Today, the commitment of beef production systems is to enhance their sustainability. Therefore, improving animal welfare and improving feed efficiency are both important challenges. Citrus flavonoids are polyphenols that have previously shown promising effects on reducing feed intake and modulating animal behavior in bulls fattened under commercial conditions. Moreover, increasing fat during the finishing phase in beef cattle diets has been used to increase dietary energy content and to reduce the risk of rumen acidosis. Therefore, the present study supports the hypothesis that a high dietary fat content does not interfere in the mode of action of these flavonoids and that the positive effects on feed efficiency and animal behaviors should be observed. Nevertheless, the mode of action of flavonoids is not known; it is speculated that digestive tract health (rumen) and changes in the expression of behavior- and inflammation-related genes (rumen and duodenum) may be involved, and dietary fat supplementation may also affect, antagonistic to flavonoids, these mechanisms. However, the present study data support that citrus flavonoid supplementation in high dietary fat diets may be a good strategy to face current beef production system challenges such as improving feed efficiency and animal welfare.Citrus aurantium, Bioflavex CA, HTBA, S.L.U., Barcelona, Spain, 0.4 kg per ton of Bioflavex CA). At the finishing phase, the dietary fat content of the concentrate was increased (58 to 84 g/kg DM). Concentrate intake was recorded daily, and BW and animal behavior by visual scan, fortnightly. After 168 d, bulls were slaughtered, carcass data were recorded, and rumen and duodenum epithelium samples were collected. Performance data were not affected by treatment, except for the growing phase where concentrate intake (p < 0.05) was lesser in the BF compared with the C bulls. Agonistic and sexual behaviors were more frequent (p < 0.01) in the C than in the BF bulls. In the rumen epithelium, in contrast to duodenum, gene expression of some bitter taste receptors  and other genes related to behavior and inflammation was higher (p < 0.05) in the BF compared with the C bulls. Supplementing citrus flavonoids in high-fat finishing diets to Holstein bulls reduces growing concentrate consumption and improves animal welfare.One hundred and forty-six bulls (178.2 ± 6.64 kg BW and 146.0 ± 0.60 d of age) were randomly allocated to one of eight pens and assigned to control (C) or citrus flavonoid (BF) treatments ( Citrus aurantium) rich in naringin , citrus flavonoids supplementation modified the eating pattern of Holstein bulls fed high-concentrate diets throughout the fattening period, reducing the large meal sizes consumed by the animals [Citrus fruits contain a wide range of flavonoids. These flavonoids are polyphenols, a category of phytochemicals with plenty of biological activities, such as anti-inflammatory, antioxidant, and antimicrobial properties . In prev animals . Further animals ,3,4. In  animals , and it  animals ,6,7 and  animals . Actuall animals ,10. Bull animals  and PPYRFinally, oral non-nutritive behaviors, agonistic interactions  and sexual behaviors  were also reduced in bulls supplemented with flavonoids independently of the feeder or concentrate presentation  ,3,4. OurThus, the present study was designed to evaluate the effects of citrus flavonoid supplementation on concentrate consumption, growth rate, concentrate efficiency, macroscopic rumen wall health, carcass characteristics, and animal behavior in Holstein bulls fed high-fat concentrate diets in commercial conditions. Furthermore, the present study also aimed to study deeper how citrus flavonoid supplementation in high-fat concentrate diets could affect the expression of some genes involved in gut–brain crosstalk mechanisms in the rumen and duodenum epithelium, such as bitter taste receptors and inflammation regulators.Citrus aurantium) of the whole fruit, rich in naringin (24%) . Bioflavex was incorporated into the concentrate during the concentrate manufacturing. Concentrates were manufactured from a 9000 kg master batch, of which 4500 kg were C, and the other 4500 kg BF. Each treatment concentrate was transported to the farm with the same truck and stored into two different silos under the same conditions.This study was conducted in accordance with the Spanish guidelines for experimental animal protection . One hundred and forty-six Holstein bulls (178.2 ± 6.64 kg BW and 146.0 ± 0.60 d of age) were fattened under commercial conditions in a farm . The study was divided into growing (0 to 112 d) and finishing (113 to 168 d) phases, after 168 animals were fed the same treatments until slaughter day. During the study, animals were not vaccinated. Animals were randomly allocated in one of eight pens and assigned to one of the two treatments , either control (C) or supplemented (BF) with 0.04% of bitter orange extract , deep-bedded with straw, and equipped with a three-space feeder . The feeder of each pen weighed the concentrate continuously as described by Verdú et al. [ m × 6 m,Animals were fed a commercial concentrate in meal form, formulated to cover their nutritional requirements . The firA visual scan procedure at days 13, 28, 44, 56, 72, 83, 100, 114, 128, 143, 153, and 167 of the study was performed to study the general activity  and social behavior  of the animals in every pen. The visual observation was made for 2 pens at the same time from 8:00 to 10:30 a.m., as described by Verdú et al. [p” to a poor conformation. The fat cover was classified according to the EU Regulation no. 1208/81, which utilizes a classification system by numbers, 1, 2, 3, 4, 5, where 5 explains a very high degree of covering fat and heavy fat deposits in the thoracic cavity, and 1 is classified as a low degree, with no fat cover.After 168 d of study, the bulls were transported to the slaughterhouse , located 15 km from the farm. Animals were slaughtered in 2 weeks, four pens per week, two pens from the control and two from the BF bulls each week. The time spent waiting before slaughter was less than 6 h. Animals were weighed before loading. They were slaughtered by commercial practices and following the EU Regulation 1099/2009 on the protection of animals at the time of killing or slaughtering. After slaughtering, the hot carcass weight (HCW) was registered for every animal. The dressing percentage was calculated by dividing the HCW by the BW recorded before slaughtering. Moreover, following the (S) EUROP categories described by the EU Regulation nos. 1208/81 and 1026/91, the conformation of carcasses was classified, where “E” corresponded to an excellent conformation, “U” to very good conformation, “R” to good conformation, “O” to fair conformation, and “All animals’ rumen and the liver of every animal were macroscopically evaluated at the slaughterhouse. Rumens were classified depending on the color by a visual evaluation, from 1 to 5, “5” being a black-colored rumen and “1” a white-colored rumen . They we2 section of rumen wall  and duodenum epithelium was sampled. Ruminal papillae from the rumen wall section was excised, and both ruminal papillae and duodenum epithelium samples were rinsed 2 times with chilled PBS after sampling and immediately incubated in RNAlater  to preserve the RNA integrity. After 24 h of incubation with RNA later at 4 °C, the liquid was removed, and the tissue was frozen at −80 °C until further RNA extraction and subsequent gene expression analysis.Additionally, a liquid sample from the rumen was obtained from homogeneous contents strained with a cheesecloth from 18 animals per treatment randomly selected from two pens per treatment, immediately following slaughter. Following the procedures of Jounay , 4 mL ofDuring the study, samples of concentrate were collected at days 0, 42, 84, 126, and 168 and analyzed for DM , ash (meNaringin was determined for every sample of concentrate (C and BF) as a Bioflavex CA marker for the BF group and was used as a marker confirming the adequate inclusion of citrus flavonoid extract in the diets by Laboratory of Interquim S.A. An internal method for naringin quantification using HLPC developed by Interquim S.A. was used .Ruminal VFA concentration was determined with a semicapillary column  composed of 100% polyethylene glycol (PEG) esterified with nitroterephtalic acid, bonded and crosslinked phase , using a CP-3800-GC .For the gene expression analyses, total RNA was extracted from rumen papillae and duodenum epitheliums homogenizing tissues in Trizol  by Polytron Instrument . Isolated mRNA was reverse-transcribed to cDNA using a PrimeScript RT Reagent Kit  following the manufacturer’s instructions. The RNA purity was assessed by a NanoDrop instrument  at 260, 280, and 230 nm. The criteria used to consider a good RNA quality was a ratio 260/280 and 260/230 in the range of 2.0 and 2.0–2.2, respectively. The quantification of the expression of genes at the mRNA level coding for: (1) the tight-junction protein claudin 4 (CLDN4); (2) the production, expression, and turnover of neurotransmitters: free fatty acid receptor 2 (GPR43) and free fatty acid receptor 3 (GPR41), pancreatic polypeptide receptor 1 (PPYR1); actual name neuropeptide Y receptor Y4 (NPY4R), α2-adrenergic receptor subtype C (ADRA2C), and cholecystokinin receptor 4 (CCKBR); (3) proinflammatory cytokines TNF-a (TNFa) and cytokine IL-25 (IL-25), pattern recognition receptor Toll-like receptor 4 (TLR4), and antimicrobial peptides released by intestinal cells (beta-defensins and lactoferrin); (4) bitter taste receptors type 2 members 7, 16, 38, and 39  were performed by quantitative PCR (qPCR). The qPCR was performed using gene codifying for ribosomal protein subunit 9 (RPS9) as a housekeeping gene, which was checked for stability following Vandesompele et al. , EquatioOnly the pen was considered the experimental unit and animals within the pen were considered sampling units in some parameters.p-values correspond to the ANOVA of the transformed data. daily. Then, these data were transformed into natural logarithms to achieve a normal distribution. The frequency of each social behavior was calculated by summing by day, pen, and scan, and transformed into the root of the sum of each activity plus 1 to achieve a normal distribution. The ANOVA was performed with transformed data, and the means shown in the tables correspond to the backtransformed data.The concentrate efficiency data were log-transformed to achieve a normal distribution. The means presented in the tables and figures correspond to nontransformed data and the SEM and The unification of performance, animal behavior, and concentrate consumption data averaged by pen and period were analyzed using a mixed-effects model . The model included the initial BW as a covariate, the treatment, period (14 d period), and the interaction between treatment and period as fixed effects, and the interaction between treatment and pen and the 3-way interaction between treatment, pen, and period as random effects. The period was considered a repeated factor, and for each analyzed variable, the animal nested within the interaction between treatment and pen (the error term) was subjected to 3 variance–covariance structures: compound symmetry, autoregressive order one, and unstructured. The covariance structure that yielded the smallest Schwarz’s Bayesian information criterion was considered the most desirable analysis.In the case of the rumen gene expression and VFA data, the pen was considered the experimental unit and the animals the sampling units, and data were analyzed using ANOVA where the model included treatment (as there were no repeated measures) as the main effect. For the categorical variables analyses  a Chi-squared test was used.p < 0.05, and trends were discussed at 0.05 ≤ p ≤ 0.10 for all models.Differences were declared significant at Two animals did not finish the study, one from the C treatment due to enterotoxaemia and one from the BF treatment due to chronic respiratory problems. p < 0.05) for the C than for the BF bulls, whilst these differences disappeared for the finishing phase  for the BF compared with C bulls, and the proportion of animals ruminating tended (p < 0.10) to be greater as well for the BF bulls than for the C bulls in this phase. During the finishing phase, as in the growing phase, no differences during the visual observation period were found in the proportion of animals per pen standing, lying, and drinking water, and in this phase, between treatments, no differences in animals eating straw. As observed in the growing phase, in this phase, the proportion of animals eating concentrate and ruminating were greater (p < 0.01) in the BF bulls compared with the C bulls.In the growing phase, no statistical differences were found in the percentage of animals per pen standing, lying, and drinking water during the visual observation period. Otherwise, the percentage of animals eating concentrate and eating straw were greater (p < 0.01) performed by the C compared with the BF bulls, as well as all agonistic behaviors  except displacement were also more frequently (p < 0.05) exhibited by the C bulls than the BF bulls. Regarding sexual behaviors, flehmens and attempts to mount were more frequent (p < 0.05) in the C compared with the BF bulls, although complete mounts were not affected by the treatments. During the finishing phase, no differences between treatments were observed for social behavior, whilst the bulls from the BF group performed (p < 0.05) more self-grooming than the C bulls. Additionally, during this phase the C bulls also performed more (p < 0.01) oral non-nutritive behaviors than the BF bulls. Again, differences among treatments in agonistic behaviors were found in this phase. Fighting, butting, and displacement behaviors were more frequent (p < 0.01) in the C bulls compared with the BF bulls, and the C bulls also tended (p < 0.10) to show more chasing-up behaviors than the BF animals. In regard to sexual behaviors, the C bulls performed more (p < 0.01) flehmen behaviors and tended (p < 0.10) to perform more attempted and complete mounts compared with the BF bulls.In the growing phase, during the visual scan observation period, self-grooming and social behavior were not affected by the treatment. On the other hand, oral non-nutritive behaviors were more frequently (p < 0.01) in the BF bulls (72.22% classified as color <“3”) compared with the C bulls (46.58% classified as color <“3”). The number of baldness regions in the rumen also tended (p < 0.10) to be greater in the C bulls (16.44%) compared with the BF bulls (6.94%). In the remaining macroscopic parameters analyzed at the slaughterhouse  no differences among treatments were found.The results of the macroscopic evaluation at the slaughterhouse of rumens and livers are shown in p < 0.01) in the BF than in the C bulls, and on the contrary, the total VFA concentration was greater (p < 0.01) for the C bulls compared with the BF bulls. The molar proportion of acetate was also affected by the treatments, being greater (p < 0.01) in the BF bulls compared with the C bulls, whereas the molar proportion of propionate was greater (p < 0.01) for the C bulls than for the BF bulls. Furthermore, the butyrate molar proportion was also greater (p < 0.05) in the BF than in the C bulls. The remaining VFAs analyzed  were not affected by the treatment. As for the acetate and propionate molar proportions, the acetate:propionate ratio was greater (p < 0.05) for the BF bulls than for the C bulls. The data of ruminal liquid parameters analyzed are presented in p < 0.01) in the rumen of the BF compared with the C bulls. The relative expression of all receptors related with the neurotransmitter signaling studied  was greater (p < 0.05) in the BF bulls compared with the C bulls. Furthermore, the relative expression of some receptors related with inflammation such as IL-25, TLR4, and defensin, was also greater (p < 0.05) for the BF bulls than for the C bulls.The data of the relative gene expression in the rumen epithelium are presented in p < 0.001) in the C bulls compared with the BF bulls. The relative expression of some receptors related with the neurotransmitter signaling also differed among treatments. The FFAR2 expression (p < 0.01) was more expressed in the C bulls compared with the BF bulls. Furthermore, the relative expression for PPYR1 and CCKBR was greater (p < 0.01) as well for the C bulls than for the BF bulls. Additionally, the relative expression of the receptors related with inflammation, such as IL-25, TLR4, and defensins, were again greater (p < 0.05) for the C bulls than for the BF bulls, contrary to the rumen epithelium results.Data of the relative expression of duodenum epithelium genes are presented in Citrus flavonoid supplementation clearly reduced concentrate intake in bulls during the growing phase, although the remaining performance parameters analyzed were not affected throughout the study. Moreover, when the bulls were supplemented with citrus flavonoids, they spent more time eating concentrate and straw than the C bulls, and also devoted more time to performing ruminating activities as observed during the visual scan procedure for the growing phase. In all our previous studies ,3,4, bulButyrivibrio spp. hydrolyzed the glycosidic bond, metabolizing naringin to naringenin in rumen. Interestingly, recent research has shown that Butyrivibrio spp. growth was clearly inhibited by the presence of oils and fats at low concentrations [Butyrivibrio spp., that would explain the higher gene expression of TAS2R in ruminal epithelium of bulls supplemented with citrus flavonoids. Due to this increase in the gene expression of TAS2R, the production, expression, and turnover of neurotransmitters studied were probably also more expressed in the bulls supplemented with citrus flavonoids.In the present study, the fat content was increased during the finishing phase; this strategy is commonly used to increase energy density in finishing diets of fattening bulls ,26. Howetrations . ConsequThe differences in the expression of the following genes would support a decreased feed intake: (i) as previously mentioned, the activation of TAS2R triggers the release of anorexigenic molecules, such as CCK and PYY ,37,38, aAs previously mentioned, citrus flavonoid supplementation clearly decreased the total VFA in ruminal liquid at the slaughterhouse and increased ruminal pH. Moreover, the acetate molar proportion was increased and the propionate molar proportion reduced, so that the acetate:propionate ratio was higher for the BF bulls. Conversely, the gene expression of FFAR2 and FFAR3 was higher for the BF bulls as previously explained, probably due to bitter and lipid senses interaction, being then completely independent of the ruminal VFA content. Previous research carried out with citrus flavonoids also showed an improvement in ruminal pH probably as consequence of rumen microflora modulation, but in previous studies  the concIn the present study, in agreement with our previous research ,3,4, citIn summary, animal and eating behavior results were similar to those of previous studies ,3,4; howIn conclusion, during the growing phase, feeding bulls with high-concentrate diets supplemented with citrus flavonoid decreased concentrate intake. Moreover, when bulls fed high-concentrate diets were supplemented with citrus flavonoids, bulls reduced oral non-nutritive behaviors, agonistic interactions, and sexual behaviors, potentially improving animal welfare. Ruminal pH and rumen wall parameters macroscopically studied suggested that rumen health was better in the BF than in the C bulls. Finally, flavonoid supplementation in a high-fat finishing diet differently modified the gene expression of genes in the rumen and duodenum epithelium, and further research is needed to relate these data with potential regulation mechanisms of eating pattern and animal behavior.


Regarding the cost of hospitalization for combinations of comorbidities, cerebral infarction + chronic pulmonary disease was the most costly (US $1718.90), followed by cerebral infarction + hypertension + chronic pulmonary disease (US $1530.60). In the results of multiple linear regression analysis, cerebral infarction with chronic pulmonary disease had significant effects on hospitalization costs , drug costs  and diagnosis costs . Conclusions: Comorbidities are significantly associated with high hospitalization costs for cerebral infarction patients. Furthermore, relevant health departments should build preventative and control systems to reduce the risk of comorbidities, as well as to improve hospital clinical pathway management and to strengthen and refine the cost-control management of cerebral infarction from the perspective of comorbidities.Objective: This study aimed to study the influence of comorbidities on hospitalization costs for inpatients with cerebral infarction. Methods: The data from the medical records pertaining to 76,563 inpatients diagnosed with cerebral infarction were collected from public hospital records for the period between 1 January 2020 and 30 December 2020 in Gansu Province. EpiData 3.1 software was used for data collation, and SPSS 25.0 was used for data analysis. Numbers and percentages were calculated for categorical variables, the chi-squared test was used to compare differences between groups, and multiple independent-sample tests  and multiple linear regression were used to analyze the influence of different types of comorbidity on hospitalization costs. Results: Among the 76,563 cerebral infarction inpatients, 41,400 were male (54.07%); the average age of the inpatients was 67.68 ± 10.75 years (the 60~80-year-old group accounted for 65.69%). Regarding the incidence of varied chronic disease comorbidities concomitant with cerebral infarction, hypertension was reported as the most frequent, followed by heart disease and chronic pulmonary disease. The average hospitalization cost of cerebral infarction inpatients is US $1219.66; the hospitalization cost increases according to the number of comorbidities with which a patient suffers (H = 404.506,  Cerebral infarction (CI), also known as ischemic stroke, is an acute cerebrovascular disease characterized by sudden blockage of an artery, preventing blood supply to a specific area of the brain, constituting the main type of stroke ,2. In 20The data were acquired from the “Direct Reporting System of Health Statistics Information Network” of the Health Commission of Gansu Province. We extracted data from the medical records pertaining to 76,563 inpatients with cerebral infarction who were discharged from public hospitals between 1 January 2020 and 30 December 2020 in Gansu Province. The Chinese National Bureau of Statistics showed that the average annual exchange rate between USD and RMB was 6.8996 RMB/USD in 2020 [(1)According to the International Classification of Diseases, patients with the primary diagnostic disease code I63.900 were included.Inclusion criteria:(1)Total hospitalization cost was zero;(2)Data were missing or incorrect (which could not be corrected);(3)Length of hospital stay was less than 1 day or more than 60 days.Exclusion criteria:(1)Basic inpatient information: gender, age, ethnicity, marital status, hospital level, and the patient’s chosen medical payment method;(2)Clinical information: discharge diagnosis, history of comorbidities, length of hospital stay, discharge and admission dates, and operation status;(3)Information relating to medical costs: total hospitalization costs, drug costs, diagnosis costs, treatment costs, costs of medical consumables, medical service costs, rehabilitation costs, and other costs.p < 0.05 indicated statistical significance.In this study, EpiData 3.1 software was used for data collation, whilst SPSS 25.0 was used for data analysis. The hospitalization costs, drug costs, and diagnostic costs were logarithmic and distributed in an approximately normal manner. Numbers and percentages were calculated for categorical variables. The chi-squared test was used to compare differences between groups. Multiple independent-sample tests  and multiple linear regression were used to analyze the influence of different types of comorbidity on hospitalization costs. In our study, all tests were two-tailed, and 2 = 0.080, p < 0.001); males had a significantly higher risk of stroke than females . All differences were statistically significant  and 35,163 females (45.93%). The average age of the inpatients was 67.68 ± 10.75 years; 65.69% were 60~80 years old. Han ethnicity accounted for 94.01%, and married patients accounted for 82.22%. Only 12,519 patients (16.35%) did not have any comorbidities, while 64,044 patients (83.64%) suffered with comorbidities. The risk of comorbidity for patients aged 60–80 years was the highest, followed by those aged 40–60 years (χnificant .p < 0.001), drug costs , and diagnosis costs  increased with the number of comorbidities. Statistically, these differences were significant, as shown in The average hospitalization cost of a patient suffering from a cerebral infarction was US $1219.66, and the average drug cost and diagnosis cost were US $391.77 and US $340.04, respectively. The average hospitalization cost of cerebral infarction patients without any comorbidities was estimated to be US $1174.31. On the other hand, the costs for patients suffering from one, two, three, and four or more chronic diseases were US $1224.41, US $1237.90, US $1247.30, and US $1322.27, respectively. Drug costs and diagnosis costs were the main components of hospitalization costs. The hospitalization costs  was the highest, followed by heart disease  and chronic pulmonary disease ; the hospitalization cost of cerebral infarction comorbid with cancer was the highest (US $1934.02), followed by chronic pulmonary disease (US $1533.02), asthma (US $1488.22), memory-related diseases (US $1439.01), and diabetes (US $1391.31)  .The 20 most frequently related comorbidities accompanying cerebral infarction are listed in p < 0.001), followed by cancer . Cerebral infarction along with emotional and psychiatric disorders had the greatest effect on drug costs , followed by chronic pulmonary disease . Cerebral infarction along with chronic pulmonary disease had the greatest effect on diagnostic costs , followed by emotional and psychiatric disorders   .β = 0.181, p < 0.001). Relevant studies have shown that cerebral infarction patients are often associated with stroke-related pneumonia in the clinic, and the rate of comorbidity has been reported as high as 10~47% [Among the types of chronic disease, hypertension and heart disease were more prone to occurring in patients alongside cerebral infarction. Related research showed that hypertension was a hallmark comorbidity of stroke. Moreover, large-artery atherosclerosis (LAA) stroke and small-vessel occlusion (SVO) stroke were closely related to hypertension. Moreover, studies have shown that cardioembolic stroke is associated with atrial fibrillation, valvular heart disease, and ischemic heart disease ,11. Hypes 10~47% . Chronics 10~47% . Patients 10~47% ,16,17. AIn terms of medical costs, the hospitalization costs of patients suffering from a cerebral infarction along with one or more comorbidities were significantly higher than those not suffering with a comorbidity. The greater the number of comorbidities, the higher the hospitalization costs of cerebral infarction patients. Studies have shown that the superposition, coexistence, and combination of multiple chronic diseases can exacerbate a patient’s disease, leading to additional overutilization of health services and the consumption of medical resources, ultimately increasing medical expenditures . RelevanAmong the types of comorbidities commonly associated with cerebral infarctions, the hospitalization cost of cancer was the highest, followed by chronic pulmonary disease.Among combinations of comorbidities, cerebral infarction + chronic pulmonary disease resulted in the highest hospitalization costs; thus, chronic lung disease is a disease that cannot be ignored in the context of our discussion. In addition, although the number of cerebral infarction patients who also suffered with hypertension and heart disease was the greatest, the cost of their hospitalization was relatively low, which might be closely related to Chinese medical insurance policies that are implemented in relation to diabetic and hypertensive drugs. As part of China’s healthcare reformation, hypertensive drugs have been uniformly included in Medicare payments, and the associated reimbursement rate is now above 50%. The implementation of the “basic medical insurance + serious illness insurance system” policy has also greatly reduced the out-of-pocket payment ratio of hospitalization costs for cerebral infarction patients also suffering with hypertension and heart disease ,24. In cMultiple linear regression analyses between hospitalization costs and types of comorbidities related to cerebral infarction revealed that chronic pulmonary disease had a significant influence on hospitalization and drug costs. On the one hand, chronic pulmonary disease is a progressive and irreversible disease, manifesting as a persistent cough or as asthma, which greatly affects patients’ quality of life, resulting in higher clinical medical needs as well as drug dependence. On the other hand, chronic pulmonary disease is characterized by polypharmacy and pharmacotherapeutic complexity. A patient’s medication compliance, drug-related side effects, and drug interactions all result in increased healthcare costs . CerebraComorbidities are significantly associated with high hospitalization costs for cerebral infarction patients. Furthermore, relevant health departments should build prevention and control systems to not only reduce the risk of patients developing comorbidities but also improve the clinical management pathways and hospitalization cost-control system, as well as better refining the cost-control management of cerebral infarction from the perspective of comorbidities.


About a hundred mono- and combination antiviral drugs have been already approved, whereas thousands are in development. Here, we briefly reviewed 7 classes of antiviral agents: neutralizing antibodies, neutralizing recombinant soluble human receptors, antiviral CRISPR/Cas systems, interferons, antiviral peptides, antiviral nucleic acid polymers, and antiviral small molecules. Interferons and some small molecules alone or in combinations possess broad-spectrum antiviral activity, which could be beneficial for treatment of emerging and re-emerging viral infectionsThe online version contains supplementary material available at 10.1007/s00018-022-04635-1. The COVID-19 pandemic associated with the severe acute respiratory syndrome coronavirus 2 virus (SARS-CoV-2) has stimulated the development of known and the discovery of new antiviral measures. According to CDC and WHO, protective clothing, face masks, washing hands, cleaning surfaces with detergents, social distancing, travel restrictions, and isolating the infected people have all helped to limit the spread of the respiratory virus. Vaccination has improved population protection from severe SARS-CoV-2 infection . The AVPs can also serve as delivery vehicles for other antivirals.Antiviral peptides (AVP) are polymers that have been experimentally verified to interfere with virus replication Fig. e. They c viruses . AltogetAntiviral nucleic acid polymers (NAPs) can directly inhibit viral entry or replication by binding to the virus particle, its building blocks, or RNA/DNA replication intermediates Fig. f. SeveraSmall-molecule or small molecular-weight antivirals attenuate viral replication Fig. g. Some sAntivirals can target one or more viruses. Agents that inhibit the replication of many viruses are called broad-spectrum antivirals (BSAs). BSAs that inhibit the replication of viruses from the same or different viral families are called intra- or inter-family antivirals, respectively  , 46, 47.Immediately after the SARS-CoV-2 was isolated the virus interactions with host cells were analyzed using systems biology approaches . ResearcViruses still infect millions and kill hundreds of thousands of people. To date, about a hundred mono- and combination antiviral therapies have been approved, while thousands are in pre- or clinical development , 60. HerSupplementary file1 (XLSX 283 KB)Below is the link to the electronic supplementary material.


Nanocomposite hydrogels are highly porous colloidal structures with a high adsorption capacity, making them promising materials for wastewater treatment. In particular, magnetic nanoparticle (MNP) incorporated hydrogels are an excellent adsorbent for aquatic pollutants. An added advantage is that, with the application of an external magnetic field, magnetic hydrogels can be collected back from the wastewater system. However, magnetic hydrogels are quite brittle and structurally unstable under compact conditions such as in fixed-bed adsorption columns. To address this issue, this study demonstrates a unique hydrogel composite bead structure, providing a good adsorption capacity and superior compressive stress tolerance due to the presence of hollow cores within the beads. The gel beads contain alginate polymer as the matrix and MNP-decorated cellulose nanofibres (CNF) as the reinforcing agent. The MNPs within the gel provide active adsorption functionality, while CNF provide a good stress transfer phenomenon when the beads are under compressive stress. Their adsorption performance is evaluated in a red mud solution for pollutant adsorption. Composite gel beads have shown high performance in adsorbing metal  and non-metal (sulphur) contaminations. This novel hybrid hydrogel could be a promising alternative to the conventionally used toxic adsorbent, providing environmentally friendly operational benefits. Global water pollution management involves decisive and innovative materials and technologies that can successfully remove pollutants from wastewater ,2,3. TheRegarding the adsorbent materials, functional composites with collective physicochemical properties could lead to an effective adsorption solution. In recent years, nanocomposite hydrogels have been broadly utilised in many adsorption-based water purification applications ,20,21 whAlthough MNP-incorporated hydrogels are incredibly beneficial for adsorption applications, these composite materials are significantly brittle and structurally unstable under compression stress ,33. For 3O4) MNPs was carried out on the surface of the nanofibres through the co-precipitation process. The physical, mechanical, and microstructural properties of the composite beads are investigated, and their adsorption performance is evaluated in aqueous media for pollutant removal.In this paper, sustainable nanocomposite hydrogel beads are demonstrated that comprise CNFs decorated with MNPs as the filler and alginate as a hydrogel matrix. Bleached sugarcane pulp is used to extract the CNF, and the synthesis of iron oxide (Few/w) (Sigma-Aldrich), ferric (III) chloride hexahydrate 96% (Sigma-Aldrich), and ferrous (II) chloride 98% (Sigma-Aldrich); and materials for the gel composite matrix: sodium alginate (Sigma-Aldrich). Deionised (DI) water was used in all experimental works.The raw sugarcane mulch was received from Sugar Research Australia Ltd. . Materials used in this study include chemicals for pulping sugarcane: sodium hydroxide , glacial acetic acid , and sodium chlorite-Technical Grade 80% ; chemicals for the in situ synthesis of iron oxide: ammonium hydroxide 25% (Hybrid hydrogel beads were prepared in a three-step process: (1) preparation of the CNF, (2) in situ precipitation of the MNPs on the CNF, and (3) solution mixing of the CNF and MNP–CNF with alginate for preparing CNF/alginate and MNP–CNF/alginate gel beads separately. w/v) sodium hydroxide solution using a 10:1 solvent to sugarcane ratio at 80 °C for two hours, followed by rinsing with 60 °C water. Alkali-treated fibres were then bleached twice using an acidic solution of 1% (w/v) sodium chlorite  at 70 °C for one hour at a 30:1 solvent to fibres ratio. CNF were prepared by passing a 0.7% (w/v) dispersion of bleached sugarcane pulp through a high-pressure homogeniser  for one pass at 800 bar and three passes at 1100 bar.For preparing the cellulose nanofibres, bleached sugarcane pulp was produced using the method reported before ,39. Wash3O4) MNPs were synthesised on the surface of the CNF using the co-precipitation of two iron salts: ferric (III) chloride hexahydrate and ferrous (II) chloride. A similar method was also demonstrated in our previous publication on magnetic nanocellulose /[Fe2+] was kept at 2. All experiments were performed at room temperature. The average size of our MNPs is in the range of 9- 14 nm with a narrow distribution. The presence of CNF during the synthesis of MNPS facilitates the formation of small nanoparticles with a narrow size distribution, possibly by evenly distributing the metal ions precipitated into each nanoparticle.Iron oxide (Feellulose . For 30 w/w) was initially mixed with 0.84% (w/v) CNF and 2.15% (w/v) MNP–CNF solution separately at 50:50. An ultrasonic probe  for 20 min  was used at 40% amplitude while keeping the solution in an ice bath to prevent an unwanted temperature rise. Afterwards, composites were dripped with a syringe pump at a 1 mL/min flowrate  into an aqueous 1.1% (w/w) CaCl2 solution for around 15 min to form spherical gel beads [2+ from the surface. The composite beads were dried in a convection oven at 60 (±0.5) °C for 24 h. A 1.27 mm dispenser needle was used that allowed the gel droplet to expand spherically at the exit to produce a hollow core within the sphere.For preparing the nanocellulose and magnetic nanocellulose incorporated alginate hydrogels , an aqueous alginate solution of  was used to study the opacity of the dry beads that provides basic information on the qualitative extent of the monodispersity of the nanomaterials within the gel. Dark-field microscopy was used to evaluate the bead size before and after swelling tests. A scanning electron microscope  was used  to understand the nanomaterials’ interaction with the alginate matrix, which is crucial to have a better filler–matrix stress transfer capability. Before SEM observations, samples were sputter-coated with platinum (~15 nm coating thickness). Section-cut beads were also microscopically observed to confirm the presence of a hollow core within the beads. A highly porous matrix structure is desirable for better volumetric transport of the adsorbates that are magnetically attracted by the MNPs.The swelling ratio of the gel beads was determined at 22 °C by measuring the weight change. The beads were immersed in water for 60 h at 22 °C, and the swelling ratio −1 under the flow of air. The mechanical characterisation of the hydrogels was conducted under compression. A universal mechanical tester  and an advanced video extensometer were used to perform uniaxial compression testing equipped with 100 N load cell and compression plates. The hydrogel samples were placed within the compression plates and compressed with a crosshead speed of 1 mm/min until ~82% compression strain was reached, and then the compression force was gradually released.Thermogravimetric analysis (TGA) was performed on a thermal analyser  having a range 40–800 °C at a heating rate of 10 °C minThe adsorption of pollutants in the wastewater was carried out in the shaking water bath (100 rpm) at room temperature. About 1 mg of the gel beads was poured into 15 mL wastewater and shaken for 24 h. After that, the filtrate was separated from the gel beads with a 0.45 µm syringe filter. Finally, the metal concentrations in the wastewater before and after adsorption were characterised using ICP-AES by ALS Environmental Division, Brisbane, Australia.2 at −196 °C and CO2 at 0 °C were measured with a TriStar II 3020 apparatus . The samples were degassed at 150 °C and a pressure of 10−5 torr for 72 h before the adsorption tests. The N2 isotherms were used to determine the Brunauer-Emmett-Teller (BET) specific surface area at a relative pressure in the range of P/P0 = (0.05–0.35), with the total pore (<66.4455 nm) volumes at P/P0 = 0.97. The pore size distributions were also calculated from the N2 and CO2 isotherms using the non-local density functional theory (NLDFT) model. The equivalent surface area, limiting micropore volume, and limiting micropore capacity were calculated from the CO2 isotherm with the DA method [Sorption isotherms of NA method .2 solution. The surface morphology of both nanocomposite beads appeared to be wrinkled and rougher than the pure alginate beads due to the gradual water loss adsorbed by CNF. The cross-sectional images display the internal structure of each bead. Both nanocomposite beads demonstrate a hollow core within their structure. This is attributed to the fast crosslinking of the surface of the alginate droplets in the CaCl2 solution when dispensed from the syringe pump. The hollow structure of the beads could improve the loading efficiency of the wastewater, thereby increasing the absorbance properties by trapping more wastewater contamination in the holes. In addition, the hollow structure provides a much larger surface area, allowing a higher surface reactivity and mass transfer, which also is beneficial for contamination adsorption [Both dry and swollen composite gel beads were microscopically investigated to study the surface and internal morphology. The external structure and colour changes from the dry alginate to the MNP–CNF/alginate beads as observed with an optical microscope are shown in sorption . The SEM3O4 magnetic nanoparticles into the alginate and CNF/alginate matrix. This incorporation influenced the thermal decomposition of the alginate and CNF/alginate because of the higher thermal stability of CNF due to their higher crystallinity than alginate, the higher thermal stability of iron oxide nanoparticles, and the uniform heat distribution of iron oxide across the CNF/alginate matrix [The thermal stability of the alginate and nanocomposite beads was evaluated using a TGA tool. e matrix ,46.The stress-strain properties of the three different gel beads in the swollen conditions are illustrated in The high-stress tolerance is attributed to the reinforcing effect of the cellulose nanofibres that absorb the compressive stress applied to the gel beads; therefore, it is unlikely that a certain extent of compressive stress will form cracks on the bead shell. It is also possible that the incorporation of MNPs resulted in augmented interfacial binding between the gel matrix and CNF, causing increased toughness and excellent energy dissipation capability during compressive loading  were also observed on the MNP–CNF/alginate gel beads, accounting for 21% of the total cumulative pore volume.2 adsorption capacity for the CNF/alginate and MNP–CNF/alginate gel beads is shown in 2. MNP–CNF/alginate adsorbed a higher amount of CO2 because of the larger surface area and porosity. The reflection of the adsorption and the filling of gas in micropores can be used to analyse the micropores [The COcropores ,58. The The pollutant concentrations in the wastewater before and after adsorption were characterised using ICP-AES at ALS Environmental Division, Brisbane, Australia . The conHybrid nanocomposites of CNF/alginate and MNP–CNF/alginate hydrogel beads were prepared and studied by analysing their morphology, swelling behaviour, thermal and mechanical strength, and pore structure. The lowest water swelling achieved for the MNP–CNF/alginate beads is owing to the addition of magnetic nanoparticles onto the reinforcing cellulose nanofibres.2 adsorption/desorption isotherm, the MNP–CNF/alginate gel beads adsorbed a maximum amount of N2 compared to the CNF/alginate gel beads in the relative high-pressure region. Both gel beads had pores smaller than 10 nm , and the CNF/alginate gel demonstrates a more homogeneous pore structure than MNP–CNF/alginate.The composite beads have a high compressive stress resistance, particularly the MNP–CNF/alginate beads, providing > 3.5% stress tolerance at ~82% compressive strain when a usual alginate bead breaks. A fully reversible stress-strain curve was obtained upon loading and unloading for both CNF/alginate and MNP–CNF/alginate beads, indicating the reusability of the gel beads in a packed column-based continuous water treatment process. According to the NThe adsorption capacity of both gel beads was found to be competent at a lower concentration of pollutants, including aluminium, potassium, selenium, sodium, vanadium, and sulphur. A higher adsorption capacity was observed for the MNP–CNF/alginate gel beads than the CNF/alginate beads due to their higher active centres for attachment of the pollutants. It is worth noting that the current work uses 50:50 volume ratio (as a basis) when mixing alginate with CNF or MNP–CNF solution. The high filler concentration resulted in a high diffusion barrier for water penetration into the nanocomposite beads. Lowering the filler concentration in the gel beads would give better adsorption results but at a compromised compressive strength. Compositional optimisation is hence needed, which we anticipate performing in our future research work.Overall, composite gel beads emerged with promising features for their application in the wastewater treatment process due to their physical and mechanical properties as well as their adsorption of pollutants . In contrast to conventional pollutant adsorbents that are toxic, the developed gel adsorbent is safe for users as well as environmentally friendly. While the strength of the current article lies in fundamental studies on pollutant adsorption and the robustness of nanocomposite beads, our future work will focus more on applied research, including packed column-based adsorption and regeneration studies. We also intend to utilise several analytical  and X-ray  techniques to evaluate pre- and post-adsorption and regeneration experiments. It is also essential to assess the antimicrobial properties of the gel beads to enhance user confidence in safety, and this is one of our future research goals.


Phlebotomus chinensis sandfly is a neglected insect vector in China that is well-known for carrying Leishmania. Recent studies have expanded its pathogen repertoire with two novel arthropod-borne phleboviruses capable of infecting humans and animals. Despite these discoveries, our knowledge of the general pathogen diversity and overall microbiome composition of this vector species is still very limited. Here we carried out a meta-transcriptomics analysis that revealed the actively replicating/transcribing RNA viruses, DNA viruses, bacteria, and eukaryotic microbes, namely, the “total microbiome”, of several sandfly populations in China. Strikingly, “microbiome” made up 1.8% of total non-ribosomal RNA and comprised more than 87 species, among which 70 were novel, including divergent members of the genera Flavivirus and of the family Trypanosomatidae. Importantly, among these microbes we were able to reveal four distinguished types of human and/or mammalian pathogens, including two phleboviruses (hedi and wuxiang viruses), one novel Spotted fever group rickettsia, as well as a member of Leishmania donovani complex, among which hedi virus and Leishmania each had > 50% pool prevalence rate and relatively high abundance levels. Our study also showed the ubiquitous presence of an endosymbiont, namely Wolbachia, although no anti-viral or anti-pathogen effects were detected based on our data. In summary, our results uncovered the much un-explored diversity of microbes harboured by sandflies in China and demonstrated that high pathogen diversity and abundance are currently present in multiple populations, implying disease potential for exposed local human population or domestic animals. Arthropod-borne diseases, such as dengue fever, forest fever, malaria, and leishmaniasis, pose a constant threat to public health across the globe, with newly emerging pathogens or variants occurring periodically and causing a greater number of human illnesses and deaths . To keepLeishmania endemic in over 90 countries and causing > 50,000 deaths per year across the globe [Phleboviruses mainly reported in countries along the Mediterranean coast [Bartonella and Coxiellaceae [Vesiculovirus and Orbivirus [Leishmania until a recent effort to look into viruses [Phlebovirus, suggesting their potential role in causing diseases in mammalian hosts, including humans [Sandflies  are flying, piecing, and blood-sucking vectors well known for transmitting diseases such as leishmaniasis  and papphe globe , whereasan coast . Other pellaceae ,8, and v viruses  and bactg humans . DespiteRecently, a whole transcriptome shotgun sequencing approach, namely meta-transcriptomics, has transformed our understanding of the RNA virosphere . To datePh. chinensis sandflies sampled in Shanxi province of China. The object of this study is to first reveal the entire diversity of virome, bacteriome, and eukaryotic microbiome, and from there further analyses were carried out to identify and characterize potential pathogens of humans or other mammalian species. And with that, our data provided a comprehensive evaluation of the disease risk of this neglected arthropod vector.In this study, we used the meta-transcriptomics approach to survey the “total microbiome” of https://www.globalsources.com/si/AS/Guangzhou-Changsheng/6008849913119/Homep-age.htm#) during the night (from 6.00am to 7.00pm next morning) [From June to July 2019 and 2020, we collected sandflies in four counties of Shanxi Province, China: HeJin, RuiCheng, WuXiang, and XiangNing. Sandflies were sampled using Wentaitai MM200 traps  . The colTo preserve the integrity of the RNA within the samples, the collected sandflies were first quickly identified by a magnifying lens at the subfamily (Phlebotominae) level using morphological characteristics listed below: (i) The adult head has large compound eyes and a pair of multiple antennae that are very long. Each antenna has 16 nodes: the base node is short, the second node is spherical, and the remaining 14 nodes are whip-shaped. And the mandible is very long, with 5 sections each, arranged on both sides of the beak and bent backward; (ii) a pair of wings on the chest is shaped like peach leaves. When resting, the wings and the chest form a "V" shape; and (iii) there are erect tufts or prone hairs on the back of the second to sixth sections of the back of the abdomen . The speSandfly specimens were grouped into 10 pools based on sampling location date, with each pool containing 50–100 sandflies. After being ground manually in the MEM medium, the homogenized samples were centrifuged at 12,000 rpm for 30 min at 4 °C to remove sandfly debris. Total RNA extraction and purification were carried out using the RNeasy Plus universal mini kit  according to the manufacturer’s instructions. RNA sequencing library construction and ribosomal RNA depletion were performed using the Zymo-Seq RiboFree™ Total RNA Library Kit . Paired-end (150 bp) sequencing of the 10 dual-indexed libraries was performed on a Novaseq platform at Novogene, China.https://sourceforge.net/projects/bbmap/), and the resulting clean reads were then assembled de novo into contigs using Megahit (version 1.2.8) [E-value threshold of 1E-5. Potential viral contigs were obtained based on the taxonomic information of the blast hits and were then merged into longer genomes using the seqman program implemented in the Lasergene software package version 7.1 (DNAstar) [Adintoviridae), and nonstructural protein 1 (Parvoviridae). To avoid misassembly, reads were mapped back to the virus genome with Bowtie2 (version 2.3.5.1) [Low-quality reads and adaptor sequences were first removed from the raw sequencing data using bbmap (n 1.2.8) . The assn 1.2.8)  with an DNAstar) . Host co2.3.5.1)  and insp2.3.5.1) .https://www.arb-silva.de/) using Bowtie2 [Clean reads associated with ribosomal RNA were first removed by mapping against the ribosomal RNA database downloaded from the SILVA website  were used to conduct similarity comparisons and phylogenetic analyses in order to determine which species or evolutionary group these microbes belong to. To estimate the relative abundance of each taxon in each library, the complete reference genome sequences of each genus were downloaded from GenBank and used as a mapping template for abundance estimation (RPM). To avoid bias in abundance estimation, highly conserved regions, such as rRNA genes, were removed from reference genome sequences before mapping analyses were carried out.Bacteria and eukaryotic microbes were firstly identified using MetaPhlAn2 . FurtherAdintoviridae) and NS1 protein (Parvoviridae) were used for phylogenetic analyses as described above. For bacteria and eukaryotes, nucleotide sequences of marker genes  were used for phylogeny inference.To determine the evolutionary history of the microbiome discovered here, we performed phylogenetic analyses based on protein and/or nucleic acid sequences of the conserved genes. For RNA viruses, RNA-dependent RNA polymerase (RdRp) of these viruses was aligned with those related viruses retrieved from GenBank using mafft . Ambiguovegan package version 2.5-6 implemented in R. The overall significance level was evaluated using ANOVA. Beta diversity was estimated based on the covariance matrix and evaluated using the Principal Component Analysis [pheatmap and ggplot2 packages [For each library, the alpha diversity was estimated by the Shannon Wiener index at the species level using the Analysis , and theAnalysis . Graphs packages .de novo assembled to form 69,337–215,424 contigs (Table S1). Among these, contigs associated with cytochrome c oxidase subunit 1 mitochondrial gene (i.e. cox1 gene) were used to confirm the morphologically identified sandfly species, that is, Phlebotomus chinensis. Importantly, the cox1 sequences obtained from the ten sequencing pools were highly similar to each other, with 97.6-100% nucleotide identities. Phylogenetic analyses revealed that these sequences formed a single cluster nested within the diversity of Ph. chinensis and related to a single sample, that is, hnf1 , sampled from the Henan province . As for bacteria, four genera, namely Acinetobacter, Pseudomonas, Rickettsia, and Wolbachia, had higher abundance levels and genome coverage rates than other bacteria so they were revealed with great certainty by our RNA sequencing. Among these, Wolbachia, an endosymbiont found in many insect species [Leishmania was identified from five of the ten pools, and its abundance level varied from 10 to 1472 RPM. Other pools also contain Leishmania-associated reads, although they were below the threshold to be considered positive (i.e. 10 RPM). Overall, the alpha-diversity of microbes, including viruses, bacteria, and eukaryotic microbes, was quite even across all libraries   contained the highest number of species, followed by Supergroups Toti-Chryso (n = 11), Mono-Chu (n = 9), and Narna-Levi (n = 7). In comparison, only three species of DNA viruses were identified, among which two belonged to the family Adintoviridae and one to Parvoviridae.This is the first time that the total virome of sandfly is systematically characterized. More than 78 species of RNA viruses were discovered in this study, the majority of which (66/78) were new viruses based on the demarcation criteria of <90% amino acid identity at the replicates (Table S3). The Supergroup Partiti-picobirna  and were found in more than half of the pools sequenced in this study (Table S3), suggesting high prevalence within the sandfly population examined here. Therefore, for new viruses with larger than 1000 RPM, host information was added to their naming because they are most likely to be associated with principal host Within each defined supergroup or family, the majority of the viruses were clustered within the viral groups harboured by other insect or arthropod hosts, and some shared close phylogenetic relationship (> 90% amino acid identity) with viruses identified from mosquitoes , aphids (Aphis citricidus meson-like virus), spiders (Hubei macula-like virus 3 and Guiyang nodavirus 2), and amongst others . Nevertheless, a few viruses with low abundance levels were bona fide fungal pathogens, including Fusarium graminearum hypovirus 2 (Hypo), Alternaria alternata partitivirus 1 (Partiti-Picobirna), Botrytis cinerea partitivirus 2 (Partiti-Picobirna), and Fusarium poae dsRNA virus 3 (Toti-Chryso). Furthermore, several newly identified species  under Supergroup Partiti-Picobirna were clustered within the diversity associated with viruses in basal eukaryotes , suggesting their potential association with the non-sandfly microbial host.Flavivirus, Alphavirus, Phlebovirus, Orbivirus, amongst others . Nevertheless, it did not cluster with mosquito-borne or tick-borne virus groups, which were known arboviruses but was presented as a divergent lineage clustered with a classic insect-specific flavivirus, which included a newly identified flavivirus associated with the Japanese horse fly (44.78% amino acid identity) [Among the discovered viruses, we further searched for viruses that can infect humans or other mammalian hosts, namely, arthropod-borne viruses or arbovirus, based on the phylogenetic relationship with the established arthropod-borne virus genera or groups, including genera t others . Only twll lines . Interesdentity)  as well Acinetobacter, Pseudomonas, Rickettsia, and Wolbachia. The Rickettsia identified in this study belonged to the well-established Spotted fever group (Rickettsia felis (97.37% identity at GroEL gene), the causative agent of flea-borne spotted fever in humans [Rickettsia discovered here to merit the assignment of a new species. On the other hand, Wolbachia was detected in all pools examined in this study and was genetically quite closely related to a group containing most of the reproductive parasites associated with insect hosts, including wMel, wAu, wRi, amongst others, although it is still unclear based on current phylogeny that how many different strains were present in the pooled samples , suggesting that these were biologically active microbes within the host.We have identified four bacteria of relatively high abundance levels from meta-transcriptomics: n humans . Given tLeishmania, an intra-cellular parasite associated with visceral leishmaniasis in humans. It was detected in at least five of the ten pools examined in this study, each with slightly different transcriptomic profiles and abundance levels  and Leishmania mexicana (92.15%) respectively based on hsp70 and beta-tubulin genes, phylogenetic analyses placed this newly identified microbe as orphan lineage sister to the entire subfamily Leishmaniinae, suggesting a highly distinctive organism.Our data revealed at least two species of eukaryotic microbes. One was Rickettsia sp., and a member of the Leishmania donovani complex. Among these, hedi virus was quite prevalent and distributed in Changzhi, Linfen, and Yuncheng, whereas the other three pathogens were distributed in Changzhi and Yuncheng  in mosquitoes [Wolbachia and the total virome, hedi virus, wuxiang virus, and Leishmania, and none resulted in a significant and strong relationship  had high prevalence and abundance in the region investigated. In addition to our findings, another study based on 16S metagenomic sequencing of sandflies collected from Henan, a neighbouring province of Shanxi, has also identified bacteria close related to Rickettsia bellii [Ph. chinensis in Northern China. Furthermore, the diversity and overall abundance of the pathogen in sandflies estimated in this study is much higher than another important insect vectors, mosquitoes, which mainly harboured viral pathogens with 0-0.0059% abundance levels based on the same meta-transcriptomics sequencing [Our study reveals that the sandfly is an important but neglected vector that harbours multiple types of infectious agents relevant to human health. Indeed, meta-transcriptomics sequencing has demonstrated with compelling molecular evidence that the presence and active biological activities of four pathogens or potential pathogens, including two viruses, one bacterium, and one eukaryotic microbe in a bellii , suggestquencing ,22,41, aLeishmania has been frequently reported in Shanxi province, which recorded 21.7% of the total mountain-type zoonotic visceral leishmaniasis cases between 2015 and–2019 in China, and, therefore, it is viewed as one of the hotspot regions for visceral leishmaniasis [Leishmania was 1.98% in female Ph. chinensis, indicating that the parasites were endemic within Ph. chinensis population [Leishmania parasites was well-recognized, the impact of other pathogens, namely, Rickettsia, hedi virus, and wuxiang virus discovered in this study, is unclear. One possibility is that these pathogens may cause sporadic self-limiting disease with non-specific symptoms, such as fever, rash, nausea, etc., which was reported in some infections caused by phleboviruses [The disease caused by maniasis . Furtherpulation . While toviruses  and rickoviruses . Withoutoviruses . TherefoPh. chinensis sandflies. Like what has been revealed in other arthropod species, such as mosquitoes [Ph. chinensis) based on the abundance levels, pool prevalence rate, and/or their phylogenetic positions, only a very small fraction is relevant for human infections (i.e. hedi virus and wuxiang virus), which is similar to the observations made in other blood-sucking arthropods, such as mosquitoes [In addition to those causing human disease, our study shows the entire virome of squitoes ,22, ticksquitoes , flies [squitoes , bees [4squitoes  and termsquitoes , the virsquitoes ,22,41, tsquitoes , midges squitoes , and batsquitoes . Also besquitoes .Ph. chinensis sandflies virome revealed here has substantial diversity overlap with the mosquito virome. Indeed, some viruses discovered here belonged to well-established mosquito virus species, such as the Zhee Mosquito virus [Also of interest is the observation that the to virus , Hubei mto virus , and Wuhto virus , etc. ItPh. chinensis sandfly populations in Shanxi province, mainly through their non-ribosomal transcriptomic profiles. Nevertheless, the bacteriome obtained here is strikingly different from those described in a previous survey based on the 16S sequencing, and the latter study was carried out on the same sandfly species sampled in the neighbouring provinces [Wolbachia was detected in all libraries whose abundance levels are orders of magnitude higher than the other bacterial taxa, whereas the 16S-based approach reveals Pseudomonas, Enterobacteriaceae, and Coxiellaceae as the dominant taxa. While it is possible that such discrepancies are caused by sampling locations or subpopulations of sandflies, it could also be due to differences in the target nucleic acids, namely, DNA or RNA, sequenced in these two studies. The latter is more likely given that RNA sequencing is prone to reflect active microbial populations and hence results in much higher abundance levels for endosymbionts than those estimated by DNA sequencing [Our study also revealed the diversity of bacteria and eukaryotic microbes from the rovinces . On the rovinces ,54. On tquencing .Wolbachia, a reproductive parasite affecting a wide range of invertebrate hosts [Ph. chinensis sandfly populations in Shanxi. The organism has gained increasing interest in its potential use as a vector-borne disease control agent, owing to its capability to reduce the vector population by a mechanism of cytoplasmic incompatibility [Wolbachia infection and the presence and abundance of arthropod-borne pathogens, namely, hedi virus, wuxiang virus, and Leishmania. One explanation is that not all Wolbachia strain is protectively against virus replications, as has been demonstrated based on a variety of Wolbachia strains in Drosophila [Our study reveals that te hosts , is prestibility  as well tibility ,57. Neveosophila ,59. Alteosophila . Indeed,osophila .Our study presents an efficient yet comprehensive “total microbiome” approach for pathogen discovery in disease vectors such as sandflies. With a single meta-transcriptomics sequencing, it carries out a thorough investigation of viral, bacterial, and eukaryotic microbes under active biological processes within vectors, with further information from genome/transcriptome comparisons, and abundance estimation as well as phylogenetic analyses to identify potential pathogens and evaluate their threat to human health. This is especially relevant for sandflies where viral, bacterial, and eukaryotic pathogens were simultaneously present in the same region. Therefore, using the “total microbiome” approach it will prevent repeated efforts to discover different types of pathogens one at a time and provide the much-needed speed to detect emerging pathogens from a large number of vector samples.Rickettsia in human infections was only assessed with sequence data, which remains to be confirmed with data from tissue culture, animal experiments, and/or serological surveys. Nevertheless, despite the limitations, our study presents the first effort to characterize the total microbiome in sandflies, and such applications can be extended to other vector species, allowing more efficient discovery and characterization of potential pathogens in vectors.Finally, our study had several limitations: first, the sampling scope is limited in terms of sandfly species and geographic regions covered, and a more systematic meta-transcriptomics survey is needed to fully capture the microbial diversity within different sandfly species and across a broader geographic range in China. Furthermore, the host association remains unclear for a number of low abundance level microbes with distinguished phylogenetic positions, and it is highly likely that they might be associated with food/symbionts instead of the sandflies. Finally, the potential role of the newly identified Click here for additional data file.


Tobamovirus causes serious losses in the quantity and quality of tomato production. The management of plant protection is very difficult, mainly due to the vector-less transmission of ToMV. Resistant breeding generally has low effectiveness. The most practical approach is the use of a rapid diagnostic assay of the virus’ presence before the symptoms occur in plants, followed by the eradication of virus-infected plants. Such approaches also include serological detection methods (ELISA and Western immunoblotting), where antibodies need to be developed for an immunochemical reaction. The development and characterization of polyclonal antibodies for the detection of ToMV with appropriate parameters  were the subjects of this study. A new polyclonal antibody, AB-1, was developed in immunized rabbits using the modified oligopeptides with antigenic potential  derived from the coat protein of ToMV SL-1. the developed polyclonal antibody. AB-1, showed higher sensitivity when compared with commercially available analogs. It also detected ToMV in infected pepper and eggplant plants, and detected another two tobamoviruses (TMV and PMMoV) and ToMV in soil rhizosphere samples and root residues, even two years after the cultivation of the infected tomato plant.Plant viruses threaten agricultural production by reducing the yield, quality, and economical benefits. Tomato mosaic virus (ToMV) from the genus  Solanum lycopersicum L.) is a member of the Solanaceae family that includes more than 3000 plant species. It is an economically important crop cultivated world-wide in almost any climate and environment. Tomato plants constitute a host for many pathogens, including fungi, bacteria, nematodes, and viruses [Tobamovirus genus, family Virgaviridae, including 37 species with a monopartite positive sense ssRNA(+) genome [The tomato  genome .Tobamoviruses naturally infect the tomato, such as Tomato mosaic virus (ToMV), Tobacco mild green mosaic virus (TMGMV), Tobacco mosaic virus (TMV), Tomato brown rugose fruit virus (ToBRFV), and Tomato mottle mosaic (ToMMV) [Tobamoviruses are considered as unusual plant viruses due to their ability of transmission between host plants without living vectors [Tobamoviruses are known as very resilient and remain infectious in contaminated soil and plant debris [Many  (ToMMV) . Tobamov vectors . Tobamovt debris , irrigatt debris , and alst debris . Among tt debris . Besidest debris .Tm-1, Tm-2, and Tm-22, have been found in Lycopersicon hirsutum L. and Lycopersicon peruvianum L. and were introduced into commercial lines of tomato [There are two main strategies for alleviating the problems of ToMV in tomato growth. One is the genetic resistance of tomato in protecting plants against ToMV. Three dominant resistance genes, f tomato ,16. Howef tomato ,18.Accurate and sensitive diagnostics of ToMV are the second-most-essential tool for virus disease management in tomato cultivation. Highly specific detection methods are based on the polymerase chain reaction (PCR) and, together with high-throughput sequencing technologies, provide accurate identification of viruses, multiplex virus detection, virus quantification, as well as the opportunity to discover new emerging viruses ,20. The Tobamoviruses. Additionally, the International Seed Federation (2019), representing the global seed industry, has drawn attention to the cross-reactivity of polyclonal antibodies for ToMV with other Tobamoviruses  [Tobamoviruses, and tested the antibody for the analysis of the persistence and resilience of ToMV in soil and plant residues.ELISA is usually associated with lower specificity than nucleic acid-based detection . It is a ToBRFV) . Howeverhttps://www.ncbi.nlm.nih.gov/nucleotide/, accessed on 27 May 2022). This analysis showed that the source tomato plant was infected solely by ToMV without co-infection with other viruses.The ToMV isolate SL-1 was obtained from the collection kept at the Research Institute of Plant Production . The virus originated from a tomato plant manifesting symptoms of virus infection at a local tomato grower in Western Slovakia. Leaves from this plant were collected and stored at −80 °C. The nucleotide sequence of the complete genome of the ToMV SL-1 isolate was determined previously by high-throughput sequencing  and inclThe tomato variety Monalbo was used for mechanical artificial inoculation with the original ToMV SL-1 isolate. ToMV SL-1-positive plants were maintained under insect-free conditions, and collected leaves were stored at −80 °C for further experiments. Subsequent artificial infections were conducted in the commercial tomato varieties Mobaci, Monalbo, and Niki Zel F1, and in four breeding lines, 706/15, 730/15, 756/15, and 762/15, developed by Zelseed spol. s r. o. .Leaf samples from two ToMV SL-1-infected tomato lines (730/15 and 762/15) were used for the confirmation of isolate identity. RNA was isolated using the NucleoSpin RNA Plant Mini kit for RNA from plants  and reversely transcribed to cDNA using the RevertAid First Strand cDNA Synthesis Kit . Selected regions of the ToMV genome  were ampThe ORF4 encoding the coat protein of ToMV was selected for antibody development as the most used for antibody development .https://www.uniprot.org/uniprot/, accessed on 27 May 2022). Homology in the coat protein sequences within them was in the range of 98.6–99.1% [2-RFPGDVYKVYRYNAVLDC-COOH and Epi 2: NH2-CESMSGLVWTSAPAS-COOH, were used for the immunization of a New Zealand White rabbit by injection with 3 mg of epitope conjugated with keyhole limpet hemocyanin protein .Specific ToMV epitopes were designed by evaluating the antigenic potential of the amino acid sequences of coat proteins from ToMV SL-1 and fifteen other ToMV isolates retrieved from the UniProt knowledgebase   at 405 nm wavelength using alkaline phosphatase-conjugated Goat anti-Rabbit IgG (H + L) , whereas immunized sera consistently showed higher absorbance levels, confirming the successful production of polyclonal antibody capable of recognizing ToMV SL-1 coat protein epitopes.−2.s−1. The control, non-infected plants were grown under the same conditions. Samples of inoculated plants were collected during the early infection phase  and also in the late infection phase (7 and 14 days after inoculation).Tomatoes were mechanically inoculated with ToMV isolate SL-1 for the comparison of the diagnostic properties of the tested antibodies using ELISA. Artificial inoculation was conducted at the stage of two true leaves by deep-frozen ground leaf samples of ToMV SL-1 isolate resuspended in Norit buffer . Mechanical wounding with sterile silicon carbide powder Merck was used for the inoculation of tomato plants. All plants were cultivated under a photoperiod of 16 h/8 h (light/dark) at 24–25 °C, with light intensity of 152 μmol.mThe sensitivity and specificity of the newly developed polyclonal antibody (AB-1) were tested by a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA)  and Westw/v) containing 0.05% Tween-20 and 2% polyvinylpyrrolidone, and subsequently applied to the ELISA plate. All used and compared primary antibodies  were diluted at ratios of 1:100; 1:500; 1:1000; 1:5000; and 1:10,000 (v/v) starting from the same concentration of 5 μg/μL of total protein. The plant sap was diluted for sensitivity testing at ratios pf 1:10; 1:100; 1:1000; and 1:10,000 (v/v). Chromogenic reactions were measured by absorbance at 405 nm . All ELISA results that were compared with each other were measured in samples at the same time and on the same ELISA plate.Extracts from fully developed leaves (0.15 g) of the inoculated plant were homogenized in PBS buffer . A standard curve prepared using bovine serum albumin standards was applied for the calculation of the protein concentrations determined using the Pierce TM BCA Protein Assay Kit . The specific protein of interest, representing the ToMV coat protein, was detected by the Western blotting technique [TM Prestained Protein Ladder 10 to 180 kDa and PageRuler TM Plus Prestained Protein Ladder 10 to 250 kDa, ThermoFisher Scientific, Waltham, MA, USA). The immunoreactive protein was visualized using the 1-step TM Ultra TMB-Blotting Substrate Solution , recorded using a Nikon D3100 camera , and evaluated using the Image Studio Lite Quantification Software .Proteins for Western immunoblotting analysis were extracted from the tomato plants using the P-PERechnique . All anaThe cross-reactivity of the AB-1 antibody with TMV and PMMoV viruses was tested in Niki Zel F1 plants mechanically inoculated with five ToMV isolates , one TMV isolate (PV-0107), and one Pepper mild mottle virus . All were obtained from the collection of viruses of the Leibniz Institute DSMZ—German Collection of Microorganisms and Cell Cultures .Capsicum annum L.) and eggplant (Solanum melongena L.). Host plants with 3–4 developed leaves were inoculated with the ToMV SL-1 isolate and analyzed by Western immunoblotting using the AB-1 antibody 21 days after inoculation.Systemic infection was tested in pepper .The two-way ANOVA and Tukey honest significant difference tests were performed using the software R version 4.0.4 , none of the antibodies were able to detect the presence of ToMV .v/v) was the strongest in the newly developed AB-1 antibody (p < 0.05) of the AB-1 antibody against all others in both dilutions.Different dilutions of all used primary antibodies were also tested by ELISA. The concentrations of all antibodies used in analyses, commercial as well as AB-1, were adjusted to the same value of 5 µg/µL of total proteins. The sensitivity at antibody dilutions of 1:100; 1:500, 1:1000; 1:5000; and 1:10,000  were tested by ELISA for the ability to detect the virus in ToMV-positive plants of the Niki Zel F1 variety as soon as possible after virus infection. A different leaf from the plant than the one infected with the virus was always used for analysis. Only the AB-1 antibody was able to confirm the presence of ToMV very early on, only 24 h after artificial infection. Late infection was tested one and two weeks after inoculation, and all antibodies were able to detect the virus. The basic parameter of any antibody is the ability to detect all isolates of the given virus, but at the same time, not to cross-react with other, albeit very similar, viruses. Altogether, six ToMV isolates , one TMV isolate (PV-0107), and one PMMoV isolate (PV-0166) were used for the artificial infection of tomato plants (variety Niki Zel F1). Plants were scored for systematic infection by Western immunoblotting analysis using the AB-1 antibody. All ToMV isolates as well as TMV and PMMoV reacted with the AB-1 antibody . The sizThe cross-reactivity of the AB-1 antibody was tested in pepper and eggplant. The pathological symptoms that occurred demonstrated the aggressiveness of the ToMV isolate SL-1 in all three plant species. The infected plants exhibited stunted growth and curling and mottling of the leaves. Symptoms began to manifest as a slight veining of the leaves, which led to a typical mosaic of green and yellow chlorotic spots that subsequently led to necrosis. The fruits of the infected tomatoes ripened later than those on the healthy control plants, were smaller, and had a variety of symptoms, including uneven coloration, a roughened fruit surface, and brown necrotic spots. The symptoms gradually led to the death of the infected tomato plants. The infected pepper and eggplant plants also showed symptoms associated with the presence of ToMV, such as leaf mosaic on eggplant and a hypersensitive reaction inducing necrotic lesions on the pepper plant, followed by the discoloration and shedding of leaves A–C. WestTm-1 resistance gene [p < 0.05). Among the genotypes, tomato line 762/15 accumulated a statistically significantly higher level of virus than all of the others (p < 0.05).Two tomato varieties and four breeding lines infected by ToMV SL-1 were tested by Western immunoblotting for the presence of the virus in roots and seeds. The presence of ToMV was confirmed both in seeds and roots of all infected tomato genotypes . The AB-nce gene . BreedinTobamoviruses are known to be persistent genetic elements that are able to survive long-term in the environment where they previously infected plants [Tobamoviruses also have extremely stable virions [d plants . The vir virions . The resAntigens for specific antibody development can be prepared by the purification of native virus particles isolated from an infected plant, subsequent cloning of a relevant gene from the viral genome (most commonly encoding the coat protein of the virus), immunization of an animal, and activation of the immune response, and by the purification of a desired antibody. However, this process is tedious and time-consuming. The simplified process is the in silico approach, where initial experiments are omitted and antibody production in an animal is induced using information from databases of viral genome and protein sequences, synthesizing of the protein for animal immunization, and subsequent purification of the antibody. Another approach, suitable mainly for the commercial production of antibodies, is the production of recombinant antibodies by the expression of the relevant gene from the virus in the host organism. These three main approaches produce monoclonal, polyclonal, or recombinant antibodies .Tobamoviruses, e.g., with Tobacco Mosaic Virus (TMV). However, most of the developed mono-clonal antibodies for ToMV detection did not cross-react with TMV [Monoclonal antibodies for ToMV detection are being developed with the aim to eliminate cross-reactivity with other tobamoviruses invading tomatoes . Howeverwith TMV .Polyclonal antibodies are easier to develop and apply for fast and routine diagnostics in phytopathology and agricultural practice by ELISA and Western immunoblotting detection assays. They recognize multiple similar epitopes of an antigen of interest, resulting in better sensitivity of routine assays. Their polyclonality allows the binding of multiple antigenic determinants of the target, better sensitivity in certain assays, and greater stability . GeneralCommercially available ToMV antibodies with an unknown recognition site origin are widely used in virological studies and in agricultural practice. The reason for the development of a new polyclonal antibody for ToMV detection within this study resulted from the absence of detailed information about the recognition site of commercially available polyclonal antibodies. Data regarding the epitope design were not available for commercially available antibodies and thus could not be compared. A common problem during the development of plant virus antibodies is acquiring purified protein for the immunization of laboratory animals . The synNewly developed polyclonal antibodies must have similar or better diagnostic properties, detection limit, and desired cross-reactivity than those of commercial ones. The antibody presented in this work was developed by designing an epitope-based peptide antigen (coat protein) of a specific ToMV isolate, SL-1. The high functionality of the developed polyclonal antibody (AB-1) and detection properties comparable to or better than those of commercially available analogs were confirmed by Western immunoblotting and ELISA analyses. The AB-1 antibody generated a stronger signal in extracts from the ToMV-infected plant in comparison with commercially available antibodies. It was the only antibody capable of detecting virus particles in the plant sap diluted at the ratio of 1:1000, which indicated its detection sensitivity. At the highest used dilution of antibody , the AB-1 antibody generated the strongest signal. It should be repeated here that the sensitivity of all antibodies compared  was tested at the same protein concentrations in the antibodies.In addition, AB-1 was the only one of the tested commercial antibodies for detection that was capable to detect ToMV in the very early phase of plant infection, as early as 24 h after virus inoculation. This may be a result of the epitope design aiming for a high antigenic response ,33. ThroBegomovirus [Tobamoviruses. The antibodies developed for Pepper mild mottle virus (PMMoV) specifically detected another four Tobamoviruses (including ToMV) in an antibody dilution of 1:1000 and infected plant sap dilution of 1:5120 [In addition to tomato plants, the AB-1 antibody was also capable of clearly detecting the presence of ToMV SL-1 in artificially infected pepper and eggplant plants. Polyclonal antibody AB-1 proved to be capable of detecting all available ToMV isolates and another two tested tobamoviruses (TMV and PMMoV). The cross-reactivity of the ToMV antibody with PMMoV observed here was not reported for the commercial antibodies tested in this study. Generally, the sensitivity and other parameters of non-commercial polyclonal antibodies developed by researchers for their specific applications vary. For example, antibodies developed for the detection of Pepper yellow leaf curl Thailand virus (PepYLCTHV) showed positive cross-reaction with Tomato leaf curl New Delhi virus (ToLCNDV), both belonging to the genus omovirus . Polycloomovirus . The samomovirus  and othef 1:5120 . The polf 1:5120 . The titThe polyclonal antibody, AB-1, developed in this study is suitable for use in agricultural practice and research. Its parameters are the same or better than those of commercial ToMV antibodies. This antibody, by immunochemical detection techniques (ELISA and Western immunoblotting), can also be applied in the wide screening of ToMV in tomato germplasm , parental genotypes and breeding lines (tomato breeding programs), and in tomato seeds during the seed certification process, i.e., in activities where the number of samples is high . Simple


Xc) were greater than 97%. Rising the bed height or reducing the flow rate enhanced the theoretical adsorption performance metrics, such as theoretical breakthrough time  and theoretical breakthrough adsorption capacity , whereas increasing the inlet concentration resulted in tB,th shortening and QB,th rising. As demonstrated in this paper, the highest tB,th and QB,th were 69.60 min and 324.50 mg g−1, respectively. Meanwhile, the spent SSA/MCM-41 could be desorbed and regenerated for cyclic reuse. Moreover, two recoverable adsorbed products, 4-isopropylbenzenesulfonic acid and 4, 4′-sulfonyl bis(isopropyl-benzene), were successfully separated and identified using FTIR and 1H/13C NMR characterization. Accordingly, the relevance of a reactive adsorption mechanism was confirmed. This study suggests that the SSA/MCM-41 has remarkable potential for application as an adsorbent for the resource treatment of cumene pollutants.Efficient removal of cumene from gaseous streams and recovery of its derivatives was accomplished using a MCM-41-supported sulfuric acid (SSA/MCM-41) adsorbent. The results indicated that the removal performance of the SSA/MCM-41 for cumene was significantly influenced by the process conditions such as bed temperature, inlet concentration, bed height, and flow rate. The dose–response model could perfectly describe the collected breakthrough adsorption data. The SSA/MCM-41 adsorbent exhibited a reactive temperature region of 120–170 °C, in which the cumene removal ratios ( Volatile organic compounds (VOCs) have a significant role in the formation of secondary organic aerosols and the destruction of atmospheric ozone. Moreover, their toxicity and pathogenicity are also constantly warned ,2,3. Amoo-xylene simulated waste gas stream [Cin), bed height (h), and flow rate (νg) to investigate the cumene adsorption behavior; and (3) to characterize the adsorbed species to explore the mechanism underlying cumene removal from the gaseous stream. We believe that the obtained results will be helpful to further understand the treatment potential of this novel gas–solid adsorption approach for benzene derivatives.Among various traditional methods for VOCs purification, destructive purification techniques, such as catalytic oxidation ,8,9,10, s stream ,22,23,24Xc − T) curve presented an inverted U-shaped profile, indicating that the temperature range of 120–170 °C was apt for the reactive adsorption of cumene on MCM-41-supported sulfuric acid (SSA/MCM-41). The two sides of the U-shaped profile represent the activation (upward trend) and the deactivation regions (downward trend). This adsorption behavior is similar to o-xylene on various supported sulfuric acid systems such as SSA/MCM-41, SSA/SBA-15, SSA/SG [o-xylene, cumene was likely removed from the gas stream by the sulfonation reaction with the anchored H2SO4 molecules on SSA/MCM-41. Therefore, the cumene removal process was similarly referred to as reactive adsorption. As shown in , SSA/SG , and two, SSA/SG . As a reXc ≥ 95%), a middle breakthrough phase of rapidly decaying adsorption (5% < Xc < 95%), and an ending phase of inefficient reactive adsorption (Xc ≤ 5%). This behavior is consistent with typical physisorption or chemisorption in gas–solid systems [−1 for the tB and QB metrics, respectively.In order to further understand the temperature dependence of SSA/MCM-41 in the reactive adsorption of cumene, dotted plots were developed based on the dose–response model-fitting curves. These curves help describe the dynamic adsorption breakthrough behavior at different bed temperatures, as depicted in  systems ,26,27. I systems . FurtherR2) higher than 0.995 , gas flow rate (v), and bed height (h) on the cumene adsorption performance. The experimental dotted plots and the corresponding dose–response model-fitting curves obtained from the seven experiments are shown in Under the optimum temperature of 160 °C, the cumene breakthrough curves in the SSA/MCM-41-filled bed were studied at different process conditions  to analy(1)All breakthrough profiles presented typical S-like curves, indicating the presence of a strong interaction between the cumene molecules and the SSA/MCM-41 surface, attributable to their sulfonation reaction (see the next section).(2)v = 50 mL min−1 and Cin = 9.2 mg L−1, when h rose from 10.5 to 20.0 mm (Exp. nos. 1–3), the curves shifted significantly to the right-hand side, the tB,th was highly extended, and the QB,th values increased, indicating that a prolonged residence time could improve the total removal amount of cumene and enhance its specific capacity. This finding was backed up by another set of experiments  in which the residence time was prolonged by reducing v from 75 to 25 mL min−1 under the same conditions of Cin = 9.2 mg L−1 and h = 14.3 mm. These experiments demonstrated the same response trend regarding both tB,th and QB,th metrics.At the stable conditions of (3)Cin from 5.3 to 14.6 mg L−1  resulted in tB,th shortening and increased QB,th. These changes could be attributed to the relative shortage of the SSA adsorption sites and the enhancement of cumene reaction driving force caused by the Cin increase [When controlling the residence time at the same value in the third experiment, changing increase .(4)tB,th (69.60 min) and QB,th (324.50 mg g−1) were obtained in Exp. No. 6, and the corresponding optimal process conditions were 9.2 mg L−1 for Cin, 14.3 mm for h, 25 mL min−1 for v, and 160 °C for T. The QB,th value identified that SSA/MCM-41 outperformed a silica-alumina material  and activated carbon  on the adsorption capacity of cumene [In the experiments mentioned above, it was found that both maximum values of f cumene ,30, whic1H/13C NMR for the adsorbed products I and II and their mixture products are depicted in 1H NMR characterization of the mixture products obtained  and the MCM-41 support from the spent SSA/MCM-41. Thus, it enabled recovering the adsorbed products and regenerating the support. The regenerated MCM-41 could be reused to prepare the novel SSA/MCM-41 via wet impregnation. The chemicals and reagents used in the study were of analytical grade, which included sulfuric acid  and cumene . In addition, MCM-41 (28–60 nm) was purchased from Nanjing Nanotechnology Co., Ltd., Nanjing, China.−1 sulfuric acid solution at room temperature for 1 h. The sample was then dried in an oven at 80 °C for 48 h to obtain an adsorbent with sulfuric acid loading of 9.25 mmol g−1, which was regarded as an optimized sample identified from the preliminary studies [The supported sulfuric acid, SSA/MCM-41, was typically prepared by wet impregnation in an appropriate volume of sulfuric acid solution. Specifically, 8 g of MCM-41 was pulverized and passed through a 0.15 mm sieve and was impregnated with 14.8 mL of 5.0 mol L studies . Finally1H NMR and 13C NMR; Bruker AVIII500, Brucker, Karlsruhe, Germany) were performed to confirm the molecular structure of the adsorbed products.The adsorbed products were characterized by two techniques. First, Fourier-transform infrared spectra  based on the KBr tablet method were employed to identify the molecular composition. Proton and carbon nuclear magnetic resonances ((1)Xc) was measured while heating gradually from room temperature up to 300 °C at 5 °C min−1 under v = 50 mL min−1, Cin = 9.2 mg L−1, and h = 14.3 mm. First, the Xc values were calculated via Equation (1). Then, the values were used to plot an Xc − T curve to analyze the reactive adsorption behavior of cumene upon the variation of the adsorption bed temperature.The removal rate of cumene ((2)h), flow rate (v), or inlet concentration (Cin) with the other two variables kept constant  in the gas flow at time t were determined experimentally. The extensive data collected were treated to draw the experimental breakthrough adsorption curves. These curves were then used to demonstrate the reactive behavior of SSA/MCM-41 toward cumene adsorption at various process conditions and to determine the experimental adsorption performance metrics such as breakthrough time  and breakthrough adsorption capacity . Here, tB was the time corresponding to the ratio Cout,t/Cin = 0.05 on the curve, and QB was calculated from Equation (2).A series of transient adsorption experiments for the cumene/SSA/MCM-41 system was performed by independently changing the bed height (tant see . The outReactive adsorption tests for removing gaseous cumene were carried out in a continuous flow glass column (6 mm i.d. × 40 cm length). SSA/MCM-41  was mixed with quartz sand  and packed into the glass column reactor with inert glass fiber plugs at both ends. The gas flow rate was controlled with a mass flowmeter , and the cumene concentration was monitored on a GC7900 gas chromatograph  equipped with a flame ionization detector (FID) and a polyethylene glycol (PEG)-20M capillary column (30 m × 0.32 mm). In order to explore the adsorption behavior and performance of SSA/MCM-41 for cumene pollutants, a three-pronged study was performed:(3)tB,th/min and QB,th/mg·g−1).Based on our previous work ,29, the q0 and a are constants of the dose–response model.1:V2 = 3:1) as eluent. Finally, the structures of the products were analyzed by FTIR and 1H/13C NMR.The SSA/MCM-41 sample exposed to cumene reactive adsorption is labeled as the spent adsorbent. First, the spent SSA/MCM-41 (5 g) was extracted with 20 mL absolute ethanol at room temperature. Then the product formed in the adsorption process was extracted by rotating evaporation of the extracted solution. In the second step, the solid filtered out from the previous step was washed with distilled water and dried at 100 °C to attain the regenerated MCM-41, which was reused for preparing the SSA/MCM-41 according to the same procedure described above. Next, the product obtained in the first step was dissolved in a small amount of water and then extracted with ethyl acetate (20 mL × 3). The resulting organic phase was dried with anhydrous sodium sulfate overnight, after which the solvent was filtered and concentrated. After that, the product was purified by silica gel column chromatography using a mixture of petroleum ether and ethyl acetate . Therefore, it can be concluded that cumene removal from the gaseous stream by SSA/MCM-41 followed a reactive adsorption mechanism, which combined the adsorption with sulfonation reactions between the cumene and anchored H2SO4. In addition, the spent SSA/MCM-41 could be desorbed and regenerated for repeated use.The SSA/MCM-41 sample with sulfuric acid loading of 9.25 mmol g