Brassica napus. Despite the agronomic relevance of this issue, the molecular factors involved in the interaction between SW and SN are largely unknown in crops. In this work, we performed a detailed transcriptomic analysis of 48 seed samples obtained from two rapeseed spring genotypes subjected to different source–sink (S–S) ratios in order to examine the relationship between SW and SN under different field conditions. A multifactorial analysis of the RNA-seq data was used to identify a group of 1014 genes exclusively regulated by the S–S ratio. We found that a reduction in the S–S ratio during seed filling induces the expression of genes involved in sucrose transport, seed weight, and stress responses. Moreover, we identified five co-expression modules that are positively correlated with SW and negatively correlated with SN. Interestingly, one of these modules was significantly enriched in transcription factors (TFs). Furthermore, our network analysis predicted several NAC TFs as major hubs underlying SW and SN compensation. Taken together, our study provides novel insights into the molecular factors associated with the SW–SN relationship in rapeseed and identifies TFs as potential targets when improving crop yield.A critical barrier to improving crop yield is the trade-off between seed weight (SW) and seed number (SN), which has been commonly reported in several crops, including  Brassica napus L.) is the third most important oilseed crop, after palm and soybean, used for oil production, animal feed, and biofuel [Research on the physiological and molecular clues in controlling seed weight (SW) has increased in recent years in model plants and staple food crops, driven by the challenge of developing high-yield varieties in order to attain food security and quality seed traits under sustainable production systems . Therefo biofuel . In oil  biofuel . This gr biofuel . However biofuel ,7,8. TheSeed weight is a conservative plant trait across grain crops, showing high heritability ,9,10. InBrassicas are the most closely related crops to the model plant species Arabidopsis thaliana, one of the most extensively studied species in the world. The sequencing of the genome of several Brassicas has provided a key opportunity to harness the rich knowledge obtained in Arabidopsis and to transfer it to staple food crops .The evidence that a reduction in SN can be fully or partially compensated by SW support the high plasticity of rapeseed, previously reported ,15,16. TIn order to gain new insights into the molecular factors underlying SW plasticity in rapeseed under field conditions, we performed a detailed transcriptomic analysis of seed samples obtained from two spring genotypes (Lumen and Solar) subjected to different S–S ratios . We extrThen, we intersected all the significantly regulated genes uncovered by multivariate analysis to find the genes exclusively regulated by each factor. In addition, we filtered out all genes that were regulated by sowing date to reduce the environmental effect on the main factors. In this manner, we identified 9227, 6698, and 1014 genes exclusively regulated by T, G, and SS, respectively . The numThe experimental factor with a higher impact on the rapeseed transcriptome of seeds was developmental time. A detailed analysis of these genes is relevant because it provides information about the biological processes that are regulated during seed filling under field conditions. To identify genes preferentially expressed at the initial and middle stages of seed development, we performed a hierarchical clustering analysis of the genes exclusively regulated by time using the Pearson’s correlation to measure the similarity among genes. In this manner, two clusters with similar sizes were identified . ClusterOn the other hand, the expression levels of genes from cluster 2 were increased at 14 DAF. The most enriched biological processes were related to vacuole organization and Golgi vesicle-mediated transport . In addiSWEET 11 and SWEET 12. We selected three genes of the GO term “sucrose transport” to show the regulatory effect of shading treatment on the expression of these genes  a list of well-known genes involved in SN and SW determination [SWEET genes, receptor-like protein kinase FERONIA (FER), or the RING-type protein with E3 ubiquitin ligase activity (DA2) [Manipulations of the S–S ratio at the beginning of seed filling is a well-known experimental factor that impacts the seed yield components in rapeseed ,15,16,30se genes B. Interemination , which ity (DA2)    with allp-value < 0.01) with at least one physiological trait  except for the case of the salmon and cyan modules B. An intp < 0.01) with at least one physiological trait , BnaA07g38140D (CRF6), and BnaC01g44850D (NGAL2) at the top TFs , indicating that these TFs might be involved in the control of the SW and SN relationship.The 7 DAF co-expression network showed that three modules positively correlated with SW and negatively with SN , BnaA05g24050D (NAC3), BnaA03g05670D (bZIP3), and BnaC01g02120D (HB40) were identified as top TFs in this module  were only affected by one experimental factor, with developmental time and genotype being the most influential factors on the rapeseed ,48 whilerapeseed . These rAtSWEET11/12/15 in Arabidopsis thaliana severely affects seed development, including reduced seed weight, and reduced starch and lipid content [OsSWEET11 and 15 genes results in a complete loss of endosperm development [GmSWEET10a) has been selected and conferred simultaneous increases in soybean seed size and oil content [We have previously shown that shading treatment negatively affects SN and positively affects SW, allowing for key compensation in seed yield . In orde content . A similelopment ,54. More content .BnaA01g19380D, an ortholog of Arabidopsis DA2. This gene encodes a ubiquitin ligase that interacts with the ubiquitin receptor DA1 to synergistically regulate seed size in Arabidopsis [DA1 in Brassica napus, BnDA1, resulted in a 21% increase in seed weight and 13% increase in seed yield per plant under field conditions [ZmDA1 improves the sugar imports into the sink organ and starch synthesis in maize kernels [DA2 could also be involved in the regulation of sugar translocation in response to changes in the S–S ratio.Another remarkable example of a gene associated with seed weight and induced by S–S treatment is bidopsis . Moreovenditions . A similnditions ,57, indi kernels . TherefoOsNAC25 and OsNAC26 bind to the promoters of important genes involved in the control of seed size in rice such as GW2, GW5, and DR11 [OsNAC020, OsNAC023, and OsNAC026) have been associated with SW in rice since their expression profiles during seed development vary among different accessions with contrasting seed size [Our study aimed to use bioinformatics analysis to identify genes related to agronomic traits with a special focus on SW–SN regulation, which might be a helpful target in yield improvement and yield stability in rapeseed and other crops. Specifically, we performed a WGCNA analysis from 48 RNA-seq samples obtained from seeds at 7 DAF and 14 DAF to identify gene co-expression modules significantly correlated with SW and SN . The seeds of the Arabidopsis IDD1 over-expressor lines were larger than those of wild-type plants due to an enlarged endosperm, higher seed weight in all the stages before seed maturity, and a significant delay in seed development [It is important to note that several key regulators of seed size are also present at the top of our ranking of highly correlated TFs with SW–SN in the yellow module of the 7 DAF network. For instance, the orthologous gene of ge seeds . In facte of DA1 , which ie of DA1 . Anotherelopment .NGAL2. The characterization of these TFs may improve the understanding of the regulatory mechanisms underlying the interaction between SW and SN in rapeseed and other crops, creating new pathways for crop yield improvements and yield stability via SW compensation to environmental conditions affecting SN.Taken together, our study provides new insights into the molecular factors that regulate the SW and SN interaction in rapeseed. We identified several new TFs that have not been previously associated with SN and SW and were co-expressed with key regulators of seed size, such as ratio with black nets intercepting 75% of solar radiation from the beginning of flowering (BBCH 61) to 15 days after flowering (DAF)). The treatments were arranged in a split–split plot design with three replicates, where the sowing date was assigned to main plots, the S–Sratio was assigned to subplots, and the genotypes were assigned to sub-sub plots [−2.The field experiment and conditions were described in detail by Verdejo and Calderini . Brieflyub plots . The rapCrop development was followed twice a week in both experiments according to the BBCH phenological scale for rapeseed . In ordeThe oil concentration of seeds was determined by near infrared reflectometry (NIR)  and the nitrogen concentration of seeds was measured using the Kjeldahl procedure . The proBrassica seeds [® Gel and PCR columns; Macherey-Nagel, Düren, Germany) in accordance with previously described protocols [In spring rapeseed, SW plasticity is maximized by shading within a time window from 0 to 15 days after the start of flowering . Therefoca seeds  with 100rotocols .The RNA samples were shipped in dry ice to Novogene facilities in Sacramento, CA USA. RNA quality analysis, library construction, and sequencing were performed by Novogene . The samples were sequenced using a 2  ×  150 bp kit on an Illumina Novaseq 6000 aiming for 6 Gb per sample. The average Q20 per sample was 97.7%, and that for Q30 was 93.8%.Brassica napus transcriptome obtained from Ensembl Plants using kallisto (v0.46) [https://plants.ensembl.org/Brassica_napus/, accessed on 23 March 2021). A multivariate linear model to test whether the expression of a given gene could be explained by the S–S ratio, genotype, sowing date, or the interaction of these factors using the R package sleuth (v.0.30.0) [RNA-seq data analysis was performed as described by . Briefly (v0.46) . The tra.0.30.0) . This R .0.30.0) .Co-expression networks were built for two separate sample sets (7 and 14 DAF) using the method of weighted gene co-expression network analysis (WGCNA) . For eacB. napus genes were assigned based on A. thaliana orthologous genes according to Ensembl Plants in order to gain more informative enrichment results. BiNGO was then used to identify the significantly enriched GO terms  using a hypergeometric test [Gene ontology (GO) terms for all of the ric test , and redric test .


The incidence of immature teratomas is highest in young adults aged 18 to 39. The prognosis heavily depends on the International Federation of Gynecology and Obstetrics (FIGO)We report a 36-year-old female who presented with a 17.6cm x 10.5cm x 24.2cm intraabdominal mass and ascites. Upon tumor resection, she was found to have a stage IIIa, grade 2 immature teratoma of the left ovary, with glial tissue being the metastatic cell type. Disease progression continued despite treatment with BEP. She was then treated experimentally with six months of bevacizumab and temozolomide, given its rarity and targeted therapy for glial tissue. Despite monoclonal antibody therapy, the tumor progressed again and was treated with docetaxel and gemcitabine. A repeat CT of the chest, abdomen, and pelvis demonstrated scattered peritoneal implants that were increasing in size. Chromosome analysis was performed and revealed somatic mutations of MLH1, MSH2, MSH6, and PD-L1. The patient has requested a break from chemotherapy but will be treated with direct immunotherapy when she restarts.This case’s importance lies in its rarity because fewer than 10 cases of immature teratomas with metastatic glial tissue are noted in the world’s literature. Furthermore, this is the first reported case of this cell type being treated with immunotherapy in the world literature. Teratoma originates from the Greek word “teras,” which signifies monster. Immature teratomas are malignant tumors of the ovary and represent 1% to 3% of all germ cell tumors and 20% of malignant ovarian germ cell tumors [We report a 36-year-old female with a medical history of Hashimoto’s thyroiditis on levothyroxine who presented with complaints of progressively worsening abdominal distention and clothes fitting tighter over the past two months. She also endorsed left lower quadrant abdominal and suprapubic pain. She reported her symptoms were associated with early satiety, constipation, nausea without emesis, and not being relieved by prescription probiotics. These symptoms prompted her to visit the emergency department. On physical examination, she had a distended abdomen with tenderness to palpation and a low-grade fever. Complete blood count was significant for anemia with a hemoglobin of 8.4 g/dL and hematocrit of 26.9%. Complete metabolic panel noted hypocalcemia of 7.5 mg/dL. Computed tomography (CT) of the abdomen and pelvis was significant for a bulky mass arising in the anterior pelvis and extending cephalad above the level of the umbilicus. The mass measured 17.6 cm transversely, 10.5 cm in the anteroposterior dimension, and 24.2 cm in the craniocaudal dimension. The lesion consisted of fluid and solid components with some calcifications and fat present. Furthermore, there was small to moderate free fluid in the posterior pelvis and lower abdomen bilaterally. The patient agreed to exploratory laparotomy and underwent left salpingo-oophorectomy, appendectomy, omentectomy, left pelvic and aortic lymph nodes sampling, and tumor debulking. Operative findings showed a large left-sided ovarian mass that was fleshy in nature. Frozen sections noted possible teratoma with stromal components concerning for malignancy; therefore, the patient was staged. Pathology results of specimens collected during the surgery showed grade 2 immature teratoma of the ovary, acute appendicitis, gliomatosis peritonei, peritoneal tumor and omentum, aortic and pelvic lymph nodes negative for metastatic tumor. The clinical stage showed the International Federation of Gynecology and Obstetrics (FIGO) stage IIIA1(i) calculated as stage IIIA2 . The pathology results of the left ovary were notable for grade 2 immature teratoma of the ovary. The right pelvic peritoneum, omentum, abdominal fluid, and peritoneal tumor were remarkable for the presence of immature glial tissue. The left fallopian tube, appendix, left pelvic, and aortic lymph nodes were negative for tumor. Immunohistochemical stains were positive for glial fibrillary acidic protein, which is consistent with immature teratoma. In addition, mesothelial cells were positive for pankeratin, calretinin, and vimentin. As seen in Figures She was started on apixaban for deep venous thrombosis (DVT) prophylaxis and oxycodone as needed for pain. Additionally, she was started on bleomycin, etoposide, and cisplatin chemotherapy regimen every 21 days for four cycles. Repeat CT scan showed progression of disease with multiple soft tissue masses seen within the mesentery representing recurrence of the patient’s disease at three months post exploratory laparotomy and start of chemotherapy. It also noted ill-defined density within the pelvis posterior to the uterus, also indicating disease recurrence. To obtain a target, molecular testing was ordered. Subsequently, molecular testing and chromosome analysis were done to direct treatment. Approval was obtained, and the patient was started on temozolomide. Results showed that immunohistochemistry of the mass was positive for MLH1, MSH2, MSH6, and PD-L1 and variation of undetermined significance of BRCA1 and AXIN2. The patient was started on immunotherapy with bevacizumab through port-a-cath. At this point, she reported symptoms of shortness of breath accompanied by chest pain both at rest and with exertion, as well as bilateral pedal swelling. She also developed chemotherapy-induced hypertension. Following cardiology recommendations, the patient was started on carvedilol, amlodipine, and as-needed Lasix for bilateral pedal edema. An echocardiogram was also ordered as bevacizumab is known to cause hypertension and left ventricular dysfunction toxicity. The transthoracic echocardiogram showed mitral and tricuspid valve regurgitation. It also noted a normal left ventricular systolic function with an ejection fraction of 55%-60%. The patient’s current chemotherapy regimen, which included IV bevacizumab and temozolomide, was stopped, and she was started on gemcitabine and docetaxel. The patient was also informed of the palliative nature of the treatment. Three months later, the patient decided she would like to take a break from the chemotherapy, and the treatment was stopped. Immature teratomas are malignant tumors that occur in the first few decades of life. Tumor grading of immature teratomas is based on the amount of immature neural tissue present. The prognosis of immature teratomas depends mainly on the staging system provided by the International Federation of Gynecology and Obstetrics (FIGO) . The othAccording to Iavazzo et al., fertility-sparing management is suggested as the standard of care of immature teratomas in young female patients . Young fThe patient was first started on bleomycin, etoposide, and cisplatin chemotherapeutic regimen. Etoposide inhibits DNA synthesis by interfering with the activity of topoisomerase II. It is predominantly active against cells in the late S and G-2 phases of the cell cycle. Cisplatin is an alkylating agent that acts in a cell-cycle nonspecific manner. Bleomycin is an antineoplastic antibiotic that binds to DNA and causes scissions of DNA strands . Tumor pUnlike the previous treatment, bevacizumab is immunotherapy aiming to prevent pathologic angiogenesis. According to Wilson, pathologic angiogenesis enhances the growth of solid tumors and causes chronic inflammation, cartilage damage, atherosclerotic plaque formation, and scarring of the eye . Wilson Tumor progression continued despite bevacizumab, and the patient was started on gemcitabine and docetaxel. As reported by de Sousa Cavalcante et al., the most common mechanism of action of gemcitabine is by inhibiting DNA synthesis . Per FarImmature teratomas mainly affect young female patients. Prognosis worsens with delay in diagnosis of disease at later stages. The fertility-sparing approach is the standard of care, and various treatment options should be thoroughly discussed with patients. These options commonly involve surgery and chemotherapy. Immunotherapy with drugs such as Bevacizumab is promising and most effective when initiated early. The five-year survival rate is excellent for stage I immature teratomas, but this survival rate decreases drastically for grade 1, 2, and 3 tumors.


Origanum vulgare ssp. hirtum) infusion (OV) followed by osmotic treatment of chicken fillets at 15 °C. Chicken fillets were immersed in an osmotic solution consisting of 40% glycerol and 5% NaCl with (OV/OD) and without (OD) prior antioxidant enrichment in a hypotonic oregano solution. A comparative shelf life study of all the samples  was then conducted at 4 °C in order to assess the impact of this process on the quality and shelf life of chilled chicken fillets. Microbial growth, lipid oxidation and color/texture changes were measured throughout the chilled storage period. Rates of microbial growth of pretreated fillets were significantly reduced, mainly as a result of water activity decrease (OD step). Rancidity development closely related to off odors and sensory rejection was greatly inhibited in treated fillets owing to both inhibitory factors (OD and OV), with water-soluble phenols (OV step) exhibiting the main antioxidant effect. Shelf life of treated chicken fillets exhibited a more than three-fold increase as compared to the untreated samples based on both chemical and microbial spoilage indices, maintaining a positive and pleasant sensory profile throughout the storage period examined.The scope of this work is the study of a combined process including a dipping step into an oregano ( Besides confronting serious microbial spoilage, fresh poultry is readily contaminated by pathogenic bacteria such as Salmonella spp., Campylobacter jejuni, Escherichia coli O157:H7, Listeria monocytogenes, etc. [Production and consumption of chicken fillets has shown a significant increase in recent years, mainly due to the low cost compared to other meat products and the nutritional and sensory profile of such foods, which are a good source of proteins, vitamins and minerals ,2, exhibes, etc. . All thees, etc. , high-pres, etc. ,9,10, moes, etc. ,12,13, ves, etc. ,15, in-pes, etc. ,17, irraes, etc. , edible es, etc. ,19, acties, etc. ,21, etc.Origanum vulgare) frequently selected in the case of poultry not only for its well-established antioxidant activity, but also for its antimicrobial properties and its sensory affinity with chicken taste. In a study by Garavito et al. [Origanum vulgare) and thyme (Thymus vulgaris) oils followed by vacuum packaging under chilled storage of chicken breast fillets was examined, and the results showed that this treatment led to a significant reduction of all groups of microorganisms measured [Listeria monocytogenes on chicken breast fillets as compared to controls or samples only treated with oregano essential oils. From an economic perspective, using dried ground leaves of oregano could be advantageous in an effort to reduce the necessary processing and the high costs of oil extraction as long as the antimicrobial properties of such dry matter could be established. Sobczyk et al. [Origanum vulgare leaves, previous studies of our research team reported identification of their phenolic substances by high-performance liquid chromatography (HPLC) coupled with multi-wavelength ultraviolet-visible (UV-vis) spectroscopy as well as NMR metabolomics and spectrophotometric studies  to characterize their infusions and decoctions [One preservation step frequently applied in the case of poultry products is related to the use of natural antioxidants incorporated within the meat tissue with a variety of methods, such as edible coatings, active packaging or by simply dipping in relevant infusions. As demonstrated in many published works, phytochemicals  may act as reducing agents, free radical scavengers, metal chelators, and singlet oxygen quenchers , offerino et al. , oreganoo et al.  studied measured . In anotmeasured , edible measured  demonstrk et al.  showed tk et al.  where auk et al. , the totcoctions ,35. AccoOsmotic dehydration (OD), a well-established mild non-thermal technique, includes product immersion in a hypertonic solution of different solutes, aiming at targeted mass transfer through a selectively permeable cell membrane. The result is simultaneous dehydration and tissue enrichment with preselected desirable compounds. Therefore, depending on the desired attributes of the final processed product, it is possible to optimize the main process parameters based on an appropriate experimental design followed by a corresponding kinetic study. One of the most important issues in OD implementation is related to the selection of the osmotic agent depending on its water activity-lowering force, effect on the final product’s organoleptic profile and safety, as well as economical aspects. In this context, a tertiary solution containing sodium chloride and glycerol (an effective humectant and plasticizer that enhances product texture) is often used in muscle foods ,37,38,39Bearing in mind current consumer needs for fresh or minimally processed, microbiologically safe and healthy foods of high quality, a concept called hurdle technology has been proposed. This approach is based on the careful combination of traditional and novel preservation methods called ‘hurdles’ in order to inhibit growth of microorganisms and other deterioration reactions ,52,53,54The aim of this research is the application of a combined technique, including the enrichment of chicken fillets’ tissue with bioactive compounds, followed by an OD step, mainly aiming at water activity reduction. The methodology is based on the measurement of microbiological, chemical and sensory changes of air-packaged, refrigerated chicken fillets pretreated in a hypertonic osmotic solution with and without prior immersion in an oregano infusion. The ultimate goal was to determine the shelf life improvement obtained during the subsequent chilled storage. This study represents an example of hurdle technology implementation by coupling two mild preservation steps, namely, impregnation with a natural extract so as to enhance the antioxidant and antimicrobial activity of the product and a water activity reduction process (mainly through water loss from the interior of chicken fillets).Origanum vulgare) infusion measured by the Folin–Ciocalteu method was found to be 3260.40 ± 32.53 mg GAE/L. Fotakis et al. [Origanum vulgare, where a total phenolic content of 215.24 ± 7.92 (mg of GAE/100 mL), antiradical activity of 132.16 ± 0.36 /100 mL) and antioxidant activity of 82.74 ± 5.06 (mg FeSO4∙7H2O/100 mL) are reported. As far as specific types of phenolics and flavonoids present in dry oregano leaves are concerned, in 2013, Proestos et al. [Origanum vulgare infusion led to a significant enrichment of meat flesh with bioactive compounds as depicted in The total phenolic content of oregano  water loss (WL) from chicken muscle, combined with a significant (p < 0.05) solid gain (SG). Moreover, aw changes showed very similar trends in both alternative procedures taking into account that in the first 60 min of OV impregnation, virtually no mass transfer was recorded , caused a significant (recorded a–c. Sampp < 0.05) change during neither of the procedures (OV/OD and OD) (Origanum vulgare infusion. From p < 0.05).Color expressed as the relative loss of the initial lightness (L*/Lo*) showed no significant ( and OD) . When sahttp://www.combase.cc/index.php/en/ (accessed on 1 March 2021)), and the growth kinetic parameters at each processing condition were calculated (R2 = in the range between 0.901–0.999). No lag phase was observed in the growth of any of the measured bacteria. The microbial growth rate , i.e., the rate at the linear phase of the measured microorganisms, the initial population (No in log CFU/g) and the final population (Nmax in log CFU/g) are presented in Before its use in the OV/OD procedure, the presence of any initial microbial load of the oregano infusion was assessed via the total viable count, and it was found below the detection limit. The growth curves of the tested spoilage bacteria  in chicken samples  stored at 4 °C were fitted to the Baranyi growth model  than that of the untreated counterparts, and that could be attributed to the aforementioned treatments. Additionally, the growth rates  at 4° C were significantly lower in the OD and OV/OD chicken samples compared to the raw ones  was approximately 3.5 log cycles lower. Psychrotrophic bacteria are the dominant spoilage microorganisms in aerobically packed chicken samples, which agrees with [w (OD step) to a level of < 0.95 acts as an effective barrier to psychrotrophic species growth [On the other hand, from the results illustrated in ees with ,67,68. Is growth ,70, leadRegarding lactic acid bacteria also analyzed in chicken samples, the results were coherent with those of total viable counts and psychrotrophic population. More specifically, a rather rapid growth was observed in untreated samples, with microbial load starting from 4 log CFU/g at day 0 and reaching final loads of 7.9 log CFU/g after eight days of refrigerated storage. The OD step resulted in a significant delay of LAB growth, with the final load measured not exceeding 6.5 log CFU/g during the eight-day chilled storage. These results are in agreement with ,72.w lowering (through the OD procedure) and the synergistic effect of the bioactive compounds’ impregnation (OV/OD process) since the limit of 8 log CFU/g was not exceeded even after 50 days of chilled storage. Nonetheless, no statistically significant difference in the antimicrobial activity was found between the two alternative procedures (OD and OV/OD), as was also confirmed by ANOVA statistics (p > 0.05). The results obtained in this study showed that the OD step and the subsequent aw decrease obtained had a more pronounced effect on microbial growth, with bioactive compounds of oregano introduced within chicken flesh having a milder non-significant antimicrobial impact.The obtained results  demonstratistics . SimilarOriganum vulgare extracts are associated with the observed antimicrobial activity of this spice [In general, there is a large body of scientific data that supports the antimicrobial activity of polyphenolic components, including poultry meat ,74,75,76is spice . The antis spice .One should notice, however, that a common point in the majority of those studies was that the extracts contained essential oils, which very often led to food with unusual and often undesirable change of organoleptic characteristics .p < 0.05) in untreated samples exceeding the acceptability limit of 1 mg MDA/kg sample within six days (p < 0.05), exceeding the limit of 1 mg MDA/kg sample after 20 days and reached a value of 1.27 mg MDA/kg after 50 days. Interestingly, TBA values of OV/OD samples showed insignificant variation, without exceeding the value of 0.63 mg MDA/kg even after 50 days of chilled storage. Regarding the above results, it is evident that the synergistic effect of impregnation with bioactive oregano compounds with the aw lowering obtained by the OD step suspended the rate of lipid oxidation, which constitutes a major factor of spoilage of chicken muscle.Besides microbial spoilage, chemical deterioration and, most particularly, lipid oxidation is a predominant factor limiting the shelf life of muscle food . Contentsix days . This liOriganum vulgare) in raw chicken meat during chilled storage were studied in [In the recent literature, there are several studies investigating the protective role of different types of extracts rich in bioactive compounds on the oxidation of chicken tissue. Aziz et al.  providedudied in , and theudied in . As detaudied in  investigp > 0.05) variations among samples as a consequence of the osmotic treatment. Results on both a* and b* values of OD and OV/OD samples revealed their stabilization throughout the storage period (data not shown). As far as the hue angle is concerned, OV/OD chicken samples exhibited good retention of their yellowish to reddish color  obtained after OV impregnation, whereas OD samples retained a more yellowish hue . All the treated samples exhibited a brighter yellow/white color, with lower hue values than the untreated samples that obtained values close to 75°  (data not shown). The above results regarding L* value decrease are in agreement with those reported in the literature , as the e to 75° a. In thiAs far as texture changes are concerned, elasticity of samples seemed to be abruptly increased immediately after the OD treatment, as expected due to the mass transfer flows occurring b. The apThe purpose of the sensory testing was mainly to evaluate consumer acceptability towards the modified chicken products immediately after the combined treatment and prior to the chilled storage. Therefore, the sensory characteristics  of raw untreated, OD and OV/OD chicken samples were assessed at time zero of chilled storage, with samples impregnated with an OV infusion exhibiting lower scores in appearance a as expe8 CFU/g of microbial count in accordance with [Regarding shelf life determination, the determining criteria set were either based on microbial growth .tN and oN are the final (limit of acceptability) and the initial microbial population and k is the relative growth rate of TVC ):e of TVC . TherefoIf rancidity development is the crucial factor for shelf life evaluation and is based on the limit of acceptability of 1 mg MDA/kg chicken flesh, one could graphically estimate the shelf life of the different pretreated samples, namely, six days for the untreated samples, 15 days for the OD-treated ones and a much longer time period (not perceived from the plot) for the OV/OD ones. Therefore, it can be concluded that at the particular storage temperature (4 °C) studied, chicken samples tend to deteriorate more rapidly in terms of lipid oxidation , with microbial growth showing slightly lower rates. This observation is in agreement with , where i3, 13 ± 2 g) in a laminar flow hood. Regarding the water-soluble bioactive compounds impregnation step, an oregano infusion (Origanum vulgare ssp. hirtum) commonly known as ‘Greek oregano’ from the cultivar ‘Greek Kaliteri’ was prepared by dissolving under continuous agitation a pre-weighed quantity of dehydrated oregano leaves in distilled water at 80 °C for 15 min in a ratio of about 1:20. The solution was microbiologically analyzed by plate counting (PCA/30 °C/72 h) and the total viable count (TVC) was below the detection limit of the method. The mixture was then strained off, filtered, and this solution also served as the basis for the OD solution (step 2) after the impregnation procedure was completed. The osmotic solution (step 2) was prepared by dissolving pre-weighed quantities of food grade glycerol  at a concentration of 40% and 5% NaCl  eithThree series of chicken fillet samples were prepared: untreated, fillets directly submitted to the osmotic dehydration procedure (OD) and fillets initially immersed into a hypotonic oregano infusion for 60 min in a food/infusion ratio of 1:2 before adding the pre-weighed osmotic agents of glycerol and NaCl (OV/OD) . This sequential two-step procedure instead of a simultaneous immersion process was selected based on previous observations  and on oFor OD and OV/OD series, the osmotic dehydration process was performed at 15 °C for a duration of up to 420 min with a food/solution ratio of 1:4. To perform the osmotic treatment, coded beakers filled with pre-weighed osmotic solutions were placed in an incubator  at 15 ± 1 °C. Pre-weighed chicken samples were kept immersed within the hypertonic solution by means of a grid. At predetermined timepoints, one beaker was removed, the samples were gently blotted with a tissue paper to remove the excess solution and then weighed.M0 is the initial mass of fresh material before the osmotic treatment, M is the mass of chicken samples after time t of the osmotic treatment, m is the dry mass of chicken after time t of the osmotic treatment and m0 is the dry mass of ‘fresh material.’ Abbreviation i.d.m. stands for ‘initial dry matter,’ t.s.—for ‘total solids.’Mass transfer kinetics were evaluated by calculating WL (water loss) and SG (solid gain). The measurements were performed in triplicate and the average values were taken. Moisture content was measured by sample drying at 70 °C under vacuum (Heraeus Instruments Vacutherm) for 24 h.w-meter (AquaLab Dew Point Water Activity Meter 4TE), and °Brix of the osmotic solution was measured with a hand-held refractometer .Water activity was determined using an aThe total phenolic content of chicken fillets during impregnation and the subsequent osmotic step was measured indirectly by assessing the TPC of the hypotonic/hypertonic solutions according to the modified micromethod of the Folin–Ciocalteu’s assay as described in . The TPCThe main purpose was to design and perform a comparative stability study of all series of samples  in order to compare deterioration of their quality during chilled storage. The purpose was to evaluate their shelf life and assess the effect of the different treatments imposed. The samples were stored at controlled isothermal conditions of 4 °C in a low-temperature incubator  after being air-packaged and thermally sealed in a laminate PET/Al/LDPE film. At predetermined time intervals, namely, at time 0, days 3, 5, 8, 16, 20, 30, 37, 42 and 50 of isothermal cold storage, the samples were withdrawn from the incubator and analyzed for a number of quality indices as detailed in the following sections. The approximately two-month analysis, although considered too extended for control samples, was deemed necessary, as some indices  remained at remarkably low levels in the case of their treated counterparts (OV/OD samples).®, Interscience, Saint-Nom-la-Bretèche, France), and then ten-fold serial dilutions were made with the same solution. Aliquots of each dilution (1 mL) were added to Petri dishes, to which an appropriated agar medium was then poured. The enumeration of total viable counts (TVC) and lactic acid bacteria (LAB) was made in plate count agar  plates incubated at 30 °C for 72 h and in the double-layered de Man, Rogosa and Sharpe Agar  incubated at 37 °C for 96 h, respectively. Regarding the enumeration of psychrophiles, 0.1 mL of each serial dilution was spread-plated on the plate count agar and incubated at 8 °C for 120 h.For microbiological analyses, 10 g of representative chicken flesh were homogenized with 90 mL of sterilized Ringer solution  in a sterile bag for 60 s using a Stomacher (BagMixermax) were estimated at the processing conditions investigated.Microbial growth modeling was performed using the Baranyi growth model  by fittiOxidation in chicken flesh was evaluated with the TBARS method, which is preferably applied to foods of animal origin since it is well-associated with sensory perception . Lipid oAt time zero of chilled storage, the sensory attributes of both raw and roasted chicken fillets from all the categories, namely, control, OD and OV/OD, were evaluated by a 15-member experienced and trained sensory panel. Testers were recruited and followed a training session on the basic organoleptic properties regarding the particular product. Appearance and odor of raw samples and color, flavor, taste, texture, juiciness, sweetness and overall impression/acceptability of their roasted counterparts were evaluated. A similar study is presented in ,67, wherIn our work, roasting took place in a domestic oven at 200 °C for 15 min, and all the samples were wrapped in thin aluminum foil to avoid extensive dehydration. After their thermal treatment, the samples were left to cool down to approximately 25 °C before being coded and evaluated by the panelists. Ratings were assigned separately to each parameter on a 1-to-9 hedonic scale (where 9 = like extremely and 1 = dislike extremely). A sensory score of 5 was taken as the average score for minimum acceptability. During the subsequent chilled storage of all the samples, no systematic sensory evaluation was performed; appearance and odor of all the raw samples was merely recorded in order to gain some information on the potential correlation of sensory rejection with microbial growth/lipid oxidation measured throughout this period.Color of chicken samples was instrumentally assessed with a tristimulus chromatometer  calibrated with a white standard plate , where CIELAB color scales were used, with coordinates  being directly read from the chromatometer. Bearing in mind the light white-to-yellowish hue of chicken muscle, lightness (described by the L* parameter) and the hue angle (h* = tan−1(b∗/a∗)) were chosen as the most representative color factors to be recorded. Hue angle is a very useful attribute since it describes the hue of the sample, and therefore it is found to represent adequately consumer perception of color. Actually, hue angle value defines the difference of a particular color vs. the grey color with the same lightness .Texture measurements were performed with a texture analyzer , and a TPA  test was carried out using chicken muscle samples of cylindrical shape. For this test, a non-lubricated flat platform, a 60-mm cylindrical compression probe and a 25 kg load cell were used. The samples were twice compressed using a fixed rate (1 mm/s) at 50% deformation. Amongst the different parameters calculated, elasticity was found to be the more representative. All measurements were performed in triplicate.® 7.0 . Duncan’s multiple range test was used for the evaluation of significant differences (a = 0.05).The statistical analysis of the results was performed by analysis of variance (ANOVA) at a 95% significance level using STATISTICAw-decreasing treatment  as an effective alternative to partially «substitute» the antimicrobial action of polyphenols in essential oils, avoiding the unpleasant modification of sensory attributes that is frequently caused; at the same time, this research confirmed the importance of water-soluble polyphenols as a significant antioxidant factor. In any case, a thorough and systematic sensory analysis should be simultaneously performed in order to optimize impregnation conditions , aiming at obtaining a novel, stable and organoleptically superior chicken fillet product with extended shelf life under chilled storage.The objective of the present study was to study the benefits of applying an osmotic dehydration step, with and without a prior step of bioactive compounds impregnation into chicken fillet muscle. Osmotic dehydration procedure as a sole technique caused a significant water activity decrease (down to 0.91) and improved quality stability during subsequent chilled storage in terms of both microbial decay and rancidity development. On the other hand, the enrichment of chicken samples with antimicrobial/antioxidant compounds after dipping in an oregano infusion seemed to have a more pronounced effect on lipid oxidation inhibition compared to microbial growth of spoilage bacteria. In order to assess the shelf life of refrigerated chicken flesh, criteria based on total microflora growth and TBARS increase were applied, the latter being directly associated with sensory perception of undesirable odors. As a concluding remark, the combined treatment of impregnation with a phenolic-rich oregano infusion followed by a water activity lowering step (osmotic dehydration) can be considered the most effective technique according to hurdle technology principles for the preservation of chicken fillets. In fact, one of the main findings of this work is that it might be possible to apply a mild a


Compared with the lowest median albumin category (Group 1), the beta coefficients for the median MMSE score were significantly and gradually increased in Group 2 , Group 3 , and Group 4 , respectively. In the analysis of repeated albumin measures, similar patterns were observed in cognitive function. All regression coefficients were greater in ε4 carriers than in non-carriers. Our findings suggested that sustained lower serum albumin levels were associated with lower MMSE scores. This observation may be modified by APOE polymorphisms.Previous studies have found an association between serum albumin levels and cognitive function. However, the results of this association are inconsistent, and the effect of Apolipoprotein E (APOE) on the association is less clear. Using retrospective cohort data (2008–2020), we investigated whether chronic serum albumin was associated with cognitive performance in older adults. We further assessed how the APOE genotype modifies its relevance. A total of 2396 Korean veterans and their families who were aged 65 years or older in 2008 and who had both data of serum albumin and cognitive performance  were included for the current study. The serum albumin levels were divided into four groups by quartiles: Group 1 (<4.0 g/dL), Group 2 (4.0–4.19 g/dL), Group 3 (4.2–4.49 g/dL), and Group 4 (≥4.5 g/dL). APOE ε4 carriers were defined as the presence of at least one ε4 allele . After adjusting for age, sex, and medical conditions, serum albumin levels  were significantly associated with increases in the median MMSE scores (beta = 3.30,  Cognitive function refers to mental abilities, including thinking, remembering, learning, judging, and problem-solving , which pThe single most important genetic risk factor for Alzheimer’s’ disease (AD) and cognitive decline is Apolipoprotein E (APOE) . APOE isGiven that poor nutritional status increases the risk of cognitive decline, and APOE interacts with age to modify the rate of cognitive decline, we hypothesized that low serum albumin levels are associated with poor performance in cognitive function, and whether the presence of APOE ε4 may modify its relevance. This study analyzed retrospective cohort data from the Veterans Health Service Medical Center (1) to verify the hypothesis that chronic serum albumin levels are associated with cognitive performance and (2) to assess the effect of the APOE genotype, as a genetic susceptibility to cognitive deficits, on these associations. Cognitive performance was assessed by the Mini Mental State Examination (MMSE). The MMSE is the most frequently used global assessment of cognitive status in clinical evaluation and epidemiological studies of dementia .Data were extracted from the electronic medical records (EMR) of the Veterans Health Service Medical Center  between 2008 and 2020. The EMR data included a patient’s medical history, diagnostic codes ), surgical operation, clinical examinations including laboratory testing, and treatment details. For the current study, we initially included 121,781 veterans and their families who were aged 65 years or older in 2008 and who were not diagnosed with dementia (ICD-10: F01-03) between 2008 and 2020. Of these patients, 116,647 who did not complete the MMSE or who had only one MMSE test were excluded. An additional 2158 individuals who did not have serum albumin tests measured at least twice during outpatient visits were excluded. Since the albumin levels were influenced by clinical conditions, 580 patients who were diagnosed with hepatic failure, liver cirrhosis, and renal failure were further excluded. Thus, 2396 subjects were included in the final study sample.Baseline information included patients’ age, gender, and medical conditions. Diagnosed medical diseases included hypertension, diabetes mellitus, dyslipidemia, heart diseases , and stroke.The study protocol was approved by the Institutional Review Committee of the Veterans Health Service Medical Center . Informed consent was waived by the committee because of the retrospective nature of this study.During the outpatient visits, blood samples were drawn from the forearm vein with the subjects in the seated position. Analyses of serum albumin and APOE genes were performed in the Department of Laboratory Medicine in the hospital. Serum albumin levels were measured using a bromocresol green dye-binding assay . Since the included study subjects underwent multiple measurements of the serum albumin level over a long-term follow-up period, we calculated the median serum albumin levels, which were considered chronic serum albumin levels during the study period.To determine APOE alleles, genomic DNA was extracted from the whole blood sample and subjected to APOE genotyping using the LightCycler 480 II Instruments Kit . APOE ε4 carriers were defined as the presence of at least one ε4 allele .Cognitive performance was assessed using the Korean version of the MMSE. The MMSE is the most widely used cognitive functioning test, including attention, language, memory, orientation, and visuospatial ability. It was scored on a scale of 0–30, with higher scores indicating better cognitive performance. The Korean translation of the instrument has been validated in clinical settings and in the elderly community ,32. WithWe calculated the median serum albumin levels during the study period. The serum albumin levels were divided into four groups by quartiles: Group 1 (<4.0 g/dL), Group 2 (4.0–4.19 g/dL), Group 3 (4.2–4.49 g/dL), and Group 4 (≤4.5 g/dL). We also calculated the median MMSE scores and used the median value during the study period as the dependent variable.p ≤ 0.05 was considered statistically significant.To evaluate the association between chronic serum albumin levels and cognitive performance, we conducted the logistic regression models with categorized serum albumin levels by quartiles. Repeated albumin measurements were modeled using a mixed effect model with a random component on the within-individual variation in albumin measurements. The logistic regression models generated the beta coefficient (β) and standard error (SE) with R-squared. We also conducted generalized additive model (GAM) regression, assuming a nonlinear association between serum albumin levels and MMSE scores. Since the functional form of GAM regression does not assume linearity between serum albumin levels and MMSE scores, it may allow a better fit than models assuming a strict linear association. The regression models of logistics and GAM were adjusted for age (continuous), sex, and disease history of hypertension, diabetes mellitus, dyslipidemia, heart diseases, and stroke. Subgroup analyses with 94 subjects who had APOE genotyping data were performed to explore effect modification in terms of the presence of the APOE ε4 allele. All analyses were performed using the Statistical Analysis System (SAS) version 9.2 , and For the current study, 2396 subjects were included, and their descriptive characteristics are shown in To evaluate the association between chronic serum albumin levels and cognitive performance, we performed logistic regression analysis. The results are shown in p < 0.0001), Group 3 , and Group 4 , respectively. In Model 2, adjusted for age, sex, medical conditions, and APOE gene, all regression coefficients for the median MMSE scores were highly increased in each albumin category: Group 2 , Group 3 , and Group 4 , respectively.Compared with the lowest median albumin category (Group 1 as a reference), the adjusted beta coefficients (in Model 1) for the median MMSE score were significantly and gradually increased in Group 2 , Group 3 , and Group 4 , respectively. In Model 2, all regression coefficients for the median MMSE scores were highly increased in each albumin category: Group 2 , Group 3 , and Group 4 , respectively.p = 0.0190), Group 3 , and Group 4 , compared with the lowest albumin category (Group 1 as a reference). After adjustment for the covariates, significant associations were found in ε4 non-carriers: Group 1 as a reference vs. Group 2 , Group 3 , and Group 4 , respectively. All regression coefficients were greater in ε4 carriers than in non-carriers.We performed the above regression analyses by stratifying the presence of the APOE ε4 allele . For ε4 The GAM regression was used to explore nonlinear associations between serum albumin and cognitive function. The estimated smooth effect curves demonstrating the associations between the median serum albumin levels and the median MMSE scores are shown in We found that chronic serum albumin was associated with poor cognitive performance in Korean veterans and their families, specifically with chronically lower serum albumin levels and lower MMSE scores over a follow-up period. In older adults with the APOE ε4 allele, the observed association was greater than in those who had no APOE ε4 allele. Our findings suggest that low serum albumin may be an important indicator of cognitive dysfunction in the aging brain. APOE ε4 carriers may be vulnerable to the negative effects of lowered albumin levels on cognitive function.Despite many previous studies showing a significant association between serum albumin and cognitive impairment, relatively little work has been conducted to address the longitudinal association between serum albumin and cognitive impairment. In cross-sectional studies with clinical samples, a low serum albumin level was associated with cognitive impairment in hospitalized patients with heart failure , rehabilThe underlying mechanism for the association between serum albumin and cognitive function has not yet been identified, but the properties of albumin involved in inflammation have been proposed ,34,35. SAPOE is a polymorphic lipoprotein that is a major cholesterol carrier in the brain ,38. APOEThis study has certain limitations. We utilized a retrospective cohort study design with the EMR database, in which each individual underwent multiple measurements of serum albumin levels and cognitive tests assessed by MMSE from the time they entered the registry of this current study until the time they exited the database. This suggests that since all serum albumin levels and MMSE scores are used to determine whether the serum albumin level is associated with the MMSE score, the target comparisons of “low serum albumin” vs. “high serum albumin” become the comparisons of “average low serum albumin” vs. “average high serum albumin”. The cognitive outcome of “MMSE score” is also the average cognitive outcome of “average MMSE score”. This leads to an unclear interpretation and potential selection bias . In addiIn conclusion, we found a significant association between serum albumin levels and MMSE scores through a retrospective analysis. This observation may be modified by APOE polymorphisms. Our findings indicate that lower serum albumin levels identify not only poor cognitive outcomes but also greater cognitive impairment in APOE ε4 carriers, although additional studies are needed to further understand the role of serum albumin in cognitive function and to optimize the prevention and management of cognitive decline.


Acute myeloid leukemia (AML) intensive chemotherapy combined with broad-spectrum antibiotics, leads to gut microbiota dysbiosis promoting pathological conditions and an increased incidence of complications. Here we report findings from a phase II single-arm, multicenter study evaluating autologous fecal microbiota transfer (AFMT) in 25 AML patients treated with intensive chemotherapy and antibiotics . The co-primary outcomes of the study are to evaluate the efficacy of AFMT in dysbiosis correction and multidrug-resistant bacteria eradication. The main secondary outcomes are to define a dysbiosis biosignature, to evaluate the effect of dysbiosis correction on patient clinical status, to assess the short and mid-term safety of AFMT in this immunocompromised population, and to evaluate the feasibility of the AFMT procedure and acceptability by the patient. Intensive induction chemotherapy induces a dramatic decrease of α-diversity indices, and a microbial dysbiosis with a significant shift of the microbial communities and domination of pro-inflammatory families. After AFMT treatment, α-diversity indices return to their initial mean levels and the similarity index shows the restoration of microbial communities. The trial meets pre-specified endpoints. AFMT appears to be safe and may be effective for gut microbiota restoration in AML patients receiving intensive chemotherapy and antibiotics, with an excellent gut microbiota reconstruction based on both richness and diversity indices at the species level. The combination of chemotherapy and broad-spectrum antibiotics induces gut microbiota (GM) dysbiosis in acute myeloid leukaemia (AML) leading to additional complications. Here, the authors report the efficacy in GM restoration and safety of autologous faecal microbiota transfer in treated AML patients in a phase II clinical trial. The different treatment phases of AML require prolonged hospital stays in a protected environment and multiple courses of antibiotic treatment, due to the high risk of life-threatening infectious complications3.Acute myeloid leukemia (AML) is a rare but potentially fatal blood cancer. The standard treatment for AML relies on conventional chemotherapy with or without allogeneic hematopoietic cell transplantation . Eligible patients first undergo an intensive induction chemotherapy (IC). If complete remission is achieved, consolidation therapy is performed to deepen response and achieve long-lasting remission. Several intensive chemotherapy cycles may be necessary. Finally, depending on the patient risk profile, HCT is undertaken6. The induced so-called dysbiosis is characterized by a reduction of overall microbial diversity, a disruption of beneficial bacteria which support host defenses, and a rise in dominance of bacterial species usually subdominant, including some pathogens, pathobionts, and multidrug-resistant bacteria (MDRB)8. The use of carbapenems during IC is associated with 4 times increased the risk of developing an infection within the following 90 days6. Thus, chemotherapy and antibiotic treatment disrupt the mutualistic relationship between host and microorganisms and promote pathological conditions involving impaired gut barrier function, uncontrolled local immune responses and, potentially, systemic inflammation9.Such treatments have been demonstrated to dramatically alter the rich and diverse composition of the human gut microbiota in AML patients11. The intestinal consequences of AML treatments can, thus, interfere with the patient’s optimal care through the increase of infectious-related complications, bad nutrition status, longer duration of hospitalization, interruption, or delayed consolidation courses due to treatment toxicity and MDRB carriage preventing alloHCT13.Moreover, the IC is known to have dramatic consequences on the gastrointestinal epithelium, which can lead to colitis with severe abdominal pain, diarrhea, and/or hematochezia5. The development of strategies such as fecal microbiota transfer (FMT) to restore the diverse microbial communities lost during disease treatment and, consequently, to suppress, decrease or even prevent – in the case of subsequent alloHCT - treatment-related complications in AML patients, could offer novel therapeutic possibilities15. Here we report the results of a single-arm study evaluating autologous FMT (AFMT) in AML patients treated with intensive chemotherapy and antibiotics. In this work, AFMT is safe, reduces the count of antibiotic resistance genes and may be effective for gut microbiota restoration in patients receiving intensive chemotherapy and antibiotics for AML, with an excellent gut microbiota reconstruction based on both richness and diversity indexes at the species level. Our findings suggest that therapeutic manipulation of gut microbiota may become a treatment option to restore gut microbiota diversity and potentiate cancer management.Recent studies demonstrated that a high gut microbial diversity is associated with improved clinical outcome and reduced infectious complications in AML patientsA total of 62 AML patients were screened in 7 different centers, 25 were treated with AFMT, and 20 were considered as the per-protocol population. The baseline characteristics of treated and per-protocol patients are listed in Table n = 1) and colitis (n = 1)), logistical organization in 2 others] or delayed 35 days after hematopoietic recovery from aplasia, in one patient, due to an infectious event requiring prolongation of antibiotics.AFMT feasibility and acceptability were evaluated as a secondary outcome. Twenty-eight patients fulfilled all inclusion and exclusion criteria, three eligible patients refused the AFMT procedure and 25 patients were treated and received the two AFMT one day apart. Nevertheless, only 20 patients were able to receive the two AFMT as planned after IC and hematopoietic recovery from aplasia , lymphocytes count and platelets count were 9.35 g/dL , 4.8 G/L , 1.78 G/L , 0.88 G/L  and 66 G/L  respectively, at time of AFMT  was evaluated throughout the clinical study. The data showed that the results of the questionnaire after AFMT (V3) were similar or tended to improve, compared to those at V2 before AFMT , which highlights the absence of a negative impact of AFMT on the overall health of patients  and median inverse Simpson index 21.5 versus 5.1 (p = 0.0009) respectively    with no significant difference between values at V1 and V3     and V2 (after IC and before AFMT), at the metagenomic species level: median Shannon index 4.0 versus 2.4;  Fig. . After A V3 Fig. . The nump = 0.0008) for per-protocol patients . The count of antibiotic resistance genes at V4  was also not significantly different compared to that in V3, suggesting that AFMT had a beneficial effect in alleviating the microbiota disruption induced by consolidation chemotherapy and antibiotics  were observed  . During the AFMT treatment, no harmful changes in vital signs of treated patients    and between V3 and V4  , one a clinically defined infection (CDI), eight had fever of unknown origin and five had no fever. There was no difference in the microbiota diversity after AFMT between these three groups  received antibiotic with large anti-anerobic coverage , while five patients received third or fourth generation cephalosporine and five patients did not received antibiotics. Interestingly, patients receiving antibiotic with a large anti-anerobic coverage had a significantly lower alpha diversity after AFMT, compared to patients that received antibiotic with less anti-anerobic coverage or no antibiotics  during the first three visits  and a return to baseline at V3 after AFMT . Systemic neopterin levels followed the same variations with an increase at V2 and a return to baseline at V3 , we also measured inflammatory parameters both locally in the gut and systematically in plasma of per-protocol patients , highlighting the expected inflammatory intestinal status of patients after IC and antibiotic therapy. Levels were significantly decreased and returned to baseline following AFMT . These variations were in line with C-Reactive Protein variations. As a mirror of local immunity, secretory IgA was also measured in feces, and similar trends were observed . Altogether, these data clearly point out to the absence of deleterious inflammatory reaction, both locally and systematically, after AFMT.We next sought to assess the local immunity and inflammation in the gut by measuring fecal neopterin and secretory IgA Fig. . Neopter18 and is also associated with gut dysbiosis. Oxidative stress occurring during inflammation is a factor amplifying dysbiosis by strongly decreasing the microbial diversity in the gut, and by promoting the outgrowth of specific aerotolerant and proinflammatory bacterial taxa19. Therefore, maintaining a functional antioxidant response is important. We measured the total antioxidant status (TAS), and we observed no significant variation of TAS levels between V1 and V2 , but a significant increase after AFMT , which could be associated with gut microbiota restoration , Bifidobacterium that cross-fed butyrate, Akkermensia and Enterococcus. We find a strong negative correlation between fecal neopterin and alpha diversity, butyrate producing bacteria and Bifidobacterium. Similarly, some butyrate producing bacteria correlate negatively with other inflammatory parameters, including CRP, ferritin and CD14+ monocytes. Furthermore, IgA, that is associated with inflammation and gut microbiota dysbiosis in irritable bowel syndrome, also correlate negatively with butyrate producing bacteria in you study. In contrast the pro-inflammatory bacteria Enterococcus correlates positively with fecal neopterin and ferritin and negatively with the butyrate producing bacteria Roseburia. Finally, Akkermansia that has been inversely associated with obesity, diabetes, cardiometabolic diseases and low-grade inflammation21, but also in response to cancer immunotherapy22, positively correlate with butyrate producing bacteria and TGFb2 and negatively correlate with inflammatory parameters .Finally, in order to establish a dysbiosis biosignature (secondary outcome), we sought to investigate the link between microbiota parameters , immune and inflammatory parameters  by performing a Spearman correlation analysis, all visits combined Fig. . We focun = 25). The median follow-up time for surviving patients was 29.9 (range 27.5–37.0) months. Among the treated patients, all but one, had responded to chemotherapy at V3 (96%), including 23 complete response and one partial response according to the 2017 ELN recommandation23. Seven patients relapsed at a median of 16.8 (range 3.9–27.8) months. The leukemia-free survival (LFS) rate was 88% at 6 months and 60% at 2 years , including three with gastrointestinal involvement . In the entire cohort of patients, 8 died, the causes of death being relapse in two patients, GVHD in one, sepsis in two , CMV disease in one, multiorgan failure in one patient and thromboembolic event in one patient. The overall survival (OS) rate was 92% at 6 months and 72% at 2 years  are described for all patients having received the AFMT (n = 1 and diarrhea n = 2) within few days after AFMT. Most importantly, none of the patients developed SAE and, in particular, infectious complications within the month after AFMT. Only one patient experienced asymptomatic feces colonization by multidrug resistant E. coli at V3, 22 days after the second AFMT, followed by an E. coli sepsis 71 days later. Nevertheless, this SAE occurred 3 months after AFMT, raising the question of its link to the administered treatment. Safety was further confirmed by the low level of inflammatory parameters after AFMT. The good safety profile is in accordance with previously published results of FMT in immunocompromised patients with hematologic malignancies26.The results of the current phase II study demonstrate that AFMT after IC in AML patients was feasible. Among the 28 patients that fulfilled the inclusion and exclusion criteria, only 3 patients refuse the procedure, and the remaining 25 patients received the AFMT. Of note, five patients could not receive the AFMT as planned after IC and hematopoietic recovery from aplasia for logistical reason or transient contraindication, but the AFMT was successfully administered after resolution of the issues. We also found the approach to be safe in immunocompromised patients, as they manifested minimal toxicity after AFMT administration. Only three patients experienced transient gastrointestinal symptoms . Importantly, Lachnospiraceae and Ruminococcaceae have been associated with reduced mortality after alloHCT28, while Enterobacteriaceae and Enterococcus were associated with a decreased OS29 and an increased GVHD-related mortality29. Therefore, our findings suggest that our current strategy of AFMT and gut microbiota restoration after IC allow AML patients to get to alloHCT with a protective microbiota. In fact, 18 patients received an alloHCT and only three developed gastrointestinal GVHD, raising the question of a possible long-term protective effect of our approach. The cumulative incidence of clinically significant grade II-IV aGVHD was 22%, and microbiota α-diversity after AFMT has no impact on the cumulative incidence of aGVHD. Our results compare favorably with an incidence of 34.8% we reported previously30.Disruption of the intestinal microbiota by intensive chemotherapy and antibiotics is well established in patients with hematologic malignancies31. This is particularly important given that bloodstream infections from MDRB are associated with a poor outcome and an increased mortality32. Eradication of antibiotic resistance genes after AFMT may have partially contributed to the good outcomes reported in our study with a 2-year OS of 72%. Importantly, OS compared favorably with previously published studies. Therefore, lower two-years OS ranging from 41.9% to 60% have been reported in randomized phase 3 studies with a similar induction chemotherapy (cytosine arabinoside + anthracycline)34. Furthermore, although not statistically significant, two-years OS and LFS were higher in patients with intermediate-high Shannon index, further suggestion a long-term protective effect of our approach on OS and AML relapse.Another important finding was the significant reduction of the median number of reads mapped against antibiotic resistance genes observed at V3 after AFMT. We and others had reported that FMT is an effective strategy for MDRB eradication in patients with hematologic malignancies26 or of readily available pooled microbiota biotherapeutic, as it is currently investigated in GVHD35, may be a promising strategy to overcome this limitation. Also, one could object that gut microbiota composition will spontaneously restore back to normal without AFMT and the absence of a control group treated with placebo FMT prevent us from drawing any definitive conclusions on our findings. Additional immunological and microbiota analysis during follow-up, in particular at 6 months or before/after alloHCT would also have been useful to evaluate the long-term effect of our approach.One of the main limits of our study was the high incidence of screening failure, owing to the exclusion of a significant number of patients during screening prior to production of the AFMT inocula. This was due to strict, rigorous guidelines regarding microbiological testing and amount of feces required for the IMP production being followed. Nevertheless, these guidelines are indispensable to guarantee product safety. Use of third party donors as it has been done after alloHCT26. Furthermore, the development of allogenic FMT from multiple donors in order to increase the amount of different bacterial species and, thus, enhance the global microbial richness of the treatment is of interest. This approach is currently being evaluated with FMT capsules for colon delivery in order to ease the administration of the product in a similar setting . Further clinical investigations will be required to evaluate the beneficial impact of this strategy implemented after each cycle of chemotherapy and HCT conditioning.Finally, AFMT may not be appropriate in patients with previous antibiotic therapies or other treatments altering the composition of the gut microbiota. Therefore, the use of allogenic FMT may be more effective, and has already been shown to be feasible, safe, and associated with the expansion of recipient microbiome diversity after alloHCTOverall, AFMT appears to be safe and effective for gut microbiota restoration in patients receiving IC and antibiotic for AML. Besides this particular setting, providing patients with a drug that may modulate inflammation and better potentiate other anti-cancer treatments is of considerable interest.ODYSSEE was a phase I/II single-arm, multicentre, prospective, interventional trial in hospitalized patients with AML/ high-risk myelodysplastic syndrome to assess the feasibility and tolerance of AFMT in the setting of IC and antibiotic therapy given during aplasia. All patients were pre-evaluated within clinical practice before the official start of enrollment. All enrolled subjects gave their written informed consent to participate in the study. The study was approved by the ethics committee “Comité de protection des personnes Ile de France V” on April 5th, 2016, was conducted in full compliance with ICMJE guidelines and was registered in ClinicalTrials.gov (Identifier: NCT02928523). While study was submitted for registration on May 26, 2016, before the first patient inclusion on June 2016, there was a delay in the approval during the registration process while some innovative aspects were clarified and the study was finally registered on October 10, 2016. The full study protocol is available as Supplementary Note Clostridium difficile, parasites, noroviruses and/or rotaviruses , or who received antibiotic therapy until 4 days before study inclusion were excluded from this trial . The AFMT was planned at the end of IC, after aplasia completion (ANC > 0.5 G/L) and 24 h after antibiotics discontinuation. AFMT was performed irrespective of platelet level, nevertheless platelet transfusion to increase platelet count before AFMT procedure was allowed based on the physician decision. In addition, patients with the following condition: persistent mucositis, colitis, or hemorrhoids were not eligible for AFMT according to exclusion criteria (Supplementary Table n = 1) and colitis (n = 1)) and for logistical organization in two others. For one patient, the period between V2 and AFMT was 35 days, due to an infectious event requiring prolongation of antibiotics, with a potential impact on the primary endpoint. Twenty patients were thus considered as per protocol. All figures and tables will present the data obtained from these patients, unless specified.Among the 25 treated patients, a major protocol deviation was reported in five: four patients could only receive AFMT after the first consolidation chemotherapy due to AFMT contraindication in two patients , and at the end of the hospitalization (Visit V4). The quality of life of patients was assessed at each visit V1 to V4), using an EQ-5D-5L questionnaire . Finally, after 6 months (Visit V5) and 1 year (Visit V6), clinical information and safety assessment were recorded  to the manufacturing facility and processed following Good Manufacturing Practices. Feces were processed within 72 h with a patented cryoprotective diluent into a proprietary device (MaaT Pharma reference: 5M001), filtered, conditioned, and stored frozen at −80 °C until transplantation36. This kit allows the distinction between live and dead bacteria based on the integrity of their membranes, through two fluorophores, SYTO9® and propidium iodide (PI). SYTO9® enters in all cells, undamaged or not, binds to the DNA (deoxyribonucleic acid) molecule and emits at 540 nm (green) after excitation at 470 nm (blue laser). PI also targets DNA molecules but only penetrates cells with damaged membranes (emission at 635 nm (red) after excitation at 470 nm.Bacterial viability of the IMP was assessed by flow cytometry using the kit LIVE/DEAD® BacLight™ Bacterial Viability Kit from ThermoFisher ScientificSuspension samples were thawed at room temperature and immediately stained with a mix of the two fluorophores. Samples were then incubated for 15 min at room temperature protected from light, and next analyzed by flow cytometry. A control sample was processed in parallel to achieve a 0% viability and validate the test.C. difficile and MDRB  was performed using PCR (polymerase chain reaction) and culture on specific isolation media, respectively. Parasites were detected using PCR  or microscopy  after feces concentration. Noroviruses and Rotaviruses were identified by PCR and immunochromatography, respectively, and pathogenic bacteria were detected using PCR (Campylobacter) and culture   guidelineBiochemical and immunological analyses were performed on the different blood and feces samples collected during the first three visits. Neopterin and secretory IgA (sIgA) were measured from feces supernatants using the Neopterin ELISA  and IgA Secretory Human ELISA sa (EUROBIO) kits, respectively. Neopterin was also measured from plasma using the same Neopterin ELISA . Total Antioxidant Status was measured from plasma using the Hitachi 912 kit (RANDOX Laboratories), and C-Reactive Protein levels were measured from plasma/serum samples in the different medical centers according to their own internal procedures.Genomic DNA was extracted from the fecal samples collected during the first four visits using the Qiagen QIAamp Fast DNA stool mini kit and following a beat-beating procedure. A sequencing library was constructed for each DNA sample using the TruSeq Nano DNA Library Prep kit (Illumina) according to the manufacturer’s instructions. Libraries were then sequenced in 2 paired-end (2 × 125 bp) HiSeq2500 v4 (Illumina) runs. Raw sequencing data was downsized to 40 million reads/sample when possible.® platform with the in-house MgRunner v1.1.2 pipeline. In brief, after quality filtering using Trimmomatic v0.3638, host sequence decontamination was performed using Bowtie2 v2.3.039. Between 936,060 and 37,212,124 pairs of reads  were obtained from the different samples. To ensure comparability, all samples were rarefied to the same sequencing depth i.e. 1,500,000 paired-end sequences per sample. Taxonomic profiling was then performed with Kraken v.0.10.5-beta40 and the RefSeq genomic database . The measure of α- and β-diversity indexes was performed in R Statistical Software  using vegan v2.4-2 and phyloseq 1.19.1 packages. Gene-based analyses were performed using the Integrated Gene Catalogue (IGC)41. Finally, antibiotic resistance analyses were performed through gene mapping with Bowtie 2 v2.3.0 on MEGARes v1.0.1 (https://megares.meglab.org/) database.Bioinformatics analyses were performed on Gut PrintThe co-primary outcomes of the study were to evaluate the efficacy of AFMT in dysbiosis correction and MDRB eradication. The main secondary outcomes were (a) to define a dysbiosis biosignature using combination of biological parameters, (b) to evaluate the effect of dysbiosis correction on patient clinical status, (c) to assess the short and mid-term safety of AFMT in this immunocompromised population and (d) to evaluate the feasibility of the AFMT procedure and acceptability by the patient. Secondary outcomes b, c, and d only appear in the study protocol and do not appear on the registration site . An individual patient Meta-analysis for a controlled result of efficacy was planned as an exploratory objective and will be conducted in a separate study.R  is at least R = 0.5. Without an accurate estimate of the standard deviation of Sd and Sf, we consider in this first-in-man trial that a change observed as at least half of its standard deviation should be considered as clinically meaningful as defined by Cohen’s rules. In these conditions, a sample size of at least 30 patients should provide a power of 0.875 to detect a difference of a clinically relevant difference Δ = | Sf−Sd | , at a one- tailed significance level of 0.05. Nevertheless, in order to limit the number of cases, given the expected recruitment difficulties, sample size was evaluated according to beta values and effect size. When the effect size is at least 0.6 and with a beta risk of at least 0.20, 20 patients should constitute an acceptable size. Therefore, a sample of 20 patients constitutes an acceptable sample size for this pilot study.By assuming repeated measurement between Simpson index post AFMT (Sf) and the two previous Simpson index values (Sb before antibiotherapy and chemotherapy and St post treatment) we assume that the correlation 42. The correlation R between St and Sf was 0.83 (Spearman correlation), higher than the requested (0.5). Furthermore, measurement of clinical relevance was based on the standardized mean difference or cohen effect size. The Standard Mean Deviation was 1.063 [(mean Sf -mean St)/pooled standard deviation]. Therefore, based on this large effect size it was not necessary to increase the sample size. Based on the result of the interim analysis performed on the first 9 patients, it was not necessary to adapt the sample size and futility analysis was not performed.A planned interim analysis was performed on the first 9 patients fulfilling inclusion and exclusion criteriaFor most visit and parameter, the normality distribution, including after a log transformation, was rejected by Shapiro–Wilk test (Supplementary Table Further information on experimental design is available in the Supplementary InformationReporting summary


Serum and nasal secretion samples were collected before administration of treatments and an intranasal vaccine against respiratory viruses , and once weekly for the first four weeks of age. Systemic redox balance was determined in serum. Immunoglobulin A specific for bovine herpesvirus 1 and bovine syncytial respiratory virus was quantified in nasal secretions as a proxy to intranasal vaccine responsiveness. Our results showed that parenteral administration of antioxidants at birth improved calves’ redox balance. Additionally, calves receiving antioxidant supplementation had higher concentrations of immunoglobulin A in their nasal secretions than calves in the control group. Thus, we conclude that supplementation of calves with antioxidants at birth could be a practical strategy to improve intranasal vaccine response. Future larger studies should evaluate the extent to which this increased mucosal response to intranasal vaccination could result in decreased calf morbidity and mortality.Newborn calves experience oxidative stress throughout the first month of their life, which is known to decrease lymphocyte functions relevant to vaccine responsiveness. Thus, this study aimed to determine the extent to which parenteral antioxidant supplementation given at birth improves the response to an intranasal viral vaccine in the first month of life of newborn dairy calves. For this, 21 calves were randomly assigned at birth to one of two commercially available antioxidant micronutrient supplements or a placebo group receiving 0.9% sterile saline ( Neonatal calf morbidity and mortality have been reported at elevated risks in several countries, including the USA, Canada, and Australia ,2,3. InfIntranasal vaccination of calves against respiratory viruses at birth has become a common practice to circumvent inference of passive immunity through the stimulation of mucosa-associated lymphoid tissue . This reParenteral supplements containing micronutrients with antioxidant capabilities are licensed in the USA for administration to cattle and could be used to assuage oxidative stress in newborn calves. To the best of our knowledge, however, the effect of improving antioxidant capacity on the calves’ response to intranasal vaccination has hitherto not been investigated. We hypothesized that calves receiving a parenteral antioxidant supplement would have an improved redox balance, resulting in a greater response to intranasal vaccination. Thus, we aimed to investigate the extent to which the two commercially available parenteral antioxidant supplements could change the systemic redox balance and nasal secretion antigen-specific IgA concentrations in newborn dairy calves throughout their first month of life.® 3, Merck Animal Health, Madison, NJ, USA) against the respiratory viruses parainfluenza 3, bovine herpesvirus type 1, and bovine respiratory syncytial virus; and received the study treatments. Calves received another 2 L of colostrum at approximately 6 h of life. Calves were housed in individual in-house stalls and fed 3 L of milk replacer  at 14% solids, 3 times/day throughout the first month of life. Water was available ad libitum from the first day, and starter concentrate was offered starting at week 1 of age. For this study, we used 21 Holstein calves from a commercial farm associated with the Michigan State University Training Center for Dairy Professionals . Calves were enrolled at birth. After calving, calves were left with their dams in the individual calving pen for 30 min. Subsequently, calves were weighed; had their navels dipped in an iodine solution; received 4 L of > 22% Brix warm thawed colostrum via orogastric tube, a commercial antibody oral supplement against enteric pathogens , and a commercial live intranasal vaccine  using randomization software , stratifying by sex. The treatment groups consisted of the two parenteral antioxidant micronutrient supplements commercially available in the USA and a placebo control group. Supplement A  contained selenium (2.19 mg/mL sodium selenite) and Vitamin E , whereas Supplement B  contained zinc (60 mg/mL), copper (15 mg/mL), selenium (5 mg/mL), and manganese (10 mg/mL). Supplements were administered subcutaneously at their labeled dosage of 0.07 mL/kg for Supplement A, and 0.022 mL/kg for Supplement B. Calves in the placebo group received 3 mL of sterile 0.09% saline subcutaneously. Farm staff but not researchers were blinded to treatment allocation. Two calves of the study died between week 1 and week 2 samplings, and the samples collected prior to death were retained in the study as calves were clinically healthy when samples were collected.Calves were allocated to 1 of the 3 treatment groups  and weekly thereafter during the first month of life (median (range) age at sampling: 7 (6–8), 14 13–15), 21 (20–22), and 30 (29–31) days). For serum collection, blood was collected via jugular venipuncture using 10 mL vacuumed tubes without anticoagulant . Tubes were allowed to clot for 15 min and centrifuged at 2000× –15, 21  Holstein cows in second to fourth lactation housed in the Michigan State University Dairy Cattle Teaching and Research Center . These samples were collected immediately before and 5 days after administering the same intranasal vaccine that calves received . The samples from each time point were pooled together to serve as negative and positive reference samples, respectively, for assay quality-control purposes to ensure that the ELISA assays could accurately detect low and high antigen-specific immunoglobulin A concentrations. Mid-lactation cows were selected because they are expected to have a robust immune response .To assess passive immunity transfer from the dam to the calf via colostrum, the concentration of serum total protein was estimated in the sample collected at week 1 of age using a digital refractometer . This method has a high accuracy for identifying animals with failed passive immunity transfer  and is rRedox balance was assessed in serum as previously described . A commeThe serum’s antioxidant potential (AOP) was measured using the Trolox equivalent antioxidant capacity, as previously described . BrieflyRedox balance was assessed as the ratio of pro-oxidant to total antioxidant defenses (RONS/AOP), namely the oxidant status index (OSi), as it accurately detects changes in redox balance during the transition period of dairy cattle . An incr50/μL and was diluted 1:100 with coating buffer . In contrast, the BRSV stock solution concentration was 600 TCID50/μL and was diluted 1:50 with the same coating buffer. All ELISA plates were prepared at the same time. For this, 96-well plates were coated with diluted solutions and incubated overnight at 4 °C. After incubation, plates were washed three times with 1X ELISA wash buffer . Then, plates were blocked and incubated at room temperature for one hour with ELISA Blocker Blocking Buffer  to prevent non-specific binding of antibodies. Subsequently, the blocking buffer was removed, and the plates were left to dry uncovered inside a biosafety cabinet for 2 h at room temperature. Once dry, the plates were sealed and stored with a desiccant at −20 °C until analysis. The detection antibody was also prepared ahead of analysis. An anti-bovine IgA HRP-conjugated sheep polyclonal antibody  was used as detection antibody and was diluted 1:50,000 with 1X PBS containing 0.2% bovine serum albumin. The detection antibody solution was stored at 4 °C until analysis. All analyses were completed within 1 week after preparation.Nasal secretion samples were analyzed for BHV1 and BRSV-specific IgA using an ELISA assay as previously described ,14. Brie®20 . All samples were analyzed in duplicate. Blanks and negative and positive reference samples were included in duplicate in each plate. Prior to analysis, coated plates were removed from −20 °C storage and washed three times with 1X ELISA wash buffer. Diluted nasal secretions (100 μL) were pipetted into each well, and plates were sealed and incubated at room temperature for 1 h. Subsequently, plates were washed 4 times with 1X ELISA wash buffer, 100 μL of detection antibody solution was added to each well, and plates were sealed and incubated at room temperature for 1 h. Following incubation, plates were rewashed 4 times with 1X ELISA wash buffer, 100 μL of TMB Stabilized Chromogen  was added, and plates were incubated at room temperature for 20 min. After incubation, 100 μL of stop solution  was added to all wells, and optical density at 450 nm (OD450) was determined immediately in a Synergy H1 Hybrid plate reader . Thus, OD450 readings are proportional to the concentration of IgA present in the nasal secretion samples. The ELISA cut-off was calculated as the mean + 3 standard deviations of the OD450 reading of the negative control samples.Nasal secretion samples were thawed on ice for 30 min and diluted 1:10 with 1X PBS containing 0.1% Tweenp < 0.05.Statistical analyses were conducted using JMP Pro 15 . Kruskal–Wallis test was used to compare birth weights and the concentration of serum total protein at week 1 of age among treatment groups. Sex distribution across groups was compared with Fisher’s exact test. Mixed models with repeated measures were built for each of the outcome variables of the study . Treatment , time , and their interaction were the main effects. Sex was included as a random effect. Five covariance structures were tested , and the one resulting in the lowest Akaike information criterion was chosen. The degrees of freedom were approximated with the Kenward–Roger method. Model assumptions were assessed by evaluating the homoscedasticity and normality of residuals. To ensure homoscedasticity of residuals, the data of BHV1-IgA and BSRV-IgA were log-transformed. Data are presented as least squares means . Tukey’s honest significance test was used for post hoc pairwise comparisons. Statistical significance was declared at By design, sex was equally distributed among treatment groups. Furthermore, there were no differences among groups in birth body weight or serum total protein concentrations at week 1 of age . All stuCalves receiving supplement A or B showed lower serum RONS concentrations, higher serum AOP, and lower OSi values than calves in the control group , thus inExamining changes over time, supplements A and B were effective in improving AOP throughout the first 4 weeks of life compared to the control placebo group b. HoweveNewborn calves have been reported to experience oxidative stress throughout the first month of life ,11, whicp = 0.061 and p = 0.017, respectively), as the AOP of control calves remained unchanged throughout the study. The literature on the redox biology of neonatal dairy calves is scarce, and findings from mature cattle cannot be directly translated to newborns [Interestingly, despite improved AOP throughout the study period, supplemented calves only showed improved redox balance, as assessed by OSi, during the first 2 weeks of life. This is attributed to a decrease in the pro-oxidant load of control calves in weeks 3 and 4 of age, as indicated by lower RONS concentrations at these time points than in week 2  = − 1.15 and − 2.03 for BHV1 and BRSV, respectively). A significant treatment effect was identified for anti-BHV1 and -BSRV IgA , with increased anti-BHV1 IgA responses in the calves receiving either antioxidant supplement starting at week 2 of age. In contrast, an increased response in anti-BRSV IgA in supplemented calves was already detected at week 1 of age. Interestingly, the anti-BHV1 IgA response in nasal secretions in the control calves was similar between weeks 1 and 2 (p = 0.97), whereas calves receiving an antioxidant supplementation showed statistically higher responses between the same timepoints (p < 0.0006). Similarly, control calves had similar anti-BRSV IgA responses between birth and week 1 (p = 0.87), whereas supplemented calves already exhibited increased IgA at week 1 compared to the basal sampling at birth (p < 0.02), thus indicating that the administration of an antioxidant supplement at birth resulted in a faster and greater response to the BHV1 and BRSV components of the vaccine. No differences in their capacity to improve vaccination response were detected between commercial antioxidant supplements (In this study, we investigated the effect of improving the antioxidant capacity of newborn calves on their response to intranasal vaccination. Prior to vaccine and colostrum administration at birth, calves in all groups had similar anti-BHV1 and anti-BSRV IgA responses in their nasal secretions a,b, withplements .Previous studies in calves have documented improved immune responses to vaccines delivered parenterally when antioxidant supplements were administered at the time of vaccination . HoweverThe sample size utilized in this proof-of-principle study was limited, and we just monitored the effect of parenteral antioxidant supplementation throughout the first month of life because this is the period where newborn calves have been reported to show oxidative stress  and the The administration of parenteral antioxidant supplements simultaneously with an intranasal viral vaccine to newborn calves resulted in an increased response to the vaccine as assessed by antigen-specific immunoglobulin A concentrations in nasal secretions throughout the first month of life. Thus, antioxidant supplementation of newborn calves as undertaken in this study could be a practical strategy to optimize calves’ immunity.