for the utilization of RP in asphalt concrete was proposed after comparing the mineral phases and chemical compositions of RP to those of LP.According to the analysis results presented in Moisture damage resistance. The moisture damage resistance of asphalt concretes was investigated by a retained Marshall stability (RMS) test and tensile strength ratio (TSR) test according to Chinese technical specifications T0709 and T0729, respectively [S0 is the average Marshall stability of all samples in the blank group, and Si is the average Marshall stability of all samples in each conditioned group after immersion in a hot water bath for i h.In the RMS test, each conditioned group was first immersed in a water bath at 60 °C for varying durations; then, the Marshall stability of each sample in the blank group and the conditioned groups was measured. The RMS can be further computed by Equation (1). A higher RMS value is associated with reduced loss of Marshall stability and improved resistance of asphalt concrete to hot water damage.SS0 is the average splitting strength of all samples in the blank group, and SSh is the average splitting strength of all samples in each conditioned group after freeze–thaw damage for h cycles.In the TSR test, each conditioned group was subjected to freeze–thaw damage for a varying number of cycles; then, the splitting strength of each sample in the blank group and conditioned groups was measured. One freeze–thaw cycle consists of freezing at −18 °C for 16 h, followed by thawing in a 60 °C water bath for 24 h. The TSR can be further computed by Equation (2). A higher TSR value indicates reduced loss of the splitting strength and improved resistance of asphalt concrete to freeze–thaw cycle damage.High-temperature deformation resistance. The high-temperature deformation resistance of asphalt concrete was evaluated by the wheel tracking test according to Chinese technical specification T0719 [s is the wheel speed (42 pass/min); and l1 and l2 are the rutting deformation depths of the slab sample when the test time reaches 45 min and 60 min, respectively (mm).on T0719 . Slab saLow-temperature crack resistance. The low-temperature crack resistance of asphalt concrete was evaluated by three-point bending beam test according to Chinese technical specification T0728 [σ(t) is the real-time flexural strength of the sample (MPa); P(t) is the real-time load borne by the sample during the bending process (kN); l, b and h are the spanning length, width and height of the sample, respectively (mm); ε(t) is the real-time strain of the sample (με); d(t) is the real-time vertical deflection of the sample (mm); fS is the flexural stiffness modulus of the sample at failure (MPa); σmax is the flexural tensile strength of the sample at failure (MPa); and fε is the flexural tensile strain of the sample at failure (με).on T0728 . The tesFour basic technical properties with significant differences between RA and limestone are shown in The property deficiencies are related to the macro- and microstructure of RA. Crushed stone aggregate and cement mortar are the main components of concrete. Therefore, the components of RA particles produced by crushing waste concrete blocks are variable and can be distinguished according to their appearance. The macroscopic appearance of RA is presented in The micromorphology of RA was further analyzed by SEM. Particles consisting of crushed stone and mortar were used because the morphologies of crushed stone, mortar and their contact area can be observed simultaneously. The SEM images are displayed in The macropores and micropores within mortar and the microcracks on the interface between crushed stone and cement result in RA with high water absorption ability (563.3% higher water absorption than limestone aggregate), low density (10.1% lower apparent specific gravity than limestone aggregate) and poor mechanical properties  relative to limestone aggregate.The poor properties of RA make it unsuitable for direct use in asphalt concrete as aggregate. Technical measures are usually required to strengthen the properties of RA, such as an organic modification method . HoweverThe micromorphologies of LP and RP are shown in The XRD analysis results of RP and LP are shown in 2. The kaliophilite and mica mainly contained K2O, Al2O3 and SiO2. The hydration and carbonization products of cement, including C-S-H gel, calcite, ettringite and calcium silicate, contained large amounts of CaO, SiO2, SO3, etc. According to 2O3 in RP was also relatively high, which may be related to the tetracalcium ferric aluminate contained in cement. Therefore, SiO2, CaO, Al2O3, Fe2O3, K2O and SO3 were the main chemical compositions of RP in oxide form. The main chemical composition of LP was CaO according to XRF analysis results. In addition, RP and LP displayed large loss on ignition (LoI), mainly caused by the decomposition of CaCO3 during XRF analysis.XRD results can explain the chemical composition results obtained with XRF. The main chemical compositions of RP and LP are listed in 2 in RP makes it strongly acidic, which is not conducive to in asphalt concrete as a filler. Therefore, RP cannot be used alone as a filler in asphalt concrete. As previously stated, using highly alkaline powder to partially or completely replace filler is a common technical measure employed to strengthen the bonding performance between various components in asphalt concrete when acidic materials are involved [The high content of SiOinvolved ,26,27. TGiven that RP produced from waste concrete containing acidic crushed aggregate can result in satisfactory performance of asphalt concrete when used in combination with highly alkaline PC, RPs produced from waste concrete containing neutral or alkaline crushed aggregate can achieve similar results. This is the original intention behind the use of waste concrete blocks containing granite crushed stone to prepare RP in this research.The RMS results of asphalt concretes are displayed in The TSR results of asphalt concretes shown in RMS and TSR results suggest that the use of RP alone cannot provide asphalt concrete with satisfactory moisture damage resistance. The combined use of RP and highly alkaline PC can improve moisture stability. This conclusion is inconsistent with some previous research findings. Chen et al. stated that asphalt concrete with RP from recycled fine aggregate showed good moisture damage resistance . Hasan aThe rutting test results of asphalt concretes are shown in According to the most rigorous requirement of the Chinese technical specification , the dynThe variation rules of rutting depths of asphalt concretes shown in The low-temperature three-point bending beam test results are listed in Unlike the strain, the flexural strength and flexural stiffness modulus of asphalt concrete with filler composed of 2%RP+2%PC were both higher than those of asphalt concrete with filler composed of 4%LP. In particular, the flexural strength and flexural stiffness modulus of the former were 29.2% and 8.9% higher, respectively, than those of the latter. Therefore, compared with asphalt concrete with common LP filler, the strength and stiffness modulus suggest that asphalt concrete with filler composed of 2%RP+2%PC has better low-temperature load-bearing capacity, although its strain was slightly lower.Compared with common limestone aggregate, recycled aggregate (RA) presented 10.1% lower apparent specific gravity, 563.3% higher water absorption, 59.9% higher crush value and 55.1% higher abrasion value. These four basic technical properties do not meet the requirement of the Chinese technical specification. Therefore, RA cannot be directly used in asphalt pavement as aggregates, and convenient and low-cost utilization methods are needed for waste concrete.2 content mainly contributed by quartz minerals makes RA strongly acidic. Acidic mineral materials are not suitable for asphalt concrete. Therefore, RP is recommended for use in combination with other highly alkaline fillers.Compared with common limestone powder (LP), RP showed a coarser microtexture and contained rich floccules. These characteristics are beneficial for the interaction between RP and asphalt in asphalt mastic. The high SiORMS and TSR results suggest that the appropriate blending ratio of RP and highly alkaline Portland cement (PC) in a hybrid filler system is 1:1. Preparing asphalt concrete with hybrid filler composed of 2%RP+2%PC can result in satisfactory moisture damage resistance, high-temperature deformation resistance and low-temperature crack resistance.In this work, waste concrete mainly composed of granite crushed stone aggregates and cement mortar were processed into powder, and the feasibility of using this recycled powder (RP) as a filler in asphalt concrete was evaluated. According to the discussed results, the following conclusions can be drawn: