Linear correlations (R2 > 0.9041) between steady shear (viscosity) and oscillatory  values of the dough rheology with bread quality parameters (volume and firmness) were obtained. Results confirmed that the yogurt addition led to a significant improvement on bread quality properties, increasing the volume and crumb softness and lowering the staling rate, with a good nutritional contribution in terms of proteins and minerals, to improve the daily diet of celiac people.Absence of gluten in bakery goods is a technological challenge, generating gluten-free breads with low functional and nutritional properties. However, these issues can be minimized using new protein sources, by the addition of nutritional added-value products. Fresh yogurt represents an interesting approach since it is a source of protein, polysaccharides, and minerals, with potential to mimic the gluten network, while improving the nutritional value of gluten-free products. In the present work, different levels of yogurt addition (5% up to 20%  Celiac disease is an immune enteropathy caused by the ingestion of gluten in genetically susceptible individuals, and it is estimated to affect about 1–3% of the population worldwide [Currently, the gluten-free (GF) diet is the only option for people suffering from gluten-related disorders. For other reasons, a high number of nonceliac individuals are also adopting this diet. Subsequently, there has been a significant increase of research work to improve the functional and nutritional properties of GF products ,3. HowevGluten-free products, especially breads being mainly based on refined flours and starches, are generally characterized by poor technological quality attributes, including dry, crumbling texture, color, and mouth feel , and undIn terms of nutritional profile, normally they present deficient values of protein and minerals and higher carbohydrates and fat content than recommended .Rice flour has been widely proposed as an alternative for making gluten-free breads (GFB) due to its hypoallergenic protein, soft taste, and white color . HoweverIn fact, gluten possesses unique viscoelastic properties which are crucial for the water-holding capacity of the dough and gas retention during fermentation . To mimiAddition of new protein sources, e.g., dairy dry powders incorporation  was shown to improve the volume, appearance, and sensory aspects of the loaves ,17. A prYogurt (Yg), is considered the most popular dairy product worldwide for its nutritional and health benefits, since it is a source of protein (casein), exopolysaccharides (EPS), vitamins , and minerals , representing an interesting alternative for new bakery products ,19. In tThe aim of the present work was to explore the potential of fresh yogurt addition on GF bread making and to overcome the technological challenge involved in gluten removal, improving the GFB quality. Different levels of Yg addition to a GF dough formulation, previously optimized, was tested, and the impact on dough, based on steady shear behavior and oscillatory measurements, was assessed. The effect on GFB quality, by the evolution of the crumb firmness and staling rate, during the storage time, as well as from bread quality parameters and nutritional profile, was also evaluated.Gluten-free bread was prepared using rice flour , buckwheat flour , and potato starch .The fresh yoghurt (Yg) used was a commercial product from LongaVida, Portugal . The dry extract of Yg was determined from the standard Portuguese method: NP.703-1982 (Standard Portuguese Norm), corresponding to 11.5% of dry matter.Commercial white crystalline saccharose , sea salt , baker’s dry yeast , vegetable oil , and xanthan gum  were also used.2) produced and retained during this bread making step. Bread baking conditions: Oven with convection at 180 °C during 30 min.Gluten-free bread dough was prepared in a thermo-processor , according to the procedure earlier described , with soDifferent GFB formulations tested are summarized in w/w (weight/weight) in overall percentage. Replacements were based on gluten-free flours basis, substituting the dry extract of each Yg percentage on 100 g of flour [Fixing the viscosity torque values of the gluten-free control dough (16.0 ± 2.0 milliNewton meter - mNm), previously optimized, the water flour absorption was determined for each Yg bread formulation tested (based on 14% of moisture basis), considering the water coming from Yg additions, applying the mixing curves procedure by MicrodoughLab assays . Control dough and doughs obtained with Yg incorporations, were prepared considering the levels 10, 20, 30, and 40 g of Yg into dough, corresponding to 5% up to 20% of flour .All rheological measurements were carried out in a controlled stress rheometer , with a universal temperature control Peltier system to control temperature, using a serrated parallel plate sensor system (PP35 and 1-mm gap), to overcome the slip effect ,22.The rheology features of the dough were evaluated after 20 min of fermentation time, and all the assays were conducted at 5 °C of temperature to inactivate the yeast fermentative activity.−6 to 1.0 × 103 s−1.The impact of the yogurt addition on dough viscosity behavior was assessed by flow curves, under shear steady conditions ranging the shear rate from 1.0 × 100 is the zero-shear rate viscosity (Pa s), γ.c is a critical shear rate for the onset of the shear-thinning behavior (s−1), i.e., the value corresponding to the transition from Newtonian to shear-thinning behavior, and s is a dimensionless parameter related to the slope of this region.The Carreau model was used to model the flow curves obtained, applying the Equation (1):Frequency sweep was applied to evaluate the impact of the Yg addition on dough structure, and the evolution of the viscoelastic functions, storage (G’) and loss (G’’) moduli, were obtained ranging the frequency from 0.001 Hz to 100.0 Hz, at a constant shear stress (10 Pa), within the linear viscoelastic region of each sample, previously determined (at 1 Hz).All rheology determinations were repeated at least three times to ensure the reproducibility of the results.Bread texture was evaluated using a texturometer TA-XTplus  in penetration mode, according to the method earlier described ,23.Comparison of the bread texture, with different contents of Yg, was performed in terms of firmness, and the staling rate of the breads was evaluated, measuring the firmness during a storage time, during 96 h (4 days).Staling bread rate was described as a function of the Yg incorporation by a linear Equation (2)3/g), as earlier described [Resulting gluten-free breads were evaluated based on after baking quality parameters, such as moisture, water activity (aw), bake loss (BL), and specific bread volume (SBV) (cmescribed .3/g) was calculated as the ratio between the volume of the bread and its weight. Weight loss during baking (baking loss) was assessed by weighing the bread forms before and after baking. These measurements were carried out in triplicates.Bread moisture was determined according to the standard method ). Water activity (aw) variations were determined at room temperature . Bread volume was measured by the rapeseed displacement standard method AACC 10-05.01, after two hours of bread cooling down. Specific volume  after calibration with a white calibration plate . The data collected from three slices of each bread measured at three different locations of the slices were averaged and expressed using illuminative D65 by L* a* b* scale, where: L* indicates lightness, a* indicates hue on a green (−) to red (+) axis, and b* indicates hue on a blue (−) to yellow (+) axis.Nutritional profile characterization of the gluten-free breads was based on protein ), lipids (NP 4168), ash (AACC Method 08-01.01), carbohydrates , and total minerals contents  as described in a previous study . All theThe experimental data were statistically analyzed by determining the average values and standard deviation, and the significance level was set at 95% for each parameter evaluated. Statistical analysis  was performed by applying variance analysis, the one factor (ANOVA), and post hoc comparisons (Tukey test). Experimental rheology data was fitted to nonlinear Carreau model, using the TA Instruments’ TRIOS software.The effect of the yogurt addition, at different levels, on steady shear behavior of the gluten-free doughs was evaluated, and the flow curves obtained are presented in 2 > 0.967), and the values of the main parameters that characterize the flow behavior are summarized in The experimental data presented in 0), higher dough viscosities were obtained for the control dough (CD) and yoghurt dough at 5% . According to previous works [As it can be observed from the zero-shear rate viscosity values , varying from 290.00 k Pa s (CD) to 9.50 k Pa s (YgD20%), representing a reduction of around 96.60%. One can suggest that the Yg incorporation promoted a dilution effect of starch–cereal protein–xanthan gum interaction density, decreasing the dough viscosity under shear conditions. In addition, the presence of the casein and of the exopolysaccharides (EPS) coming from Yg (produced by lactic acid bacteria) impacted the system, improving the lubrication and flexibility of the dough network. These effects can also be explained by the ability of EPS to bind water and retain moisture, contributing to the increase in the water-holding capacity [Nevertheless, increasing the amounts of Yg resulted in a significant decrease of dough viscosity  and viscous (G´´) moduli, of the gluten-free doughs, with different levels of Yg addition, were evaluated on fermented doughs, by oscillatory frequency sweep measurements.The comparison of the mechanical spectra of control dough (CD) and doughs obtained with Yg additions (YgD) are represented in 5%) were observed, comparing with CD.These results are aligned with those obtained by steady shear flow curves, discussed above, where a significant reduction of dough viscosity was registered from 10% on of Yg addition. Similar results were obtained by other researchers , using dAnalyzing in detail the results  at low f20% (higher level tested). Based on these results, and although the Yg addition promoted significant changes on dough structure, some reinforcement of the molecular linkages on the dough matrix should be considered. This may be explained by the presence of casein and exopolysaccharides coming from Yg that are acting on the system through three probable mechanisms:(1)Improving the orientation and disentanglement of the molecular linkages on the dough matrix, under oscillatory conditions , by lubr(2)Reducing the stiffer network structure formed by xanthan gum, giving more flexibility to the dough network , and(3)Reinforcing the dough molecular bonds, by additional casein and exopolysaccharides interaction, giving more stability to the dough ,17.It can be observed that the frequency values of the G´ crossing over the G´´ were reduced by the Yg addition to dough, varying from 0.025 Hz for CD to 0.004 Hz for YgDThese results showed that the Yg additions promoted considerable changings in viscoelastic properties that resulted in the improvement of the dough network capacity to incorporate and retain gas bubbles produced by yeast´s fermentative activity, consequently, reducing significantly the viscoelastic properties of the dough under oscillatory conditions. This observed behavior resulted in better texture properties and higher specific volumes of the bread .From previous work, it was already stated that interactions between protein and polysaccharides led to similar changes in the viscoelastic functions (G´ and G´´) profile of gluten-free bread doughs . These fThe texture of the GFB produced with different contents of Yg addition was evaluated based on bread crumb firmness by a puncture test, and subsequent bread staling rate obtained from the evolution of the bread firmness, during the storage time of 96 h at room temperature ,23.p ≤ 0.05) positive impact to increase the bread crumb softness: Initial and final values of control bread firmness were higher than all levels of Yg tested, varying from 2.25 Newton  to 6.50 Newton  for CB and 0.82 Newton  to 1.35 Newton  for higher levels of Yg tested on breads (YgB20%), representing a decrease of 64% and 80% of crumb firmness values, respectively.From 2 > 0.974). The linear parameters presented in Bread staling rate was described as a function of time (R20%), representing a reduction of 90%. Similar results were obtained by other authors [The staling rate of the Yg breads were significantly lower than for the (CB), ranging from 0.044 N/h (CB) to 0.005 N/h for higher level of yoghurt tested yoghurt bread , bake loss (%), and specific bread volume (SBV), was evaluated. Results obtained are summarized in Bread crumb color was significantly changed by Yg addition, giving a lighter color  with more pronounced yellow tone  while the red tone became less dominant .20% (higher level tested), corresponding to an increase of 20%. Related to water activity, no significant (p ≥ 0.05) differences were registered for all levels tested, compared to CB.In terms of bread moisture, an increase in moisture content was observed , varying from 42.0 for CB to 50.0 for YgB2 and other volatiles) lost during baking [p ≤ 0.05), corresponding to an increase of 27% on bread yield, comparing CB with YgB20%. Presumably, as discussed above, the dough network formed by the Yg addition, probably via starch molecules, vegetable protein, casein, and EPS (coming from Yg), had a higher ability to trap water and it may cause an increase in water-holding capacity, consequently, a decrease on the BL percentage [The bake loss (BL) represented the amount of water and organic material , a significant improvement was observed by Yg addition from 10% on, varying between 1.80 cmg baking ,41. In ag baking ,39, imprOur results agree with those published by other authors  reportinResuming, it can be stated that the Yg addition improved the bread quality of the gluten-free bread in all baking quality parameters evaluated.Bread crumb texture and volume are considered the most important baking properties by the consumers  and the 2 > 0.9041) between the bread firmness (BF) and specific bread volume (SBV) with steady shear (dough viscosity (DV)) and oscillatory  values of dough rheology were obtained. The linear correlations are illustrated in The results presented along this work suggested a relationship between bread quality parameters and dough rheology properties. Linear correlations  and elastic modulus (G´ at 1 Hz) by the Yg addition to dough resulted in breads with lower values of crumb firmness (B1) and staling rate and with higher specific volumes (B2) than control bread.These linear correlations support the results presented along this work, showing a strong correlation between bread quality parameters and dough rheology properties. It can be stated that the dough system was improved by Yg addition, which resulted in softer breads with better volumes, as aforementioned.These findings disagree with those obtained by other researchers ,17 evaluThese relations between the dough rheology and bread quality can be useful to predict the behavior of the dough and provide important information for the GF bread making industry.p ≤ 0.05) reinforcement on protein and ash content was observed for both levels of Yg tested. However, a remarkable effect was obtained for 20% (w/w) of Yg addition in both cases, representing an increase of 52% and 55%, respectively. Nutritional composition and minerals profile of the gluten-free breads are summarized in The nutritional composition of the gluten-free breads, including the mineral profile, were determined for control bread (CB) and breads obtained with 10% and 20% of Yg addition (YgB). A significant (p ≥ 0.05) differences were obtained. Based on carbohydrates content, a significant decrease for higher levels of Yg tested (20% w/w) was observed, representing a decrease of 25%, due to the dilution effect of the starch content.In terms of lipids, no significant (p ≤ 0.05) of major  and trace minerals  was obtained, representing, in general, more than 15% of the recommended daily values (Regulation (European community)), No. 1924/2006; Directive No. 90/494 (CE). Considering the higher level of Yg tested (YgB20%), a significant increase in Ca (158.0%), K (25.1%), P (7.0%), and Mg (4.1%) can be noticed as well as in Fe (55.3%), Cu (30.4), and Mn (37.3%), compared to control bread (Regarding the minerals profile, a significant improvement (ol bread . SimilarThese results showed that the Yg addition can be used to enhance the nutritional value of the GFB, increasing the amount of protein and minerals profile with a good contribution to reducing the carbohydrates intake.Gluten-free bread formulations, with different levels of yogurt addition, were evaluated using dough rheology measurements and baking quality parameters.The results of the present work showed that the functionality of gluten-free breads, in terms of bread making performances, quality parameters, and nutritional profile can be successfully improved by the addition of fresh yogurt.Although, the yogurt incorporation significantly reduced the rheology properties of doughs, such effects resulted in significant improvements in the overall quality of the corresponding breads. Good linear correlations between bread firmness, specific volume with flow behavior, and viscoelastic functions were found, supporting the results obtained.Resuming, the Yg showed to be a potential ingredient to improve the quality of gluten-free breads, resulting in softer breads with higher volume and lower staling rate, compared to control bread.Related to the nutritional composition, the addition of Yg revealed to be an attractive ingredient to enhance the nutritional value of GF breads, increasing the protein and mineral contents and reducing the carbohydrates intake, with a good contribution to improve the daily diet of the celiac people.


STING1 (TMEM173) gene cause a rare autoinflammatory syndrome, which is known as STING-associated vasculopathy with onset in infancy (SAVI) and which was initially described in 2014 , and the evidence of typical extrapulmonary symptoms of early-onset systemic inflammation  were minimal except for the proband, who was diagnosed with arthritis 8 years after onset. In addition, a younger sibling showed no symptoms. Such reports are rarely related to mutations in STING1. The proband was examined with bronchoscopy and alveolar lavage to determine the cause. This study emphasizes that, in the clinical assessment of interstitial pneumonia in children, the possibility of STING1 mutation should be considered, especially in patients with arthritis in addition.Mutations that affect the  STING1, termed STING-associated vasculopathy with onset in infancy (SAVI), was initially reported. SAVI is characterized by early onset systemic inflammation related to cutaneous vasculitis and tissue damage in addition to interstitial lung disease. Because fewer relevant cases have been reported and the associated phenotype is highly variable, more studies should be conducted to delve into the disease. Furthermore, the phenotype of ILD and rheumatoid factor positive (RF+) polyarticular arthritis is reported even less frequently. Accordingly, this study reports three familial cases of this new phenotype, which is expected to expand the spectrum of this disease.In childhood, ILD refers to a heterogeneous group of rare pulmonary conditions with complex etiologies. Some gene mutations have been found to be related to systemic autoinflammatory diseases that cause ILD. In 2014, a novel autoinflammatory syndrome, a novel type I interferonopathy attributed to mutations in The study was approved by the institutional review board of the Children's Hospital of Hebei Province, and informed consent was obtained. Consent for minors was signed by their parents.The index case is a boy, age 9 when enrolled, who presented with a persistent cough after physical activity over the course of more than 8 years and joint pain for 4 months. He was the first full-term child born via vaginal delivery of unrelated parents of Chinese ethnic background with a body weight of 4.6 kg at birth. Shortly after birth, the patient showed fatigability after light activity and activity-induced tachypnea and cough. A chest X-ray and lung computed tomography (CT) showed bronchial pneumonia with interstitial changes, and percutaneous oxygen saturation was 95% in room air. Severe febrile attacks were rare, and the patient did not seek treatment regularly. At the time of enrollment, the patient weighed 32 kg and was experiencing no failure to thrive (FTT), no obvious abnormality in growth and development, and no tissue lesions involving the skin.Four months before admission, the patient developed migratory polyarthralgia, affecting the bilateral fingers and toes, wrists, knees, and other joints with swelling at the proximal joints of the middle finger. After the administration of the musk rheumatism capsule , the patient's symptoms were alleviated. However, the pain worsened 3 days before admission. Laboratory tests showed a rheumatoid factor (RF) of 47.9 IU/ml  and erythrocyte sedimentation rate (ESR) of 75 mm/h (prior to admission).The proband's only sibling is a 4 year-old boy, who is asymptomatic; however, CT of the chest showed interstitial changes.The proband's father presented with shortness of breath post-exercise at the age of 18 after an episode of illness. Chest radiographs and CT showed persistent pathological changes of interstitial pneumonia. At the time of enrollment, the patient's percutaneous oxygen saturation was 93% in room air.None of the patients needed supplemental oxygen or experienced tachypnea in daily life, but the father experienced mild dyspnea during heavy physical activity.The proband's mother is healthy. The proband's father has a sister from the same parents who has no similar symptoms. The proband's grandmother and grandfather were both healthy.The proband and his father and brother underwent chest X-ray or CT. In addition, the proband's pulmonary function and indicators of systemic inflammation were examined.Targeted exome sequencing (TES) was performed on DNA from peripheral blood cells. Genomic DNA was fragmented, ligated with paired-end adaptors, amplified, and purified. A total of 6,110 gene exons and their 50 bp adjacent introns were captured by a TES Kit . A DNA library was established by postcapture amplification and purification and then sequenced on Illumina HiSeq X Ten . NextGene V2.3.4 software  was used for sequencing data alignment to the human genome reference (hg19) and variant calling. The mean read depth was 134.01 ×, but reads reached up to 200 × for 96.194% of the target sequences. Meanwhile, annotation information, including conserved nucleotides and amino acids; prediction of biological functions; frequency in healthy population ; and data from HGMD, Clinvar, and OMIM, was added in NextGene V2.3.4, using self-constructed scripts in our lab.STING1 gene analysis. Potentially pathogenic mutations were verified using Sanger sequencing with forward primer 5′-GGACTCTATCGTTACAGGCTGAGG-3′, reverse primer 5′-GCTCCATAGCCCCTTCTGACTCT-3′, and a product length of 414 bp.Variants of pathogenicity were chosen according to standards and guidelines for the interpretation of sequence variants published by ACMG in 2015 with HGVS nomenclature. We used NM_198282.3 as the reference sequence for After birth, chest X-ray or CT of the proband showed progressive pulmonary interstitial fibrosis. In addition, there were multiple cystic changes and emphysema in the lobular center of the lung .Pulmonary function tests indicated mixed ventilation dysfunction . Broncho*10∧9/L LY4.01*10∧9/L). During hospitalization, the child developed fever, and the blood routine was reviewed; it showed WBC 12.3*10∧9/L NE 5.79*10∧9/L LY 5.83*10∧9/L systemic inflammation with elevations of C-reactive protein (CRP), high-sensitivity CRP (hs-CRP), and ESR levels . Blood CD4+ T was normal. CD8+ T was 38.4% . The ratio of CD4+ to CD8+ cells in the lymphocytes was normal. Total B cells (CD19+) were 18.6% , which was slightly elevated, and natural killer (NK 1.7%) lymphocytes decreased. Hypergammaglobulinemia of IgA and normal IgM and IgG were detected. All autoantibodies were negative . Indicators of vasculitis  were all negative except for p-ANCA. Anticyclic citrullinated peptide antibody was 10.33 RU/ml  . Liver, renal, and thyroid functions were normal.Chest imaging of the proband's brother and father showed interstitial changes , 7.STING1 (NM_198282.3), e.g., the gene that encodes the stimulator of interferon genes, consistent with SAVI.Whole genome sequencing of samples from the index patient was performed, and genetic analysis by Sanger sequencing confirmed a heterozygous mutation (c.842G>A p.Arg281Gln substitution of arginine to glutamine) in the exon 7 of Mutation analysis revealed that the father and younger brother of the proband both carry the same p.Arg281Gln variant , and hisThe proband's aunt, grandmother, and grandfather are healthy, and no DNA samples were available.The diagnosis of SAVI was made due to the presence of interstitial lung disease, systemic inflammation, and genetic analysis results.After 13 days of non-glucocorticoid therapy for phlegm (acetylcysteine was used for atomization) and 7 days of anti-infection (cefathiamidine) treatment—on the seventh day of hospitalization the patient experienced fever and increased leukocytes in the blood—the patient's symptoms improved, but mild shortness of breath was still present when physically active. However, no oxygen therapy was required.The proband's younger brother showed no signs of discomfort, but their father could not tolerate heavy physical activity; none of them received treatment.So far, we have followed the family for more than 3 years. There was no significant change in the proband and his brother with no signs of hypoxia in their normal activity, but his father died of sudden respiratory failure in the third year, which was unanticipated.STING1 and other novel genes, such as COPA, MIM612374, or MIM601924, have been shown to cause systemic autoinflammatory diseases involving the lungs.Pediatric ILD is a heterogeneous group of rare pulmonary diseases presenting with chronic respiratory pathologies, including inflammatory and fibrotic changes. ILD detected in infancy is often related to genetic mutations . At presSTING1 encodes the STING protein, which is an adaptor molecule linking the sensing of foreign DNA  to the production of type 1 IFNs. Its mutations are responsible for type-I IFN overproduction, which has been recently identified as a new cause for interferonopathy, such as SAVI. SAVI is characterized by systemic inflammation  associated with cutaneous vasculitis and tissue damage in addition to interstitial lung disease. To date, 13 identified mutations in the STING1 gene  (STING1 point mutation (c.842G>A p.Arg281Gln) in Chinese patients.STING1 that led to a constitutive production of high levels of type I IFNs without infectious triggers  mutation as previously reported by Melki in a 7 year-old, white, European ethnicity girl in 2016. The authors used a luciferase-based IFN-β transcription reporter assay and detected robust IFN-β activation Common laboratory features of SAVI include raised IgG and IgA and positive autoantibodies—not all of which were observed in our patients. In the index case, IgA was raised, but IgM and IgG were normal, and autoantibodies were negative. SAVI is characterized by systemic inflammation with elevations of CRP, hs-CRP, and ESR levels, which were also characteristic of our patients.As one of the type I interferonopathies, SAVI has clinical manifestations that are variable even within familial cluster. A literature review reveals that the age of clinical presentation is typically in infancy between birth and 6 months (17/26) . ClinicaSTING1 gene may result in a decrease in the expression or function of the haplotype, resulting in a mild clinical phenotype, we further checked the exome sequencing of variants INFAR1 and 2, JAK-STAT, TBK1, and IRF3 to see if the variants were affecting the pathway, and the results were negative.However, genotype–phenotype correlations may vary. Compared with this disease as previously reported and the same mutation reported by Melki et al. , the symSTING1. They found that these JIA patients had a relatively frequent incidence of autoinflammatory syndromes  who was treated with another Janus kinase inhibitor, tofacitinib, and the patients' skin lesions improved but the pulmonary defects remained unchanged  are not available.Written informed consent was obtained from the individual(s) and/or minor(s)′ legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.JL wrote the manuscript under the direction of SA and ZD and his classmate modified the language. We thank them for their efforts. All the authors designed the study and analyzed experimental results together.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Evans’ syndrome (ES) is defined as the concomitant or sequential association of warm auto-immune haemolytic anaemia (AIHA) with immune thrombocytopenia (ITP), and less frequently autoimmune neutropenia. ES is a rare situation that represents up to 7% of AIHA and around 2% of ITP. When AIHA and ITP occurred concomitantly, the diagnosis procedure must rule out differential diagnoses such as thrombotic microangiopathies, anaemia due to bleedings complicating ITP, vitamin deficiencies, myelodysplastic syndromes, paroxysmal nocturnal haemoglobinuria, or specific conditions like HELLP when occurring during pregnancy. As for isolated auto-immune cytopenia (AIC), the determination of the primary or secondary nature of ES is important. Indeed, the association of ES with other diseases such as haematological malignancies, systemic lupus erythematosus, infections, or primary immune deficiencies can interfere with its management or alter its prognosis. Due to the rarity of the disease, the treatment of ES is mostly extrapolated from what is recommended for isolated AIC and mostly relies on corticosteroids, rituximab, splenectomy, and supportive therapies. The place for thrombopoietin receptor agonists, erythropoietin, immunosuppressants, haematopoietic cell transplantation, and thromboprophylaxis is also discussed in this review. Despite continuous progress in the management of AIC and a gradual increase in ES survival, the mortality due to ES remains higher than the ones of isolated AIC, supporting the need for an improvement in ES management. Evans’ syndrome (ES) was first described by Evans in 1951 . AutoimmThis review will focus on ES in adults. However, as clinicians managing adults will have to take care of patients diagnosed with ES during infancy, a specific paragraph is dedicated to ES in paediatrics. Moreover, ES in children can be part of a more complex clinical situation due to primary immunodeficiencies (PID) that will be discussed, as diagnosis of such syndromes might sometimes be suspected in adults.Moreover, it must be specified that due to the rarity of the disease, there is almost no clinical trials comparing treatment modalities and that most of the recommendations that are given here are extrapolated from those of isolated ITP or isolated AIHA.The knowledge about ES epidemiology in adults has been refined by a recent nationwide study in Denmark reporting on 242 patients managed for the last 40 years (1977–2017). The rarity of the disease was confirmed by an annual incidence of 1.8/million person-years, and an annual prevalence of 21.3/million persons ,6.In adults, autoimmune cytopenia (AIC) arise concomitantly in 30–57%, while ITP precedes AIHA in 27–44% [ES is often diagnosed during the 5th–6th decades  associated with reticulocytosis and with markers of haemolysis, i.e., elevated lactate dehydrogenase, low haptoglobin and elevated indirect bilirubin, with a positive direct antiglobulin test (DAT) for IgG with or without complement (C3d) as cold agglutinins are excluded from ES .ITP remains a diagnosis of exclusion suspected in case of rapid onset thrombocytopenia not related to liver diseases , splenomegaly , drug-related thrombocytopenia, bone marrow deficiency  or inherited thrombocytopenia . Due to AIN is suspected when facing a neutrophil count <1.5 G/L, after exclusion of other causes of neutropenia , Epstein Barr Virus (EBV), Human Immunodeficiency Virus (HIV), parvovirus B19, and influenza; myelodysplastic syndrome or leukaemia) as there is no specific test for its diagnosis . AntineuTo avoid difficulties in the interpretation of biological tests, it must be kept in mind that some of these investigations must be performed before treating patients. Notably, intravenous immunoglobulins (IVIg) preclude the correct quantification of serum IgG, and immunosuppressants interfere with T and B cell phenotyping .Due to the increased mortality in secondary ES compared to primary ES, diseases that are associated in up to 50% of ES in adults must be clearly identified at diagnosis .ES has been reported to be associated with non-Hodgkin lymphoma (NHL) in 7% of a cohort of 68 adult ES, thus representing 15% of secondary ES , which iBased on a prospective population-based registry of our area department, we confirmed that the frequency of ES associated with lymphoproliferative malignancies was low, representing 10 patients among a cohort of 3499 patients (0.29%) managed from 1995 to 2015. The frequency of ES depends on the underlying malignancies, occurring in 0.44% (4/911) of CLL, 0.43% (1/231) of T cell lymphoma, 0.24% (5/2078) of B cell lymphoma, while not observed among the 279 patients with Hodgkin lymphoma. Thus, the standardized incidence rate was 0.037/100,000 person-years for CLL, 0.041/100,000 person-years for B cell lymphoma and 0.007/100,000 person-years for T cell lymphoma. To characterize the clinical presentation of ES associated with lymphoproliferative malignancies, the 10 patients identified in the registry were gathered with 7 other patients managed in our institution during the same period, identified by the diagnosis-related group medical information system. The median age at diagnosis was 62  with 76% of male (13/17). The most frequent malignancies were CLL , marginal zone B cell lymphoma , or another low-grade B cell lymphoma . AIC occurred after the diagnosis of the lymphoproliferative malignancy by a median of 5 years (IQR:0.3–10 years) in 65% of the cases (11/17), occurred synchronously in 29% cases (5/17), while AIC preceded the diagnosis of the malignant hemopathy in only one case (6%). ES-AIC appeared simultaneously in most of the cases , while ES-thrombocytopenia preceded ES-anaemia in 29% of the cases (5/17), by a median of 0.5 year (IQR: 0.1–13.4). The treatment of the haematological malignancies was required in most of the cases , associated with a specific treatment of ES-thrombocytopenia in 88% (15/17) or ES-anaemia in 94% (16/17). A response was achieved in 70.5% for ES-thrombocytopenia (12/17) and 76.4% for ES-anaemia (13/17) after a median follow-up of 4.7 years (IQR: 0.4–27.9). After 5 years follow-up, 35% (6/17) of the patients had died. The death was related to the malignant hemopathy or its treatment in 4 cases , while related to ES in 1 case  and of unknow origin for the remaining. At the time of death, 5 patients were under treatment for their malignant hemopathy and the remaining had relapsed. Concerning ES, 3 patients were responders for both anaemia and thrombocytopenia, 2 were responders for anaemia but not for thrombocytopenia and 1 was a non-responder for both.In the diagnosis procedure of ES , serum pAID have been reported in 18.2% of an adult ES cohort . SystemiThus, at the diagnosis of ES, it is recommended to measure antinuclear antibody to not undermine SLE (anti-dsDNA antibodies) or Sjögren disease (anti-SSA or SSB antibodies), while lupus anticoagulant assay and antiphospholipid antibodies should be considered in case of a previous history of thrombosis or obstetrical morbidity suggesting antiphospholipid syndrome . In caseFew papers reported on the association of ES with various viral infections (hepatitis C virus (HCV), Epstein Barr Virus (EBV), cytomegalovirus (CMV), and Varicella Zoster Virus (VZV)) that should be ruled out even though this situation seems rare. More recently SARS-CoV-2 has been reported as a potential cause of ES .Due to their potential association with ES, specific viral serology testing and/or blood PCR for EBV and CMV should be considered when clinical exam is suggestive or in case of atypical lymphocytosis on the blood smear. Moreover, testing for Human Immunodeficiency Virus (HIV), HCV, and hepatitis B virus (HBV) must be performed to prevent reactivation or uncontrolled progression of infection upon immunosuppressive therapy .Even though PID is most often diagnosed in children with ES, it should also be considered in adults when the clinical presentation is suggestive . Due toOne publication reported on the association of ES and pregnancy based on a systematic review of the literature, including 8 papers, consistent with 10 patients and 11 pregnancies . ES is rThrombotic microangiopathies (TMA) represent rare diseases with a devastating prognosis without treatment . TMA areEven though severe bleedings are rare during isolated ITP, they can lead to gastro-intestinal bleedings responsible for anaemia. In these cases, a careful examination of the full blood count will rule out ES as anaemia is normocytic and non-regenerative in case of acute bleeding or microcytic in case of chronic bleedings. Moreover, haemolytic parameters , if measured, will be in normal ranges, with a negative DAT.Anaemia due to vitamin B12 deficiency is often associated with intramedullary haemolysis responsible for high level of LDH and low haptoglobin and sometimes the presence of numerous schistocytes on the blood smear, and can also be associated with thrombocytopenia . The nonMyelodysplastic syndromes (MDS) are responsible for cytopenia that can affect multiple lineages, most particularly RBC and platelets . ES is eParoxysmal Nocturnal Haemoglobinuria (PNH) is a rare, acquired bone marrow failure due to somatic mutation in phosphatidylinositol glycan class A (PIGA), one of the genes involved in glycosylphosphatidylinositol (GPI) anchor biosynthesis. The complement inhibitors CD55 and CD59 are both GPI-anchored proteins that are deficient during PNH, thus leading to complement activation on the surface of RBC and to their haemolysis. Because PNH can be associated with aplastic anaemia, patients can also present with other cytopenia that can mislead to the diagnosis of ES. However, during PNH, TDA is negative and when aplastic anaemia is associated, the reticulocyte count is lower than what is observed during isolated AIHA. The diagnosis of PNH is confirmed by flow cytometry using a specific reagent  that binds to GPI and allows the determination of the deficit. Bone marrow aspiration and biopsy are of interest and show hypoplastic bone marrow in case of associated-aplastic anaemia . CorticoDue to the rarity of ES, no clear therapeutic regimen has been established. However, treatments are mostly extrapolated from those commonly used for isolated ITP and isolated AIHA, and are summarized in Corticosteroids represent the cornerstone therapy, used at a daily dose of 1 mg/kg of prednisone. The duration of treatment is determined by the AIC: 3–4 weeks with a brutal discontinuation or a rapid tapering over one week for ES-thrombocytopenia  and a slInitial response rates are as high as 80% but the one-year-remission rate after corticosteroid as monotherapy is low for isolated ITP (20–30%) and for isolated AIHA (33%) ,32. ImpoDexamethasone (40 mg/day for 4 days) has been used for isolated ITP, leading to a faster response but a similar long-term response compared to prednisone ,28. No dConcerning ITP, IVIg should be restricted to patients with low platelet count <30 G/L) associated with important bleeding symptoms, best assessed by using a bleeding score . IVIg ar G/L assoOnly one study reported on IVIg during isolated AIHA and showed a low efficiency (12/37 patients (32%) increased their haemoglobin ≥2 g/dL, and only 15% achieved a partial response defined by haemoglobin ≥10 g/dL with an increase of haemoglobin ≥2 g/dL) , which pIn case of symptomatic anaemia, RBC transfusions are required. The challenge during isolated AIHA and ES-anaemia is to not dismiss alloantibodies that can be masked by autoantibodies, notably in patients who have previously been transfused or women who have been pregnant. As autoantibodies usually lead to panagglutination of RBC by targeting antigens widely expressed on RBC such as glycophorin, protein band 3, and rhesus, specific techniques are needed to unmask potential alloantibodies . These tPlatelet transfusion is not recommended during isolated ITP and by extension during ES-thrombocytopenia, due to the short half-life of platelets after transfusion and the fact that it does not improve outcome in most of the patients . HoweverRituximab is a drug of choice in ES as its efficacy has been clearly demonstrated in both isolated AIHA, with response rate of 75% at 1 year follow up ,32 and iData regarding the use of rituximab in secondary ES are scarce. One study specifically assessed rituximab in SLE-associated AIC in 71 patients, among which 11 had ES . An overConcerning haematological malignancies-associated ES treated with rituximab, there is only one study that reported specifically on CLL . Among tSplenectomy is an efficient treatment of both isolated ITP and isolated AIHA leading to response rates of 88% (66% complete response)  and 70% Data concerning splenectomy in ES are derived from small series showing response rates that are quite similar than those observed in isolated AIC with an initial response rate of 78–85%, with long-term response ranging between 42–62% ,66. SpleAs in isolated AIC, various immunosuppressants have been used in ES, mostly before the availability of rituximab. Cyclophosphamide, azathioprine, ciclosporin, or mycophenolate have been used, usually in association with corticosteroids, and allowed a response in 50–100% . NowadayIn the coming years, the use of immunosuppressants will probably decrease due to novel therapies that should be efficient in both isolated AIHA and isolated ITP, and by extension in ES, such as fostamatinib (a syk inhibitor that blocked phagocytosis), inhibitors of the neonatal Fc receptor, or inhibitors of the classical complement pathway ,69.High dose chemotherapy followed by hematopoietic stem cell transplantation (HSCT) is rarely performed for AIC compared to other AID and is restricted to patients who are refractory to multiple lines of treatments. The results of a study assessing the tolerance and efficacy of autologous lymphocyte-depleted peripheral blood stem cell transplantation conducted by the National Institute of Health (NIH) including 14 patients (9 ITP and 5 ES) will not be detailed here as only 2 adult patients with ES were treated . BrieflyData from the European Group of Blood and Marrow Transplantation (EBMT) that concerned 36 patients  with 38 transplant procedures were first reported in 2004 . AutologIn this cohort, 7 patients had ES: 2 received an autologous transplantation leading to prolonged response in one patient and transient response for the other, while 5 patients had allogeneic transplantation leading to prolonged remission in only one case, 2 being not evaluable and 2 dying from complications related to transplantation or disease progression.Data from the EBMT were updated in 2008, with 65 transplantations  performed in 59 patients. Considering all AIC, the 5-year survival was 79 +/− 14% for autologous and 58 +/− 25% for allogeneic transplantation.Twelve patients with ES were treated, 4 with autologous and 8 with allogeneic transplant, but their specific evolution and follow-up are not provided in the article .Thus, considering that a sustained response could be achieved in only up to 25% of the patients, in our opinion, HSCT should be restricted to patients who are refractory to multiple line therapies and those that could not participate in clinical trials assessing new innovating drugs.The efficiency of TPO-RA in ES is only supported by case reports ,62. TPO-It has recently been shown in a multicentric retrospective international study including 51 patients with isolated AIHA that erythropoietin (EPO) could be of interest . Most ofIt is now clearly established that isolated AIHA enhances the risk of thrombosis ,57,73, mIn our opinion, this approach could be extrapolated to ES, except in case of profound thrombocytopenia. Thus, we recommend prophylactic anticoagulation by low molecular weight heparin for ES patients that are hospitalized or with one general VTE risk factors such as age above 70, past history of VTE, reduced mobility, active cancer, known thrombophilia, recent surgery or trauma, acute infection, heart or respiratory failure, and who have an active ES-anaemia with a platelet count above 50 G/L.The management of ES during pregnancy is challenging as most of the drugs that are usually used in ES are not recommended, notably rituximab and TPO-RA, even though few reports showed favourable outcomes ,75.During pregnancy, corticosteroids remain the cornerstone therapy due to their high efficiency and short delay of action.IVIg are also useful to treat ES-thrombocytopenia but are not recommended for ES-anaemia.Azathioprine can be efficient on both ES-thrombocytopenia and ES-anaemia and could be maintained in case of ES prior to pregnancy. However, due to its long delay of action, azathioprine is of poor interest in case of ES emerging during pregnancy.Splenectomy can be efficient on both ES-anaemia and ES-thrombocytopenia but is challenging during pregnancy and should be performed during the second trimester when needed.The median survival of patients with ES has increased over time and is around 7 years. Importantly, the survival is poorer in secondary ES compared to primary ES (1.7 vs. 10.9 years) , with a The causes of death are bleedings, with a similar frequency than observed in isolated ITP on the same period, and haematological neoplasms, notably for secondary ES .An increase in mortality was also observed in a large Italian cohort of AIHA with an adjusted hazard ratio of death of 6.8 (95%CI: 1.99–23.63) for ES compared to isolated AIHA [Although life-threatening complications are rare during ES, they need prompt recognition and management. ES-thrombocytopenia responsible for life-threatening haemorrhage, i.e., intracranial, visceral haemorrhage, or gastrointestinal bleedings responsible for profound anaemia should be managed as recommended in isolated ITP, although these recommendations are based on expert committee reports, opinions, or clinical experiences (grade C) .High dose of corticosteroids (daily pulses of methylprednisolone at a maximum dosage of 15 mg/kg per day for 3 days and no more than 1g/day) are used in association with IVIg (1 g/kg/day during two consecutive days) .In order to obtain a rapid haemostasis, platelet transfusions are recommended in situation of severe uncontrolled bleeding and can be repeated every 8 h until bleeding resolved, and immunomodulatory drugs are efficient. In a retrospective review of 40 patients treated with IVIg and platelet transfusions, with a mean pre-treatment platelet count of 10 G/L, an increase to 55 G/L and 69 G/L was observed at day 1 and 2, respectively . After 12 vincristine) are also recommended. In a prospective study enrolling 35 patients with isolated ITP who were not responders to corticosteroids and IVIg, the combination treatment of pulses of high dose methylprednisolone with IVIg and vinca alkaloids showed a response in 71% [Weekly infusions of vinca alkaloids  added to corticosteroids and IVIg was of interest to improve the response rate . Among tDespite its long delay of action (3 to 4 weeks), early administration of rituximab must also be considered in life-threatening situations in order to shorten the critical condition .Rarely, emergency splenectomy should be considered with a response usually rapidly observed during the days following surgery .Data concerning the management of life-threatening haemolysis are scarce, particularly in ES, and the recommendations are based on isolated AIHA.Due to the delay of effectiveness of immunomodulatory drugs  used to manage isolated AIHA and by extension ES anaemia, RBC transfusions remain essential to avoid severe hypoxemia. As alloantibodies can account for up to 30% of patients with AIHA previously transfused or who had a pregnancy , in emerHigh doses of methylprednisolone are recommended as previously mentioned for ITP, based on expert recommendations .Rituximab should also be performed as early as possible to shorten the life-threatening period.Despite few data concerning the efficiency of IVIg in isolated AIHA, with a response observed in one-third of patients , it can To remove pathogenic autoantibodies, plasmapheresis has been used but with contradictory results in severe isolated AIHA. A review reported on plasmapheresis as adjunctive therapy with a response observed in 4/6 of isolated AIHA and 4/4 of ES . A monocInhibition of the classical complement pathway represents a new therapeutic approach in AIC. The efficacy of eculizumab, a C5b inhibitor, has been reported in few case reports of severe isolated AIHA ,81 and cIn children, ES is also a rare situation, as reported in a recent Danish nationwide population-based study that identified 159 AIC among which 21 were ES, between 1981 and 2015 in children less than 13 years of age . The incIn this study, the mean age at diagnosis was 4.7 years 3.3–6), which is close to the one reported in the largest cohort of 156 paediatrics ES (<18 year-old) prospectively included since 2004 in 26 French centres with a mean age at diagnosis of 5.4 years (0.2–17.2) . Contrar.3–6, whiThe frequency of secondary ES is difficult to determine in children. In the French cohort, only 30% of ES were considered as primary and only 8% were associated with SLE . Of noteThus, contrary to what is observed in adults, a specific genetic background of PID should be considered in the majority of ES in children (detailed in paragraph 3.2.2.). Moreover, the early-onset of SLE also raises the question of interferonopathies, diseases that are due to type-I interferon dysregulation and that mimic SLE . To daten = 13, 8.3%) or to ALPS .Based on the data of the French cohort including 156 children with ES, after a mean follow-up of 6.5 years (0.1–28.8), 30% were considered as primary and 10% were proven to be secondary, i.e., due to SLE (n = 52), most of them having a pathogenic mutation in genes known to be involved in PID (n = 32) [FAS mutation in ALPS, CTLA4 and LRBA deficiency) will be detailed below. Other mutations were less frequent and involved STAT3  [PIK3CD  [CBL and KRAS (that are implicated in RAS-associated autoimmune leukoproliferative disorder (RALD) responsible for splenomegaly, AIC, monocytosis, pericarditis and skin manifestations) [RAG1  [ADAR1  [TNFR2, IFNAR1, TGFBR2), for molecules involved in intracellular signalling , for proteins involved in apoptosis pathways , or for transcriptional factors  [Thus, 60% of ES were considered to be associated to a still undefined-genetic background due to their association with other immune abnormalities such as polyclonal lymphoproliferation, hypogammaglobulinemia, or other AID . This wa(n = 32) . The PIDfailure) ,86, PIK3 levels) , CBL andtations) ,89, RAG1sorders) ,91, and fection) . For 20  NFATC1) . Importa NFATC1) .CVID is the most frequent PID characterized by a deficit in the humoral immune response responsible for a low level of immunoglobulins, most particularly IgG and IgA. CVID patients have recurrent bacterial infections, mostly of the ears, nose, throat, and respiratory systems. They can also display lymphoproliferation (either benign or lymphoma) and are more prone to AIC. In an American registry involving 990 CVID patients, AIC accounted for 10.2%, with ES being diagnosed in 1.6%. Importantly, AIC were associated with non-infectious CVID complications such as interstitial lung disease, enteropathy, hepatic disease, lymphoproliferation, and granulomatous disease that are known to worsen the prognosis . Infecti+TCRαβ+CD4−CD8−) that are increased, and to the measurement of soluble FasL, IL-10 and IL-18 that can be increased. ALPS is due to various mutations of the FAS/FAS ligand pathway , FASLG, and CASP10 (caspase 10)) leading to inefficient FAS-mediated apoptosis of T cells responsible for the survival of activated T cells and the emergence of autoreactive T cells. When performed, lymph node biopsy shows reactive follicles. The diagnosis criteria were refined in 2010 and are based on the associations of the two required criteria  with at least one accessory criterion, among primary criteria  and secondary criteria (1. Elevated plasma sFASL levels (>200 pg/mL) or elevated plasma interleukin-10 (>20 pg/mL) or elevated serum or plasma vitamin B12 levels (>1500 ng/L) or elevated plasma interleukin-18 levels (>500 pg/mL), 2. Typical immunohistological findings as reviewed by an experienced haematopathologist, 3. AIC  and elevated IgG levels  [CASP8  or NRAS  [ALPS is a rare disease generally revealed during the first decade, associating lymphoproliferation , AID, mostly AIC, and is associated with an increased risk of malignancies . Biologimmunity) . Similare, RALD) . ALPS pae, RALD) .n = 5) or associated with ALPS (n = 11), mycophenolate mofetil led to a high complete response rate that was similar in ALPS-associated ES (82%) and primary ES (80%) [Treatment of ALPS is not always required, and a wait-and-watch strategy can be applied. When needed, corticosteroids could be used. AIC during ALPS are often refractory and require second line therapies . In a reES (80%) . SimilarES (80%) . ImportaES (80%) .CTLA-4 is expressed on activated T cells and competes with the activating receptor CD28 for binding to the costimulatory molecules CD80 and CD86 expressed on antigen-presenting cells, thus leading to the inhibition of T cells. LRBA is a molecule involved in the intracellular recycling of CTLA-4, its deficiency thus decreasing the expression of CTLA-4. Deficiencies of CTLA-4 or LRBA lead to a quite similar phenotype associating lymphoproliferation, lymphoid infiltration of various organs , AID, notably AIC, predisposition to infections due to hypogammaglobulinemia and to lymphoma ,98. DespOf the 156 children of the French cohort, 69% required at least one second line treatment, and almost half needed more than one second line therapy. First line therapies relied on steroids and IVIg for ES thrombocytopenia, and steroids for ES anaemia. The main second line therapies were rituximab (31%), azathioprine (15%), splenectomy (12%), cyclosporin (9%), and mycophenolate mofetil (2.5%). At the last follow-up, 74% of the children had a complete response, while 10% died from infections (70%), that were favoured by ES treatments, while death was related to ES, most particularly bleeding due to uncontrolled thrombocytopenia for the others (30%) . This unES is a rare combination of AIHA and ITP that is associated in 50% of adult cases with various diseases such as SLE, haematological malignancies, or PID, the latter being the most frequent in children. Its prognosis is poorer than the one of isolated AIC and is particularly worse when associated with haematological malignancies. Its management is mostly empirical and extrapolated from guidelines for both isolated AIHA and isolated ITP. Corticosteroids remain the first line therapy with a short course duration for ES-thrombocytopenia and of six months for ES-anaemia. Second line treatments are usually required and the ones that are efficient in both isolated AIHA and isolated ITP such as rituximab, immunosuppressants, and splenectomy are recommended. In specific situations such as ALPS, mycophenolate mofetil or sirolimus should be preferred. Treatments that could be required for managing isolated ITP and that are associated with an increased risk of thrombosis such as IVIg and TPO-RA should be used with caution in ES as ES-anaemia probably increases the risk of thrombosis, as observed in isolated AIHA.


How auxin transport regulates organ abscission is a long-standing and intriguing question. Polar auxin transport across the abscission zone (AZ) plays a more important role in the regulation of abscission than a local concentration of this hormone. We recently reported the existence of a spatiotemporal sequential pattern of the indole-3-acetic acid (IAA) localization in the area of the yellow lupine AZ, which is a place of flower detachment. In this study, we performed analyses of AZ following treatment with an inhibitor of polar auxin transport ). Once we applied TIBA directly onto the AZ, we observed a strong response as demonstrated by enhanced flower abscission. To elucidate the molecular events caused by the inhibition of auxin movement, we divided the AZ into the distal and proximal part. TIBA triggered the formation of the IAA gradient between these two parts. The AZ-marker genes, which encode the downstream molecular components of the inflorescence deficient in abscission (IDA)-signaling system executing the abscission, were expressed in the distal part. The accumulation of IAA in the proximal area accelerated the biosynthesis of abscisic acid and ethylene (stimulators of flower separation), which was also reflected at the transcriptional level. Accumulated IAA up-regulated reactive oxygen species (ROS) detoxification mechanisms. Collectively, we provide new information regarding auxin-regulated processes operating in specific areas of the AZ. Abscission is a developmentally programmed phenomenon that facilitates the shedding of damaged, senescent, or unnecessary organs, such as leaves, petals, or in some cases, infected parts of a plant. Under certain circumstances, e.g., unfavorable environmental or endogenous conditions, such as drought or nutritional limitations, even necessary organs are shed. Importantly, in a similar way, whole flowers or young fruit detachment may occur. A serious consequence of this is reduced yielding in many crop species . RegaArabidopsis thaliana 1) transcription factor, which is a repressor of KNAT2 and KNAT6 expression. KNAT2/KNAT6 mediates the activation of genes involved in the dissolution of pectin-rich middle lamellae, and consequently in the disintegration of AZ tissues [In recent years, our knowledge about separation processes has been improved mainly due to advances in genetic, molecular, and biochemical methods. Next to hormones, many additional molecules have been identified as playing a crucial role in abscission-accompanying events. Among them, a small signaling peptide (inflorescence deficient in abscission (IDA)) and its receptor HAESA/HAESA-like function in a genetic pathway that also involves MAP kinases, which belong to the most important plant-signaling networks involved in response to changing environmental conditions. MPK-mediated protein phosphorylation determines their activity, stability, and localization, which in turn modulates gene expression and triggers phytohormone pathways . It has  tissues ,8,9.2−), hydrogen peroxide (H2O2), hydroxyl radical (OH−), and singlet oxygen (1O2), are important signaling messengers produced in the plant cell wall to modify its components via processes of stiffening/softening. It has been established that exogenous ROS can induce organ abscission, while the application of antioxidants and ROS scavengers inhibits this process [Reactive oxygen species (ROS), e.g., superoxide anion . SODs f process . Lupinus luteus L.) is an excellent model for studying abscission-related changes. It is an agronomically important species that is cultivated for food and feed in many regions of the globe [Yellow lupine . Morblade-on-petiole (LlBOP), inflorescence deficient in abscission-like (LlIDL), receptor-like protein kinase HSL (LlHSL), and mitogen-activated protein kinase 6 (LlMPK6) transcripts [LlBOP and LlIDL expressions are regulated by ET and ABA which are strong stimulators of flower detachment [zeaxanthin epoxidase (LlZEP) gene in ABA biosynthesis and aminocyclopropane-1-carboxylic acid synthase (LlACS) and aminocyclopropane-1-carboxylic acid oxidase (LlACO) genes in ET biosynthesis [2O2), the CAT activity, and the localization significantly changed, which indicated modifications in the redox balance [We have already indicated that the flower separation process in lupine is associated with a substantial accumulation of nscripts ,21,28,29ynthesis ,23. The ynthesis . Analyseynthesis ,29. FollSummarizing, a multilevel and complex mechanism of phytohormonal networking is active in the regulation of organ abscission events. However, the mechanisms governing specific differences between distal and proximal AZ areas are not yet clear. Although the involvement of auxin in abscission-related events has been analyzed for the last few decades, still little is known about the molecular, biochemical, and cellular changes caused by the disturbance of polar auxin transport (PAT), which is a factor responsible for the organ maintenance of the plant. For this study, we decided to continue our previous considerations related to auxin balance in the AZ area and answer another scientific question. Using an inhibitor of PAT, namely 2,3,5-triiodobenzoic acid (TIBA), we analyzed the molecular events related to flower abscission that were evoked by the disruption of auxin movement through the AZ. Importantly, we described changes related to TIBA-induced abscission in the different areas of the flower AZ. The present paper provides completely novel information about tissue-specific changes within the AZ, which is a tiny but extremely significant fragment of a plant.L. luteus [Based on previous reports, it is known that it is not the total IAA content, but rather the maintenance of a proper ratio of this phytohormone between different areas of the AZ that is the most relevant factor determining the time of abscission ,32. Our . luteus . In thisFor further examination of the TIBA action, we divided the whole AZ area into two parts: distal  and proximal , as illustrated in LlBOP, LlIDL, LlHSL, and LlMPK6 are those of major importance [LlBOP showed an opposite pattern of expression in both regions in the presence of TIBA (LlBOP remained unchanged in both parts of the non-treated AZ (0 h)  and the proximal region (6 h) in response to the TIBA treatment , and then it dropped (6 h). In the proximal part, it was negatively regulated over time  A. The saIDL mRNA B. When treatment B. It seereatment C. There t . We observed an interesting pattern of ABA localization in the immunofluorescence experiments  in the flower AZ of yellow lupine [LlACS down-regulation in the distal part (6 h) and up-regulation in the proximal region (4 h)  than in the distal one  A. The lestal one B. qPCR af the AZ A,B. We tf the AZ C,D. In tf the AZ D. Such af the AZ C. Collec2O2 production. In-gel assays of SOD activity revealed the presence of different isoforms: one identified as Mn-SOD and two as CuZn-SODs , whose activity was up-regulated in the distal part of the AZ in the fourth and sixth hours after the TIBA application. In the case of the proximal part, the activity was shifted in time, with their maximum values measured at the last two time points considered (6 and 8 h after TIBA application). APX activity in the inactive areas of the AZ did not display noticeable changes between the proximal and distal parts . We alsoGossypium hirsutum [Geraniaceae, Primulaceae, Ranunculaceae, Fabaceae, and Rosaceae [Abscission is a genetics-dependent program but the crucial factor determining the time of organ separation is the changing level of phytohormones. During the last few decades, significant progress has been made toward understanding their mode of action following abscission. Initially, ABA was pointed to as a dominant factor responsible for organ detachment because of its role it stimulating flower abscission in hirsutum . Nowadayhirsutum ,38,39,40hirsutum . The stiRosaceae ,42,43,44TIR) and auxin efflux carriers (AEC)) [MiETR1 and MiERS1) is enhanced, and consequently, the sugar content in fruit decreases, and hence the abscission is stimulated [L. luteus, IAA also affects ET biosynthesis genes  and causes an accumulation of the ACC, which is a precursor of ET in the flower AZ [The activation of the AZ and the consequent organ separation requires dynamic hormonal interactions. Physiological and molecular analyses demonstrated that the time of abscission depends on the antagonistic action of ET and auxin . The inhs (AEC)) ,47. The s (AEC)) ,48. On timulated . It shouimulated ,51,52. Wlower AZ . Howeverlower AZ . As rapidly growing tissues, flowers are abundant sources of auxins, which are transported from the organ through the AZ. Such a distribution of auxins across the AZ area is important for the completion of abscission. When the auxin concentration at the distal side is lower than at the proximal one, abscission is favored, while the opposite pattern of this phytohormone distribution delays separation processes ,32,45,54LlBOP, LlIDL, LlHSl, and LlMPK6 in the distal area of the AZ was observed, while most of them were expressed less efficiently in the proximal area when compared to the control  was up-regulated in response to the TIBA treatment, especially in the proximal area of AZ  in the proximal part . The positive feedback loop in ET biosynthesis has been already reported in the literature [MdACS and MdACO expression in active fruit AZ correlates with intensive ET production in apple [ACO activity, the accumulation of ACC, and ET production accompanied grape flower abscission [Pelargonium, it was up-regulated in the whole abscised sepals [Molecular changes in the lupine AZ were associated with spatial and temporal modulation of the homeostasis of ABA and ET , which aea of AZ A. We premulation . A similobserved . Neverths fruits . Interespression B,C. An ely shown . Given tmal part A,B, whicmal part B, and wamal part A could bmal part D. In this period B, which s period D for an terature ,81,82,83in apple , while hscission . During d sepals ,87,88. SOeSOD1, OeCAT2, and OeCAT3 [2O2, which is further scavenged by CAT and POX. As expected, we observed a strong, gradual accumulation of H2O2 in the proximal area of the AZ , such a scenario cannot be excluded during the flower abscission evoked by the disruption of auxin transport  Taper cultivar. Plants were grown in phytotrons under controlled light and temperature conditions, as previously described [The experiments in this study were carried out on yellow lupine  was performed. The solution was applied precisely with a tiny brush on the inactive AZ fragments situated at the base of the flower pedicel. At the same time, each inactive AZ (IN) was treated with 0.05% Tween 20 only, which served as a control. Following subsequent hours after application of the substances, the flower pedicel and stem fragments containing the AZ were excised by hand under a binocular microscope using a razor blade (to a size of approx. 2 mm). Then, the harvested AZ was divided into two small pieces. The first, namely the distal region, was situated closer to the pedicel, and the second, namely the proximal region, was positioned below the AZ, near the stem A,B. The To assess the influence of the inhibition of PAT on flower shedding, a parallel set of lupines  were left for the TIBA treatment (0.05 mM in 0.05% Tween 20). The solution was applied directly onto the IN AZ. The control IN AZ was treated with 0.05% Tween 20. All plants were further monitored for the abortion rate. The number of detached flowers was counted 120 h after the application of the solutions. The experiment was performed using three biological replicates.For the RNA extraction, the distal and proximal AZ fragments were collected from the IN AZ (Tween-treated), as well as those treated with TIBA. Three biological replicates were harvested for each time point. The RNA was isolated and reverse transcribed while following a previously applied procedure . After tTo perform the anatomical analysis, the AZ fragments were collected 24 h after treatment with TIBA or Tween 20. Next, they were fixed, dehydrated, embedded, sectioned, and placed on slides, as previously described . The sliTissue sections of AZ were stained with Toluidine blue, following an earlier procedure . The staThe sections on slides were subjected to immunofluorescence studies using antibodies provided by Agrisera . In brief, sections were blocked in bovine serum albumin and incubated with primary antibodies. For anti-CAT (AS09 501), the protocol of Wilmowicz et al. was applied . In the −2 to the one obtained for the Cu/Zn-SOD reference pattern of the defined activity of 4.4 U mg−1 protein  was applied to calculate the activity of a different SOD isoform band. In turn, the APX activity was evaluated in the extracts using the spectrophotometric method of Nakano and Asada [−1 protein . Densitometric analysis was performed using Quantity1D  software. The CAT activity was determined spectrophotometrically according to Aksmann et al. [2O2 (μmol min−1 mg−1 protein). The reproducibility of all results was confirmed by making three independent replicates.Frozen AZ fragments (−80 °C) collected 0, 2, 4, 6, and 8 h after the TIBA treatment were subjected to enzyme assays. The control IN AZ was dissected and frozen 8 h after the Tween application. To perform the SOD activity measurements, the method based on nitroblue tetrazolium (NBT) reduction was applied. We obtained protein extracts and identified isoforms in PAGE, as described previously ,106. A cnd Asada , modifiend Asada . In brien et al.  and the 2O2 was analyzed, as described previously [2O2 per gram of fresh weight. The concentration of Heviously . The res2-IAA).The endogenous level of IAA in frozen (−80 °C) AZ fragments was examined using GC-MS according to a previously published methodology . GC-MSSIt-test were evaluated at significance levels of p < 0.05 and p < 0.01.The obtained data were subjected to statistical analysis performed using SigmaPlot 2001 v.5.0. The standard error (SE) and Student’s


Objective: In this study, we built a mobile continuous Blood Oxygen Saturation (SpO2) monitor, and for the first time, explored key design principles towards daily applications. Methods: We firstly built a customized wearable computer that can sense two-channel photoplethysmogram (PPG) signals, and transmit the signals wirelessly to smartphone. Afterwards, we explored many SpO2 model building principles, focusing on linear/nonlinear models, different PPG parameter calculation methods, and different finger types. Moreover, we further compared PPG sensor placement principles by comparing different hand configurations and different finger configurations. Finally, a dataset collected from eleven human subjects was used to evaluate the mobile health monitor and explore all of the above design principles. Results: The experimental results show that the root mean square error of the SpO2 estimation is only 1.8, indicating the effectiveness of the system. Conclusion: These results indicate the effectiveness of the customized mobile SpO2 monitor and the selected design principles. Significance: This research is expected to facilitate the continuous SpO2 monitoring of patients with clinical indications. Photoplethysmography (PPG) is a convenient, low-cost technology that has been applied to various aspects of cardiovascular monitoring, such as blood oxygen saturation, heart rate, respiration, blood pressure, cardiac output, microvascular blood flow, endothelial function, arterial aging, and autonomic function . The optPrior studies on PPG-based SpO2 monitoring usually relied on inconvenient methods, such as wired solutions, or wireless methods ,20,21,22In this study, we have developed a highly convenient, continuous SpO2 monitor, and explored all the above mentioned key design principles. To the best of our knowledge, it is the first time a study thoroughly investigates all these key aspects towards medical-grade, continuous, wireless SpO2 monitoring. More specifically, our contributions include: (i) The building of a two-channel PPG wearable prototype for wireless optical signal sensing, (ii) the investigation of three finger types for PPG model calibration: Index, middle, and ring fingers, (iv) the investigation of different PPG parameter calculation methods for the PPG model building: Filtering-based approach and FFT-based approach, (v) the probe of different SpO2 estimation models: From linear and non-linear regressions models to the machine learning model, (vi) the comparison of left and right hands, and the comparison of different fingers, for SpO2 monitoring.The human studies were ethically approved by the institutional review board of the Massachusetts General Hospital (Protocol#: 2018P003132), and all participants provided oral informed consent. All studies were performed in accordance with relevant guidelines and regulations.To evaluate the wearable computer prototype and the signal processing algorithms, we have collected data from eleven (M/F: 10/1) healthy human subjects of: 39 ± 17 years old, height 5.7 ± 0.3 ft., and weight 169 ± 22 lbs. During data collection, the PPG finger-cuff was placed on the index/middle/ring fingers on the left and right hand, respectively, while subjects were still in a sitting position. Thus, for each participant, there were six recordings, each one being 1 min long. We have developed a state-of-the-art signal acquisition, display, and processing system, which supports the acquisition, display, and real-time analysis of 16 analog signals sampled at 1000 Hz by a multi-channel 16-bit data acquisition card  ,27,28. TAnalog PPG signals were acquired from a Tram-rac 4A (Marquette) and a SOLAR 8000 (Marquette) patient monitoring system as well as the Masimo SpO2 finger cuff and used as each subject’s gold-standard SpO2 measurement. For each finger, we firstly performed a gold-standard SpO2 measurement, and then, immediately thereafter, performed the wearable, battery-powered device  measurement to minimize the chance of SpO2 fluctuations. In this section, the mobile SpO2 monitor prototyping and design principles are detailed. We have built a wireless prototype for two-channel PPG sensing, as shown in Thereafter, we built an app to calculate the SpO2 in real-time, as shown in More specifically, the app receives two channel PPG signals (red and infrared) continuously sent by the wearable monitor. The app then performs the SpO2 calculation based on the algorithms described below. The estimated SpO2 value is displayed on the phone screen in real-time C. Both rTo calibrate the system, i.e., to train the PPG model parameters, we have collected PPG signals using a Fluke  SpO2 tester, which generates artificial PPG signals in a plastic finger according to a predetermined configuration. We have considered three figure type configurations: Index, middle, and ring fingers. These three finger types correspond to different pre-stored R-curves in the Fluke tester, aiming to provide corresponding PPG signals for calibration purposes. We have collected and used PPG signals corresponding to different SpO2 values in order to determine the SpO2 model parameters. We have configured the Fluke to deliver SpO2 values ranging from 70% to 100% and then used our wearable device to collect the simulated PPG signals. The emulated PPG signals and the SpO2 values were used to calibrate different SpO2 models (introduced below). Combing the finger types with the four SpO2 models to be introduced later, there are in total 12 SpO2 models to be calibrated and compared. Pulse Oximeter testers like the Fluke ProSim-8 measure the output of the pulse oximeter’s LEDs and recreate the light modulated as though it had passed through a human finger. These testers store R-curves calibrated for particular brands of pulse oximeters and use these curves to create light output that will cause a pulse oximeter of the matching brand to output an SpO2 value corresponding to the tester’s settings. They are used by biomedical engineers to test individual pulse oximeters in clinical settings to confirm that each device under test shows the same SpO2 output as the reference device that was used to create the R-curve. They cannot be used to check the accuracy or calibration of a pulse oximeter, instead, they are ‘transfer standards’ that compare the device under test to a reference device that was calibrated by other means, usually a desaturation study with human volunteers. Using a pulse oximeter tester to calibrate a novel pulse oximeter allows the developers to match their R-curve to the manufacturer’s chosen in the tester, but because they use different LEDs and photosensors and likely different filtering algorithms, this does not ensure accuracy for human use.SpO2 is defined as,To estimate SpO2 from two-channel PPG signals, we firstly calculate a key parameter, the ratio of ratios We have compared two approaches to estimate the parameter T method . For theSince oxyhemoglobin (HbO2) has a much higher attenuation coefficient at the red-light wavelength compared to that of the IR light, and hemoglobin (Hb) has a higher attenuation coefficient for the IR light, the key parameter Several models have been considered in the SpO2 estimation, such as linear/quadratic/cubic polynomials, and a regression tree: We aim to evaluate how the complexity of the SpO2 model affects the SpO2 estimation accuracy. While the linear polynomial has been used in prior studies ,25, a noWe further take into consideration different finger types , in building the SpO2 model. In the calibration phase, we aim to select the best combination of SpO2 model, finger type and approach for PPG parameter estimation. The selected combination will be finally used to evaluate the SpO2 estimation performance in the human study.In a study in humans, we first evaluated the SpO2 performance by comparing the left hand and right hand, and reported the root mean square error , respectively, using the Wilcoxon signed-rank test.We have further evaluated the calibrated model performance across fingers (index/middle/ring), using the Wilcoxon signed-rank test. In With respect to the finger type, we observe that the middle finger type is superior to the ring and index finger types, exhibiting the lowest RMSE of 2.2%–2.8%. The box plots also show that the middle finger type has a much lower error than the other two finger types. The ring finger and the index finger have an RMSE as high as 43.1% and 46.5%, respectively. Therefore, the middle finger type is selected to calibrate the SpO2 model. With respect to the model type, both model 2 and model 3 correspond to the lowest RMSE, but considering model 2 is of lower complexity and therefore it is easier to implement in the smartphone, we finally choose model 2 as the final SpO2 model. Outliers are likely to be due to high sensitivity to finger movement, and therefore poor contact and signal quality. This is very common for PPG signal acquisition since even a small finger movement is expected to cause sensor to skin contact changes. R, which is fed into the SpO2 model for SpO2 estimation. Therefore, this calibration step involves the method to calculate R, not to train parameters for the SpO2 model training. Therefore, in the calibration step, we have selected the optimal model as: Middle finger type, model 2 , and filtering method-based parameter R calculation. We have further compared two methods to estimate the PPG parameter We have found that the mean and standard deviation of the R value for the middle finger, to be 0.58 and 0.04, respectively.We used the calibrated SpO2 model, i.e., the middle finger type, second-order polynomial, and the filtering-based parameter calculation method, to measure the SpO2 on 11 human subjects. p = 1.00). This observation indicates that, as expected, both hands have similar blood perfusion conditions and are almost equivalent in terms of SpO2 measurement, which demonstrates the effectiveness of the calibrated SpO2 model.Firstly, we compared the SpO2 model between hands. For each hand, we tested the model on three fingers: Index, middle, and ring fingers, and then averaged the performance across fingers. The RMSE (%) of the right and left hands , shown in p = 0.13). This has demonstrated that the calibrated SpO2 model can be used on not only different hands but also different fingers. Moreover, the RMSE (%) of the left index finger is as low as 1.8, much lower than the FDA standard that requires the RMSE (%) to be less than 3. Overall, we observe that both index and middle fingers can be used in mobile SpO2 monitoring. To further evaluate the SpO2 model, we have compared its performance for different fingers, as shown in We recognize that anatomical differences among fingers may result in different contact conditions within the probe, which may induce artifact and outliers, as seen in Despite the availability of a multitude of evidence-based therapies for the treatment of chronic diseases, the burden of these diseases on the US population remains unacceptably high, with an estimated one million admissions per year, primarily involving the elderly . At the In this paper, we introduce a wireless, smartphone-based, continuous SpO2 monitoring system that aims to supplement cvrPhone, a medical-grade, smartphone-based cardiac and respiratory monitoring platform ,7,8. To There are several major findings in this study. First, our two-channel PPG wearable device can robustly acquire PPG signals for SpO2 estimation, second, in terms of SpO2 model calibration, the middle finger (the most common finger type) is more appropriate than the index or ring finger types, third, the filtering-based method is very effective to calculate the ratio of ratios—A SpO2-relevant parameter, fourth, the second-order polynomial model provides the best performance, fifth, our wearable device performs equally well for both hands and different fingers in measuring the SpO2. Current SpO2 technologies ,24,25 usIn conclusion, SpO2 measurement is an important vital sign of cardiopulmonary monitoring, not only in a routine in-hospital assessment of patients’ physiology, but also in out-of-hospital follow-up patients’ treatment. Ambulatory, Bluetooth/smartphone-based monitoring of SpO2, together with 12 lead ECG, respiration rate, and tidal volume, provide to the end-user (both the patient and patient-care-team) the benefits of: (i) Extensive, continuous respiratory monitoring never before possible to the non-ventilated patient to assess the incidence, severity, and progression of chronic conditions, (ii) assisting patients and clinicians in the immediate evaluation of the patient’s respiratory system and changes in respiratory state that can precede respiratory depression and death, (iii) the ability to quickly assess an underlying ischemic episode and an-arrhythmic risk in a trend graph and assist patients and clinicians in determining whether immediate therapy is needed in order to prevent life-threatening arrhythmias and death, (iv) it will permit the care-team to constantly monitor the high volume of patients under their care. Study Limitations An important aspect of the present study is that the evaluation of the proposed mobile, continuous blood oxygen saturation monitoring system, presently, has been evaluated only in normal subjects, with a limited SpO2 range (95%–100%). Another limitation involves the use of a commercial device as the gold standard and not a blood test. Outliers due to high sensitivity to finger movement may affect contact and signal quality, therefore, an improved probe design is expected to reduce sensor to skin contact changes. Finally, the small number of tested subjects limits the power of this study.


Recently, the interest in recovery bioactive compounds from food industrial by-products is growing abundantly. Olive oil by-products are a source of valuable bioactive compounds with antioxidant and antimicrobial properties. One of the most interesting by-products of olive oil obtained by a two-phase decanter is the olive paste, a wet homogeneous pulp free from residuals of the kernel. To valorize the olive paste, ready-to-cook cod sticks breaded with dried olive oil by-products were developed. Shelf-life tests were carried out on breaded cod sticks and during 15 days of storage at 4 °C pH evolution, microbiological aspects, and sensory properties were also monitored. In addition, the chemical quality of both control and active samples was assessed in terms of total phenols, flavonoids, and antioxidant activity. The enrichment with olive paste increased the total phenols, the flavonoids, and the antioxidant activity of the breaded fish samples compared to the control. Furthermore, the bioactive compounds acted as antimicrobial agents, without compromising the sensory parameters. Therefore, the new products recorded a longer shelf life (12 days) than the control fish sample that remained acceptable for nine days. Pseudomonas fluorescens and putida, Aeromonas hydrophila, and Shewanella putrefaciens [Seafood is an important part of a healthy diet, being an excellent source of high biological value proteins, omega-3 polyunsaturated acids, vitamins, and mineral salts . Among tefaciens .In addition, its time-consuming preparation contrasts with the trend of the modern consumer, who is looking for ready-to-eat fish products or ready-to-cook products. In this context, the idea of developing new cod fillets could gain consensus among consumers ,6.Among the preservation methods reported in the abundant literature dealing with seafood shelf life ,8,9,10, However, modern consumers are increasingly looking for sustainable, safe, and healthy products . TherefoIn the context of food sustainability, several pieces of research have been conducted with the aim to valorize industrial by-products. In fact, natural preservatives from fruit and vegetable by-products could be valid agents to guarantee the shelf-life extension, since they are rich in bioactive compounds with antimicrobial and antioxidant properties ,17,18,19The purpose of our study was to develop breaded ready-to-cook sticks of cod, implementing new and effective combinations between fish and olive-oil by-products. To the aim, the breading was done with and without the olive paste, properly distributed, and then applied on the fish surface. During refrigerated storage, a shelf-life test was carried out, monitoring the quality parameters related to pH evolution, microbiological aspects, and sensory properties to verify if the active breading was able to extend the shelf life of the product. For completeness, the chemical quality of the samples was also assessed, to highlight the difference in terms of phenolic compounds, flavonoids, and antioxidant activity between the control and active products.Refrigerated salted cod fillets were purchased from a local market . The olive paste from Cellina di Nardò cultivar was obtained at the beginning of the year 2020, in January, from a local olive mill  using a Pieralisi Leopard with Multi-Phase Decanter, which combines modern extraction technology without water addition and recovers a certain quantity of by-product paste, named olive paste, made up of wet pulp without any traces of kernel. The olive paste was immediately dried at 35 °C in a dryer  for 72 h. The dried olive paste was reduced to a fine powder (<500 μm) by a hammer mill  and then stored at 4 °C until its utilization in February 2020. All the ingredients to prepare the cod stick breaded fish, such as the breading, the spices, the potato flakes, and the fresh milk were purchased at a local market .w/w water content and about 1% w/w NaCl) were cut in sticks of approximately 12 g. Two different mixtures were prepared: (i) the control mixture contained 180 g of breading with fish spices, 180 g of potato flakes; (ii) the active mixture prepared with 180 g of breading with fish spices, 180 g of potato flakes, 90 g of olive paste. The amount of olive paste represents the best optimization with other ingredients to give a final product acceptable after cooking. Control samples (Ctrl) were obtained as follows: after dipping in a solution of water and milk (1:1), samples were breaded in the control mixture, by repeating the passage twice, then were manually compacted, placed above a food tray with a pad and packaged in air using high-barrier film bags (multi-layer film Nylon/Polyethylene) with a thickness of 150 µm, provided by Biochemia  and kept under refrigeration (4 ± 1 °C). Active samples (Active) were prepared using the same procedure as for Ctrl samples, using the active mixture. Two sticks were packaged in each bag. A total of 64 sticks were prepared from two different batches: 32 Ctrl samples and 32 Active samples. All samples were stored at 4 ± 1 °C for 15 days.Cod fillets were coarsely desalted, soaked, and stored at 4 ± 1 °C for five days, changing the water every day. On the sixth day, cod fillets were drained to eliminate excess water for about half an hour and the skin was removed. Then, the fillets (about 67% 2S2O8), Trolox , aluminum chloride (AlCl3), sodium nitrite (NaNO2), sodium hydroxide solution (NaOH), quercetin, were supplied from Sigma-Aldrich . Anhydrous sodium carbonate (Na2CO3) was supplied from Carlo Erba . For the preparation of the phosphate-buffered saline (PBS), the following salts were used: sodium phosphate dibasic heptahydrate (Na2HPO4·7H2O) and sodium phosphate monobasic monohydrate (NaH2PO4·H2O). All reagents were of analytical grade.Folin–Ciocalteu reagent, gallic acid monohydrate, ethanol, ABTS  diammonium salt), potassium persulfate  and cooked samples (C-Ctrl and C-Active) , were firstly subjected to drying , milled to obtain a powder, and then subjected to extraction as reported by Cedola et al. [2 = 0.9997).The evaluations of total phenols and total flavonoids were carried out on both raw and cooked breaded cod sticks. All the chemical analyses were performed the day after the sample preparation. Total phenol content was determined according to the Folin–Ciocalteu method as reported by Cedola et al. . The col2 = 0.9955).Total flavonoid content was determined using the aluminum chloride colorimetric method, according to . The mea2 = 0.9977) and the antioxidant activity was expressed as milligrams of Trolox equivalents for gram of dry weight (dw). All tests were carried out in triplicate.The antioxidant activity of breaded cod sticks was assessed using two methods: ABTS  assay. The test is based on the ability of antioxidants to interact with the radical cation 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS·+) inhibiting its absorption at 734 nm , and thePseudomonas spp., hydrogen sulfide-producing bacteria (HSPB), psychrotolerant and heat-labile aerobic bacteria (PHAB), mesophilic and psychrotrophic bacteria, Enterobacteriaceae and lactic acid bacteria (LAB) according to Danza et al. [6 cfu/g for total viable mesophilic and psychrotrophic bacteria, 106 cfu/g for Pseudomonas spp. and Shewanella, 107 cfu/g for Photobacterium [Ctrl and Active samples (20 g) were aseptically weighed into a sterile stomacher bag, diluted with peptone water (dilution 1:10), and homogenized for 90 s with a Stomacher LAB Blender 400 . Serial dilutions were plated onto specific media in Petri dishes to enumerate a et al. . The cona et al. . All medacterium . The fitacterium .The measurement of pH was performed in triplicate on the first homogenized dilution of fish samples, using a pH meter . Two different samples were used for each measurement. Microbiological analyses and pH were analyzed at the initial time and after 2, 4, 8, 12, and 15 days of refrigerated storage at 4 °C.The quantitative descriptive analysis (QDA) was used for a sample comparison, according to the guidelines of the Codex Alimentarius Commission. To the aim, breaded cod sticks were submitted to a panel of five trained judges. The panelists have familiar eating habits with fish and fish products and already had experience in the evaluation of burgers and fillets based on fish. They were retrained for two days (2 h session), to establish the appropriate attributes for sensory evaluation and in order to minimize individual differences and ensure repeatability of results. The panelists were asked to give judgments on odor, color, appearance, texture, and overall quality using a nine-point scale. In the scale, 9 corresponded to excellent, 8 to very good, 7 to good, 6 to reasonable, 5 to not good (acceptable limit), 4 to disliked, 3 to bad, 2 to very bad, and 1 to completely unacceptable . Before p < 0.05), was carried out to determine significant differences among samples. STATISTICA 7.1 for Windows  was used.Fitting of experimental data provided us MAL and SAL parameters. The lowest value among the MAL and SAL parameters gave us the product shelf life. The experimental data were compared by one-way analysis of variance (ANOVA). A Duncan’s multiple range test, with the option of homogeneous groups , flavonoids (mg QE/g dw), and antioxidant activity  as shown in Pseudomonas spp. (Pse), Shewanella (Shew.) and Photobacterium (Phot.). The evolution of total mesophilic and psychrotrophic bacteria was reported in 6 cfu/g. In contrast, a significant difference was observed for psychrotrophic bacteria, since the cell concentration in the Ctrl samples on the 15th day of monitoring was higher compared to the Active samples. In fact, the Ctrl exceeded the microbial limit (5 × 106 cfu/g) after 9.05 days, while the Active samples maintained the microbial concentration below the limit until the 15th day of storage.In general, the quality of raw fish depends on microbial and sensory quality. The microbial quality decay of the developed breaded cod sticks (Ctrl and Active) was determined by monitoring the viable cell concentration of the total mesophilic and psychrotrophic bacteria , 2S off-odors and off-flavors, caused by specific microbial groups [Pseudomonas spp., hydrogen-sulfide-producing bacteria (HSPB-Shewanella), and psychrotolerant and heat-labile aerobic bacteria (PHAB-Photobacterium phosphoreum) were also monitored.It is well known that the deterioration of fish products could be characterized by the presence of Hl groups . For thiPseudomonas spp. as a function of storage days. A substantial difference between the Ctrl and the Active samples was also found. Although both samples exceeded the microbial limit set at 106 cfu/g, the microbial concentration of Active samples, for almost the entire monitoring time, remained below the control by about one log cycle. As can be seen in Pseudomonas spp. growth was slower in the Active samples compared to the control, thus suggesting that the bioactive compounds present in dry olive paste acted as antimicrobial agents [Pseudomonas spp. [l agents . Therefonas spp.  and thisShewahella and Photobacterium, the microbial growth was very similar in both Ctrl and Active samples (Shewanella (6 cfu/g) almost on the same day of observation . On the other hand, the cell concentration of Photobacterium in Ctrl and Active samples was below the microbiological acceptability limit (107 cfu/g) for the entire storage time.As regards the behavior of  samples a,b. In bewanella a the ActPhotobacterium may be considered of less importance for the evaluation of the microbial quality of this fish product, with their cell concentrations lower than those required to spoil raw fish [Pseudomonas ssp., Enterobacteriaceae and Photobacterium.Based on the results obtained by the microbiological analyses, the fitting of experimental data allowed calculating the microbiological acceptability limit (MAL). All the MAL values were summarized in raw fish . For theraw fish ,41. Our During the 15 days of storage, in the Ctrl the pH remained roughly between values of 7.12 and 6.96, while in the Active samples between 7.0 and 6.40 (data not shown). This slight difference in pH between Ctrl and Active samples could not be attributed to relevant differences in microbial proliferation, but it is most probably ascribed to the olive paste itself which is slightly acidic ,43.Pseudomonas spp., HSPB, and PHAB, keeping their growth lower than the control up to 13 days of storage. On the other hand, Del Nobile et al. [The antimicrobial effect exerted by olive by-products has also been well evaluated by Kuley et al. . These ae et al.  evaluateThe expert panel judged the general appearance, the color, the odor, the texture, and the overall quality of breaded cod sticks. Scores recorded from Ctrl and Active samples for the four specific sensory attributes are reported in In Pseudomonas spp. and Shewanella affected the shelf life of the Ctrl samples, while the growth of Pseudomonas spp. and Shewanella influenced the shelf life of the Active cod sticks. These results highlighted that the shelf life of Active samples (12.23 days) was longer than the Ctrl, for which a shelf life of 9.05 days was recorded. Similar results have been reported by Martínez et al. [To determine final product shelf life, both fitting values of microbiological (MAL) and sensory quality (SAL) were taken into account. To this aim, data collected in z et al. , who alsz et al.  also notBased on our findings, breading enriched with olive paste is a useful approach to valorize an industrial by-product, enhance the nutritional properties of cod sticks, and extend their shelf life because the bioactive compounds entailed better stability of the product from both the microbiological and sensory point of view ,40. It iIn this study, dried olive paste, as a valuable by-product of the modern olive oil production process, was applied as breading to cod sticks to prolong their shelf life. To this aim, the microbiological, sensory, and chemical qualities of the breaded cod sticks were assessed during two weeks of refrigerated storage. The results show that the olive paste used in the breading increased the phenol and flavonoid content of cod sticks and, as a consequence, its antioxidant activity. In addition, the fortification maintained the microbiological quality of the active samples better than the control by more than three days and the sensory acceptability of the product was not compromised. In fact, an extension of the shelf life of active samples was observed. While the control cod sticks remained acceptable for about nine days, the active fish products were acceptable up to more than 12 days. Therefore, this was a concrete example of sustainable reuse of olive oil by-products to develop a breaded cod stick with healthful properties.