In the Experimental procedures of the original article , the concentrations of stock solutions for taurine, hydrocortisone, and triiodothyronine were mistakenly reported rather than the final concentrations of those components in the medium. Additionally, nonessential amino acids (NEAA) were mistakenly omitted from the medium recipe.Corrected text is provided below.taurine (0.25 mg/ml) (Sigma–Aldrich), hydrocortisone (20 ng/ml) (Sigma–Aldrich), triiodothyronine (0.013 ng/ml) (Sigma–Aldrich), 0.1 mM NEAA (Fisher Scientific), and 10 mM nicotinamide (Sigma–Aldrich).”“Briefly, the cells were cultured in MEM-Nic , 1% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin, 1% N2 supplement (Sigma–Aldrich), The authors state that the corrections do not affect the results and conclusions of the paper.


The most dispensed antibiotic was oxytetracycline, a Watch antibiotic, mainly without prescriptions. Good knowledge of ABU and AMR was associated with work experience for more than one year (p = 0.016), while good practices were associated with male gender (p = 0.039) and work experience of more than one year (p = 0.011). The study found moderate knowledge, positive attitudes, and moderate practices of pharmacy professionals on poultry ABU and AMR. There was high dispensing of poultry antibiotics without prescriptions, which calls for strict implementation of antimicrobial stewardship and surveillance programs in poultry production in Zambia to reduce AMR.Globally, the inappropriate dispensing and use of antibiotics in animals has contributed to the development of bacterial antimicrobial resistance (AMR). In Zambia, there is insufficient information among community pharmacy professionals on antibiotic use (ABU) and AMR in food-producing animals. This study assessed community pharmacy professionals’ knowledge, attitudes, and practices regarding poultry antibiotic dispensing, use, and bacterial AMR in the Lusaka district of Zambia. A cross-sectional study was conducted among 178 community pharmacy professionals between February and April 2022 using a semi-structured questionnaire. Data were analyzed using Stata version 17. Of the total participants ( In recent years, there has been an increase in the use of antibiotics in poultry farming for improved egg production and growth promotion, as well as the prevention and treatment of infections across countries arising from increased intensification of farming ,2,3,4,5.Poultry has been reported to be a source of proteins and income for many people across the globe, leading to increased demand for poultry products ,3,20. DuOverall, the access to antibiotics among poultry farmers, often without prescriptions, has worsened the problem of bacterial AMR ,35. AntiGlobally, the use of antibiotics in both layer and broiler production systems has been reported ,44,45. MAMS and surveillance programs may help reduce bacterial AMR by promoting the rational use of antibiotics ,52,53,54Understanding the knowledge, attitudes, and practices (KAP) of professionals who prescribe and dispense antibiotics used in poultry is crucial in promoting rational ABU and curbing AMR ,15,62. ACurrently, Zambia, a country in sub-Saharan Africa, faces the challenge of AMR in human and animal medicine ,66,67,68Overall, 178 participants participated in the study, of which 91 (51.1%) were pharmacists and 87 (48.9%) were pharmacy technologists. The largest proportion, 79 (44.4%) of participants were between 26 and 33 years old, and 91 (51.1%) had more than five years of working experience. More than half, 103 (57.9%) of the participants were males, 95 (53.4%) were married, and 136 (76.4%) resided in urban areas .The most dispensed antibiotics in retail pharmacy outlets for poultry use were oxytetracycline (33.9%) followed by sulfadimidine (9.3%), gentamicin/doxycycline (gentadox) (8.5%), and amoxicillin (8.0%) .Most (96.6%) of the participants were aware of AMR, knew about the use of antibiotics in poultry (54.5%), discouraged the addition of antibiotics to chicken feed (73.6%), and encouraged farmers not to stop administration of antibiotics until the course was complete (86.5%). Most (73%) participants dispensed antibiotics without prescriptions .p = 0.049) higher for pharmacy technologists, 83.0 (12.1), than for pharmacists, 79.7 (10.6). Furthermore, there was a significant difference in knowledge scores between male pharmacy technologists and pharmacists, with the former recording higher scores (69.3 vs. 61.9). In addition, pharmacy technologists aged between 26 and 33 years had higher scores for attitude than pharmacists of a similar age group (83.2 vs. 78.2), staying in urban areas (83.5 vs. 79.3) and with less than one-year working experience (90.6 vs. 72.9).The results  demonstrp = 0.002), knowledge and practice  and attitude and practice   .To the best of our knowledge, this is the first study in Zambia to assess community pharmacy professionals’ knowledge, attitudes, and practices (KAP) on ABU and AMR for antibiotics used in poultry production. Overall, there was moderate knowledge, positive attitudes, and moderate practices regarding poultry ABU and AMR. The most dispensed antibiotics used in poultry production were oxytetracycline, followed by gentamicin+doxycycline (gentadox) and amoxicillin. A brief review of KAP studies in different countries presented various findings in relation to our study.The overall knowledge and practices concerning ABU and AMR among our participants were moderate, contrasting with a study in the Kingdom of Bhutan in which most veterinarians and veterinarian assistants had good knowledge of AMR building on the Bhutan National Plan of AMR . A multiThe low knowledge of ABU and AMR reported in these studies could be due to a lack of participation in AMR activities, including awareness weeks, continuous professional development, and not attending refresher courses. Our study found that the pharmacy professionals generally had positive attitudes regarding ABU and AMR. This is important as positive attitudes can influence the practice and help improve the future use of antibiotics in poultry. Similarly, favorable attitudes were reported in the Kingdom of Bhutan among veterinarians and para-veterinarians . In BangThe moderate practices reported in our study can be improved by adding more information regarding veterinary medicine into the pharmacy curriculum and promoting greater collaboration  between the two professions . In addiOf concern is that the indiscriminate use of antibiotics in the poultry sector in Zambia was similar to other studies ,84,85, wThere are also gaps in the awareness of AMR and the links between ABU and AMR in our study, similar to other studies ,71. ThisMost participants in our study felt that restricting the use of antibiotics in poultry would be beneficial, along with instigating guidelines to improve the prescribing and dispensing of antibiotics. This is because continuous access to antibiotics in poultry production without prescriptions is a global challenge requiring appropriate action and policies ,91,101. On a positive note, most participants in our study encouraged poultry farmers to maintain the withdrawal period and not to sell poultry products when still using antibiotics, which, as mentioned, is important in preventing the passage of resistance to humans ,112. TheEncouragingly as well, our findings indicated that the majority (84%) of pharmacy professionals referred poultry farmers to animal health workers for specialized services, indicating a good collaboration between pharmacy and veterinary professionals to help reduce the inappropriate use of antibiotics among poultry . There iThis study also demonstrated that knowledge of ABU and AMR among pharmacy professionals was influenced by years of work experience. Professionals who had worked for one year and above had better knowledge of ABU and AMR. However, a study among veterinarians reported that good knowledge of ABU and AMR was reported among those that read the National Action Plan on antibiotics and AMR . AnotherFurther, our study found that participants’ knowledge, attitudes, and practices on ABU and AMR were related. Increasing participants’ knowledge through education and training activities on ABU and AMR should improve their attitudes and practices leading to improved use of antibiotics in the future. However, using a cross-sectional study to predict the interventions’ outcomes is impossible. Additionally, our study did not include questions whether pharmacy professionals had asked poultry farmers on previous access to antibiotics for use in their flocks. Nevertheless, this study provides vital information that can be used to help develop context-specific strategies more likely to restrict the prescribing and dispensing of antibiotics for poultry use in the future. As an ongoing study, we recommend future studies that may predict outcomes of introducing educational campaigns and training activities on antibiotic use in poultry and AMR among pharmacy professionals. Furthermore, we recommend the implementation of evidence-based practices that focus on behavior change of all stakeholders  involved in handling and administration of antibiotics used in poultry. This will be based on strategies that have been successfully introduced in a number of sectors and situations to improve future ABU .A cross-sectional study was conducted among community pharmacists and pharmacy technologists in Zambia’s Lusaka province from February 2022 to April 2022. Lusaka district was purposively selected because it had the largest number of poultry farmers at the time of the study . MoreoveThis study was conducted among community pharmacy professionals (pharmacists and pharmacy technologists) working in the Lusaka district. Community pharmacies and the pharmacy professionals registered with the Zambia Medicines Regulatory Authority (ZAMRA) and the Health Professions Council of Zambia (HPCZ) were eligible for the study. No community pharmacist was excluded due to their age. However, we excluded community pharmacies that did not stock poultry antibiotics at the time of the study. We also excluded veterinarians and agrovets as they do not practice in community pharmacies. In Zambia, every registered community pharmacy is under the responsibility of a pharmacist and assisted by pharmacy technologists. The number of registered community pharmacies under the responsibility of a registered pharmacist in Lusaka was obtained from the ZAMRA website . Thus, aThe data collection tool was adapted from a similar study by Kalam and colleagues , reviewet-test was used to compare the mean differences across explanatory variables since the knowledge, attitude, and practice scores were normally distributed. The normality test was done graphically using QQ-plots and Shapiro–Wilk test.Descriptive statistical methods were used to assess socio-demographic characteristics by the mean score for knowledge, attitude, and practice of ABU and AMR. Student p < 0.20 in univariable analysis were included in the multivariable linear regression model, which included specialty (Pharmacists vs. Pharmacy technologists). Stepwise regression and backward elimination algorithms were used with a liberal p-value for exclusion (p < 0.15) to fit the multivariable model. Only the specialty was fixed in the multivariable models because it was set as a priori variable.Three general linear regression models were fitted with knowledge, attitude, and practice scores as outcomes, respectively. First, a univariable linear regression model was fitted with socio-demographic characteristics. Candidate variables with p < 0.05.Studentized residuals and the Cook–Weisberg test for heteroskedasticity were used to assess the goodness of fit of the models. Finally, we checked for linear relationships  under model-checking procedures and the variance inflation factors (VIF) to check for multicollinearity. In the final model, none of the factors reached a VIF value of 5; thus, multicollinearity was not a problem in this case. Stata/BE version 17  was used in all the statistical analyses. Significance was considered when This study found that the most dispensed antibiotics for poultry use in Zambia are tetracyclines, sulfonamides, aminoglycosides, and penicillins. The pharmacists and pharmacy technologists had moderate poultry ABU and AMR knowledge and practices. Conversely, their practice demonstrated some gaps that require a multi-sectoral one-health approach in tackling AMR, as well as continuous professional development (CPDs) training on the use of antibiotics in poultry. Further, based on our findings with most young professionals having poor practices, there is a need to strengthen the pharmacy training curriculum on poultry diseases and ABU may improve their practice. Alongside this, there is a need to develop and strengthen strategies that promote the prudent use of antibiotics in poultry. The development of AMS and surveillance programs may be one way forward to address the AMR challenges faced in Zambia and other nations. Community pharmacy professionals should be included in any future AMS programs given their importance in this and other areas, and we will be monitoring this in the future. We will also be following up with veterinarians and agrovet personnel in the future, especially those working in rural areas, as key stakeholders with improving future ABU among poultry.


Enterocytozoon hepatopenaei (EHP) in cultured giant freshwater prawns (Macrobrachium rosenbergii) in 2021. While the prawn farms exhibited no clinical signs of EHP or mortality, histopathological and molecular biological analyses detected EHP during disease monitoring. The prevalence of EHP in infected prawn farms ranged from 4.9% to 18.2%. This is the first case of EHP infection in giant freshwater prawns cultured in the Republic of Korea.To our knowledge, this is the first reported detection of Enterocytozoon hepatopenaei (EHP) was first isolated from Pacific whiteleg shrimp in April 2020; however, there are no existing reports of EHP infection in other shrimp or prawns. Here, we aimed to investigate EHP infection and its prevalence in giant freshwater prawn farms in the Republic of Korea. We tested prawns from 22 farms for EHP infection, and samples from eight farms showed positive EHP infection results in 2021. In EHP-infected prawn farms, the prevalence ranged from 4.9% to 18.2%. The prevalence of EHP infection in the Republic of Korea, derived from the prevalence in prawn farms, was estimated to be 0.8% in 2021. The proliferation of EHP was observed within the hepatopancreatic epithelial cells of prawns using H&E and Giemsa staining. Mature EHP was observed in the sinus between epithelial cells of the digestive tubules. Phylogenetic analysis revealed a clade distinct from the previously reported EHP in Pacific whiteleg shrimps. This is the first report of EHP infection in a giant freshwater prawn in the Republic of Korea, where the prevalence of EHP infection is not high, but it is recognized as an emerging disease that requires periodic monitoring and quarantine management in giant freshwater prawns.In the Republic of Korea,  Enterocytozoon hepatopenaei (EHP) with intracellular parasitic capacity. HPM is a wasting disease that results in economic loss due to growth retardation and reduced production  ranged from 99.6% to 99.7%.The is study . BLAST assu rRNA gene sequences revealed that the EHP sequence obtained from the giant freshwater prawn (designated NFQS_MR_EHP1) was clearly distinct from the previously reported EHP sequences and formed a monophyletic clade  sequence detected from Pacific whiteleg shrimp that was previously reported in 2021 in the Republic of Korea. Artanto et al. (2019) also found that the nd China .EHP ranging from 0.8 to 1 µm were observed in epithelial cells and sinuses of the hepatopancreas in giant freshwater prawns; other lesions, including regressive and inflammatory changes, such as necrosis of epithelial cells, hemocytic infiltration, and serous exudate accumulation in the hepatopancreas, were absent ,35. The Vibrio sp. and EHP [In this study, the incidence of EHP infection in prawn farms was relatively low, ranging from 4.9 to 18.2%, in contrast to incidence ranging from 30 to 93% in other shrimp farms ,42,43. I and EHP . Aquacul and EHP . Giant fThe lack of any drug treatment for HPM ,16, tranM. rosenbergii) in the Republic of Korea. EHP was detected in eight farms between April and November 2021, and the prevalence of HPM within each farm ranged from 4.9% to 18.2% using PCR results. EHP was detected histopathologically in epithelial cells and sinuses of hepatopancreas without inflammation, such as hemocytic infiltration. Hence, it is crucial to monitor and control the status of HPM in M. rosenbergii in the Republic of Korea.In conclusion, this is the first report of EHP infection in giant freshwater prawns (


To genetically dissect the contribution of HYDs to carotenoid metabolism and wheat growth and yield, we isolated loss-of-function mutants of the two homoeologs of HYD1 (HYD-A1 and HYD-B1) and HYD2 (HYD-A2 and HYD-B2) from the sequenced ethyl methanesulfonate mutant population of the tetraploid wheat cultivar Kronos, and generated various mutant combinations. Although functional redundancy between HYD1 and HYD2 paralogs was observed in leaves, HYD1 homoeologs contributed more than HYD2 homoeologs to carotenoid β-ring hydroxylation in this tissue. By contrast, the HYD2 homoeologs functioned toward production of lutein, the major carotenoid in mature grains, whereas HYD1 homoeologs had a limited role. These results collectively suggested subfunctionalization of HYD genes and homoeologs in different tissues of tetraploid wheat. Despite reduced photoprotective responses observed in the triple hyd-A1 hyd-B1 hyd-A2 and the quadruple hyd-A1 hyd-B1 hyd-A2 hyd-B2 combinatorial mutants, comprehensive plant phenotyping analysis revealed that all mutants analyzed were comparable to the control for growth, yield, and fertility, except for a slight delay in anthesis and senescence as well as accelerated germination in the quadruple mutant. Overall, this research takes steps toward untangling the functions of HYDs in wheat and has implications for improving performance and consumer traits of this economically important global crop.The genetic diversity provided by subgenome homoeologs in allopolyploid wheat can be leveraged for developing improved wheat varieties with modified chemical traits, including profiles of carotenoids, which play critical roles in photosynthesis, photoprotection, and growth regulation. Carotenoid profiles are greatly influenced by hydroxylation catalyzed by The online version contains supplementary material available at 10.1007/s00122-023-04276-3. Triticum turgidum; AABB subgenome) is an allopolyploid formed by the hybridization of two diploid species: T. urartu  and an unknown Sitopsis species of genus Aegilops (BB) about half a million years ago   are important precursors for vitamin A biosynthesis in humans.Carotenoid profile is one trait that can be fruitfully altered in wheat for improving plant performance and nutritional quality. Carotenoids can be divided into two groups: carotenes and xanthophylls, with the latter being oxygenated derivatives of carotenes. Carotenoid compounds collectively are important for processes central to plant physiology including photosynthesis, photoprotection, phytohormone biosynthesis, pigmentation, and stress response  carotenoids and the non-heme di-iron type β-hydroxylases (HYDs) that prefer the β-carotene-branch  carotenoids  and HYD2 (HYD-A2 and HYD-B2) from a sequenced ethyl methanesulfonate (EMS) mutant population of tetraploid wheat cv. Kronos  setting to infer the potential effect of these combined mutations on plant performance.To genetically dissect the contribution of HYDs to carotenoid metabolism, photosynthesis, and the growth and yield of wheat, we isolated loss-of-function mutants in each homoeolog of −2 s−1 for 3 days. The seedlings were transferred to soil and grown in a temperature-controlled greenhouse. Light intensities in the greenhouse varied throughout the day but averaged around 400 μmol m−2 s−1. Supplemental lighting was supplied to plants to extend the photoperiod to a 16 h light and 8 h dark cycle. For greenhouse experiments, individual plants were grown in pots that were approximately 3 L in volume, with a top diameter of 16 cm, a bottom diameter of 13 cm, and a height of 18 cm. Plants were watered twice weekly with nutrient water (Table S1). Every fourth watering was done with distilled water to ensure that salts did not build up in the soil. Watering was stopped at the hard dough stage.Wheat seeds were surface sterilized using a 1% (w/v) sodium hypochlorite and 0.1% (v/v) Triton X-100 solution for 35 min and rinsed at least 5 times with sterile distilled water. For genotyping and whole-plant phenotyping, as well as carotenoid and photosynthesis analyses, sterilized seeds were stored at 4 °C in dark for 3–4 days, then kept at room temperature under a constant light intensity of 120 μmol mHYD1 and HYD2 homoeologs. Putative nonsense mutants for each HYD homoeolog were functionally characterized to verify loss-of-function. To clone the hyd/HYD homoeologs, leaves of the mutant and wild-type plants were collected and total RNA was extracted from 100 mg of leaf tissue using a GeneJET RNA Purification Kit ; the total RNA was subsequently treated with DNase I (Thermo Fisher Scientific) to remove the residual genomic DNA in the sample. First-strand cDNA was synthesized using SuperScript™ III reverse transcriptase . The open reading frame of the wild-type HYD or the mutant hyd alleles was amplified using Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific), cloned into the pENTR/D-TOPO vector (Invitrogen), and then recombined into pDEST17 (Invitrogen) using the Gateway LR Clonase II Enzyme Mix (Invitrogen). Primers used in the TOPO cloning and in the mutant screening are listed in Table S2.A sequenced tetraploid wheat cv. Kronos EMS mutant population . The cells were grown at 37 °C for an additional 4 h with shaking at 220 rpm and then harvested. For carotenoid extraction, the bacterial pellets were resuspended in 300 μL of formaldehyde, followed by the addition of 300 μL of methanol, and then 600 μL of ethyl acetate. The mixture was centrifuged and the supernatant was transferred to a new tube. After phase separation by adding 400 μL of H2O, the upper ethyl acetate layer was transferred to a high-performance liquid chromatography (HPLC) vial, 10 μL of which was injected on a reverse phase HPLC and separated following a previously established gradient , HYD-B1 (T4-0641), HYD-A2 (T4-0870), and HYD-B2 (T4-4420) were designated as hyd-A1, hyd-B1, hyd-A2, and hyd-B2, respectively, and used for the following studies. These mutants were each backcrossed to the wild-type parental line Kronos two times to remove ~ 75% of background mutations caused by chemical mutagenesis. The homozygous, background-purified single mutants were intercrossed and genotyped to identify homozygous double mutants of the HYD1 or HYD2 homoeologs . The homozygous hyd-A1 hyd-B1 and hyd-A2 hyd-B2 mutants were crossed to produce quadruple mutant plants heterozygous for the HYD-A1, HYD-B1, HYD-A2, and HYD-B2 loci. These plants were self-pollinated to produce a segregating population for identification of the homozygous single, double, triple, and quadruple mutants used in this study. The segregating progenies were genotyped for the HYD-A1, HYD-B1, HYD-A2, and HYD-B2 loci to identify various mutant combinations using Cleaved Amplified Polymorphic Sequence (CAPS) and derived CAPS (dCAPS) markers generated for each of the four selected EMS mutations. Primers and restriction enzymes  used for the dCAPS markers of HYD-A1 and HYD-B1 are shown in Table S2. The CAPS and dCAPS markers used for HYD-A2 and HYD-B2 were previously described  and whole grain (~ 300 mg) samples and HPLC analysis were conducted using established procedures  was determined with the first saturating pulse.Non-photochemical quenching (NPQ) induction and relaxation was measured using the LI-6400XT Portable Photosynthesis System . Leaves from 12-week-old plants grown under greenhouse conditions were dark-adapted overnight by wrapping leaves completely in aluminum foil. The flag leaf of the primary tiller was used for measurement whenever possible. Flag leaves of other tillers were also used if the flag leaf of the primary tiller was beginning to senesce or was otherwise unsuitable. Between 4 and 6 biological replicates were measured for each genotype. Dark-adapted leaves were clamped into the fluorometer chamber and allowed for a further dark adaptation of 5 min prior to measurement. The leaves were exposed to an actinic light intensity of 1000 μmol mPlant height was measured during the grain filling stage (~ 14 weeks old) as the distance from the soil line to the top of the highest spike (not including awns). Anthesis time for each plant was defined as the number of days from planting to the time when the first anthers became extruded. Senescence in wheat follows a characteristic pattern in spikes where green awn tissue browns gradually, and this pattern of browning proceeds down the spike. In this study, senescence was defined as the point where half of the spikes had no green tissue remaining between flag leaf ligule and the tip of the awns. The number of days from planting to this senescence was measured for the control plants and each of the mutant lines.For pot yield assessment, plants were harvested after no green aerial tissues remained. Spike number, total spikelets per spike, and fertile spikelets per spike were determined manually for 4–6 plants of each genotype at harvest. Spikes were dried for 3 days at 37 °C and weighed after drying. Grains were counted and weighed after being manually separated from chaff. The chaff weight for each spike was computed as the difference between the dried spike mass and the total grain mass. Spike fertility index was defined as the number of grains per gram of spike chaff for each plant . Grains were spread across the scanner bed and placed with their crease facing downward. The glass bed of the scanner was covered with a transparency to prevent scratching and black paper was laid on top of the grains to improve contrast in the image. Scans were acquired at 300 dpi and stored as jpeg files. No color correction options were enabled during image acquisition. Grain parameters including length, width, perimeter, and greyscale values were measured from the images of 11–12 plants of each genotype using GrainScan  were used.Germination of seeds and loss of water in leaves were determined as previously described with modification  were imaged after 4 days using a document scanner (Epson). Prior to imaging, condensation was removed from the inside of the transparency using a Kimwipe . Images were acquired as 400 dpi TIF files.Seminal root phenotypes were analyzed on 4-days-old wheat seedlings. Sterilized seeds were placed in transparent sheet protectors on a sheet of germination paper  soaked with 40 mL of sterile distilled water. Germination paper was aligned with the bottom of the sheet protector; a single seed was placed in each sheet protector with its crease facing the germination paper (and with embryo facing downward) in the midpoint of the sheet and 2.5-cm from its top edge. Seeds were stratified at 4 °C for at least three days inside the sheet protectors. After stratification, sheets with seeds of different genotypes were placed randomly in a 3-ring binder and positioned vertically at room temperature (~ 22 °C) under long-day conditions  with a light intensity of 120 μmol mSeminal root traits were assessed from images using RhizoVision Explorer . Plants of each genotype  were grown in a liquid growth solution consisting of the same nutrient concentrations as used for the soil-grown plants (Table S1). These plants were also grown under similar, semi-controlled greenhouse conditions described above. Wheat seeds for this analysis were germinated in the germination sleeves as previously described for 4-days-old seedlings, transferred to cylindrical sponges roughly 2.5 cm in length and diameter, then mounted in a foam board floating in the nutrient solution. The tanks of nutrient solution had dimensions 24 cm × 24 cm × 16 cm, with each tank holding 4 plants that were spaced 5 cm apart. The nutrient solution was filled up to 3 cm from the top of the tank (approximately 9.2 L) and was changed twice weekly to ensure sufficient dissolved oxygen. Root length and shoot fresh weight were assessed at harvest. Root and shoot dry weight were measured after drying for 5 days at 37 °C. The root: shoot ratio, defined as the ratio of the dry mass of the root compared to the shoot, was also computed.t test was used when only two genotypes were being assessed. All tests were carried out using JMP software .When all genotypes were being evaluated, one-way analysis of variance (ANOVA) was performed followed by pairwise mean comparisons using Tukey’s Honestly Significant Difference (HSD) test. Student’s hyd-A1, 60 hyd-B1, 50 hyd-A2, and 67 hyd-B2 mutants were identified. Selected mutant line T4-2017 (hyd-A1) contains a G to A mutation at the exon–intron junction (donor splice site) of the second intron, leading to a 19-amino acid (AA) insertion followed by a premature stop codon, thus a net result of a 155-AA truncation of HYD-A1  contains a G to A mutation in the third exon, resulting in a premature stop codon (W158*), and a 160-AA truncation of HYD-B1 , and the G to A mutation in the third exon of HYD-B2 in selected mutant line T4-4420 (hyd-B2) lead to premature stop codons in HYD-A2 (Q113*) and HYD-B2 (W149*), respectively  and the mutant alleles of hyd-A1, hyd-B1, hyd-A2, or hyd-B2, indicating that the proteins encoded by these truncated mutants are compromised for HYD function .To investigate the contribution of HYDs to carotenoid metabolism and plant growth in wheat, a library of 1536 sequenced tetraploid wheat cv. Kronos EMS mutants , lutein, and β-carotene represent the carotenoids that accumulate in leaves of the control and all mutant genotypes  from those of the control  in leaves of hyd-A1 hyd-B1 hyd-A2 hyd-B2 than those of the control. However, total carotenoids decreased by only 8% in the quadruple mutant when compared to the control , and NPQ induction and relaxation were determined (Tables P < 0.05) was observed in chlorophyll a (Chl a), chlorophyll b (Chl b), total chlorophylls (Chl a + b), or chlorophyll a/b ratio (Chl a/b) relative to the control  between any of the mutant lines (including the quadruple mutant) and the control  reduced in hyd-A1 hyd-B1 hyd-A2 and marginally reduced in hyd-A1 hyd-B1 hyd-B2 (Table S3). The quadruple mutant hyd-A1 hyd-B1 hyd-A2 hyd-B2 displayed slower NPQ induction and lower max NPQ (a ~ 34% reduction) than the control  had similar levels of lutein and β-carotene to the control, suggesting that the HYD1 homoeologs, unlike the HYD2 homoeologs, do not play a major role in the production of these carotenoids in mature tetraploid wheat grains  were examined for their growth and pot yield traits  among the hyd mutants and the control at both flowering stage and grain filling stage  were observed in the root length, dry weight, or root:shoot ratio (Table S4) across any of the mutant genotypes examined relative to the control.To evaluate the effects of hyd mutations on plant productivity, several pot yield components were examined in the hyd mutants and the control. These traits included grain morphology, grain weight and yield, harvest index , fertile spike numbers per plant, spike fertility index (grain numbers/spike chaff mass), spikelet fertility rate , and grains per spikelet  in the β, β-branch and lutein in the β,ε-branch are the three xanthophylls produced and accumulated in leaves  relative to hyd-A1 hyd-B1 hyd-A2 suggests a relatively more important role of HYD-B2 than HYD-A2 in β-xanthophyll biosynthesis in leaves  to produce zeaxanthin , which is subsequently converted to violaxanthin and neoxanthin; such activities of CYP carotenoid hydroxylases were also suggested by the analysis of the Arabidopsis thaliana cyp97a3 mutant  and lutein  , which is subsequently converted to lutein  was shown to be the major HYD activity for the conversion of β-carotene to zeaxanthin in maize endosperm through RNAi-mediated silencing of ZmBCH2  in vegetative and reproductive tissues was also studied in other plant species, such as maize  genes from plant and bacterial sources in transgenic and transplastomic tomato plants changed their carotenoid profiles and resulted in rebalancing of biomass partitioning in vegetative and reproductive tissues Supplementary file2 (PDF 338 KB)Below is the link to the electronic supplementary material.


For people with advanced-stage Kaposi’s sarcoma (KS), a common HIV-associated malignancy in sub-Saharan Africa, mortality is estimated to be 45% within 2 years after KS diagnosis, despite increasingly wide-spread availability of antiretroviral therapy and chemotherapy. For advanced-stage KS, chemotherapy in addition to antiretroviral therapy improves outcomes and saves lives, but currently, only ~50% of people with KS in western Kenya who have an indication for chemotherapy actually receive it. This protocol describes the evaluation of a multicomponent patient navigation strategy that addresses common barriers to service penetration of and fidelity to evidence-based chemotherapy among people with advanced-stage KS in Kenya.This is a hybrid type III effectiveness-implementation study using a non-randomized, pre- post-design nested within a longitudinal cohort. We will compare the delivery of evidence-based chemotherapy for advanced-stage KS during the period before (2016–2020) to the period after (2021–2024), the rollout of a multicomponent patient navigation strategy. The multicomponent patient navigation strategy was developed in a systematic process to address key determinants of service penetration of and fidelity to chemotherapy in western Kenya and includes (1) physical navigation and care coordination, (2) video-based education, (3) travel stipend, (4) health insurance enrollment assistance, (5) health insurance stipend, and (6) peer mentorship. We will compare the pre-navigation period to the post-navigation period to assess the impact of this multicomponent patient navigation strategy on (1) implementation outcomes: service penetration (chemotherapy initiation) and fidelity (chemotherapy completion) and (2) service and client outcomes: timeliness of cancer care, mortality, quality of life, stigma, and social support. We will also describe the implementation process and the determinants of implementation success for the multicomponent patient navigation strategy.This study addresses an urgent need for effective implementation strategies to improve the initiation and completion of evidence-based chemotherapy in advanced-stage KS. By using a clearly specified, theory-based implementation strategy and validated frameworks, this study will contribute to a more comprehensive understanding of how to improve cancer treatment in advanced-stage KS.The online version contains supplementary material available at 10.1186/s43058-022-00281-7. Contributions to the literatureFor advanced-stage Kaposi’s sarcoma, chemotherapy in addition to antiretroviral therapy improves outcomes and saves lives.Despite the evidence for chemotherapy, currently only approximately 50% of people with advanced-stage Kaposi’s sarcoma in western Kenya who should receive chemotherapy actually receive it.This study will contribute to our understanding of whether a multicomponent patient navigation strategy is effective in improving chemotherapy service penetration (chemotherapy initiation) and fidelity (chemotherapy completion) for advanced-stage Kaposi’s sarcoma in sub-Saharan Africa.Kaposi’s sarcoma (KS) continues to be one of the most common HIV-associated malignancies in sub-Saharan Africa, and unfortunately, KS also continues to be deadly . MortaliThe reasons for suboptimal chemotherapy initiation and completion for advanced-stage KS are multifactorial including individual , inter-personal , community/social , and environmental factors  , 8. TogePatient navigation is a community-based strategy  that proIn high-income countries, patient navigation has been shown to be effective and cost-effective for improving outcomes across the cancer care continuum, including cancer screening rates . It alsoBased on our work describing major barriers to accessing KS care  and the This study addresses an urgent need for evidence-based strategies to improve service penetration of and fidelity to cancer treatment in KS, which continues to be a common, deadly, and debilitating disease in sub-Saharan Africa. It also incorporates the strength of a type III effectiveness-implementation hybrid study that evaluates primary implementation outcomes as well as secondary clinical effectiveness outcomes, thus potentially accelerating the process of implementing evidence-based interventions into routine practice .Throughout this protocol, we use Proctor et al.’s taxonomy of implementation outcomes as the framework to define and distinguish implementation, service, and client outcomes . We alsoThe following illustrates this distinction for service penetration, which Proctor et al. define as “the number of eligible persons who use a service, divided by the total number of persons eligible for the service.”  (1) ServEvaluate the impact of a multicomponent patient navigation strategy on service penetration of, and fidelity to, evidence-based chemotherapy for people with advanced-stage KS by comparing the pre-navigation period (2016–2020) to the post-navigation period (2021–2024). We hypothesize that the multicomponent patient navigation strategy will increase service penetration of and fidelity to evidence-based chemotherapy post-navigation as compared to pre-navigation among people with advanced-stage HIV-associated KS.Evaluate the impact of a multicomponent patient navigation strategy on (a) service outcomes, including timeliness (time to oncology consultation), and (b) client outcomes, including mortality, quality of life, stigma, and social support. We hypothesize that a multicomponent patient navigation strategy will decrease time to oncology consultation, decrease KS-associated mortality, increase the quality of life, decrease stigma, and increase social support post-navigation as compared to pre-navigation among people with advanced-stage HIV-associated KS.Describe the implementation process and degree to which the multicomponent patient navigation strategy was successfully implemented, focused on service penetration, fidelity, acceptability, feasibility, appropriateness, and client satisfaction.Identify the determinants of successful implementation of the multicomponent patient navigation strategy, specifically focused on determinants of service penetration of and fidelity to the multicomponent patient navigation strategy.This study is a hybrid effectiveness-implementation type III study,  using a This study will take place at the Academic Model Providing Access to Health (AMPATH) in Eldoret, Kenya. AMPATH is an academic medical partnership between Moi University School of Medicine, Moi Teaching and Referral Hospital and several North American and European Universities led by Indiana University. AMPATH oversees over 50 HIV primary care clinics and delivers care to ~160,000 clients in western Kenya. Dermatology and Oncology services including diagnostic biopsy and treatment services for KS are available within the AMPATH healthcare system. As part of routine clinical care, the KS Center for Excellence at AMPATH implemented a multicomponent patient navigation strategy in 2021. All clients who are diagnosed with KS are screened using clinical evaluation, AIDS Clinical Trials Group Oncology Committee (ACTG) staging criteria, and WHO staging criteria for KS. AMPATH KS Center of Excellence invites any clients with KS who meet criteria for chemotherapy  to participate in the multicomponent patient navigation strategy .The parent longitudinal cohort enrolls all clients of any age with newly diagnosed KS. This includes clients with newly diagnosed KS who have either biopsy confirmed KS or are diagnosed with KS on clinical grounds alone when a biopsy is unsafe . We will exclude clients who have a prior biopsy confirmed diagnosis of KS or those who are unable to provide consent.For this study, the control group will include all clients with advanced-stage newly diagnosed KS enrolled in the parent cohort who are eligible for chemotherapy (based on the criteria outlined above) enrolled during the pre-navigation period (2016–2020). The study group will include all clients with advanced-stage newly diagnosed KS enrolled in the parent cohort who are eligible for chemotherapy and are engaged in the multicomponent patient navigation strategy during the post-navigation period (2021–2024).We will also enroll healthcare workers who are involved in the multicomponent patient navigation strategy and/or the care of clients with KS to understand the impact of the implementation strategy on routine clinical care. This will include physicians, clinical officers, nurses, social workers, patient navigators, peer mentors, and health insurance officers.In HIV-associated KS, treatment with chemotherapy is recommended in addition to antiretroviral therapy based on consensus guidelines for clients with KS with any of the following: symptomatic visceral disease , extensive oral KS lesions that interfere with chewing or swallowing, painful or disabling tumors, life-threatening or functionally disabling disease, and progressive or persistent KS despite antiretroviral therapy . The mosThe multicomponent patient navigation strategy was designed using intervention mapping, a structured stepwise process for identifying interventions that target key factors associated with a given behavior, as the guiding framework. We used the first three steps of intervention mapping  to guideThe responsibilities of the patient navigator will include assistance arranging transportation to oncology and chemotherapy visits, meeting clients on arrival to the health center and physically guiding the clients to their first oncology and first chemotherapy appointments, oncology and chemotherapy visit reminders, guiding clients to health insurance enrollment assistance, and connection to other social services based on each patient’s needs.The educational component will include educational videos viewed at two-time points: the first clinic visit after KS diagnosis and the initial oncology consultation. The first educational video focuses on the etiology of KS, the natural disease course of KS, and KS diagnostic procedures. It was designed to address the key barriers and facilitators of early diagnosis of KS. The second educational video focuses on the treatment of KS including antiretroviral therapy for people with HIV-associated KS, the treatment options for KS, detailed explanations of chemotherapy regimens for KS, and potential side effects from chemotherapy. The educational video on KS treatment was designed to address key barriers and facilitators to chemotherapy initiation and completion. Both educational videos were developed as part of a participatory process, and feature KS survivors, sharing a message of hope for the survival of KS through treatment with chemotherapy. Both videos were found to be acceptable and informative during field testing with KS survivors.All clients will receive a stipend to assist with the cost of their transportation for KS-related oncology care and treatment until the completion of their prescribed treatment course.Clients will be connected by patient navigators to health insurance officers who will assist them in registering for the Kenyan health insurance, the National Hospital Insurance Fund.The KS Center of Excellence will provide assistance in paying for the entire first year of health insurance for all clients.Clients will be assigned a peer mentor, who is a KS survivor from the same region in western Kenya. Peer mentors will contact the client prior to their first chemotherapy and subsequently after each chemotherapy visit to offer support and when needed connect the client back to a patient navigator or clinical health professionals for additional services or assistance.The primary outcomes of service penetration (chemotherapy initiation) and fidelity (chemotherapy completion) are defined in Table Secondary outcomes include: timeliness (time from KS diagnosis to oncology consultation), mortality, quality of life  , 34, stiIn this study, we will also evaluate the implementation process and the degree to which the multicomponent patient navigation strategy was successfully implemented because during routine implementation, and penetration may be variable and modifications  may compromise fidelity . AdditioThe process outcome measures for describing the implementation process (roll-out) of the multicomponent patient navigation strategy are outlined below.Service penetration and fidelity: A detailed description of the operationalization of service penetration and fidelity to the multicomponent patient navigation strategy is outlined in Table Acceptability, feasibility, and appropriateness: We will use quantitative questionnaires evaluating Acceptability of Intervention Measure (AIM), Intervention Appropriateness Measure (IAM), Feasibility of Intervention Measure (FIM) , and semSatisfaction: Among all clients, we will use previously validated questionnaires, which have been adapted and field-tested for this study, to evaluate satisfaction with cancer care , patientAmong clients, patient navigators, and peer mentors, we will assess the determinants of service penetration of (engagement with) and fidelity to the multicomponent patient navigation strategy, using the Theoretical Domains Framework (TDF) as a guiding framework . We haveThere are three main data sources: structured questionnaires, semi-structured interviews, and chart review of the electronic medical record. All enrolled clients and a representative sample of patient navigators, peer mentors, and healthcare workers will be asked to complete a series of structured questionnaires Tables , and 4, For chart review, we will review medical records at the client’s HIV primary clinic and all relevant oncology clinics. We will collect information on oncology and/or chemotherapy visits, provider seen, and therapy given including dates of (a) initial visit to oncology clinic, (b) initial evaluation by an oncology provider qualified to make a treatment decision, (c) first chemotherapy dose, and (d) the timing and number of subsequent chemotherapy doses. As a part of the longitudinal evaluation, clients will be contacted every 3 months for the first year after enrollment and every 6 months thereafter to evaluate the vital status and any oncology care received outside of the AMPATH health system Table . In the Data collection will be performed by either trained research staff or (where appropriate) collected by patient navigators and peer mentors as part of routine clinical care.We will estimate the cumulative incidence of chemotherapy initiation and completion accounting for death as a competing event using the Aalen-Johansen estimator . We willWe will also use a Cox regression model to compare time to oncology consultation and mortality in the post-navigation period to the pre-navigation period. For other client outcomes , we will compare the pre-navigation and post-navigation periods using generalized linear regression with a Gaussian family and identity link for continuous outcomes and binomial family with log link for binary outcomes.We will use a theory-based, framework approach to qualitative data analysis focused on acceptability, appropriateness, and feasibility of the multicomponent patient navigation strategy, while allowing for emergent themes that do not fit within the pre-defined Proctor implementation outcomes taxonomy . InterviN=50), we will not conduct hypothesis testing for differences between these groups, in accordance with best practices.We will use descriptive statistics  to describe acceptability, appropriateness, and feasibility   among heWe will report descriptive statistics  for service penetration, fidelity, dose, and patient satisfaction with the implementation strategy  associated with service penetration of the multicomponent patient navigation strategy, using generalized linear regression with binomial family and a log link for the binary outcome of service penetration (engagement).We will use a convergent design for our mixed-methods evaluation, where we triangulate the quantitative results from structured questionnaires with the qualitative semi-structured interviews. We will integrate quantitative and qualitative results, using qualitative data to provide depth of understanding –43 for tWe will recruit all newly diagnosed clients with advanced-stage KS. Between 2016 and 2020, we enrolled 367 clients, and 242 clients met the criteria for advanced KS based on either ACTG T1 or WHO “Severe KS” criteria . In 2019, there were 96 clients with newly diagnosed KS within the AMPATH network. Based on this, we estimate that we will enroll 335 clients during the post-navigation time period . Assuming we enroll 335 clients, we estimate that 221 clients will have advanced-stage KS. This makes a total of 463 clients in the pre-navigation and post-navigation period combined.For the primary implementation outcomes of service penetration (chemotherapy initiation) and fidelity (chemotherapy completion), we anticipate 242 clients in the pre-navigation period and 221 clients in the post-navigation period. If we assume a type I error of 5% and 56% initiation of chemotherapy by 1 year accounting for death as a competing event, we will have 80% power to detect a relative hazard of 0.70 or greater.Assuming that the estimated proportion of newly diagnosed clients who have an oncology consultation is similar to the proportion who initiate chemotherapy, the estimated power to detect changes in the time to oncology consultation will be similar to the primary outcome (chemotherapy initiation). For the outcome of mortality, during the pre-navigation time period between 2016 and 2020, we observed 141 deaths in the 367 enrolled clients . If we assume a similar mortality rate in the post-navigation period (2021–2024) and we enroll 335 clients, we are likely to observe around 127 deaths. Thus, assuming a type I error of 5% and a mortality of 38% at 1 year, we will have 80% power to detect a relative hazard of 0.67 or greater when comparing the pre-navigation period to the post-navigation period.In addition to enrolled clients (described above), a total of 50 healthcare workers will participate in structured questionnaires. Healthcare workers will include all patient navigators and all peer mentors as well as a representative sample of health insurance officers, social workers, HIV providers , oncology providers , and dermatology providers .We will perform semi-structured interviews with a subset of clients who are eligible to participate in the multicomponent patient navigation strategy. The sample size will be driven by reaching thematic saturation, which will likely be achieved by interviewing 20 clients who participated in the multicomponent patient navigation strategy and 20 who did not participate . For heaIn western Kenya, currently less than 50% of people with KS who qualify for chemotherapy receive it . StrategPatient navigation is well-suited to improving outcomes in HIV-associated malignancies, such as KS in LMICs. This study will build on the evidence for patient navigation for oncology care in resource-limited settings, providing evidence for an implementation strategy that can be adapted to other resource-limited contexts in sub-Saharan Africa and throughout the world. While many studies have focused on patient navigation as a strategy to improve cancer screening rates and satisfaction with cancer care, fewer studies have focused on increasing treatment initiation. There have been other efforts to establish cancer patient navigation in sub-Saharan Africa, though to our knowledge, these have focused primarily on increasing adherence to chemotherapy and do not address barriers prior to the initiation of chemotherapy . In summThis approach is also unique because the hybrid design represents an opportunity to evaluate both relevant implementation outcomes as well as important long-term client and effectiveness outcomes. A recent review found that none of the studies in low-and-middle-income countries evaluating patient navigation reported on relevant long-term clinical outcomes , and fewIt is also the first study, to our knowledge, to use implementation science frameworks to guide the evaluation of a multicomponent patient navigation strategy for cancer in a low- and middle-income country. Although several published evaluations of patient navigation in low- and middle-income countries reported on common implementation science outcomes, none reported the frameworks or theories used to guide their evaluation . By usinA limitation of this study is the lack of randomization. Randomization of the multicomponent patient navigation strategy was not felt to be ethical by the KS Center of Excellence at AMPATH because in western Kenya treatment for KS is currently suboptimal and associated with unacceptably high mortality. Additionally, there is a substantial risk of experimental contamination in the control arm with individual or cluster randomization. This is due to high levels of information sharing among individuals within a given oncology clinic, high regional population mobility, and high crossover between clinics in western Kenya. Although causal conclusions may be limited, a non-randomized approach is effective for studying the implementation of evidence-based interventions as part of routine clinical care and may offer rare insights into the efficacy of patient navigation outside of a randomized control trial , 48. In This study will provide evidence for a multicomponent patient navigation strategy to improve cancer treatment initiation and completion that may be generalizable to other low-resource contexts, including in sub-Saharan Africa. This multicomponent patient navigation strategy, which was developed using a rigorous structured stepwise process, Intervention Mapping, is designed to address common barriers to cancer care in low-resource contexts around the world. This implementation strategy could be adapted to improve cancer care in other low-resource contexts experiencing similar barriers to cancer care. Our implementation science-based approach to evaluation could also be adapted to inform the evaluation of implementation strategies to improve cancer care, which are implemented as part of routine clinical care.In summary, this type III hybrid effectiveness-implementation evaluation will provide valuable insights into the real-world implementation and impact of a multicomponent patient navigation strategy to improve chemotherapy service penetration and fidelity for people with advanced-stage KS.Additional file 1. StaRI 2017, Standards for Reporting Implementation Studies Checklist.


While organ transplantation centres around the world were coping with the severe consequences of the COVID-19 pandemic during the last years, immunological complications that occur after transplantation still represent an ever-growing medical and socio-economic problem. This Research Topic was envisioned to shed light on the growing application of new techniques and approaches, such as the molecular microscope and computational prediction of biomarkers, to improve rejection diagnosis and immunological risk evaluation in organ transplant recipients. Here, we briefly present the main highlights of the reports published in our topic.Wahrmann et al. studied the influence on kidney transplant survival of Fc gamma receptor (FcγR) IIIA single nucleotide polymorphism FCGR3A-V/F158 in a large cohort of 1.940 donor/recipient pairs selected from the Collaborative Transplant Study (CTS) database. Analysing 10-year death-censored allograft survival, no differences in relation to FCGR3A-V/F158 variations were found. The study suggests that the previously reported association of the FCGR3A V/V-genotype with microcirculation inflammation (ref) does not play a role in kidney graft survival.Trailin et al., laser capture microdissection combined with quantitative RT-PCR was used to discriminate the transcript patterns in kidney allografts of recipients at high risk of antibody-mediated rejection (AMR). The transcripts were either compartment-, AMR-, or “follow-up”-specific. The transcriptional profiles of early acute AMR shared similarities with ATN. The team also demonstrated distinct gene expression patterns in different renal compartments reflecting cellular infiltration.In the study of Sherwood et al. investigated the immune profile of a kidney transplant recipient who encountered COVID-19 infection early post-transplant. Immunological monitoring of cellular, proteomic, and other parameters during the first 4 months post-infection was performed. The authors concluded that Basiliximab induction and maintenance triple immunosuppression did not impair the recipient’s ability to mount an immune response against COVID-19 detected during the first week post-transplant.The case study of Alfaro et al. searched for differentially expressed genes in patients with or without AMR, new target drugs, etc. and analysed their biological activities. The group reported an increase in the expression of genes related to various stages of immune activation, antigen presentation and antibody-mediated cytotoxicity during AMR. Computational approaches to look for new therapeutic uses of already allowed target drugs showed that the anti-cancer kinase inhibitor imatinib might be helpful in kidney transplantation for the prevention and/or treatment of AMR. These results have to be further confirmed for the eventual detection of highly expressed genes as rejection biomarkers/or as therapeutic targets to treat rejection in patients after kidney transplantation.The team of Vietzen et al. was to investigate whether the KLRC2 gene variants, which determine NKG2C expression, affect the pathogenicity of donor-specific antibodies (DSA) and influence long-term graft survival. They genotyped the KLRC2 wt/del variants for two distinct kidney transplant cohorts: (i) a cross-sectional cohort of 86 recipients who, on the basis of a positive post-transplant DSA result, all underwent allograft biopsies, and (ii) 1.860 recipients of a deceased donor renal allograft randomly selected from the CTS database. In the DSA-positive patient cohort, the KLRC2wt/wt variant was associated with AMR, microvascular inflammation, and elevated NK cell-related transcripts. However, neither in the DSA-positive nor in the CTS cohort, the KLRC2wt/wt allele was associated with impaired long-term allograft survival.The aim of the group of Baardwijk et al. developed and validated a transplant biopsy classifier using the genes of the Banff-Human Organ Transplant (B-HOT) panel obtained from the Molecular Microscope® Diagnostic system (MMDx) microarray dataset. Gene expression data from almost 1.200 kidney transplant biopsies were used as training data for random forest models to predict kidney transplant biopsy Banff categories, including AMR, T-cell-mediated rejection and non-rejection. After performance assessment on cross-validation, the best-performing model was validated on an external independent dataset based on a different microarray version. The report demonstrates that, compared to the centralized and microarray-limited MMDx, a decentralized, open-access kidney transplant biopsy classifier can be developed that might work on data derived from different gene expression platforms and enable other centres to check and improve the tool. The multiplatform-compatible B-HOT panel proved to be a reliable predictor for classification of kidney transplant rejection.AS wrote the editorial, CS critically reviewed the editorial, ID critically reviewed the editorial. 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Waste tires are represented by natural rubber (NR), which uses cis-1, 4-polyisoprene as a repeating unit. In contrast, waste plastics are characterized by polyethylene (PE), whose optimum degree of polymerization is determined by the difference in solubility parameters. Then, the above molecular models are changed to a stable equilibrium state through the molecular dynamics process. Finally, the interaction process is analyzed and inferred using the indexes of radial distribution function, diffusion coefficient, and concentration distribution; further, the interaction mechanism is revealed. The results show that the optimal degree of polymerization of PE is 12, so the solubility parameter between PE and NR-modified asphalt is the lowest at 0.14 (J/cm3) 1/2. These models are in agreement with the characteristics of amorphous materials with the structures ordered in the short-range and long-range disordered. For NR-modified asphalt, the saturate moves fastest, and its diffusion coefficient reaches 0.0201, followed by that of the aromatic (0.0039). However, the molecule of NR ranks the slowest in the NR-modified asphalt. After the addition of PE, the diffusion coefficient of resin increased most significantly from 0.0020 to 0.0127. NR, PE, and asphaltene have a particular attraction with the lightweight components, thus changing to a more stable spatial structure. Therefore, using NR and PE-modified asphalt can change the interaction between asphalt molecules to form a more stable system. This method not only reduces the large waste disposal task but also provides a reference for the application of polymer materials in modified asphalt.This paper aims to study the interaction mechanism of waste tire/plastic modified asphalt from the microscopic perspective of molecules. Based on BIOVIA Materials Studio, a classic four-component asphalt model consisting of asphaltene (C Petroleum asphalt is an essential raw material in road engineering, especially in asphalt pavements, which are used widely. At present, petroleum is gradually becoming exhausted, so asphalt, a by-product of petroleum, will also face the problem of exhaustion. Against this background, it is indispensable and crucial to lower the consumption of asphalt in road engineering and decrease dependence on petroleum. In addition, the deteriorating environment and traffic conditions put higher requirements on the performance of asphalt pavements. Therefore, the modification of asphalt to reduce cost and improve performance is one of the leading research directions for researchers, which is very significant. The use of modified asphalt has recently increased, and its application scope and applications are becoming broader ,2,3,4,5.There are many kinds of materials that can be used as modifiers, and the current research mainly includes the following aspects. Some researchers use biomaterials as modifiers to prepare bio-asphalt ,7,8, sucIt should be noted that as asphalt modifiers, waste tires and plastics have a significant negative influence on the ecological environment . Waste tIt is a much more cost-effective and scientific to perform research using molecular dynamics. The traditional test has a long test cycle and a high cost. In addition, many conventional experiments only describe and compare the experimental results, which fails to explain some empirical phenomena in depth. Molecular dynamics, on the other hand, refers to the study of microscopic phenomena from the perspective of molecules or atoms. Currently, many researchers are studying the molecular dynamics of asphalt, such as the modification mechanism of modifiers . For exaThe purpose of this paper is to study the molecular interaction mechanism after two modifiers simultaneously using molecular dynamics. In this paper, the original asphalt and rubber-modified asphalt models were built from the perspective of molecular dynamics. Then, plastic was added to create a plastic/rubber asphalt model, and the influence of plastic molecules on rubber asphalt was analyzed. Among the models, the classical four-component molecular model was taken to represent asphalt, and rubber is expressed by natural rubber molecules composed of polyisoprene. Additionally, plastics are represented by polyethylene molecules, the optimal polymerization degree of which is measured by solubility parameters. After the molecular dynamics process, all the above models reach a state of equilibrium and stability. The radial distribution function (RDF) was used to verify the molecular model, and the mean square displacement (MSD) and diffusion coefficient were used to study the diffusion of molecules in modified asphalt. Finally, concentration distribution was taken to study the spatial distribution of different molecules.Bitumen is a highly complex mixture of hydrocarbons of different molecular weights and their derivatives of non-metallic components . Petroleum bitumen, a vital type of bitumen, is widely used in road engineering. From a scientific research perspective, the parts of petroleum asphalt with some common characteristics, such as similar chemical composition and physical properties, are divided into the same group, called components. The American Society for Testing and Materials (ASTM) D4124-09 has proposed and recommended a four-component analysis method. This method divides petroleum asphalt into asphaltene, aromatic, resin, and saturate.149H177N3O2S2), resin (C59H85NOS), aromatic (C46H50S), and saturate (C22H46), as shown in The simplified model adopted in this study consists of four representative molecules, and this method has been widely used because of its simplicity, convenience, and conformity with the actual situation . The fouRubber powder mainly comes from waste tires, and its composition is complicated. The three most common types of rubber are NR , BR (cis-polybutadiene rubber), and SBR (styrene-butadiene rubber). Since natural rubber is the most widely used, it was selected as the research object in this study.Natural rubber mainly comprises cis-1, 4-polyisoprene, accounting for more than 97% and 2–3% of the 3- and 4-bonded structure. Therefore, when building the molecular model of rubber, NR was regarded as a homopolymer, which was polymerized from the isoprene mentioned above. The relative molecular weight range of NRs is relatively wide, generally between 30,000 and 30 million, because of the different degrees of polymerization. The degree of polymerization affects the results of the experiments and leads to different simulation costs, so a proper selection is needed. Referring to previous studies ,38, the 3) 1/2, the two materials are compatible. The smaller the difference is, the more easily the two are compatible. Therefore, in this paper, the solubility parameter difference is selected to evaluate the solubilization effect of PE on rubber-modified asphalt.Polyethylene, short for PE, is a thermoplastic resin prepared by the polymerization of ethylene, including ethylene and a small amount of α-olefin copolymer. The degree of polymerization, a key factor, affects the molecular weight and the amount of simulation, and it is also closely related to the solubility parameters. The independent variables of the polymerization degree for polyethylene used in this article are 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, and there are 20 different degrees of polymerization in total. Firstly, the degree of polymerization is closely related to the amount of simulation calculations because the degree of polymerization is a direct factor affecting the molecular weight; the greater the degree of polymerization, and the greater the molecular weight, the larger the amount of simulation calculations. Secondly, the solubility parameters of polyethylene with different degrees of polymerization are different. If the difference in the solubility parameters is less than 1.3~2.1 dvidFormula (1) and (2) are a set of COMPASS is a powerful force field that supports the atomic level simulation of condensed matter materials, short for “Condensed-Phase Optimized Molecular Potential for Atomistic Simulation Study.” It is the first ab initio force field parameterized and verified by condensed matter properties and various ab initio and empirical data of isolated molecules. Therefore, the structure, conformation, vibration, and thermophysical properties of different molecules in isolated or condensed systems could be accurately predicted for a wide range of temperatures and pressures by this field ,42,43,44The specific steps of building a stable model are as follows.The establishment of initial polymer model.Geometric optimization.The simulation of annealing.−4 Gpa) and temperature of the system remain constant [In this procedure, the pressure  1/2. Therefore, the optimum polymerization degree of the PE molecule in this paper is 12.Then, the related cohesive energy index is obtained and used to estimate the solubility parameter. The solubility parameters of the PE additives with individual degrees of polymerization are shown in A PE molecular chain with a polymerization degree of 12 was adopted to establish a modified asphalt model. After the steps of dynamics simulation mentioned above were performed, the balanced model of PE/NR modified asphalt was finally obtained for subsequent analysis and calculation.RDF, an essential indicator in molecular dynamics, can be employed to verify the correctness of molecular models.The radial distribution function (RDF) can be applied to analyze the interaction and microscopic distribution of particles ,43,44. TFor the RDF of amorphous crystals, there are a few peaks with different heights and sharpness at close distances, but the peak height decreases rapidly with an increase in space. At long distances, the radial distribution function tends to be evenly distributed, The RDF of the crystal still maintains a sharp peak shape at a long distance, indicating that the crystal has a long-range ordered structure, whereas the amorphous crystal has a short-range ordered structure and a long-range disorder structure.The diffusion coefficient can represent the molecular movement rate and is employed to study the changes of diverse molecular movements in NR-modified asphalt systems with and without PE. According to Einstein’s law , if the To analyze the movement of various molecules in NR-modified asphalt, the MSD results of different molecules were obtained through molecular dynamics calculation, as shown in In addition, the molecular movement velocity of asphaltene, resin, and rubber are roughly similar, showing that their diffusion coefficients are about 0.0018, likely connected with the higher molecular weight and unique shapes to some extent.For the NR-modified asphalt system, the diffusion coefficient of NR is the lowest. More specifically, the long-chain shape of NR and its poor compatibility with the polar components of asphalt may be the main reasons restricting the movement of NR. Therefore, NR is the slowest molecule in the NR-modified asphalt system, with a diffusion coefficient of 0.00171. However, NR attracts non-polar components in asphalt (aromatic and saturate), which results in a more aromatic and saturated distribution around NR molecules. Some light components are attracted by NR molecules and transferred to the vicinity of NR molecules, so it could be noted that the light components have the highest diffusion coefficients, which are 0.0201 and 0.0039, respectively, in NR-modified asphalt.As shown in In the statistical data analysis, the Pearson correlation coefficient is adopted to reflect the linear correlation of two random variables. For NR-modified asphalt and PE/NR-modified asphalt, Concentration distribution, an important indicator, can show how concentration varies with position in a region at a given time. This parameter can be acquired through the Forcite Analysis module in Materials Studio. The spatial distribution of each molecule in the NR modified asphalt in three different directions  and the spatial distribution of each molecule in the PE/NR modified asphalt were evaluated, and the interaction between each molecule was further analyzed and predicted. In the concentration distribution curve of a molecule, the peak represents the location at which the molecule is most abundant.As shown in In this paper, two types of polymer materials, PE (polyethylene) and NR , were used as modifiers to study the interaction mechanism in the two modified asphalts. Specifically, the interaction of molecules in the original asphalt and NR-modified asphalt was analyzed before and after the addition of PE. The change of the interaction can help the modified asphalt to become a more stable in structure and thus have better storage performance, which has a reference value for changing the molecular agglomeration phenomenon. On the other hand, the reuse of PE and NR, two typical wastes, is of great significance for the protection of the ecological environment.3) 1/2.The optimal degree of polymerization of PE in this study model is 12. In this case, the solubility parameter between PE and NR-modified asphalt is the smallest at 0.14 < 1.3~2.1 (J/cmThe three models in the paper are typical amorphous substances with structures of short-range order and long-range disorder. According to the RDF diagram, when In NR-modified asphalt, the diffusion coefficient of saturate is the largest, at far higher than that of other types of molecules, and its value reaches 0.0201. In addition, the molecular velocity of asphaltenes, resins, and rubbers are about the same, showing that their diffusion coefficients are about 0.0018. NR is the slowest molecule.In PE/NR-modified asphalt, the movement of molecules changed significantly. The diffusion coefficient of resin molecules increased considerably from 0.0020 to 0.0127. The most noticeable feature is a specific adsorption phenomenon between the PE molecule and light components.The specific conclusions are as follows.


The as-described system was implemented in miniaturized devices via wax-based printing, in which their electroanalytical performance was characterized for both outer- and inner-sphere redox markers and then applied to the detection of dopamine (the peak appeared at ca. 200 mV vs f-LCNFs RE) with a remarkable LOD of 55 pM. When modified with Nafion, the f-LCNFs were highly selective to dopamine even against high concentrations of uric and ascorbic acids. Especially the integration into closed microfluidic systems highlights the strength 3D porous structures provides excellent analytical performance paving the way for their translation to affordable lab-on-a-chip devices where mass-production capability, unsophisticated fabrication techniques, transfer-free, and customized electrode designs can be realized.3D-carbon nanomaterials have proven to be high-performance transducers in electrochemical sensors but their integration into miniaturized devices is challenging. Herein, we develop printable freestanding laser-induced carbon nanofibers (f-LCNFs) with outstanding analytical performance that furthermore can easily allow such miniaturization through a paper-based microfluidic strategy. The f-LCNF electrodes were generated from electrospun polyimide nanofibers and one-step laser carbonization. A three-electrode system made of f-LCNFs exhibited a limit of detection (LOD) as low as 1 nM (S/N = 8) for anodic stripping analysis of silver ions, exhibiting the peak at ca. 100 mV The online version contains supplementary material available at 10.1007/s00604-022-05492-2. Three-dimension (3D) carbon nanomaterials, e.g., graphene foam and carbon nanofibers, possess intrinsic beneficial architectures, besides their favorable electrical conductivity and low material costs, that enable their potential use as a high-performance transducer in miniaturized electrochemical sensors . In compVarious approaches have been reported for the fabrication of 3D graphene composites. Template-based methods are known as an effective strategy to generate 3D graphene with well-controlled morphologies. However, the methods suffer from high production cost and inherent complexity, in particular the need for template removal. Template-free based strategies such as hydrothermal technique , freeze-Despite their high-performance capability, 3D-carbon nanomaterials suffer from difficulties in integrating into microfluidic analytical systems due to their irregular shape, high thickness (in micrometers to millimeters range), and rough surface. A few studies attempted such integration, for example, graphene foam was manually inserted into a polydimethylsiloxane (PDMS) microfluidic channel . Ali et 2 laser pyrolysis, namely laser-induced carbon nanofibers (LCNFs), with great flexibility in shapes and designs. Excellent electroanalytical performances were achieved apart from fulfilling all requirements stated above. In situ generation of metal oxide decorated LCNFs was recently demonstrated with highly competitive performance for glucose sensing ), and potassium ferrocyanide (K4[Fe(CN)6]) were purchased from Sigma Aldrich, Germany. N, N-dimethylacetamide (DMAc) and potassium nitrate (KNO3) were purchased from Merck KGaA Darmstadt, Germany. Silver nitrate (AgNO3) was purchased from Carl Roth GmbH Karlsruhe, Germany. Silver paint was purchased from Busch GmbH & Co. KG Viernheim, Germany. Filter paper MN616 with 90 mm in diameter and 200 µm in thickness  was purchased from Machery-Nagel Düren, Germany. Double-sided adhesive tape was purchased from tesa. Plastic sheets in A4-size were purchased from Exponent as Laser Films. Dropsens electrodes were purchased from Metrohm, Germany.A solvent-soluble polyimide (Matrimid 5218) was purchased from Huntsman Advanced Materials (Europe) BVBA Belgium. Iron (III) acetylacetonate (Fe(acac)3 (37.5 mg) (unless stated otherwise) in 5 mL DMAc. In order to obtain a homogenous mixture, the solution is sonicated for 30 min prior to stirring overnight at room temperature. The solution could be used 1 to 5 days after the preparation without significant changes in nanofiber properties.The PI nanofibers were fabricated by electrospinning the solution containing 15% (w/v) Matrimid 5218 (750 mg) and 5% Fe(acac)An electrospinning device (Spraybase®) connected to a syringe pump was used to generate PI nanofibers. Here, the solution was injected into the system by a 5-mL-glass syringe connected to a polytetrafluoroethylene (PTFE) tubing where a metal needle (inner diameter of 1.2 mm) was placed at the end. The distance between the collector and the needle was set to 15 cm. The flowing rate was chosen at 10 µL/min. Environmental conditions such as humidity and temperature play an important role in the electrospinning process. Therefore, the applied voltage, between the needle and the collector, has to be adjusted according to the two parameters. This can be usually changed from 13 to 17 kV. However, the optimum conditions for spinning the nanofiber were found to be at 20 °C and around 40% relative humidity where the optimum applied voltage was ca. 16 kV.2 plasma at 100 W (unless stated otherwise) to promote strong adhesion between the nanofibers and the paper’s surface.The nanofibers were deposited on a filter paper (9 cm in diameter with a collecting area of 6 cm × 6.5 cm) that was fixed on a metal collector. Prior to collecting the fibers, the filter paper was treated with O2 laser with 10.6 µm wavelength from Universal Laser Systems, Polytech System GmbH was used to carbonize the nanofiber substrate. Its maximum power is 30 W, and its maximum speed is 1270 mm/s. The laser process was always set to 1000 DPI and 500 PPI. For the carbonization of the nanofibers, the 2-inch lens is used. The design of the electrodes was created using CorelDRAW. The nanofiber mat is first fixed in place with tape in the laser chamber to prevent the uncontrolled movement of the mat during the scribing process. The laser conditions were varied concerning laser power and speed. The optimum conditions were found to be 4% power and 80% speed for the chosen fiber collection time. If the fiber collection time is varied, the laser parameters must be adjusted accordingly. Each electrode is scribed separately.A COIp is the peak current (mA), A is the ESA of the electrode investigated (cm2), D is the diffusion coefficient of ferricyanide (7 × 10−6 cm2/s), of RuHex (9.1 × 10−6 cm2/s), and of dopamine (6.74 × 10−6 cm2/s) [n is the number of electrons transferred in the redox reaction, C is the concentration of the analyte in the bulk solution (M), and ν is the scan rate (V/s). The value for the ESA is calculated from the slope of the Ip versus ν1/2 linear regression equation. Dopamine was detected via LSV. The resistance was measured with a four-point probe, containing a current source, Keithley 6430 SUB-FEMTOAMP REMOTE SourceMeter, and a Keithley Multimeter 2000 to measure the voltage between the inner two probes.The potentiostat PalmSens4 (the Netherlands) was employed for the electrochemical measurements. One electrochemical cell is fabricated containing a three-electrode setup, all of them are made from LCNFs. For detecting silver, anodic stripping voltammetry (ASV) was used. ASV consists of two main steps, including a deposition step and a stripping step. In the first step, silver is cathodically electrodeposited onto the surface of the working electrode by applying a constant negative potential. Then using linear sweep voltammetry (LSV) silver is stripped away from the surface by re-oxidation in the second step. Electroactive surface area (ESA) is estimated by the Randles–Sevcik equation shown below at the scan rate of 25 to 250 mV/s.6 cm2/s) , n is thFor open devices without plasma treatment, a larger volume (approx. 50 μL) of solution is required to cover the three-electrode strips while 10–30 μL is sufficient for the open and closed devices with plasma treatment in which 10 μL was used for these cases especially when f-LCNFs were employed as a reference electrode (RE). However, in case of using an Ag/AgCl external RE, 30 μL of sample was dropped on the plasma treated electrodes where the tip of RE can be inserted to the drop.The morphology of the nanofibers was characterized using a scanning electron microscope (Zeiss/LEO 1530 Germany) at 5.0 kV. Prior to imaging, the samples have been sputtered with gold for 30 s, which should create a layer of ≈ 7 nm. The confocal microscope Olympus LEXT 3D Measuring Laser Microscope OLS4000, Germany was used to determine the thickness of the mat and the side view of the device with and without wax. Images for optical measurements were taken with a light microscope (KERN optics).Tensile strength measurements were performed using Instron 5566 testing machine. The nanofibers were electrospun for 90 min without any further modification. The filter paper was peeled off, so both PI nanofibers and PI carrying LCNFs were tested in a freestanding form.The collection of fiber precursors is generally performed on a conductive substrate, e.g., ITO , to provTo achieve a suitable nanofiber substrate for f-LCNF generation, various parameters were studied. The effect of iron content and lasing condition was initially assessed suggesting optimal concentrations between 5 and 7% iron Fig. . FurtherAfter the collection of a homogenous nanofiber mat, the pattern of the electrodes was carbonized into the mat, as shown in Scheme Proper adhesion between electrospun nanofibers and the filter paper was studied as poor adhesion promoted bending and loose nanofibers which in turn led to unreliable surfaces for the lasing process Fig. -iii. OxyCharacterizing the mechanical properties of the LCNFs, their tensile strength was measured Fig. . For thivs f-LCNFs RE) was performed to determine the proper reduction potential of silver ions with respect to an LCNF pseudo-RE  for electrodeposition of silver ions  generated an anodic peak corresponding to silver at ca. 100 mV (vs f-LCNFs RE)  Fig. -iii. TheRE) Fig. . At highIn the end, a three-electrode f-LCNF compares favorably to other high-performance electrochemical transducers Table  without wb0) and resulting channel width (wc0)  regardless of fiber density. Fluid flow and hence functionality of the resulting channels was done using sulforhodamine B (SRB) dissolved in ethanol. It was found that high-density fiber mats require a minimum amount of wax ink (determined by the designed width of the wax barrier) to completely form a hydrophobic barrier  . Since dc0) Fig. -i. Lowerc0) Fig. -ii, wher mm Fig. -iii. Theier Fig. -iv.Fig. Since aqueous solutions need to be transported within the channel system for applications in point-of-care testing (POCT), the hydrophobic LCNFs/PI nanofibers were treated with oxygen plasma Scheme -v prior Further imaging the cross-section of the assembled device revealed that the proposed fabrication strategy is highly robust, resulting in intact freestanding LCNFs and PI nanofibers Fig.  to S6c wThe proposed integration strategy is by far superior to other traditional sealing methods such as bonding of channels engraved in plastic sheets or using double-sided adhesive tape to create a channel barrier and later closing the channel. The irregular structure as well as the high thickness of 3D porous carbon electrodes, including LIG, make the traditional sealing methods challenging. For example, the high thickness leads to incomplete sealing between channel and electrode pieces. Moreover, pressing the channel piece over the delicate and brittle graphene flakes increases the chance of breaking the connection of working area and electrical contact pad.Three sensor configurations were generated to investigate the analytical performance of the electrochemical devices Fig. . Open deEp)  Fig. -i when cEp) Fig. -i and ESEp) Fig. -i of RuHEp related to the pristine devices . As shown in Fig. Further studies regarding the reliability and stability of LCNF REs over a long measuring period were conducted by determining the open-circuit potential (OCP) using standard Ag/AgCl as RE of the system, and comparing to other, commonly used REs, e.g., Ag/AgCl film of Dropsens and laser-induced graphene (LIG). As can be seen in Fig. N = 3 devices), which were cycled 30 times in 1 mM of ferri/ferrocyanide or dopamine, respectively  is probably due to the redox reaction of dopamine derivative, which is commonly formed when dopamine is electrochemically measured in neutral pH [Furthermore, the stability of the three-electrode system made of LCNFs was evaluated with closed devices , which renders this simple electrochemical transducer highly potent for on-site POCT dopamine detection. The calculated LOD compares favorably to similarly developed detection strategies (Table Dopamine (DA) is an important neurotransmitter involved in several neurological disorders, which requires a highly sensitive transducer to detect the alteration from normal levels (10 to 480 pM in blood) . Considees Table .Fig. 5El2SO4 incubation, the latter being performed after device assembly. As expected, the LCNFs modified with Nafion were able to suppress any signal interferences from UA and AA even at concentrations several times higher than dopamine   and ascorbic acid (AA), common interferences for dopamine detection  Fig. S1 and S10bon) Fig. .Herein, we present laser-induced carbon nanofibers as freestanding electrodes that can be easily integrated into miniaturized devices. Ultralow detection limits for silver ions via stripping voltammetry suggest their applicability towards the detection of AgNPs use as signal generating label in (bio)sensors , 47, espSupplementary file1 (DOCX 1.66 MB)Below is the link to the electronic supplementary material.