Brucella spp. causing illness, describe non-Brucella bloodstream infections, and identify risk factors for brucellosis in febrile patients from a pastoralist community of Tanzania. Fourteen (6.1%) of 230 participants enrolled between August 2016 and October 2017 met study criteria for confirmed (febrile illness and culture positivity or ≥four-fold rise in SAT titre) or probable (febrile illness and single SAT titre ≥160) brucellosis. Brucella spp. was the most common bloodstream infection, with B. melitensis isolated from seven participants and B. abortus from one. Enterococcus spp., Escherichia coli, Salmonella enterica, Staphylococcus aureus and Streptococcus pneumoniae were also isolated. Risk factors identified for brucellosis included age and herding, with a greater probability of brucellosis in individuals with lower age and who herded cattle, sheep or goats in the previous 12 months. Disease prevention activities targeting young herders have potential to reduce the impacts of human brucellosis in Tanzania. Livestock vaccination strategies for the region should include both B. melitensis and B. abortus.Brucellosis is an endemic zoonosis in sub-Saharan Africa. Pastoralists are at high risk of infection but data on brucellosis from these communities are scarce. The study objectives were to: estimate the prevalence of human brucellosis, identify the  The Brucella species that most commonly cause human infections are B. melitensis, B. abortus, and B. suis5. These species are classically associated with small ruminants, cattle, and swine, respectively but transmission between animal hosts is possible7. Transmission from animals to people is typically via direct contact with infected animals, foodborne transmission, or indirect contact with contaminated environments2. Human to human transmission is negligible8. Human brucellosis typically presents as non-distinct acute or chronic febrile illness10, and is frequently clinically misdiagnosed as other causes of febrile illness, such as malaria or typhoid fever11. Brucellosis is seldom fatal, but chronic infection is often debilitating and severe complications may occur10.Brucellosis is a globally widespread zoonotic disease13. Within pastoral systems where people live in close contact with livestock, there is increased risk of human infection14. Approximately 16% of the population of sub-Saharan Africa practice pastoralism. Within Tanzania, it is estimated that up to 40% of the population practices exclusive pastoralism15. As is true regionally, data on brucellosis prevalence and incidence are limited for these communities17.Although not as well recognised as in northern Africa, the Middle East or central Asia, brucellosis is endemic in many regions of sub-Saharan Africa18 and 8.9% for confirmed or probable brucellosis in 2012–201419 by Brucella microagglutination testing. Serological testing cannot be used for Brucella spp. identification20. Brucella spp. can be identified through molecular diagnostic analyses of DNA obtained from culture isolates21 or clinical samples, or with phenotypic testing of isolates2. Studies in sub-Saharan Africa that have identified the infecting Brucella spp. from human infections are rare22. There are no human Brucella spp. isolates from Tanzania recorded to date. Livestock isolates from Tanzania are also scarce, but both B. melitensis and B. abortus have been isolated, from goats and cattle, respectively24. Identification of the Brucella spp. causing human illness is vital to inform understanding of the most likely animal source population(s) and the design of vaccination strategies, as brucellosis vaccines are animal host specific.Hospital-based febrile surveillance studies of a predominantly urban population in Tanzania, have generated prevalence estimates of 3.5% for confirmed brucellosis in 2007–2008Brucella spp. are typically a relatively infrequent cause of human bloodstream infections in sub-Saharan Africa as compared to other regions. In sub-Saharan Africa the predominant causes of bloodstream infections are Salmonella enterica, Streptococcus pneumoniae, Staphylococcus aureus, and Escherichia coli26. Bloodstream infection data from communities considered at a high risk of brucellosis within sub-Saharan Africa are scarce.Brucella spp. exposure29. One study, conducted within a largely urban population in Tanzania identified assisting in small ruminant births and contact with cattle as risk factors for human brucellosis19. Consumption of boiled or pasteurised dairy products was a protective factor19.There are few studies in East Africa that have identified risk factors for acute brucellosis infection as compared to Brucella spp. responsible for human brucellosis in Tanzania. The objectives of this study were to estimate the prevalence of human brucellosis, identify the Brucella spp. causing human illness, characterise additional causes of bloodstream infections, and identify risk factors for brucellosis in febrile hospital patients from a pastoralist community of Tanzania.To our knowledge, no studies have identified the 2, designated for the pastoralist activities of the local Maasai community, the conservation of wildlife, and tourism30. Livestock keeping is ubiquitous among the Maasai. Cattle, sheep, and goats are the predominant livestock species kept and are managed extensively in mixed herds30. Endulen Hospital is the only hospital within the NCA. It is a 110-bed hospital providing both outpatient and inpatient services to a population of approximately 77,000 persons predominantly resident within the NCA31.The study was conducted at Endulen Hospital in the Ngorongoro Conservation Area (NCA), northern Tanzania. The NCA is a multiple land use area of 8,292 kmAll individuals seeking care at the outpatient department of Endulen Hospital were eligible for screening, which was integrated into the patient triage processes and performed by Endulen Hospital staff. All individuals aged ≥two years with reported fever within the past 72 hours and/or a tympanic temperature of ≥38.0 °C at presentation were eligible for enrolment. After initiation of the outpatient visit and routine clinical assessment by Endulen Hospital staff, eligible patients were approached by a member of the study team for informed consent to participate in the study.Brucella serology, and malaria testing. The target blood volume at enrolment was 40 mL, enabling distribution of two 10 mL volumes into BacT/ALERT  aerobic blood culture bottles for automated culture, 10 mL into Castañeda media32 , Weybridge, UK), and 10 mL into a plain vacutainer  for serology. For study participants weighing less than 25 kg, target blood volumes were determined based on weight33 and paediatric BacT/ALERT bottles were used for automated culture. For all study participants, a fill order algorithm was followed, with sample collection prioritised as follows: first automated aerobic blood culture bottle, plain vacutainer, Castañeda culture bottle, and second automated aerobic blood culture bottle. Malaria rapid diagnostic testing was performed directly from the sample collection syringe using the SD BIOLINE Malaria Ag P.f/Pan rapid diagnostic test  or CareStart Malaria HRP2 (Pf) .After cleaning the participant’s skin with isopropyl alcohol and povidone iodine, blood was drawn from study participants for culture, A member of the study project team administered a structured, closed-ended questionnaire for each study participant (Supplementary Methods). Question topics included: demographic data, symptoms of current and recent illness, dietary practices, and animal-related activities. Data collected from study participant clinical records included the the initial clinical diagnosis recorded and any drug treatments prescribed on the day of hospital presentation .All participants were approached for convalescent-phase blood sampling at their homes four to six weeks after enrolment. Up to 10 mL blood was collected into a plain vacutainer for convalescent-phase serology.34. Tinytag Transit 2 Temperature Data Loggers  were used to monitor transport temperature. Inoculated culture bottles were transferred to the Kilimanjaro Clinical Research Institute (KCRI) in Moshi, Tanzania, for laboratory processing the day after inoculation. Time and date of inoculation and receipt at the laboratory were recorded. Prior to incubation, the bottom of the BacT/ALERT blood culture bottle was visually assessed to confirm no colorimetric change. BacT/ALERT bottles were then loaded into the BacT/ALERT 3D instrument and incubated for up to 5 days. Castañeda bottles were incubated in a CO2 incubator at 5–10% CO2 and 37 °C. Bottles were examined for growth every 72 hours for up to 35 days. For two periods during the study (first from 30 April 2017 to 16 June 2017 inclusive and second from 13 July 2017 to 30 September 2017 inclusive) BacT/ALERT bottles were also processed by manual culture methods due to technical malfunction with the BacT/ALERT system. Standard methods were used for identifying isolates36. Isolates of gram-negative coccobacilli that had positive reactions for urease, catalase and oxidase were classified as presumptive Brucella and stored on Microbank beads  at −70 °C. Culture bottles were classified as adequately filled if the blood volume added met the supplier’s recommended volume +/− 20%. The following organisms were considered likely contaminants: non-anthracis Bacillus spp., Corynebacterium spp., Escherichia vulneris, Micrococcus spp., coagulase-negative Staphylococcus spp., viridans streptococci. Additionally, Pantoea spp. was identified in three bottles of Castañeda media and was also classified as a likely contaminant in our analysis. The results of positive blood culture were shared immediately with the clinical team at Endulen Hospital to inform clinical management.Inoculated culture bottles were packed for transportation to achieve a target temperature range of 4–10 °CBrucella spp. isolates were shipped on dry-ice to APHA, UK, for confirmatory testing. Identification of Brucella species by culture was performed according to an established typing scheme37. Crude lysates for use as a template for PCR were prepared by suspending a single colony from solid media in 500 µL of nuclease-free water and heating (100 °C for 10 minutes). Molecular confirmation of culture-based species typing was performed using multiplex conventional PCR38, quantitative PCR39 and application of a nine locus Brucella spp. multilocus sequence typing scheme41.Presumptive B. abortus, B. melitensis and B. suis was used  at a working strength to give 50% agglutination with a 1/650 dilution of the International Standard Serum to B. abortus. Samples were screened in a microtitre plate at serum dilutions of 1/5, 1/10, 1/20 and 1/40, with total volume per well of 200 µl. Any sample showing agglutination in any screening titre was then retested using the tube test format to confirm the final titre.Filled vacutainer tubes were inverted five times immediately after blood collection and kept at ambient temperature for 45–60 minutes to ensure clotting prior to centrifugation at 1300–1500 g for 10 minutes. Serum was pipetted into cryovials and stored temporarily at 4 °C at Endulen Hospital before being transported at 4–10 °C to KCRI. Tinytag Transit 2 Temperature Data Loggers monitored transport temperature of serum samples. Sera were stored at −80 °C on arrival at KCRI. Sera were shipped on dry ice to the APHA, for serological testing using the Serum Agglutination Test (SAT). Standardised antigen for the detection of antibodies to Brucella spp. or a four-fold or greater rise in Brucella antibody titre between acute and convalescent serum samples. Laboratory evidence of probable brucellosis was defined as a SAT antibody titre of ≥160 in either acute or convalescent-phase serum42.Brucellosis cases were defined as meeting the study criteria for febrile illness of reported fever within the past 72 hours, and/or a tympanic temperature of ≥38.0 °C at hospital presentation, plus laboratory evidence of infection. Laboratory evidence of confirmed brucellosis was defined as a blood culture positive for Brucella blood culture positive as in a previous study in Egypt43, it was estimated that a sample of 348 individuals was needed to detect a minimum of six Brucella blood culture positive cases with 95% probability. Based on an assumed prevalence of 9%18, it was estimated that a sample of 126 individuals would be required to estimate brucellosis case prevalence with precision of 5% and 80% power.A sample size calculation was performed to estimate the number of enrolled febrile hospital participants required in order to (i) detect a minimum number of blood culture positive individuals and (ii) estimate brucellosis case prevalence . Assuming that 3% of presenting febrile individuals would be 44. Proportions were reported within exact binomial confidence intervals. Generalised linear models were used to evaluate associations between brucellosis case status  and clinical data. Univariable logistic regression models were used to investigate pairwise associations between brucellosis case status and candidate risk factors for human brucellosis. Questionnaire variables were selected for evaluation based on the previously identified or biologically plausible risk factors. The following variables were evaluated: age (years), sex, and a series of livestock-related risk variables occurring within the previous 12 months. In each case, livestock refers to cattle, sheep and/or goats. Variables evaluated were: assisting livestock parturition, occurrence of livestock abortion or still-born offspring in flock/herd, milking livestock, herding livestock, contact with livestock waste , slaughtering or butchering livestock, consumption of raw meat  from cattle, sheep and/or goats, and consumption of raw dairy products from cattle, sheep and/or goats. Univariable models were evaluated using likelihood ratio tests (LRT), with a significant p value ≤0.05. Given the small number of positive brucellosis cases and high potential for data overfitting, variable selection was performed using lasso regression using the R package glmnet45. Variables retained in the lasso regression were fitted in a multivariable logistic regression model and evaluated using LRT. Odds ratios (OR), adjusted odds ratios (aOR), and 95% confidence intervals (CI) were estimated.Data were entered using the OpenText TeleForm system  into an Access database . Data manipulation and statistical analyses were performed using RApproval to conduct the the study was granted by the Tanzania Commission for Science and Technology, Tanzania Wildlife Research Institute and the Ngorongoro Conservation Area Authority. Ethical approval was granted by the Kilimanjaro Christian Medical Centre (KCMC) Ethics Committee (698); National Institute of Medical Research (NIMR), Tanzania (NIMR/HQ/R.8c/Vol. I/1140) University of Otago Human Ethics Committee (H17/052), and University of Glasgow College of Medical, Veterinary and Life Sciences Ethics Committee (200140149). The research was performed in accordance with the guidelines and regulations prescribed by the above organisations. Written informed consent for study participation was obtained from each participant and/or their legal guardian, using forms translated into Swahili and verbal translation into Maa when needed.From 15 August 2016 to 11 October 2017, 3,473 patients were screened for enrolment into the study. Of the 3,473 screened patients, 435 (12.5%) were eligible for inclusion in the study. A total of 230 participants enrolled and contributed data for the study analyses  of 228 participants were Brucella spp. culture positive. Six other pathogenic bacterial species were identified, each in a single participant (Table Brucella spp. isolates from seven (3.0%) participants were identified as B. melitensis while B. abortus was isolated from one participant (0.4%). The results of molecular speciation assays were congruent with phenotyping. Multilocus sequence typing (MLST) identified all seven B. melitensis isolates as sequence type (ST) 12, while the single B. abortus isolate was identified as ST32.Fourteen 6.1%) of 228 participants with one or more inoculated blood culture bottle had a bloodstream infection. % of 228 Brucella spp. isolation success from adequately filled culture bottle types is given in Table Brucella spp. was identified by culture from 16 bottles from the eight culture positive participants, twelve of which had adequate fill volumes. Brucella spp. was identified in the Castañeda media bottle for all eight of the culture positive individuals. Brucella spp. was identified in one or more of the inoculated BacT/ALERT bottles for five of the eight culture positive individuals including adequate and non-adequate fill volumes. The details of Brucella spp. isolation success from the individual culture bottles used for the eight Brucella spp. positive individuals is given in Supplementary Fig. In total, 601 culture bottles were inoculated, with one or more bottles inoculated for 228 (98.3%) of 232 participants. Of the 601 culture bottles, 531 (88.4%) bottles collected from 215 participants were adequately filled and 21 (3.5%) of 601 were contaminated. The median time interval between sample collection and processing at the laboratory was 25.9 (interquartile range 24.8 to 27.0) hours. A total of 77 (85.0%) of 91 shipments of inoculated culture bottles stayed within the target temperature range, all inoculated culture bottles were processed in the laboratory. The comparison of Of 232 participants, 230 had acute and/or convalescent serum collected. Paired acute and convalescent phase samples were collected for 154 (70.0%) of 230 participants: 70 participants had acute serum collected only; and six had convalescent serum only, due to an insufficient blood volume collected at acute hospital presentation. Eleven (4.8%) of 230 participants met the case definition for probable brucellosis with one or more SAT antibody titre ≥160. One additional participant met the case definition for confirmed brucellosis based on serology data alone, showing a four-fold rise in antibody titre.A total of nine (3.9%) of 230 participants met criteria for confirmed brucellosis; eight based on blood culture and one by seroconversion. Six (75.0%) of the eight blood culture positive participants had an acute phase SAT titre ≥160, with four of the six also having a convalescent phase SAT titre ≥160. In total, 14 (6.1%) of 230 participants, met the study definition for a probable or confirmed brucellosis case. The correspondence between culture and serology results for study participants and details of the dates of culture positives are shown in Supplementary Fig. Study participants had a median (range) age of 27  years and brucellosis cases had a median (range) age of 11  years. Ten (71.4%) of 14 brucellosis cases were male and 13 (92.9%) reported their tribe as Maasai. Of 232 study participants, 226 (97.4%) reported residence in the NCA with 170 (73.3%) from Endulen village. Residence was also reported in the larger Arusha region and adjacent Simiyu region  and admission to the inpatient ward of the hospital . Sixteen (7.0%) of 230 participants, were prescribed a drug regimen consistent with treatment for brucellosis at their initial visit. All of the participants treated for brucellosis had a presumptive diagnosis of brucellosis and treatment for brucellosis was significantly associated with case status as defined by this study . Data on treatment regimens prescribed after the provision of blood culture results  are not included in our dataset. Six (2.6%) of 230 participants had a test positive for malaria. No brucellosis cases were malaria positive.The relationship between clinical symptoms and brucellosis case status, as well as details of participant clinical management are shown in Table χ2 = 20.05, df = 1, p = <0.01, n = 229), with probability of brucellosis declining with increasing age in years; sex , with a greater probability of brucellosis in male participants; and herding livestock in the last 12 months , with participating in herding cattle, sheep and/or goats associated with increased probability of brucellosis. The lasso regression indicated that herding livestock and age variables should be retained. Variables significantly associated with brucellosis in febrile hospital participants in the final multivariable model were: age of participant , with probability of brucellosis declining with increasing age in years; and herding livestock in the last 12 months , with participating in herding cattle, sheep and/or goats associated with increased probability of brucellosis  and wildebeest (Connachaetes taurinus) are known hosts of Brucella spp.50. Transmission from livestock to humans is likely to account for the majority of human infections, but transmission from wildlife to humans in this setting cannot be ruled out.The isolation of both 19. All confirmed cases in these previous studies were defined by seroconversions19. In this present study, the majority of confirmed cases were blood culture positive and the number and proportion of confirmed cases defined by seroconversion was lower than expected. This difference in the ratio of culture to seroconversion-defined confirmed cases may relate to the timing of participant presentation at hospital. Brucella spp. isolation is typically more likely early in acute infection21. Confirmation of cases based on demonstration of seroconversion is most likely when an acute phase sample is collected very soon after initial infection and a convalescent phase sample is also collected several weeks after initial infection. Interestingly, the majority of blood culture-confirmed brucellosis cases identified in this study also had acute phase sample SAT titre ≥160, indicating that they had already seroconverted by the time of presentation and also had bacteraemia that was still detectable by blood culture . Patients attending an urban referral hospital may have had increased access to antimicrobials prior to hospital presentation, again reducing chances of positive culture. Additionally, our study was designed to preferentially detect acute brucellosis in acutely febrile individuals seeking healthcare, and is thus less likely to detect chronic brucellosis cases. It has been estimated that 40% of brucellosis cases may persist with chronic disease for two years following disease onset, and 10% may persist with clinical manifestations after six years51. Further investigations of the combined health impacts of acute and chronic human brucellosis in this population are warranted to evaluate the combined burden of disease52.The overall prevalence of brucellosis identified in this study is consistent with the findings of previous febrile surveillance studies conducted in Moshi, Tanzania, providing additional evidence of the health impacts of brucellosis in northern Tanzania. Using similar diagnostic approaches these previous studies have detected serologically confirmed brucellosis at between 3.5% and 6.9%Brucella spp. exposure at an early age has been recognised for nomadic groups, with adults often more likely suffering from chronic brucellosis infection2. Within the largely Maasai community of the NCA, young boys are preferentially given responsibility for herding livestock . Herding can involve multiple activities that may put individuals at higher risk of exposure to Brucella spp. as well as many other zoonotic pathogens. These activities may include: frequent contact with livestock, assisting in animal parturition, butchering livestock and consuming specific organs raw or eating undercooked meat and blood53, and drinking raw milk directly from animals in the herd or flock53. Some of the more common risk factors for human brucellosis reported in East Africa such as: consumption of raw animal products54, assisting in animal parturition events and contact with aborted animal materials56 and slaughtering57 were not identified in this study. The small number of brucellosis cases identified and the high proportions of the study population participating in perceived high risk activities, complicate efforts to disentangle the relative importance of specific risk activities and transmission pathways in this population. Further investigation into activities conducted whilst herding that may increase exposure to Brucella spp. may identify additional specific risk factors for infection in the young herders identified as high risk in our study.Risk factors associated with brucellosis in the univariable models in this study included younger age, male sex and herding livestock. Lasso regression selected herding livestock and age variables, which were retained for the multivariable model. The fact that the sex variable was not retained in the final model is likely to be due to correlation between male sex and having herded livestock. Higher risk of 10. In the present study brucellosis case status was positively associated with night sweats but negatively associated with back pain. These findings reinforce the challenges of using clinical symptoms for brucellosis case identification. In spite of this, brucellosis was a relatively common presumptive diagnosis made by clinicians at the Endulen Hospital, indicating high clinical awareness of the disease in this setting. There was also a significant association between presumptive diagnosis of brucellosis and brucellosis case status. Not all participants with a presumptive diagnosis of brucellosis were started on a brucellosis consistent treatment on the day of hospital presentation. However, the data presented on treatments refer to treatments prescribed at initial presentation only and do not capture later decisions based on additional findings, including the subsequent blood culture results provided through the study. Overall, these data highlight the challenges faced by clinicians in areas where brucellosis is endemic but access to high quality diagnostics including blood culture and SAT serology is limited. There is a need for improved diagnostic tools and diagnostic guidelines for the management of acute brucellosis and febrile illness more generally in Tanzania, and East Africa more widely11.Diagnosis of brucellosis by clinical symptoms alone is unreliable due to varying clinical manifestations of infection58, prompting the development of a national strategy for brucellosis prevention and control in humans and animals59. These study findings, including human prevalence estimates, identification of both B. melitensis and B. abortus as causes of human illness and risk factors for acute human illness, can inform the development of evidence-based control strategies for brucellosis in Tanzania.Brucellosis has been identified as one of six priority zoonotic diseases in TanzaniaBrucella spp. are the most commonly identified bloodstream infection in this predominantly pastoralist community from the NCA, Tanzania. Our findings show that human brucellosis is caused by both B. melitensis and B. abortus consistent with transmission from multiple livestock host species. Within this pastoralist community, young livestock herders are at high risk of brucellosis infection. Brucellosis control activities in Tanzania which focus on prevention of transmission to individuals at high risk of infection, and livestock vaccination campaigns directed at both cattle and small ruminants have potential for substantial impacts in reducing human brucellosis risk.We found that brucellosis was the cause of 6.1% of febrile illness among study participants presenting for outpatient hospital care, and that Supplementary Information.


Pseudomonas fluorescens strains on yields and physiological characteristics of sweet corn (Zea mays L. var saccharata) under different levels of irrigation. Three irrigation levels i.e., 100% (I100 no stress), 80% (I80), and 60% (I60) were used during sweet corn cultivation. However, there were four rhizobacteria strains i.e., P. fluorescens P1, P. fluorescens P3, P. fluorescens P8, P. fluorescens P14 which were used in the experiment. The results showed that severe water stress (60% of plant water requirement) decreased chlorophyll a, chlorophyll b, and total chlorophyll contents, Fv/Fm ratio and nutrients uptake. A significant increase in F0, Fm, proline, total soluble sugars, catalase (CAT) and peroxidase (POX) activity led to less ear yield and canned seed yield. Combination of four strains significantly increased the yield traits of sweet corn i.e., ear and (44%) and canned seed yield (27%) over control. The highest promoting effect was observed in the combination of four strains treatment and followed by P1 strain in reducing the harmful effects of drought stress and improving sweet corn productivity. However, P14 gave minimum improvement in growth and yield indices under limited availability of water. In conclusion, combination of four strains inoculation is an efficacious approach for the achievement of better yield of sweet corn under osmotic stress.Accumulation of stress ethylene in plants due to osmotic stress is a major challenge for the achievement of optimum sweet corn crop yield with limited availability of irrigation water. A significant increase in earth’s temperature is also making the conditions more crucial regarding the availability of ample quantity of irrigation water for crops production. Plant growth promoting rhizobacteria (PGPR) can play an imperative role in this regard. Inoculation of rhizobacteria can provide resistance and adaptability to crops against osmotic stress. In addition, these rhizobacteria also have potential to solve future food security issues. That's why the current study was planned to examine the efficacious functioning of  Water stress causes economic damages in agriculture, inoculation of plants with useful microorganisms stimulates plant growth and enhances water stress tolerance2. Sweet corn (Zea mays L. saccharata) is a hybridized variety of corn specifically bred to increase the sugar content. Sweet corn has a high sugar content; mutation in genes and following that genetic change causes the accumulation of sugars and soluble polysaccharides in the endosperm of the seed3. Globally, sweet corn plays an important role, both directly and indirectly, in providing the calories, protein, and some of the vitamins and minerals needed by humans4. This crop has been cultivated for a different purpose, such as food for humans, forage for feeding, ethanol, or sugar production in an industry5. Sweet corn by producing most structural and soluble sugars, has a very high potential as an alternative raw material for ethanol production; also, the production of biofuels from sweet corn stalk syrup shows that in addition to increasing the value of agricultural losses, this plant can play a temporary role in emerging biofuel markets6.Drought is the most important environmental stresses limiting the productivity of crop plants around the world, especially in arid and semi-arid regionsPseudomonas, Azotobacter, and Azospirillum is their capability to support plants under stressed environments7. Among all, some rhizobacteria are capable to produce ACC deaminase that has the potential to decrease the stress generating ethylene in the plants. Recent studies indicated that rhizobacteria having ACC deaminase are helpful to mitigate the adverse effects of stress generating ethylene14. Enzyme 1-aminocyclopropane-1-carboxylate deaminase (ACCD), can break ACC, (an immediate precursor of ethylene) to ammonia and α-ketobutyrate15. A low level of ACC deaminase activity, namely approximately 20 nmol a-ketobutyrate mg−1 h−1 is sufficient for bacterium to grow on ACC16.Beneficial bacteria have enormous potential to facilitate plant growth and productivity, in several a number of ways. A remarkable eminence on the credit of plant growth-promoting rhizobacteria (PGPR) such as 19. Khan et al.20 reported elevated activities of CAT, POX, APX, and SOD in response to drought stress; however, under drought stress conditions, inoculation of pea plants with a combination of Bacillus subtilis, Bacillus thuringiensis, and Bacillus megaterium decreased the activity of these enzymes and was significantly increased the amount of chlorophyll, protein, starch, proline, and carbohydrates. Their results indicated that bacteria symbiosis alleviates the toxic effect of drought stress via improving the water status of plants. Danish et al.21 in the study of plant growth-promoting rhizobacteria Pseudomonas aeruginosa, Enterobacter cloacae, Achromobacter xylosoxidans and Leclercia adecarboxylata in maize plants inoculated under water stress conditions, increasing the concentration of N, P and K nutrients were confirmed by reducing ethylene accumulation and improving plant root growth.Beneficial effects of PGPR have been reported on reducing the harmful effects of water deficiency on crops growth22, however, few studies focus on physiological responses and nutrients uptake sweet corn to PGPR, especially Pseudomonas fluorescens under stressful conditions. Therefore, the objective of this study was to evaluate the water stress tolerance that induced in sweet corn by four strains of Pseudomonas fluorescens: P1, P3, P8, and P14 under different irrigation levels. The current study was conducted with hypothesis that combination of four Pseudomonas fluorescens strains inoculation may be more effective approach over sole inoculation for mitigation of adverse effects of limited availability of in sweet corn.Although numerous kinds of literature have studied the effect of PGPR on growth and drought stress tolerance of maizeP. fluorescens  were provided from a bacterial collection of Vali-e-Asr University of Rafsanjan. For analysis of ACC deaminase and auxin synthesis standard protocols were used23  on nutrient agar (0.5) and incubated at 27 °C for 48 h. Afterwards, after the transfer of bacteria to the liquid culture medium (TSB), were laid in a shaker incubator at 28 °C for 48 h, and then were centrifuged at about 7000 rpm for 5 min until bacteria precipitate. The optical density of each suspension was determined according to an absorbance at 600 nm of 0.5 in distilled water, with an estimated bacterial cell density 108 CFU ml−124. The seeds were placed in the suspensions for 24 h for inoculation with different strains of bacteria.Four strains of 25 and N, P, and K26.Soil sampling was done according to the zig-zag pattern from depth 0–30 cm sub-samples mixed within the bucket and then 1 kg of soil sample placed in a clean plastic bag and transferred to the laboratory. Soil samples were passed through a 2-mm sieve after being exposed to the laboratory at room temperature for 1 day. The samples were analyzed for soil texture (hydrometric)27. Details of the measured soil properties (0–40 cm depth) before the experiment are provided in Table 100 no stress), 80% (I80), and 60% (I60) of plant water requirement) and the subfactor included bacteria inoculation with four strains of Pseudomonas fluorescens including P. fluorescens P1 (P1), P. fluorescens P3 (P3), P. fluorescens P8 (P8), P. fluorescens P14 (P14), a combination of four strains (4 Strains) and control). The rhizobacteria were isolated from corn rhizosphere and all strains were drought tolerant (able to grow at − 0.90 Mpa). The Average daily temperature along with daily evaporation is made in Fig. 2 on 22 June in both years, which rows and plant spacing were 75 and 20 cm, respectively . Based on soil test . The previous cultivated crop was corn. This region receives a mean annual temperature of 28.5 °C with a mean annual rainfall amount of 365 mm. The soils in Marvdasht are clay-loam classified as 28.−1), Rn is net radiation at the crop surface (MJ m−2 day−1), G is soil heat flux density (MJ m−2 day−1), T mean daily air temperature at 2 m height (°C), U2 wind speed at 2 m height (m s−1), es saturation vapour pressure (kPa), ea actual vapour pressure (kPa), es-ea saturation vapour pressure deficit (kPa), ∆ slope vapour pressure curve (kPa °C−1), γ psychrometric constant (kPa °C−1). Daily weather information, consisting of a minimum and maximum temperature, precipitations, relative humidity, and sunny h were obtained from Koshkak Meteorological Station . Daily water requirement of plants was calculated as follows:Irrigation was done using a tape system and was similar for all treatments up to complete seedling establishment. Irrigation treatments were applied from 5 leaf stage until the end of experiments. The water requirement of plants was determined based on evapotranspiration using the Penman–Monteith by Eq. 28.1\docuc, KC and ET0 are evapotranspiration of plants (mm day−1), crop coefficient, and reference evapotranspiration (mm day−1), respectively. According to the FAO report28, KC for sweet corn is 1.15 at the middle stage and is 1.05 at maturity. Irrigation depth also was calculated as follows:In this equation, ETci is water requirement. The water volume of each plot was controlled by a water counter.In this equation, D is irrigation depth and ET29. Chlorophyll fluorescence parameters  were also read at the milk grain stage by using the fluorimeter (OSI-FL). The content of the free proline was determined according to the acid-ninhydrin method30 with minor modification. The determination of soluble sugars was done using Dubois et al.31. Catalase32 and peroxidase activity33 were measured at 240 and 470 nm, respectively, by using the spectrophotometer .For plant sampling, at the milk grain stage, the three last development leaf were randomly selected in each sub-plot. The samples were immediately frozen in liquid nitrogen and stored at − 80 °C for future extraction. Samples Frozen were ground to a fine powder in liquid nitrogen using a pestle and mortar. The concentration of pigments consisted of chlorophyll a & b and total chlorophyll were measured using the spectrophotometer  at three absorbances  based on Lichtenthaler and Wellburn34, phosphorus by calorimetry method (yellow colour of vanadium molybdate) by using the spectrophotometer  and potassium by the flame photometer. Micronutrients were also measured by using the dry ash method and then were dissolving in two-normal hydrochloric acid and by using the atomic absorption device (Hitachi ZCAST 2300)35.N content was measured based on the Kjeldahl method2 plants of each plot were harvested manually and ear yield and canned grain yield were determined , 3 m36.Data analysis was performed using SAS software v.9.1 . Means of three replicates were compared using the LSD at a P-value of 5%60 compared to I100, resulted in a decrease of 36.0, 32.0, and 39.0% of N, Zn and Mn accumulation of leaf, respectively, and K was increased by 21.98%  and rhizobacteria (B) were significant on nutrients accumulation in sweet corn leaf (except Cu); and the interaction (I × B) was significant only on Fe and P at the probability level of 1 and 5%, respectively Table . Drought80 and I60 were increased catalase and peroxidase activity by 53.0 and 65.50% and 55.0 and 64.00%, respectively , chlorophyll b (Chl b), and total chlorophyll, which shown in Table a concentration was significantly greater in the seeds inoculated with P1 and P8  and maximal photochemical efficacy of photosynthetic system (Fv/Fm) II and also interaction (I × B) was significant on maximum fluorescence (Fm) of sweet corn leaf  was observed at I60, and there was a statistically significant difference with other irrigation levels  was obtained from the combination of four strains treatment, which was significantly different from strains P3, P14, and control. However, it was not statistically significant with P1 and P8 strains  and canned seed yield (616.1 g m−2) were obtained in inoculated plants with a combination of four strains treatment in I100 conditions  by mineral and organic acids production.Phosphorus is an immobile element in soils. The absorbable phosphorus amount in the rhizosphere is very low. The reason for the fact is mineral phosphate firm ionic bond with soil colloid and fixation as Ferro phosphate or aluminium phosphate which causes phosphorus immobilization in both forms and water stress also reduces this nutrient transmission40. Potassium active absorption is responsible for an increase in its absorbance under water restriction, actually the plant increase potassium density in a shoot with energy consumption unlike the diffusion process for an increase in tolerance against water stress41. In the present research, the most potassium amount of leaf was observed at the combination of four strains treatment; it seems that at this level mineral and organic acids production causes more potassium solubility in soil solution by this element ease from biotite and tricalcium phosphate minerals42. The strains also impress root growth by IAA hormone production and ACC deaminase activity and finally cause an increase in root absorption and higher potassium amount.Leaf potassium rising under water deficit can because of its role in cell turgor maintenance43. These materials are exuded from the microbial cell for against Fe absorbable form deficit (< 20 µM) and make this element to the soluble chelate form, therefore iron mobilization in the rhizosphere is increased and absorbed by plant root system with special mechanisms. After that roots can absorb iron from Fe-siderophore that this process can conduct by different mechanisms such as chelate destruction follow by Fe loose, direct absorption from Fe-siderophore or ligand conversion reaction44. In this research, the most amount of Fe achieved from P1 strain and it can because of the high ability of this strain to produce siderophore  compare with other strains and mentioned strain caused more Fe absorbability by the iron chelate formation and more shoot Fe amount.Iron (Fe) is one of the necessary micronutrients for plant growth. Despite there are lots of this element in soil, it is impressed of some soil properties and its mobilization is reduced severely in dry and alkaline soils, therefore will be unavailable for plants. PGPR inoculation under low irrigation caused more Fe amount in the shoot. PGPRs exude chelators with low molecular weight  called siderophore which trends to absorb iron45. Calcareous soils (high pH) under low irrigation causes a severe reduction in micronutrients especially zinc solubility and also being the high amount of bicarbonate in water, especially under low irrigation, results from more bicarbonate concentration in cellular sap follow by more pH in sap and zinc sediment in vessels and so lower zinc transformation to the rest parts of plants43. In the present research, among different strains of P. fluorescens, the most amount of leaf zinc achieved from the combination of four strains treatment. It seems that this level of strains can change insoluble zinc forms into soluble forms by organic acids production, increase in proton (H+) and chelating compounds making. The main role of these bacteria on zinc solubility is pH reduction up to 5 or less and then more availability of the element. Also, the increasing effect of strains under combination inoculation increases root contact surface with soil and shorter element transform to roots46. Zinc and Fe compete for absorption from the root and the combination of four strains level was effective on Fe absorption more than zinc because of the most siderophore production and chelate Fe-siderophore formation.Zinc plays an important role in protein and carbohydrates synthesis and effects on saccharides, nucleic acids, and lipids metabolism47. Iran soils have deficit micronutrients because of being classical, bicarbonate water, and low organic matter, also under low irrigation Mn solubility becomes less and its absorption and transformation into plants will be lower and finally Mn amount in shoot decreases. The combination of four strains level improved plant ability for nutrients absorption like Mn by expanding the host root system and increasing root contact with soil as a result of more lateral root and root hair production. Abbaszadeh-Dehaji et al.48 reported that three PGPRs  inoculation resulted from a significant increase in sorghum growth and nutrients like Mn, Zn, Cu, and K in plant leaf in compare with control plants. Malekzadeh et al.49 showed that Fe, Zn, P and Mn absorption by maize shoot increased significantly in all inoculated treatments with Bacillus mycoides and Micrococcus roseus in compare with control.The main role of Mn in plants is in photosynthesis and oxygen production (Hill reaction). Mn plays an electron receptor transformer role in photosystem II during the primary phase of photosynthesis. Another role of Mn is participation in antioxidant enzymes which restricts free radicals activity so prevents lipids destruction like glycolipids and membrane fatty acids2 absorbance and fixation deficit, as a result antioxidant enzymes activity gets higher to eliminate oxygen active species50. P. fluorescens strains inoculation caused a reduction in catalase and peroxidase activities in leaf. Both enzymes activities reduction under strains combined inoculation can be explained by this fact that the combination of four strains level by stress tolerance related genes induction causes transpiration decrease by leaves roll and stomata regulation. Also, this level prevents high enzyme activity by cell water maintenance, photochemical efficiency improvement of photosystem II, electron final receptor reduction preventing and active oxygen species decrease51. Under water stress condition, PGPRs  using caused plant growth promotion and reduction in superoxide dismutase, catalase, peroxidase, and acrobate peroxidase activity in two species of drought-tolerant rice (Sahbhagi) and drought-sensitive species (IR-64) in comparison with control plants52.Under water stress, plants have more antioxidant enzyme activity. Under that situation oxygen active species production increases because of stomata closing and COa and chlorophyll b were significantly reduced by water stress, which intensifies stress level and increased its adverse impact. It was also shown that the sensitivity of chlorophyll b was the most. The most chlorophyll lost in response to drought stress happens in the mesophyll cells compared to the bundle sheath cells53. Reduced chlorophyll concentration mainly is known to be due to reactive oxygen species in oxidative stress54, which have been reported in different plants such as sweet corn by Singh and Shinde1, sunflower by Manivannan et al.55 and mung bean (Vigna radiata) by Farooq and Bano56. Under water deficit stress conditions, chlorophyll concentration is reduced, which may be because chlorophyll degradation is more than chlorophyll synthesis.The results showed that photosynthetic pigments consisted of chlorophyll 1 and P8 strains had the most effect and P14 had no significant effect. A combination of four strains had no advantages than individual use of them. The improved concentration of chlorophylls under water deficit stress in P. fluorescens inoculated plants might be probably due to greater synthesis of photosynthetic pigments through higher stomatal conductance and increase photosynthetic area or also through enhanced water and nutrients absorption57. Strains of P. fluorescens especially P1 and P8 enhanced concentration of Chl a and Chl b. Inoculation with P1 not only enhanced Chl a concentration but also improved water-deficit stress tolerance of plants at both water stress levels. This improvement was observed for Chl b only in moderate water stress. Prolonged growth cycle and consequently higher green stay and delaying aging might be the main reason for the enhancement of chlorophylls concentrations in inoculated plants60, which could be due to synthesized auxin as a result of inoculation with P. fluorescens strains. In the current research, all evaluated strains had a considerable activity of ACC deaminase  capacity decrease and uncompleted oxidation because of slow electron afflux in photosystem II and finally, this photosystem becomes inactivated62. It seems the combination of four strains level resulted in promotion in photosystem II electron transforming chain efficiency by synergistic effects of strains on root growth properties improvement and higher root contact surface and better water and nutrients absorption leading to lower minimum florescence63. Stomata closing after low water potential in leaf has a bad effect on CO2 entrance and decreases electron reception and transferring capacity, in a result system attains maximum florescence immediately; in response, variable florescence is reduced64. It can be concluded that under low irrigation (I80 & I60), strains combined inoculation treatment causes plant water condition improvement and alleviation in low water effects by root system expanding and more root contact surface with soil, which in response maximum florescence would decrease in comparison with no inoculated treatment.Chlorophyll florescence has been suggested as a criterion for environmental stress effects on the measurement of plants and their tolerance against stress determination. This factor can show these stresses on how much hurt photosynthetic systemsFv/Fm is an index for photosystem II photochemical quantum efficiency measurement that is a fast and useful way to investigate drought stress66. The decrease in CO2 assimilation because of stomata closing under drought stress causes no consumption for electron transferring chain outputs (ATP and NADPH) and so reduced ferodoxine is increased and it causes more active radicals production, these radicals destroy tilacoid membrane proteins and prevent electron transferring from reception place of photosystem II leads to slower electron transferring speed and lower photosystem II photochemical efficiency67. The combination of four strains treatment results in different mechanisms like IAA production which leads to root cell propagation and elongation, using ACC enzyme that applies ACC preform for ethylene production and by hydrolyzing ACC to ammonia and α-chetobutirate causing lower ethylene prevention level and more root growth. Also solubilizing of mineral phosphates and siderophore production and Fe supplement for plant causes more water and nutrients absorption for photosynthesis and because of that electron flowing from photosystem II to photosystem, I would be better and maximum photochemical efficiency of photosystem II will increase68.7, which shown in the current study. Proline and soluble sugars are acting as osmolytes sustaining cell turgor of leaves, protecting the integrity of the membrane, and preventing the denaturation of proteins70. The mechanism of bacteria inoculation on proline has not well-cleared yet. Bacteria inoculation significantly enhanced the content of FP and TSS, and this effect was varied between strains and irrigation levels. On average, all strains had positive effects; however, P1 and P8 were the most effective of those.Increased free proline, as well as total soluble sugars, were observed in stressed plants, and these changes depending on the severity of water deficit stress. One of the most important plant responses to water deficiency is an osmotic adjustment that mentions the decreased osmotic potential of the plants by accumulation solutes like proline and sugars15. The moderate activity of ACC deaminase causes a balance between ACC deaminase activity and root growth23. It seems under water stress conditions, plants inoculated invest a large amount of carbon and nitrogen resources into synthesis compatible solutions in the leaves such as proline for maintaining cell turgor by improving root growth and increasing the area of nutrient uptake.Experimental evidence indicated that the activity of ACC deaminase was the key factor in the ability of PGPR to stimulate the elongation of plant roots5. The yield of sweet corn consisted of ear yield and canned seed yield was greater in plants inoculated with P. fluorescens. The main mechanisms of P. fluorescens to facilitate plant growth and development is the lowering of the ethylene levels by hydrolysis of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene in plants19. In this study, water stress at I60 has influenced the production of assimilates and ear beginnings by reducing the absorption of water and nutrients, especially N, P and K and leaf area expansion, as a result, has led to a decrease in the number of rows in the ear, the number of seeds per row, the weight of 1000 seeds and finally ear and canned seed yields.Overall results showed that sweet corn ear and canned seed yields were decreased in moderate and severe water stress levels. Previous studies also reported that water stress has been led to less growth and yield in sweet corn72.At all levels of irrigation, the combined inoculation of strains has affected root growth characteristics by their high ability to inhibit ethylene hormone and prevent its inhibitory effect on root growth and in terms of water and nutrients, they provide a large area of soil for the plant; and increasing the concentration of photosynthetic pigments and improving the photochemical efficiency of photosystems has led to an increase in assimilates and following that an increase in the number of ears, the number of seeds per ear, the weight of 1,000 seeds, and thus the canned seed yield. It has been well-accepted that root growth, especially under water deficit conditions could lead to more photosynthesis rate and so higher yieldP. fluorescens improved physiological attributes and increased yield. Combination of four strains treatment through the synergistic effects of strains in the production of ACC deaminase, auxin synthesis, the ability in mineral phosphate solubilizing and the production of siderophore significantly improved the yield traits of sweet corn at different levels of irrigation by reducing the effects of stress ethylene and increasing water absorption and nutrients.Water scarcity is a limiting factor for agricultural productivity that causes changes in physiological characteristics and nutrients uptake in sweet corn. Inoculation with different strains of Supplementary Information.


The fitting of isothermal data with different models showed the model suitability trend as: Sips model > Langmuir model > Freundlich model, where the theoretical maximum adsorption capacity calculated from the Sips model was 4.6, 2.9, 2.7 and 2.4 mg/g for AFB1, AFB2, AFG1 and AFG2, respectively. Kinetics study revealed the fast AFs uptake by BB (50–90 min) while thermodynamics studies suggested the exothermic nature of the AFs adsorption from both, single as well as multi-toxin buffer systems, gastrointestinal fluids and model wine. Accrediting to the fast and efficient adsorption performance, green and facile fabrication approach and cost-effectiveness, the newly designed BB pomace can be counted as a promising contender for the sequestration of AFs and other organic pollutants. Blueberry (BB) and cherry pomace were investigated as new biosorbents for aflatoxins (AFs) sequestration from buffered solutions, gastrointestinal fluids and model wine. Among the tested biosorbents, BB exhibited the maximum adsorption performance for AFs and hence was further selected for the optimization of experimental parameters like pH, dosage, time and initial concentration of AFs. Material characterizations via scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, N Aspergillus parasiticus, Aspergillus nomius and Aspergillus flavus [Aflatoxins (AFs), on a worldwide scale, are the most toxic class of mycotoxins primarily produced by s flavus ,2. More s flavus  which iss flavus ,5,6. AFss flavus  with thes flavus . Frequens flavus . Pyracantha koidzumii biomasses [AFs can survive heat and physicochemical treatments in the food processing industry, hence their removal from the food is indispensable and to attain that goal, certain conventional strategies have been demonstrated such as biodegradation, irradiation and physical separation , among which adsorption is considered as the most economical and feasible approach . Classesiomasses , banana iomasses  and grapiomasses  are noteOn the other side, most of the AFs removal studies reported previously focused on AFB1 adsorption only , ignorinScanning electron microscope (SEM) images of the dried and fermented CHs and BBs are displayed in Brunauer–Emmett–Teller (BET) surface area and porosity analysis for CH and BB biosorbent were performed to assess the modification resulted from fermentation and the results are shown in These results suggest 3.6 times higher surface area and pore volume and smaller pore width and particle size of the fermented BB than the fresh one while no obvious change was observed for CH . Moreove−1. The absorption band at 1064 and 1382 cm−1 could be ascribed to C–OH and C–O–C functional groups respectively [−1 represent the aromatic C=C, olefinic C=C and C=O functionalities [−1 justified the asymmetric and symmetric stretching vibrations of C–H groups in an organic framework while stretching vibrations of -OH from alcoholic and carboxylic groups were observed around 3421 cm−1 as a broad band [Fourier transform infrared (FTIR) spectroscopy analyses of the biosorbents are displayed in ectively  whereas nalities . Peaks aoad band . Owing tThermogravimetric analysis (TGA) curves of the biosorbents are presented in During stage one, the mass loss reached up to 10% at 200 °C which would correspond to the evaporation of physically adsorbed water, impurities and volatile organic compounds ,33. The XPS analysis provides useful information about the surface elemental composition and chemical states . Thus, fpH greatly affects the adsorption process and adsorption capacity in aqueous medium by varying surface charges and adsorbent–adsorbate interactions ,39. TherAdsorbent dosage affects adsorption capacity as it can alter the number of active sites on the adsorbent surface. Kinetics study of the biosorption process is also a factor of great concern as it determines the efficiency and mechanism of adsorption process and, ideally, adsorbent is expected to exhibit fast reaction rate and enhanced adsorption capacity . Figure −1) and k2 (g.mg−1min−1) are the PFO and PSO rate constants, respectively. The simulated plots for kinetic models and associated parameters of these models are presented in In order to further verify the mechanism behind the adsorption of AFs over BB, pseudo first order (PFO) and pseudo second order (PSO) kinetics models were applied to the kinetic data using Equations (1) and (2) respectively ,44,45,462 values above 0.99 for all four types of AFs, while the qe values obtained from PSO were also closer to the experimental qe values than those obtained from the PFO kinetic model. These findings suggested that the chemisorption could be the more prevalent mechanism and rate determining step in the adsorption process [Correlation coefficient thus obtained suggested the best suitability of PSO in defining the adsorption mechanism and had R process . Isothermal study is conducted to analyze the interactions between adsorbent and adsorbate and to estimate the maximum adsorption capability of the adsorbent under optimized experimental conditions. Therefore, adsorption isotherms for the four types of AFs were studied at four different temperatures and the experimental data obtained were fitted with Langmuir, Freundlich and Sips using non-linear regression a–d. f (L/mg) and n are Freundlich isotherm constants which signify the biosorption capacity and intensity, respectively.The Freundlich isotherm model describes the reversible and non-ideal adsorption behavior and eliminates the limitation of monolayer adsorption ,47. Thisl is the Langmuir isotherm constant and is related to the adsorption energy. Although both Freundlich and Langmuir isotherm models are the most widely used isotherm models to describe the adsorption process, they have limitations to cover all aspects of adsorption process. Hence, the Sips model with three parameters intrinsically covers all the features of both, Freundlich and Langmuir isotherm models and is presented by Equation (5) [s (L/mg) and ns are the Sips isotherm constant and isotherm exponential coefficient respectively. The corresponding parameters of all the isotherm models for the adsorption of AFs on BB sample (at 15 °C) are compiled in s [s is close to 1, the adsorption would be a monolayer, following the Langmuir isotherm model, while its value near to zero employs a Freundlich-type heterogenous adsorption process [s values for the adsorption of AFs over BB data ranges from 0.77–0.86  .qe = (qtion (5) : qe = , a possible adsorption mechanism can be proposed for the adsorption of AFs by BB and is shown in The presence of plentiful OH groups in the molecular structure of cellulosic/hemi-cellulosic material can establish hydrogen bonds with the O atoms of carbonyl, methoxy and ether groups in AFs molecules, hence providing strong interactive forces for the adsorptive removal of AFs by BB. Moreover, aromatic compounds from the complex BB matrix would also be responsible for π–π stacking ,64 or thIn conclusion, this study reports the fabrication of four biosorbents obtained from industrial waste i.e., BB, BBF, CH, CHF which were in turn applied for the sequestration of AFs from multiple matrices, among which CH and BB were found to be superior adsorbents. The synthesized biosorbents were characterized by SEM, FTIR, TGA, BET surface area and XPS techniques while the adsorption of AFs on BB was confirmed by analyzing the post-adsorption BB by SEM imaging and XPS. The adsorption of AFs over BB was marginally affected by solution pH and BB outscored many reported adsorbents in the adsorption capacity for simulated gastrointestinal fluid and model wine. An isothermal study showed the best fitting of adsorption data with the Sips model  with a predominant monolayer type adsorption mechanism while kinetics and thermodynamics studies confirmed the rapid AFs uptake and the spontaneity and exothermicity of the adsorption process. The overall coverage of the adsorption performance of BB proved the high competence of this novel, economic, environment-friendly and non-toxic bio-sorbent for the efficient sequestration of AFs and could be envisaged for the remediation of AFs both in vitro and in vivo. However, further studies are required to understand the complex mechanism of the adsorption of AFs on various matrices other than aqueous medium.AFs standard solutions were purchased from Sigma-Aldrich . HPLC grade methanol and acetonitrile were supplied by Thermo-Fisher. All other chemicals used for making buffer solutions, gastrointestinal fluid and model wine were of HPLC grade and supplied by Macklin . Milli-Q quality water was used for preparing solution and HPLC mobile phase. Blueberry and cherry fruits were purchased from a local supermarket , cleaned, washed and divided into two parts i.e., one for juice making and other for fermentation. Fruit pomace was collected by filtration after juice making and washed until the filtrate became transparent. For fermentation, fresh fruits were squeezed and subjected to fermentation according to an already reported procedure . After f−1 following the KBr disc method [2 adsorption-desorption method [The chemistry and functional group characterization of the prepared adsorbents were achieved by FTIR using Thermo Fisher Scientific Nicolet 6700 instrument  in a range of 400–4000 cmc method ,69,70. Sn method . WhereasDuring initial screening all the four adsorbents i.e., BB juice waste, BB fermentation waste (BBF), CH juice waste and cherry fermentation waste (CHF) were applied for the adsorptive removal of AFs. Adsorption experiments were conducted in 10 mM phosphate buffer saline with an adsorbent dosage of 2 mg/mL and suitable adsorbate concentration (AFB1 (2 µg/mL) and AFB2, AFG1 and AFG2 (1 µg/mL)) at pH 7, 25 °C and 160 rpm. After the adsorption, the supernatant was separated by centrifugation and filtration through 0.22-micron syringe filter. The supernatants were then mixed with properly measured volume of methanol for HPLC quantification. All the adsorption experiments were conducted in triplicates and the results were given as their average value.9) and (10) respectively.A water e2695 HPLC system equipped with Waters fluorescence detector 2475 (λex 365 nm and λem 435 nm) and autosampler was used for AFs quantification. Chromatographic separations of AFs were performed on Agilent reverse phase C18 column  at 40 °C. A mixture of water:methanol:acetonitrile (60:20:20) was used as mobile phase and pumped at 1 mL/min for 21 min. This method could simultaneously quantify all the under-investigation AFs with a retention time of 9.66, 11.48, 12.78, and 15.35 min for AFG2, AFG1, AFB2, and AFB1 respectively . The adsAfter the initial screening of biosorbents, the results , suggest9) and (10) respectively. Isothermal studies were performed at variable temperature i.e., 15–45 °C under optimized reaction conditions i.e., pH 7 and dosage of 2.0 mg/mL with AFs concentration in a range of 1.0–10 mg/mL. The adsorption rate (%) and the adsorption capacity (qe) were calculated using Equations  and intestinal fluids (pH 7.0) were prepared according to a previously published method , and adsModel wine was prepared as described elsewhere , and ads


Ischemic lesions could lead to secondary degeneration in remote regions of the brain. However, the spatial distribution of secondary degeneration along with its role in functional deficits is not well understood. In this study, we explored the spatial and connectivity properties of white matter (WM) secondary degeneration in a focal unilateral sensorimotor cortical ischemia rat model, using advanced microstructure imaging on a 14 T MRI system. Significant axonal degeneration was observed in the ipsilateral external capsule and even remote regions including the contralesional external capsule and corpus callosum. Further fiber tractography analysis revealed that only fibers having direct axonal connections with the primary lesion exhibited a significant degeneration. These results suggest that focal ischemic lesions may induce remote WM degeneration, but limited to fibers tied to the primary lesion. These “direct” fibers mainly represent perilesional, interhemispheric, and subcortical axonal connections. At last, we found that primary lesion volume might be the determining factor of motor function deficits. Stroke is one of the leading causes of death and disabilities  and prodAs awareness of the importance of secondary degeneration after stroke increases, the first question that needs to be answered is where the secondary degeneration occurs and its relationship to the primary ischemic lesion. The mainstream assumption is that secondary degeneration occurs primarily on cortical/subcortical regions that have direct synaptic connections with the primary lesion area, but conclusive evidence is lacking for this assumption. This assumption primarily comes from histopathological findings in preclinical studies , but sucex vivo and in vivo studies  method  method . Then, t) method  was usedAll surgical and experimental protocols in this study were carried out in accordance with the Guide for The Care and Use of Laboratory Animals  and approved by the Animal Care Committee of Zhejiang University, China. Fourteen adult male Sprague–Dawley rats, who were 2–3 months old and weighed between 230 and 280 g, were included in this study. All rats were housed in individual cages under a 12/12 h light/dark cycle with free access to water. Food was restricted to 15 g chow per day to motivate reaching and grasping performance during the training and testing periods. Eight rats were subjected to the surgery and lesion group. Based on a protocol in a previous study , we usedThe single-pellet retrieval (SPR) task was adopted for the assessment of precise reaching and grasping behavior . Before ex vivo brains were rehydrated in phosphate-buffered saline for at least 3 days before imaging and immersed in perfluoro polyether  to avoid magnetic susceptibility artifacts during MRI scanning. Diffusion weighted image (DWI) was achieved using a three-dimensional echo planar imaging (EPI) sequence with eight segments: TE/TR = 31/800 ms, b = 3,800 s/mm2, 32 gradient directions, δ/Δ = 3/18 ms, 0.16 × 0.16 × 0.16 mm3 voxel size, and two acquisitions at b = 0 s/mm2. Here, the b values were chosen to induce similar diffusion signal attenuation as conventional DTI in vivo  as the diffusivity in ex vivo brain is around 3–4 times smaller than that in in vivo brain. Similar b values were also used by other groups for ex vivo studies  with the same spatial dimensions as DWIs: TE/TR = 4/1,000 ms and 32 echoes.All scanning was performed on a 14 T vertical Bruker Micro imaging system with a 30 mm RF coil. The  studies . DWIs wiT2-weighted structural images within MIPAV1 to provide a preliminary initialization for the following tensor template generation. The T2-weighted images after AC–PC alignments were further used as reference images for eddy current and EPI geometric distortion corrections along with the DWIs of the opposite phase encoding direction using the DIFFPREP and DRBUDDI    and 3,00b = 0 s/mm2) of each rat brain using a symmetric image normalization method (SyN) in ANTs , primary lesion volume, and the motor behavioral score at 21 days after ischemia onset. These statistical analyses were completed in GraphPad Prism 83.The median FA of fibers passing through the spatially matched ROI in six control subjects was used to perform an unpaired Student’s T2-weighted images. As shown, the ischemic lesion size is relatively small with most voxels distributed in the sensorimotor cortex. The averaged lesion size is 19.2 ± 6.8 mm3 (mean ± SD). Lesion voxels are distributed in ipsilesional M1, M2, S1FL, and S1HL with the percentage of the injured volume in the total volume of this brain region 24.5 ± 12.1, 20.8 ± 11.9, 12.3 ± 17.3, and 18.9 ± 21.3%, respectively.p < 0.05, TFCE corrected, labeled as red) primarily distributed in the ipsilesional external capsule and two remote regions including the anterior part of the corpus callosum (CC) and the contralesional external capsule. To further confirm secondary WM degeneration, these TBSS-detected ROIs were transformed back from the template space to the subject’s space using the transformation matrix obtained in the registration process. Not surprisingly, the mean FA values of those ROIs in the lesion group still indicated a significant decrease (p < 0.0001) compared with the control group  and is a highly sensitive index for detection of WM integrity properties . StudiesFurther investigation of the fibers passing through these TBSS-detected WM regions indicated, surprisingly, that secondary WM degeneration primarily occurs on fibers having direct axonal connections with the primary ischemic lesion (direct fibers) and does not expand to other fibers in the sensorimotor network having no direct axonal connections with the primary ischemic lesion (indirect fibers). Because TBSS only detects changes of FA in the WM skeleton whose values represent the maximum FA of all WM voxels perpendicular to the local skeleton, TBSS itself could not tell which fibers have significant degeneration and are the contributors of the skeleton changes. Further quantitative microstructure analysis along fibers demonstrated that the degeneration of direct fibers is the major contributor to the FA changes of TBSS-detected WM skeleton. The concept of secondary damage or degeneration of remote brain regions having direct structures tied to the primary lesion, named “diaschisis,” can be tracked back to the early twentieth century . Our resIn our results, focal unilateral ischemic lesions of the sensorimotor cortex induced secondary WM degeneration which primarily occurred on the perilesional axonal connections  and the interhemispheric axonal connections . Some also occurred on axonal connections with ipsilesional subcortical regions. The subregions in the sensorimotor cortex are strongly connected , and it In addition, we found that the primary lesion volume might be the major factor in determining the final motor function recovery level. This is not surprising. The correlation between final infarct volume and motor functional outcome at the chronic stage has been reported in other studies using a middle cerebral artery (MCAO) model in rats . A similex vivo MRIs on rat brains without skulls, which could further avoid potential MRI artifacts. Second, the dynamic observation on WM microstructure changes is lacking in this study. Besides, the sample size is relatively small. In the future, a longitudinal study combined with a larger sample size will help us further explore the evolution of secondary degeneration of WM and its dynamic relation with motor functions. What is more, functional information of connectivity would help to explore the mechanisms underlying microstructure changes and motor function outcome. In the future, longitudinal studies combined with diffusion MRI and functional MRI are planned. Due to the inherent technological limitation of diffusion tensor imaging, it cannot make a distinction between two parallel but opposite directions. In this study, WM secondary degeneration was found after focal ischemic lesion. However, the anterograde and retrograde degeneration could not be differentiated by diffusion tensor image in this study when the projection between the lesion site and remote intact sensorimotor cortex is bidirectional. It will be an interesting direction to research anterograde and retrograde degeneration along a unidirectional projection pathway in a future study. Finally, although the histological results from similar animal models support our findings, it would be desirable to compare MRI findings with histological findings in the future.Several limitations in this study and perspectives for future work should be considered. First, ideally, we should use sham animals by performing similar surgeries but with Rose Bengal solution replaced with saline solution as the control group . The purIn conclusion, by using high-resolution MRI, advanced whole-brain analysis methods, and well-controlled ischemic lesion animal models, we found broad secondary degeneration in white matter following focal ischemic lesions in the sensorimotor cortex. More importantly, secondary degeneration primarily occurs on fibers having direct axonal connections with the primary lesion, which includes the perilesional, interhemispheric, and ipsilesional subcortical axonal connections. The primary lesion volume plays the most important role in determining final motor function deficits.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Guide for The Care and Use of Laboratory Animals  and the Animal Care Committee of Zhejiang University, China.ZL, HG, KX, and RB designed the study and contributed to the data interpretation and the manuscript. HG and YJ performed the animal experiments. PZ and XK conducted the MRI scanning. ZL performed the data analysis. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Proton radiotherapy has a dosimetric advantage over photon radiotherapy. Many retrospective studies have shown promising results with proton radiotherapy in treating hepatocellular carcinoma (HCC). However, clinical evidence demonstrating the benefit of protons over photons is still limited. We therefore compared the clinical outcomes of the two modalities using medical research databases from our medical foundation.We conducted a propensity score-matched cohort study based on our multi-institution medical organization research database. From January 2007 to January 2018, a total of 413 patients  who were diagnosed with HCC and primarily treated with radiotherapy with curative intent were enrolled. Overall survival (OS) and radiation-induced liver disease (RILD) were assessed. Stratified analysis was also performed to evaluate the heterogeneous effects of the two arms.p = 0.032, HR 0.56, 95% CI 0.33–0.96). The median overall survival in the proton group was not reached and that in the photon group was 17.4 months. The biological equivalent dose of radiotherapy was significantly higher in the proton group than in the photon group . The risk of RILD was significantly lower in the proton group .A total of 110 patients  were analyzed in the propensity-matched series. The matched groups were balanced for baseline tumor risk factors. Cox regression analysis revealed a significant survival benefit in the proton group (Proton radiotherapy could deliver a higher radiation dose than photon radiotherapy without increasing the risk of RILD and result in a better overall survival rate for those diagnosed with HCC and treated with radiotherapy with curative intent. Radiotherapy is one of the crucial local treatment modalities for hepatocellular carcinoma (HCC). The delivery of a higher radiation dose to the target has been proven to result in better clinical outcomes –3. HowevThere has only been one single-institution retrospective study, from the Massachusetts General Hospital (MGH), comparing the clinical benefits of proton radiotherapy over photon radiotherapy . The stuWe conducted a retrospective analysis using deidentified data retrieved from the Chang Gung Research Database (CGRD). It is an electronic health record dataset derived from the Chang Gung Medical Foundation, which consists of a group of medical centers, regional hospitals, and local hospitals and provides approximately 13–14% of the cancer healthcare services in Taiwan . Among tPatients diagnosed with HCC and receiving local radiotherapy as a primary curative treatment modality between January 2007 and January 2018 were included in the study. To exclude patients treated with palliative intent, we excluded patients with an unknown stage, distant metastasis, or a biological equivalent dose (BED) below 50 Gray (Gy). The BED was calculated using an α/β value of 10 Gy. For proton radiotherapy, a relative biological effectiveness (RBE) value of 1.1 was used. The type of radiotherapy was further identified to group patients receiving photon and proton radiotherapy. Patients whose radiotherapy was not initiated within 3 months after diagnosis, received mixed photon and proton radiotherapy, or received isotope or brachytherapy were also excluded  stage, liver fibrosis score , tumor differentiation, hepatitis B status, hepatitis C status, alpha-fetoprotein (AFP) status, ALBI grade, Child-Pugh class, tumor number, size of the largest tumor, and portal vein tumor thrombosis (PVTT) status. AJCC stage data were originally coded according to the 6th, 7th, or 8th edition depending on the year of the diagnosis and were all transformed to be in accordance with the 8th edition for the analysis.The primary outcome of this study was the overall survival (OS) of patients who were diagnosed with HCC and treated primarily with proton radiotherapy or photon radiotherapy with curative intent. OS was defined from the date of diagnosis to the date of death as a result of any cause. Radiation-induced liver disease (RILD) was also assessed to determine the toxicity of the treatment. There are two types of RILD. A patient who presented with anicteric hepatomegaly, ascites, and elevated alkaline phosphatase  2 weeks to 3 months after therapy was defined as classic RILD positive. A patient whose liver transaminase levels were elevated more than five times the upper limit of normal  or whose Child-Pugh score worsened by two or more within 3 months after the completion of radiotherapy was defined as nonclassic RILD positive .PSM was applied to reduce selection bias between the study groups. Sex, age, clinical AJCC stage, liver fibrosis score, tumor differentiation, hepatitis B status, hepatitis C status, AFP status, ALBI grade, Child-Pugh class, tumor number, size of the largest tumor, and PVTT status were selected as independent variables. Using NCSS 10 Statistical Software , the greedy method was used for matching at a 1:1 ratio between the study groups with a caliper width 0.2-fold the standard deviation of the propensity score between the study groups. The standardized mean difference (SMD) was used to evaluate covariate balance after PSM.p-value of < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS statics v 25.0  unless otherwise noted.To compare the groups, we used the Pearson chi-square test or Fisher’s exact test for categorical variables and the Student t-test or Mann-Whitney test for continuous variables depending on the result of the Kolmogorov-Smirnov test for normality. OS was assessed using the Kaplan-Meier method and the Cox regression model. Stratified analyses for OS were performed to assess the heterogeneous effects of proton and photon radiotherapy. A two-sided p = 0.002), with more cases of hepatitis B or C infection  and a higher liver fibrosis score (75.0% vs. 66.2% for Ishak F5–6) but better liver function . At the tumor level, those treated with proton radiotherapy had an earlier clinical AJCC stage , lower AFP level , and fewer cases of PVTT .A total of 12,937 patients diagnosed with HCC were identified. A total of 413 patients met the inclusion criteria and were analyzed  . Patient characteristics before and after matching are presented in Table After 1:1 PSM, 110 patients were analyzed . The baseline characteristics were balanced in the matched groups  and after PSM  . Among 110 patients in the PSM series, 101 patients had sufficient data available to evaluate RILD . There were 18 and 6 nonclassic RILD patients in the photon and proton groups, respectively. No classic RILD was recorded in either group. The risk of RILD was significantly lower in the proton group . The distribution of the BED and cases of RILD is presented in Fig. The BED was significantly higher in the proton group than in the photon group  Gy (RBE) vs. 62.5 (58.5–76.2) Gy, Clinical data comparing proton and photon radiotherapy with curative intent for HCC are limited. In the present study, we found that after appropriately adjusting for prognostic variables, patients who received proton radiotherapy had significantly better OS, which may be driven by a higher BED and lower risk of RILD.These results echo those of the MGH report . The stup < 0.001) and 1-year survival rate  for those who received BED ≥58 Gy [p = 0.008) and 1-year OS  rates [For liver tumors, it has been well demonstrated that a higher radiation dose could result in better oncological outcomes. The prescribed dose varies among series depending on the tumor entity, radiotherapy technique, and combined treatment modalities. In the era of 3D conformal radiotherapy (3DCRT), Park et al. analyzed 158 HCC patients and found that a higher radiation dose predicted a better tumor response rate . However, the radiation dose also seemed to be a determining factor for RILD  [15) was found to be a parameter capable of predicting the deterioration of hepatic function [RILD is a major concern of using radiotherapy to treat liver tumors. The risk of RILD may be related to the liver radiation dose, the dose distribution, and underlying liver disease . Investifunction . These f30) compared to photon radiotherapy [3) who were treated with proton radiotherapy. The oncological results were a 1-year in-field local control rate of 95.5% and a 1-year OS rate of 81.8%; there were no cases of liver failure, and only one case of nonclassic RILD could be identified [n = 92) of the patients included in the PSM series had multiple tumors or a largest tumor size > 5 cm, and the risk of RILD would therefore be high with photon radiotherapy. Nonetheless, with proton radiotherapy, albeit with a higher BED, the risk of RILD was significantly lower, which is attributed to the advantages of its physical properties.The story could be changed by the superior physical properties of proton radiotherapy. A proton beam has a finite range of energy deposition with no exit dose after the target. This physical advantage, compared with photon beams, may allow a higher target dose to be achieved without the unwanted spread of low doses to the surrounding normal liver. In a dosimetric study, Wang et al. demonstrated that for liver tumors, proton radiotherapy could significantly lower the mean liver dose and volume of normal liver receiving a dose of more than 30 Gy (RBE)  protocols . BrieflyOur study does have potential limitations. Limited by the nature of the CGRD, we could only record the “size of the largest tumor” instead of the “total tumor volume.” Although the “size of the largest tumor” has been demonstrated to predict survival outcomes for HCC patients undergoing liver transplantation , this maIn this propensity-matched analysis, we demonstrated that compared to photon radiotherapy, proton radiotherapy could deliver a higher radiation dose without increasing the risk of RILD and result in a better overall survival rate for those diagnosed with HCC and treated with radiotherapy with curative intent. The physical advantages of proton therapy allow it to be used to treat HCC safely and potentially improve OS. Prospective investigations are needed to assess the role of proton radiotherapy in treating liver tumors.