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The objectives of this study were to explore the use of Fourier-transform infrared (FITR) spectroscopy on 458 goat milk samples for predicting cheese-making traits, and to test the effect of the farm variability on their prediction accuracy. Calibration equations were developed using a Bayesian approach with three different scenarios: i) a random cross-validation (CV) [80% calibration (CAL); 20% validation (VAL) set], ii) a stratified CV [(SCV), 13 farms used as CAL, and the remaining one as VAL set], and iii) a SCV where 20% of the goats randomly selected from the VAL farm were included in the CAL set (SCV 80 ). The best prediction performance was obtained for cheese yield solids, justifying for its practical application at population level. Overall results were similar to or outperformed those reported for bovine milk. Our results suggest considering specific procedures for calibration development to propose reliable tools applicable along the dairy goat chain., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The aim of this study was to quantify some environmental (individual herds, herd productivity, milking system, and season) and animal factors [individual animals, breed, days in milk (DIM) and parity] on the variability of the log-10 transformation of somatic cell count (LSCC) and differential somatic cell count (DSCC) on individual bovine milk. A total of 159,360 test-day records related to milk production and composition were extracted from 12,849 Holstein-Friesian and 9,275 Simmental cows distributed across 223 herds. Herds were classified into high and low productivity, defined according to the average daily milk net energy output (DMEO) yielded by the cows. Data included daily milk yield (DYM; kg/d), milk fat, protein, lactose, SCC, and DSCC, and information on herds (i.e., productivity, milking system). The daily production of total and differential somatic cells in milk was calculated and then log-10 transformed, obtaining DLSCC and DLDSCC, respectively. Data were analyzed using a mixed model including the effects of individual herd, animal, repeated measurements intra animal as random, and herd productivity, milking system, season, breed, DIM, parity, DIM × parity, breed × season, DIM × milking system and parity × milking system as fixed factors. Herds with a high DMEO were characterized by a lower content of LSCC and DSCC, and higher DLSCC and DLDSCC, compared to the low DMEO herds. The association between milking system and somatic cell traits suggested that the use of the automatic milking systems would not allow for a rapid intervention on the cow, as evidenced by the higher content of all somatic cell traits compared to the other milking systems. Season was an important source of variation, as evidenced by high LSCC and DSCC content in milk during summer. Breed of cow had a large influence, with Holstein-Friesian having greater LSCC, DSCC, DLSCC, and DLDSCC compared to Simmental. With regard to DIM, the variability of LSCC was mostly related to that of DSCC, showing an increase from calving to the end of lactation, and suggesting the higher occurrence of chronic mastitis in cows toward the end of lactation. All the somatic cell traits increased across number of parities, possibly because older cows may have increased susceptibility to intramammary infections., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science.)

Inbreeding depression has become an urgent issue in cosmopolitan breeds where the massive genetic progress achieved in the latest generations is counterbalanced by a dramatic loss of genetic diversity causing increased health issues. Thus, the aim of this study was to estimate inbreeding depression on productive traits in Holstein dairy cattle. More precisely, we aimed to i) determine the level of inbreeding in 27,735 Italian Holstein dairy cows using pedigree and genotype data, ii) quantify the effect of inbreeding on 305-d in milk yield (MY; kg), fat yield (FY; kg), and protein yield (PY; kg) based on different statistical approaches, iii) determine if recent inbreeding has a more harmful impact than ancestral ones, and iv) quantify chromosomal homozygosity effect on productive traits. Quality control was performed on the autosomal chromosomes resulting in a final dataset of 84,443 single nucleotide polymorphisms. Four statistical models were used to evaluate the presence of inbreeding depression, which included linear regression analysis and division of FPED and FROH into percentile classes. Moreover, FROH was partitioned into i) length classes to assess the role of recent and ancestral inbreeding and ii) chromosome-specific contributions (FROH-CHR). Results evidenced that inbreeding negatively impacted the productive performance of Italian Holstein Friesian cows. However, differences between the estimated FPED and FROH coefficients resulted in different estimates of inbreeding depression. For instance, a 1% increase in FPED and FROH was associated with a decrease in MY of about 44 and 61 kg (P < 0.01). Further, when considering the extreme inbreeding percentile classes moving from the 5th lowest to the 95th highest, there was a reduction of -263 kg and -561 kg per lactation for FPED and FROH. Increased inbreeding, estimated by FPED and FROH, had also a negative effect on PY and FY, either fit as a regressor or percentile classes. When evaluating the impact of inbreeding based on runs of homozygosity (ROH) length classes, longer ROH (over 8 Mb) had a negative effect in all traits, indicating that recent inbreeding might be more harmful than the ancestral one. Finally, results within chromosome homozygosity highlighted specific chromosomes with a more deleterious effect on productive traits., (© The Author(s) 2023. Published by Oxford University Press on behalf of the American Society of Animal Science.)

PACSIN2 variants are associated with gastrointestinal effects of thiopurines and thiopurine methyltransferase activity through an uncharacterized mechanism that is postulated to involve autophagy. This study aims to clarify the role of PACSIN2 in autophagy and in thiopurine cytotoxicity in leukemic and intestinal models. Higher autophagy and lower PACSIN2 levels were observed in inflamed compared with non-inflamed colon biopsies of inflammatory bowel disease pediatric patients at diagnosis. PACSIN2 was identified as an inhibitor of autophagy, putatively through inhibition of autophagosome formation by a protein-protein interaction with LC3-II, mediated by a LIR motif. Moreover, PACSIN2 resulted a modulator of mercaptopurine-induced cytotoxicity in intestinal cells, suggesting that PACSIN2-regulated autophagy levels might influence thiopurine sensitivity. However, PACSIN2 modulates cellular thiopurine methyltransferase activity via mechanisms distinct from its modulation of autophagy., (© 2023 Zudeh et al.)

Background: Thiopurine methyltransferase (TPMT) is a crucial enzyme for azathioprine biotransformation and its activity is higher in very early onset inflammatory bowel disease (VEO-IBD) patients than in adolescents with IBD (aIBD)., Aims: The aims of this pharmacoepigenetic study were to evaluate differences in peripheral blood DNA methylation of the TPMT gene and in azathioprine pharmacokinetics in patients with VEO-IBD compared to aIBD., Methods: The association of age with whole genome DNA methylation profile was evaluated in a pilot group of patients and confirmed by a meta-analysis on 3 cohorts of patients available on the public functional genomics data repository. Effects of candidate CpG sites in the TPMT gene were validated in a larger cohort using pyrosequencing. TPMT activity and azathioprine metabolites (TGN) were measured in patients' erythrocytes by HPLC and associated with patients' age group and TPMT DNA methylation., Results: Whole genome DNA methylation pilot analysis, combined with the meta-analysis revealed cg22736354, located on TPMT downstream neighboring region, as the only statistically significant CpG whose methylation increases with age, resulting lower in VEO-IBD patients compared to aIBD (median 9.6% vs 12%, p = 0.029). Pyrosequencing confirmed lower cg22736354 methylation in VEO-IBD patients (median 4.0% vs 6.0%, p = 4.6 ×10 -5 ). No differences in TPMT promoter methylation were found. Reduced cg22736354 methylation was associated with lower TGN concentrations (rho = 0.31, p = 0.01) in patients with VEO-IBD and aIBD., Conclusion: Methylation of cg22736354 in TPMT gene neighborhood is lower in patients with VEO-IBD and is associated with reduced azathioprine inactivation and increased TGN concentrations., Competing Interests: Conflict of interest statement All authors read and approved the final manuscript. All authors have no conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Cheeses are produced by many different procedures, giving rise to many types differing in ripening time, size, shape, chemical composition, color, texture, and sensory properties. As the first step in a large project, our aim was to characterize and quantify the major sources of variation in cheese characteristics by sampling 1050 different cheeses manufactured by over 100 producers and grouped into 37 categories (16 with protected designation of origin, 4 traditional cheese categories, 3 pasta filata cheese categories, 5 flavored cheese categories, 2 goat milk categories, and 7 other categories ranging from very fresh to very hard cheeses). We obtained 17 traits from each cheese (shape, height, diameter, weight, moisture, fat, protein, water soluble nitrogen, ash, pH, 5 color traits, firmness, and adhesiveness). The main groups of cheese categories were characterized and are discussed in terms of the effects of the prevalent area of origin/feeding system, species of lactating females, main cheese-making technologies, and additives used. The results will allow us to proceed with the further steps, which will address the interrelationships among the different traits characterizing cheeses, detailed analyses of the nutrients affecting human health and sensorial fingerprinting.

Thiopurine drugs are part of the therapeutic armamentarium for pediatric patients suffering from inflammatory bowel disease (IBD) and acute lymphoblastic leukemia (ALL). The therapeutic drug monitoring of these drugs, consisting of measurements of the thiopurine metabolites thioguanine nucleotides (TGN) and methylmercaptopurine nucleotides (MMPN) are used to optimize the effectiveness of treatment and prevent adverse effects. In this context, we developed and validated a high-performance liquid chromatography-diode array detection (HPLC-DAD) method for the simultaneous quantification of thiopurine metabolites according to the most recent International Council for Harmonisation (ICH) guidelines. The calibration curves were built in the clinically relevant range of concentrations for TGN of 300-12,000 nM and for MMPN of 3000-60,000 nM. The limit of detection and the lower limit of quantification were 100 and 300 nM for TGN and 900 and 3000 nM for MMPN, respectively. The percentage of inter-day accuracy and precision (CV%) varied between 85 and 104% and 1.6 and 13.8%. Stability was demonstrated for both of the metabolites for at least 50 days at -20 °C. The proposed HPLC-DAD method showed an appropriate selectivity, specificity, linearity, accuracy, precision and good applicability to samples from patients with IBD and ALL undergoing thiopurine treatment.

Background: To date, measurement of intracellular cystine is used for the therapeutic monitoring of patients affected by cystinosis in treatment with cysteamine. Since this method is time and sample consuming, development of a faster method to quantify cysteamine would be extremely useful in order to help clinicians to adjust dosages of cysteamine and to define better the pharmacokinetic profile of this drug. The aim of the study was to develop a liquid chromatography tandem mass spectrometry method for the quantification of cysteamine in plasma samples and to test its applicability on plasma samples derived from patients with nephropathic infantile cystinosis in treatment with cysteamine., Results: The percentage of accuracy of the developed method varied between 97.80 and 106.00% and CV% between 0.90 and 6.93%. There was no carry over. The calibration curves were built from 2.5 to 50 µM. The limit of detection and the lower limit of quantification occurred at 0.25 and 1.25 µM respectively. Cysteamine was stable up to 2 months at -20 °C. Concentrations of cysteamine and intracellular cystine of 4 patients were in line with data previously reported., Conclusion: The proposed method showed an appropriate selectivity, specificity, linearity, sensibility, accuracy, precision and good applicability to samples., (© 2022. The Author(s).)

The objective of this study was to develop formulas based on milk composition of individual goat samples for predicting cheese yield (%CY) traits (fresh curd, milk solids, and water retained in the curd). The specific aims were to assess and quantify (1) the contribution of major milk components (fat, protein, and casein) and udder health indicators (lactose, somatic cell count, pH, and bacterial count) on %CY traits (fresh curd, milk solids, and water retained in the curd); (2) the cheese-making method; and (3) goat breed effects on prediction accuracy of the %CY formulas. The %CY traits were analyzed in duplicate from 600 goats, using an individual laboratory cheese-making procedure (9-MilCA method; 9 mL of milk per observation) for a total of 1,200 observations. Goats were reared in 36 herds and belonged to 6 breeds (Saanen, Murciano-Granadina, Camosciata delle Alpi, Maltese, Sarda, and Sarda Primitiva). Fresh %CY (%CY CURD ), total solids (%CY SOLIDS ), and water retained (%CY WATER ) in the curd were used as response variables. Single and multiple linear regression models were tested via different combinations of standard milk components (fat, protein, casein) and indirect udder health indicators (UHI; lactose, somatic cell count, pH, and bacterial count). The 2 %CY observations within animal were averaged, and a cross-validation (CrV) scheme was adopted, in which 80% of observations were randomly assigned to the calibration (CAL) set and 20% to the validation (VAL) set. The procedure was repeated 10 times to account for sampling variability. Further, the model presenting the best prediction accuracy in CrV (i.e., comprehensive formula) was used in a secondary analysis to assess the accuracy of the %CY predictive formulas as part of the laboratory cheese-making procedure (within-animal validation, WAV), in which the first %CY observation within animal was assigned to CAL, and the second to the VAL set. Finally, a stratified CrV (SCrV) was adopted to assess the %CY traits prediction accuracy across goat breeds, again using the best model, in which 5 breeds were included in CAL and the remaining one in the VAL set. Fitting statistics of the formulas were assessed by coefficient of determination of validation (R 2 VAL ) and the root mean square error of validation (RMSE VAL ). In CrV, the formula with the best prediction accuracy for all %CY traits included fat, casein, and UHI (R 2 VAL = 0.65, 0.96, and 0.23 for %CY CURD , %CY SOLIDS , and %CY WATER , respectively). The WAV procedure showed R 2 VAL higher than those obtained in CrV, evidencing a low effect of the 9-MilCA method and, indirectly, its high repeatability. In the SCrV, large differences for %CY CURD and %CY WATER among breeds evidenced that the breed is a fundamental factor to consider in %CY predictive formulas. These results may be useful to monitor milk composition and quantify the influence of milk traits in the composite selection indices of specific breeds, and for the direct genetic improvement of cheese production., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Inflammatory bowel diseases (IBDs) are chronic inflammatory disorders of the intestinal tract that have emerged as a growing problem in industrialized countries. Knowledge of IBD pathogenesis is still incomplete, and the most widely-accepted interpretation considers genetic factors, environmental stimuli, uncontrolled immune responses and altered intestinal microbiota composition as determinants of IBD, leading to dysfunction of the intestinal epithelial functions. In vitro models commonly used to study the intestinal barrier do not fully reflect the proper intestinal architecture. An important innovation is represented by organoids, 3D in vitro cell structures derived from stem cells that can self-organize into functional organ-specific structures. Organoids may be generated from induced pluripotent stem cells or adult intestinal stem cells of IBD patients and therefore retain their genetic and transcriptomic profile. These models are powerful pharmacological tools to better understand IBD pathogenesis, to study the mechanisms of action on the epithelial barrier of drugs already used in the treatment of IBD, and to evaluate novel target-directed molecules which could improve therapeutic strategies. The aim of this review is to illustrate the potential use of organoids for therapy personalization by focusing on the most significant advances in IBD research achieved through the use of adult stem cells-derived intestinal organoids., Competing Interests: Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article., (©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.)

The use of infliximab has completely changed the therapeutic landscape in inflammatory bowel disease. However, despite its proven efficacy to induce and maintain clinical remission, increasing evidence suggests that treatment failure may be associated with inadequate drug blood concentrations. The introduction of biosensors based on different nanostructured materials for the rapid quantification of drugs has been proposed for therapeutic drug monitoring. This study aimed to apply atomic force microscopy (AFM)-based nanoassay for the measurement of infliximab concentration in serum samples of healthy donors and pediatric IBD patients. This assay measured the height signal variation of a nanostructured gold surface covered with a self-assembled monolayer of alkanethiols. Inside this monolayer, we embedded the DNA conjugated with a tumor necrosis factor able to recognize the drug. The system was initially fine-tuned by testing known infliximab concentrations (0, 20, 30, 40, and 50 nM) in buffer and then spiking the same concentrations of infliximab into the sera of healthy donors, followed by testing pediatric IBD patients. A good correlation between height variation and drug concentration was found in the buffer in both healthy donors and pediatric IBD patients (p-value < 0.05), demonstrating the promising use of AFM nanoassay in TDM.

We consider the archetype situation of a chemical species that diffuses in a material and irreversibly escapes through the interface. In our setup, the interface switches between two states corresponding to 'release phase' (absorbing boundary) during which the species is released to the exterior, and 'pause phase' (reflecting boundary) during which the species is not released and its concentration profile inside the material partially relaxes back to uniformity. By combining numerical solution of the diffusion equation and statistical analysis of the outcomes, we derive upper and lower bounds and an empirical approximation for the amount of species released up to a certain time, in which the only information about the release-pause alternation schedule is the number of release phases and the average duration of a release phase. The methodology is developed thinking especially to dermal exposure assessment in the case of a slab-like homogeneous material irreversibly releasing chemicals during a number of contacts. However, upon proper extensions, this approach might be useful for inspecting other situations that are encountered, for instance, when dealing with leakage of chemicals in environmental contexts and regulatory toxicology., (© 2022. The Author(s).)

Genetic diversity has become an urgent matter not only in small local breeds but also in more specialized ones. While the use of genomic data in livestock breeding programs increased genetic gain, there is increasing evidence that this benefit may be counterbalanced by the potential loss of genetic variability. Thus, in this study, we aimed to investigate the genetic diversity in the Italian Holstein dairy cattle using pedigree and genomic data from cows born between 2002 and 2020. We estimated variation in inbreeding, effective population size, and generation interval and compared those aspects prior to and after the introduction of genomic selection in the breed. The dataset contained 84,443 single-nucleotide polymorphisms (SNPs), and 74,485 cows were analyzed. Pedigree depth based on complete generation equivalent was equal to 10.67. A run of homozygosity (ROH) analysis was adopted to estimate SNP-based inbreeding (F ROH ). The average pedigree inbreeding was 0.07, while the average F ROH was more than double, being equal to 0.17. The pattern of the effective population size based on pedigree and SNP data was similar although different in scale, with a constant decrease within the last five generations. The overall inbreeding rate (ΔF) per year was equal to +0.27% and +0.44% for F ped and F ROH throughout the studied period, which corresponded to about +1.35% and +2.2% per generation, respectively. A significant increase in the ΔF was found since the introduction of genomic selection in the breed. This study in the Italian Holstein dairy cattle showed the importance of controlling the loss of genetic diversity to ensure the long-term sustainability of this breed, as well as to guarantee future market demands., Competing Interests: 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., (Copyright © 2022 Ablondi, Sabbioni, Stocco, Cipolat-Gotet, Dadousis, Kaam, Finocchiaro and Summer.)

Background: Extracellular vesicles (EVs) are a heterogeneous family of small vesicles released by donor cells and absorbed by recipient cells, which represent important mediators with fundamental roles in both physiological and pathological conditions. EVs are present in a large variety of biological fluids and have a great diagnostic and prognostic value. They have gained the interest of the scientific community due to their extreme versatility. In fact, they allow us to hypothesize new therapeutic strategies since, in addition to being cell signal mediators, they play an important role as biomarkers, drug vehicles, and potential new therapeutic agents. They are also involved in immunoregulation, have the ability to transmit resistance to a drug from one cell to a more sensitive one, and can act as drug delivery systems., Objective: The main reciprocal interactions between EVs and immunosuppressive drugs will be presented., Results: The known interactions between EVs and immunosuppressive drugs, in particular cyclosporin, glucocorticoids, rapamycin, methotrexate, cyclophosphamide, eculizumab, infliximab, certolizumab, etanercept, glatiramer acetate, and fingolimod are presented., Conclusion: This review provides relevant information on the links between EVs and immunosuppressive drugs with a focus on EVs' role as tools to assess the effects of immunosuppressants, suggesting innovative properties and new possible therapeutic uses., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The pathogenic role of the overactivated ABL1 tyrosine kinase (TK) pathway is well recognized in some forms of BCR-ABL1 like acute lymphoblastic leukemia (ALL); TK inhibitors represent a useful therapeutic choice in these patients who respond poorly to conventional chemotherapy. Here we report a novel peptide biosensor (P ABL )-ELISA assay to investigate ABL1 activity in four immortalized leukemic cell lines with different genetic background. The P ABL sequence comprises an ABL1 tyrosine (Y) phosphorylation site and a targeting sequence that increases the specificity for ABL1; additional peptides (Y-site-mutated (P ABL - F ) and fully-phosphorylated (P PHOSPHO - ABL ) biosensors) were included in the assay. After incubation with whole cell lysates, average P ABL phosphorylation was significantly increased (basal vs. P ABL phosphorylation: 6.84 ± 1.46% vs. 32.44 ± 3.25%, p -value < 0.0001, two-way ANOVA, Bonferroni post-test, percentages relative to P PHOSPHO - ABL in each cell line). Cell lines expressing ABL1-chimeric proteins (K562, ALL-SIL) presented the higher TK activity on P ABL ; a lower signal was instead observed for NALM6 and REH ( p < 0.001 and p < 0.05 vs. K562, respectively). Phosphorylation was ABL1-mediated, as demonstrated by the specific inhibition of imatinib ( p < 0.001 for K562, NALM6, ALL-SIL and p < 0.01 for REH) in contrast to ruxolitinib (JAK2-inhibitor), and occurred on the ABL1 Y-site, as demonstrated by P ABL-F whose phosphorylation was comparable to basal levels. In order to validate this novel P ABL -ELISA assay on leukemic cells isolated from patient's bone marrow aspirates, preliminary analysis on blasts derived from an adult affected by chronic myeloid leukaemia ( BCR-ABL1 positive) and a child affected by ALL ( BCR-ABL1 negative) were performed. Phosphorylation of P ABL was specifically inhibited after the incubation of BCR-ABL1 positive cell lysates with imatinib, but not with ruxolitinib. While requiring further optimization and validation in leukemic blasts to be of clinical interest, the P ABL -based ELISA assay provides a novel in vitro tool for screening both the aberrant ABL1 activity in BCR-ABL1 like ALL leukemic cells and their potential response to TK inhibitors., Competing Interests: 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., (Copyright © 2021 Montecchini, Braidotti, Franca, Zudeh, Boni, Sorio, Toffoletti, Rabusin, Tommasini, Decorti and Stocco.)

Infliximab is commonly used in inflammatory bowel disease (IBD), however, differences in clinical response among patients are common. Several studies have considered the possibility that these differences are caused by genetic variability even if no unique marker has been yet identified in pediatric patients. We evaluated the impact of two candidate single-nucleotide polymorphisms (SNPs) rs396991 in FCGR3A and rs1800629 in TNFα genes on infliximab response in an Italian cohort of 76 pediatric patients with IBD. Results showed that patients with the variant FCGR3A allele had a reduced clinical response at the end of induction (p value = 0.004), at 22 weeks (p value = 0.001), and at 52 weeks of treatment (p value = 0.01). A significant association between the FCGR3A variant and median infliximab levels measured during maintenance therapy was also observed: patients with wild type genotype had higher infliximab levels compared to patient with variant allele. Furthermore, patients with the variant allele had a higher probability to produce antidrug antibodies (ADAs). No association was found among the TNFα SNP, clinical response, and infliximab levels. This study addressed for the first time in pediatric patients with IBD, the association of FCGR3A SNP, infliximab response, and ADA production., (© 2021 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Introduction: Juvenile idiopathic arthritis (JIA) is a chronic disease characterized by persistent joint inflammation. JIA is the most common pediatric chronic rheumatic disease and no curative therapy is currently available. Methotrexate (MTX) is an important treatment for JIA even though a high inter-individual variability in response is observed in patients. Among the factors of this variability, genetics and epigenetics might play an important role., Areas Covered: This review summarizes the results of pharmacogenetic and pharmacoepigenetic studies regarding MTX response in JIA. Studies considering epigenetic factors in JIA patients are still very limited, therefore this review includes also studies performed in adult patients with rheumatoid arthritis. Moreover, the relevance of biomarkers measured in blood or urine of JIA patients in relation to MTX treatment is discussed., Expert Opinion: Nowadays, even though many pharmacogenomics studies have been published, a specific genetic marker predictor of MTX efficacy or adverse events has not yet been identified. Encouraging results are available and great expectations rely on the study of epigenetics. Future studies are needed in order to identify genetic and epigenetic biomarkers that can be implemented in the clinical practice.

Thiopurines, immunomodulating drugs used in the management of different chronic autoimmune conditions and as anti-leukemic agents, may exert in some cases gastrointestinal toxicity. Moreover, since these agents are administered orally, they are absorbed across the gastrointestinal tract epithelium. On these premises, cellular and molecular events occurring in intestinal cells may be important to understand thiopurine effects. However, quantitative information on the biotransformation of thiopurines in intestinal tissues is still limited. To shed light on biotransformation processes specific of the intestinal tissue, in this study thiopurine metabolites concentrations were analyzed by an in vitro model of human healthy colon, the HCEC cell line, upon exposure to cytotoxic concentrations of azathioprine or mercaptopurine; the investigation was carried out using an innovative mass spectrometry method, that allowed the simultaneous quantification of 11 mono-, di-, and triphosphate thionucleotides. Among the 11 metabolites evaluated, TIMP, TGMP, TGDP, TGTP, MeTIMP, MeTIDP and MeTITP were detectable in HCEC cells treated with azathioprine or mercaptopurine, considering two different incubation times before the addition of the drugs (4 and 48 h). Different associations between metabolites concentrations and cytotoxicity were detected. In particular, the cytotoxicity was dependent on the TGMP, TGDP, TGTP and MeTITP concentrations after the 4 h incubation before the addition of thiopurines. This may be an indication that, to study the association between thiopurine metabolite concentrations and the cytotoxicity activity in vitro, short growth times before treatment should be used. Moreover, for the first time our findings highlight the strong correlation between cytotoxicity and thiopurine pharmacokinetics in HCEC intestinal cells in vitro suggesting that these cells could be a suitable in vitro model for studying thiopurine intestinal cytotoxicity., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Natural variations in milk minerals, their relationships, and their associations with the coagulation process and cheese-making traits present an opportunity for the differentiation of milk destined for high-quality natural products, such as traditional specialties or Protected Designation of Origin (PDO) cheeses. The aim of this study was to quantify the effects of the native contents of Ca, P, Na, K, and Mg on 18 traits describing traditional milk coagulation properties (MCP), curd firming over time (CF t ) equation parameters, cheese yield (CY) measures, and nutrient recoveries in the curd (REC) using models that either included or omitted the simultaneous effects of milk fat and casein contents. The results showed that, by including milk fat and casein and the minerals in the statistical model, we were able to determine the specific effects of each mineral on coagulation and cheese-making efficiency. In general, about two-thirds of the apparent effects of the minerals on MCP and the CF t equation parameters are actually mediated by their association with milk composition, especially casein content, whereas only one-third of the effects are direct and independent of milk composition. In the case of cheese-making traits, the effects of the minerals were mediated only negligibly by their association with milk composition. High Ca content had a positive effect on the coagulation pattern and cheese-making traits, favoring water retention in the curd in particular. Phosphorus positively affected the cheese-making traits in that it was associated with an increase in CY in terms of curd solids, and in all the nutrient recovery traits. However, a very high P content in milk was associated with lower fat recovery in the curd. The variation in the Na content in milk only mildly affected coagulation, whereas with regard to cheese-making, protein recovery was negatively associated with high concentrations of this mineral. Potassium seemed not to be actively involved in coagulation and the cheese-making process. Magnesium content tended to slow coagulation and reduce CY measures. Further studies on the relationships of minerals with casein and protein fractions could deepen our knowledge of the role of all minerals in coagulation and the cheese-making process., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Interferonopathies are rare genetic conditions defined by systemic inflammatory episodes caused by innate immune system activation in the absence of pathogens. Currently, no targeted drugs are authorized for clinical use in these diseases. In this work, we studied the contribution of sulforaphane (SFN), a cruciferous-derived bioactive molecule, in the modulation of interferon-driven inflammation in an immortalized human hepatocytes (IHH) line and in two healthy volunteers, focusing on STING , a key-component player in interferon pathway, interferon signature modulation, and GSTM1 expression and genotype, which contributes to SFN metabolism and excretion. In vitro, SFN exposure reduced STING expression as well as interferon signature in the presence of the pro-inflammatory stimulus cGAMP (cGAMP 3 h vs. SFN+cGAMP 3 h p value < 0.0001; cGAMP 6 h vs. SFN+cGAMP 6 h p < 0.001, one way ANOVA), restoring STING expression to the level of unstimulated cells. In preliminary experiments on healthy volunteers, no appreciable variations in interferon signature were identified after SFN assumption, while only in one of them, presenting the GSTM1 wild type genotype related to reduced SFN excretion, could a downregulation of STING be recorded. This study confirmed that SFN inhibits STING -mediated inflammation and interferon-stimulated genes expression in vitro. However, only a trend towards the downregulation of STING could be reproduced in vivo. Results obtained have to be confirmed in a larger group of healthy individuals and in patients with type I interferonopathies to define if the assumption of SFN could be useful as supportive therapy.

Although the use of glucocorticoids (GC) is well established, the therapeutic response to these agents often shows important interindividual differences, in particular among young patients with inflammatory bowel diseases (IBD). Currently, GC resistance or dependence cannot be predicted by clinical or laboratory findings. The aim of this study was to investigate the association of gender and age with GC efficacy and with the expression of Glucocorticoid-Induced Leucine Zipper (GILZ). One hundred thirty patients (mean age at enrolment 12.6 years, 53 Crohn's disease, 70 males) were enrolled in this retrospective study. IBD patients with active disease despite prednisone at a daily dose of up to 2 mg/kg over a period of 4 weeks were defined as steroid resistant. Patients who initially responded but relapsed upon dose reduction were considered steroid-dependent. Total RNA was extracted from biopsies of 14 patients (9 males) and the levels of GILZ mRNA were evaluated by real-time PCR. Association between clinical response to prednisone and the considered demographic variables was evaluated using logistic regression models. After 4 weeks of treatment, 112 patients were responders to prednisone and 18 were resistant; at this time-point, resistant patients were older than responders (p=0.032). After 12 weeks, 42, 71 and 12 patients were sensitive, dependent and resistant respectively; at this time-point, females were more prone than males to develop prednisone dependence vs a good response (p=0.028) while age had no effect. Age was associated with response both at 4 and 12 weeks in the subgroups of females: resistant patients were older than sensitive ones at 4 weeks (p=0.02). Likewise, at 12 weeks of therapy, dependent patients resulted older than sensitive ones (p=0.05). No association of age with prednisone response was found in males. In a subgroup of 14 patients (5 females), GILZ mRNA expression in intestinal biopsies was higher in males (p=0.0031). Patients with unfavorable response (7) presented lower GILZ expression at disease onset in comparison to the responder group (p=0.017). Older females with IBD have a higher incidence of prednisone unfavorable response and reduced intestinal expression of the GC pharmacodynamic marker GILZ., Competing Interests: 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., (Copyright © 2021 Lucafò, Bramuzzo, Selvestrel, Da Lozzo, Decorti and Stocco.)

Introduction: Adalimumab is effective in inducing and maintaining remission in children with inflammatory bowel diseases (IBD). Therapeutic drug monitoring is an important strategy to maximize the response rates, but data on the association of serum adalimumab levels are lacking. This study aimed to assess the association of adalimumab concentrations at the end of induction and early during maintenance for long-term response. Materials and Methods: Serum samples for adalimumab level measurement were collected during routine visits between adalimumab administrations and therefore not necessarily at trough, both during the induction (week 4 ± 4) and maintenance phases (week 22 ± 4, 52 ± 4, and 82 ± 4). Adalimumab and anti-adalimumab antibodies were measured retrospectively using enzyme-linked immunosorbent assays (ELISA). Disease activity was determined by Pediatric Crohn's Disease Activity Index or Pediatric Ulcerative Colitis Activity Index. Results: Thirty-two children (median age 14.9 years) were enrolled. Sixteen, 15, 14, and 12 patients were in remission at weeks 4, 22, 52, and 82, respectively. Median adalimumab concentration was higher at all time points in patients achieving sustained clinical remission. Adalimumab levels correlated with clinical and biochemical variables. Adalimumab concentration above 13.85 and 7.54 μg/ml at weeks 4 and 22 was associated with remission at weeks 52 and 82. Conclusions: Adalimumab non-trough levels are associated with long-term response in pediatric patients with IBD., Competing Interests: 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., (Copyright © 2021 Lucafò, Curci, Bramuzzo, Alvisi, Martelossi, Silvestri, Guastalla, Labriola, Stocco and Decorti.)

To assess whether NLRP3 gene promoter methylation was able to discriminate glucocorticoid (GC)-resistant from GC-sensitive idiopathic nephrotic syndrome (INS), patients with minimal change disease (MCD) or focal segmental glomerulosclerosis (FSGS), we measured the methylation level of NLRP3 promoter in DNA from peripheral blood cells of 10 adult patients with GC-resistant FSGS already in hemodialysis and 18 patients with GC-sensitive INS (13 MCD/5 FSGS) and in 21 pediatric patients with INS with MCD/FSGS before starting any treatment. Association of NLRP3 inflammasome with GC resistance was recapitulated in vitro in monocytic cell lines (THP-1 and U937). In both adults and pediatric patients, NLRP3 promoter methylation was significantly reduced in GC-resistant compared with GC-sensitive patients. Indeed, NLRP3 methylation distinguished GC-resistant and GC-sensitive patients (area under the receiver operating characteristic curve [AUROC] 86.7% in adults, p = 0.00019, and 73.5% in children, p = 0.00097). NLRP3 knock-down augmented sensitivity to GCs in THP-1 cells, whereas NLRP3 inflammasome activation lowered GC receptor concentration, increasing GC resistance in U937 cells. Our results uncovered a new biological mechanism by which patients with INS may acquire GC resistance, that could be used in future as a novel noninvasive diagnostic tool. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? ☑ Approximately 80% of patients with idiopathic nephrotic syndrome (INS) respond to glucocorticoids, with the remaining 20% being steroid-resistant. WHAT QUESTION DID THIS STUDY ADDRESS? ☑ Whether NLRP3 gene promoter methylation was able to discriminate glucocorticoid-resistant from glucocorticoid (GC)-sensitive INS. WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE? ☑ In both adults and children, NLRP3 promoter methylation was significantly reduced in leukocytes of patients with GC-resistant compared with GC-sensitive INS. NLRP3 inflammasome activation lowered GC receptor concentration and augmented GC resistance, whereas NLRP3 knockdown increased sensitivity to GCs in cell lines representative of monocytes (U937 and THP1). HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE? ☑ Our findings uncovered a new biological mechanism whereby patients with INS may develop resistance to GCs that could be used in the future as a novel noninvasive diagnostic tool., (© 2020 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of the American Society for Clinical Pharmacology and Therapeutics.)

The prediction of traditional goat milk coagulation properties (MCP) and curd firmness over time (CF t ) parameters via Fourier-transform infrared (FTIR) spectroscopy can be of significant economic interest to the dairy industry and can contribute to the breeding objectives for the genetic improvement of dairy goat breeds. Therefore, the aims of this study were to (1) explore the variability of milk FTIR spectra from 4 goat breeds (Camosciata delle Alpi, Murciano-Granadina, Maltese, and Sarda), and to assess the possible discriminant power of milk FTIR spectra among breeds, (2) assess the viability to predict coagulation traits by using milk FTIR spectra, and (3) quantify the effect of the breed on the prediction accuracy of MCP and CF t parameters. In total, 611 individual goat milk samples were used. Analysis of variance of measured MCP and CF t parameters was carried out using a mixed model including the farm and pendulum as random factors, and breed, parity, and days in milk as fixed factors. Milk spectra for each goat were collected over the spectral range from wavenumber 5,011 to 925 × cm -1 . Discriminant analysis of principal components was used to assess the ability of FTIR spectra to identify breed of origin. A Bayesian model was used to calibrate equations for each coagulation trait. The accuracy of the model and the prediction equation was assessed by cross-validation (CRV; 80% training and 20% testing set) and stratified CRV (SCV; 3 breeds in the training set, one breed in the testing set) procedures. Prediction accuracy was assessed by using coefficient of determination of validation (R 2 VAL ), the root mean square error of validation (RMSE VAL ), and the ratio performance deviation. Moreover, measured and FTIR predicted traits were compared in the SCV procedure by assessing their least squares means for the breed effect, Pearson correlations, and variance heteroscedasticity. Results showed the feasibility of using FTIR spectra and multivariate analyses to correctly assign milk samples to their breeds of origin. The R 2 VAL values obtained with the CRV procedure were moderate to high for the majority of coagulation traits, with RMSE VAL and ratio performance deviation values increasing as the coagulation process progresses from rennet addition. Prediction accuracy obtained with the SCV were strongly influenced by the breed, presenting general low values restricting a practical application. In addition, the low Pearson correlation coefficients of Sarda breed for all the traits analyzed, and the heteroscedastic variances of Camosciata delle Alpi, Murciano-Granadina, and Maltese breeds, further indicated that it is fundamental to consider the differences existing among breeds for the prediction of milk coagulation traits., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Driven by the large amount of goat milk destined for cheese production, and to pioneer the goat cheese industry, the objective of this study was to assess the effect of farm in predicting goat milk-coagulation and curd-firmness traits via Fourier-transform infrared spectroscopy. Spectra from 452 Sarda goats belonging to 14 farms in central and southeast Sardinia (Italy) were collected. A Bayesian linear regression model was used, estimating all spectral wavelengths' effects simultaneously. Three traditional milk-coagulation properties [rennet coagulation time (min), time to curd firmness of 20 mm (min), and curd firmness 30 min after rennet addition (mm)] and 3 curd-firmness measures modeled over time [rennet coagulation time estimated according to curd firmness change over time (RCT eq ), instant curd-firming rate constant, and asymptotical curd firmness] were considered. A stratified cross validation (SCV) was assigned, evaluating each farm separately (validation set; VAL) and keeping the remaining farms to train (calibration set) the statistical model. Moreover, a SCV, where 20% of the goats randomly taken (10 replicates per farm) from the VAL farm entered the calibration set, was also considered (SCV 80 ). To assess model performance, coefficient of determination (R 2 VAL ) and the root mean squared error of validation were recorded. The R 2 VAL varied between 0.14 and 0.45 (instant curd-firming rate constant and RCT eq , respectively), albeit the standard deviation was approximating half of the mean for all the traits. Although average results of the 2 SCV procedures were similar, in SCV 80 , the maximum R 2 VAL increased at about 15% across traits, with the highest observed for time to curd firmness of 20 mm (20%) and the lowest for RCT eq (6%). Further investigation evidenced important variability among farms, with R 2 VAL for some of them being close to 0. Our work outlined the importance of considering the effect of farm when developing Fourier-transform infrared spectroscopy prediction equations for coagulation and curd-firmness traits in goats., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

A mutualistic relationship between the composition, function and activity of the gut microbiota (GM) and the host exists, and the alteration of GM, sometimes referred as dysbiosis, is involved in various immune-mediated diseases, including inflammatory bowel disease (IBD). Accumulating evidence suggests that the GM is able to influence the efficacy of the pharmacological therapy of IBD and to predict whether individuals will respond to treatment. Additionally, the drugs used to treat IBD can modualate the microbial composition. The review aims to investigate the impact of the GM on the pharmacological therapy of IBD and vice versa. The GM resulted in an increase or decrease in therapeutic responses to treatment, but also to biotransform drugs to toxic metabolites. In particular, the baseline GM composition can help to predict if patients will respond to the IBD treatment with biologic drugs. On the other hand, drugs can affect the GM by incrementing or reducing its diversity and richness. Therefore, the relationship between the GM and drugs used in the treatment of IBD can be either beneficial or disadvantageous.

Intense SERS spectra of the natural amino acid ergothioneine (ERG) are obtained on different substrates upon 785 nm excitation. A characteristic spectral pattern with a distinctive intense band at 480-486 cm -1 is conserved when substrates of different type and characteristics are used. On the basis of available literature, we propose ERG is adsorbed on the metal surface in its thiolate form via the sulphur and heterocyclic nitrogen. The same spectral pattern is obtained in SERS spectra of filtered erythrocytes lysates, confirming the presence of ERG in those cells. The occurrence of ERG bands in label-free SERS spectra of serum and plasma reported in literature by different authors is discussed, highlighting the importance of this amino acid for the interpretation of SERS spectra of these biofluids., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Different fractions of milk nitrogenous compounds (not only caseins) have different effects on the nutritional value of milk, its coagulation and curd firming properties, and its cheese-making efficiency. To assess different sources of variation, especially the cows' breed and genetic variants of the main protein fractions, milk samples were collected from 1,504 cows belonging to 3 dairy breeds (Holstein-Friesian, Brown Swiss, and Jersey) and 3 dual-purpose breeds (Simmental, Rendena, and Alpine Grey) reared in 41 multibreed herds. Beyond crude protein, casein (CN), and urea, 7 protein fractions were analyzed using HPLC, and 5 other N fraction traits were calculated. All 15 traits were measured qualitatively (% of milk N) and quantitatively (g/L of milk). The HPLC technique allowed us to discriminate between the main genetic variants of β-CN, κ-CN, and β-lactoglobulin and thus to genotype the cows for the CSN2, CSN3, and BLG genes, respectively. Data were analyzed using 2 mixed models, both including the effects of herd-date, breed, parity, and lactation stage, and only one also including the effects of the genotypes of the milk proteins. Breed of cow explained 2 to 36% of phenotypic variability for all the N fractions, with the exception of the urea and total casein contents of milk and the urea and β-CN proportions of total milk N. Lactation stage had a considerable influence on the amount (g/L) of almost all the protein fractions in milk, but neither the nonprotein N fractions nor the percentage of milk N protein profile were affected. The inclusion of the CSN2, CSN3, and BLG genotypes in the model explained a large part of the total variability in all the milk protein and nonprotein fractions except urea. It also reduced the variance explained by breed and residual factors. An exception was shown by the proportion of α S1 -CN variance explained by breed that moved from 13 to 28%. Similarly, for amount (g/L) of β-CN, the effect of breed became significant (12%), whereas it was almost null before inclusion of genotypes. In terms of percentage of milk N, the genotypes of CSN3 notably affected all the casein fractions, whereas the BLG genotypes had a much greater influence on most noncasein traits. The genotypes of the CSN2 gene exerted an appreciable effect on α S2 -CN and not β-CN, as expected. Comparing the 2 models, we were also able to discriminate the effect of the breed on a milk N fraction, both quantitatively and qualitatively, in 2 quotas: the first due to the milk protein polymorphisms (major genes) and the second due to other genetic factors (polygene), after correcting for the effect of herd-date of sampling, parity, and lactation stage. The knowledge about the detailed milk protein profile of different cattle breeds provided by this study could be of great benefit for the dairy industry, providing new tools for the enhancement of milk payment systems and breeding program designs., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Glucocorticoids (GCs) are commonly used as therapeutic agents for immune-mediated diseases and leukemia. However, considerable inter-individual differences in efficacy have been reported. Several reports indicate that the inhibitor of mTOR rapamycin can reverse GC resistance, but the molecular mechanism involved in this synergistic effect has not been fully defined. In this context, we explored the differential miRNA expression in a GC-resistant CCRF-CEM cell line after treatment with rapamycin alone or in co-treatment with methylprednisolone (MP). The expression analysis identified 70, 99 and 96 miRNAs that were differentially expressed after treatment with MP, rapamycin and their combination compared to non-treated controls, respectively. Two pathways were exclusively altered as a result of the co-treatment: the MAPK and ErbB pathways. We validated the only miRNA upregulated specifically by the co-treatment associated with the MAPK signaling, miR-331-3p. Looking for miR-331-3p targets, MAP2K7, an essential component of the JNK/MAPK pathway, was identified. Interestingly, MAP2K7 expression was downregulated during the co-treatment, causing a decrease in terms of JNK activity. miR-331-3p in mimic-transfected cells led to a significant decrease in MAP2K7 levels and promoted the reversion of GC resistance in vitro. Interestingly, miR-331-3p expression was also associated with GC-resistance in patient leukemia cells taken at diagnosis. The combination of rapamycin with MP restores GC effectiveness through the regulation of different miRNAs, suggesting the important role of these pharmacoepigenetic factors in GC response.

The marine polyether palytoxin (PLTX) is one of the most toxic natural compounds, and is involved in human poisonings after oral, inhalation, skin and/or ocular exposure. Epidemiological and molecular evidence suggest different inter-individual sensitivities to its toxic effects, possibly related to genetic-dependent differences in the expression of Na + /K + -ATPase, its molecular target. To identify Na + /K + -ATPase subunits, isoforms correlated with in vitro PLTX cytotoxic potency, sensitivity parameters (EC 50 : PLTX concentration reducing cell viability by 50%; Emax: maximum effect induced by the highest toxin concentration; 10 -7 M) were assessed in 60 healthy donors' monocytes by the MTT (methylthiazolyl tetrazolium) assay. Sensitivity parameters, not correlated with donors' demographic variables (gender, age and blood group), demonstrated a high inter-individual variability (median EC 50 = 2.7 × 10 -10 M, interquartile range: 0.4-13.2 × 10 -10 M; median Emax = 92.0%, interquartile range: 87.5-94.4%). Spearman's analysis showed significant positive correlations between the β2-encoding ATP1B2 gene expression and Emax values (rho = 0.30; p = 0.025) and between Emax and the ATP1B2/ATP1B3 expression ratio (rho = 0.38; p = 0.004), as well as a significant negative correlation between Emax and the ATP1B1/ATP1B2 expression ratio (rho = -0.30; p = 0.026). This toxicogenetic study represents the first approach to define genetic risk factors that may influence the onset of adverse effects in human PLTX poisonings, suggesting that individuals with high gene expression pattern of the Na + /K + -ATPase β2 subunit (alone or as β2/β1 and/or β2/β3 ratio) could be highly sensitive to PLTX toxic effects.

The aims of this study were to explore the variability of milk composition, coagulation properties, and cheese-making traits of the Sarda goat breed, and to investigate the effects of animal and farm factors, and the geographic area (Central-East vs. South-West) of an insular region of Italy, Sardinia. A total of 570 Sarda goats reared in 21 farms were milk-sampled during morning milking. Individual milk samples were analyzed for composition, traditional milk coagulation properties (MCP), modeled curd-firming over time parameters (CF t ), and cheese-making traits (cheese yield, %CY; recovery of nutrients, %REC; daily cheese yield, dCY). Farms were classified into 2 categories based on milk energy level (MEL; high or low), defined according to the average net energy of milk daily produced by the lactating goats. Milk yield and composition were analyzed using a mixed model including the fixed effects of MEL, geographic area, days in milk, and parity, and the random effect of farm within MEL and geographic area. Data about MCP, CF t , and the cheese-making process were analyzed using the same model, with the inclusion of the effects of animal and pendulum of the lactodynamograph instrument, allowing the measure of repeatability of these traits. Results showed that animal had greater influence on coagulation and cheese-making traits compared with farm effect. Days in milk influenced milk composition, whose changes partly reflected the modifications of %CY traits. Moreover, large differences were observed between primiparous and multiparous goats: primiparous goats produced less milk of better quality (higher fat, lower somatic cell and bacterial counts) and less cheese, but with higher recovery of fat and protein in the curd, compared with multiparous goats. The repeatability was very high, for both coagulation (84.0 to 98.8%) and cheese-making traits (89.7 to 99.9%). The effect of MEL was significant for daily productions of milk and cheese, coagulation time, and recovery of protein in the curd, which were better in high-MEL farms. As regards geographic area, milk composition and percentage cheese yield were superior in the Central-East area, whereas daily milk and cheese production and MCP were better in the South-West. This result was explainable by the phenomenon of crossbreeding Sarda goats with Maltese bucks, which occurred with greater intensity in the South-West than in the Central-East area of the island. The results provided by this study could be of great interest for the goat dairy sector. Indeed, the methods described in the present study could be applicable for other farming methods, goat breeds, and geographic areas. The collection of a wide range of phenotypes at individual animal level is fundamental for the characterization of local populations and can be used to guarantee breed conservation and the persistence of traditional farming systems, and to increase farmers' profit., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Pharmacogenomics is one of the emerging approaches to precision medicine, tailoring drug selection and dosing to the patient's genetic features. In recent years, several pharmacogenetic guidelines have been published by international scientific consortia, but the uptake in clinical practice is still poor. Many coordinated international efforts are ongoing in order to overcome the existing barriers to pharmacogenomic implementation. On the other hand, existing validated pharmacogenomic markers can explain only a minor part of the observed clinical variability in the therapeutic outcome. New investigational approaches are warranted, including the study of the pharmacogenomic role of the immune system genetics and of previously neglected rare genetic variants, reported to account for a large part of the inter-individual variability in drug metabolism. In this Special Issue, we collected a series of articles covering many aspects of pharmacogenomics. These include clinical implementation of pharmacogenomics in clinical practice, development of tools or infrastractures to support this process, research of new pharmacogenomics markers to increase drug efficacy and safety, and the impact of rare genetic variants in pharmacogenomics.

Introduction : Natural and synthetic glucocorticoids are widely employed in different diseases, among which are hematological and solid tumors. Their use is however associated with a number of serious side effects and by the occurrence of resistance. With the aim of separating their gene transactivating effect, more linked to side effects, from transrepressive properties, associated with therapeutic efficacy, a number of selective glucocorticoid modulators have been identified. Areas covered : This review summarizes the patent applications from 2014 to present in the field of selective glucocorticoid receptor modulators employed in cancer therapy. Only few patents have been identified, that concern the identification of new molecules or the method of use of already patented compounds. In addition, a discussion of the mechanism of action of these compounds is included. Expert opinion : Only a very limited number of patents have been applied that concern selective glucocorticoid receptor modulators and their use in cancer. Biological information is scarce for most of these patents; more research is necessary in this field in particular concerning clinical data in order to understand whether it is actually possible to improve the efficacy and therapeutic index of these compounds in cancer therapy.

Recent available instruments allow to record the number of differential somatic cell count (DSCC), representing the combined proportion of polymorphonuclear leukocytes and lymphocytes, on a large number of milk samples. Milk DSCC provides indirect information on the udder health status of dairy cows. However, literature is limited regarding the effect of DSCC on milk composition at the individual cow level, as well as its relation to the somatic cell score (SCS). Hence, the aims of this study were to (i) investigate the effect of different levels of DSCC on milk composition (fat, protein, casein, casein index, and lactose) and (ii) explore the combined effect of DSCC and SCS on these traits. Statistical models included the fixed effects of days in milk, parity, SCS, DSCC and the interaction between SCS × DSCC, and the random effects of herd, animal within parity, and repeated measurements within cow. Results evidenced a decrease of milk fat and an increase in milk fatty acids at increasing DSCC levels, while protein, casein and their proportion showed their lowest values at the highest DSCC. A positive association was found between DSCC and lactose. The interaction between SCS and DSCC was important for lactose and casein index, as they varied differently upon high and low SCS and according to DSCC levels.

Extracellular nicotinamide phosphoribosyltransferase (eNAMPT) is increased in inflammatory bowel disease (IBD) patients, and its serum levels correlate with a worse prognosis. In the present manuscript, we show that eNAMPT serum levels are increased in IBD patients that fail to respond to anti-TNFα therapy (infliximab or adalimumab) and that its levels drop in patients that are responsive to these therapies, with values comparable with healthy subjects. Furthermore, eNAMPT administration in dinitrobenzene sulfonic acid (DNBS)-treated mice exacerbates the symptoms of colitis, suggesting a causative role of this protein in IBD. To determine the druggability of this cytokine, we developed a novel monoclonal antibody (C269) that neutralizes in vitro the cytokine-like action of eNAMPT and that reduces its serum levels in rodents. Of note, this newly generated antibody is able to significantly reduce acute and chronic colitis in both DNBS- and dextran sulfate sodium (DSS)-induced colitis. Importantly, C269 ameliorates the symptoms by reducing pro-inflammatory cytokines. Specifically, in the lamina propria, a reduced number of inflammatory monocytes, neutrophils, Th1, and cytotoxic T lymphocytes are found upon C269 treatment. Our data demonstrate that eNAMPT participates in IBD and, more importantly, that eNAMPT-neutralizing antibodies are endowed with a therapeutic potential in IBD. KEY MESSAGES: What are the new findings? Higher serum eNAMPT levels in IBD patients might decrease response to anti-TNF therapy. The cytokine-like activity of eNAMPT may be neutralized with a monoclonal antibody. Neutralization of eNAMPT ameliorates acute and chronic experimental colitis. Neutralization of eNAMPT limits the expression of IBD inflammatory signature. Neutralization of eNAMPT impairs immune cell infiltration in lamina propria.

The aims of the present research were to quantify the effects of each coagulation trait, traditional milk coagulation properties [MCP: rennet coagulation time (RCT), curd-firming time (k 20 ), and curd firmness at 30 min (a 30 )], and modeled curd-firming over time (CF t ) parameters [estimated rennet coagulation time (RCT eq ), curd-firming instant rate constant (k CF ), and potential curd firmness (CF P )] directly on the following: (1) recovery of 3 milk components in the curd (%REC), (2) 3 measures of cheese yield (%CY), and (3) 3 daily cheese yield traits (dCY) from goat milk. Cheese-making traits were analyzed using 2 mixed different models, the first to test MCP and the second to test CF t parameters. Pearson correlations were also calculated. Significant and favorable relationships (negative for time intervals and positive for CF measures) were found between the traditional MCP and the CF t parameters and %REC and %CY traits. The effects of milk fat and protein contents were particularly important on all cheese-making traits, with the only exception being the effect of fat content on water retention in cheese (%CY WATER ). We found an optimum value of milk k 20 , associated with the highest recovery of components and cheese yield in solids (%CY SOLIDS ). In addition, a lower level of curd water retention and an increased fresh curd yield (%CY CURD ) were associated with greater recovery of fat. The collection of all available information during the process of milk coagulation and curd-firming allowed us to discover the effect of RCT eq on %REC traits and %CY SOLIDS , which had not previously been revealed for traditional RCT. Moreover, higher k CF values were associated with increased %CY CURD and %CY SOLIDS . Given that CF t parameters showed a high level of independence from one another, these can also be easily used and characterized in future applications at the industry level. Information provided by traditional and modeled coagulation properties could efficiently support the goat dairy industry and lay the foundations for a quality payment scheme for goat milk., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Adverse drug reactions (ADRs) are major clinical problems, particularly in special populations such as pediatric patients. Indeed, ADRs may be caused by a plethora of different drugs leading, in some cases, to hospitalization, disability or even death. In addition, pediatric patients may respond differently to drugs with respect to adults and may be prone to developing different kinds of ADRs, leading, in some cases, to more severe consequences. To improve the comprehension, and thus the prevention, of ADRs, the set-up of sensitive and personalized assays is urgently needed. Important progress is represented by the possibility of setting up groundbreaking patient-specific assays. This goal has been powerfully achieved using induced pluripotent stem cells (iPSCs). Due to their genetic and physiological species-specific differences and their ability to be differentiated ideally into all tissues of the human body, this model may be accurate in predicting drug toxicity, especially when this toxicity is related to individual genetic differences. This review is an up-to-date summary of the employment of iPSCs as a model to study ADRs, with particular attention to drugs used in the pediatric field. We especially focused on the intestinal, hepatic, pancreatic, renal, cardiac, and neuronal levels, also discussing progress in organoids creation. The latter are three-dimensional in vitro culture systems derived from pluripotent or adult stem cells simulating the architecture and functionality of native organs such as the intestine, liver, pancreas, kidney, heart, and brain. Based on the existing knowledge, these models are powerful and promising tools in multiple clinical applications including toxicity screening, disease modeling, personalized and regenerative medicine., Competing Interests: Conflict-of-interest statement: Authors of this manuscript have no conflicts of interest to disclose., (©The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved.)

Patients with ulcerative colitis (UC) using marijuana have been reported to experience symptomatic benefit. Cannabidivarin (CBDV) is a safe non-psychoactive phytocannabinoid able to activate and desensitize TRPA1, a member of the TRP channels superfamily, which plays a pivotal role in intestinal inflammation. Here, we have investigated the potential intestinal anti-inflammatory effect of CBDV in mice and in biopsies from pediatric patients with active UC. Colonic inflammation was induced in mice by dinitrobenzenesulfonic acid (DNBS). The effect of orally administered CBDV on macroscopic and microscopic damage, inflammatory parameters (i.e. myeloperoxidase activity, intestinal permeability and cytokine production) and faecal microbiota composition, was evaluated 3 days after DNBS administration. TRPA1 expression was studied by RT-PCR in inflamed colons of mice as well as in mucosal colonic biopsies of children with active UC, whose response to incubation with CBDV was also investigated. CBDV attenuates, in a TRPA1-antagonist sensitive manner, DNBS-induced signs of inflammation including neutrophil infiltration, intestinal permeability, and cytokine (i.e. IL-1β, IL-6 and the chemokine MCP-1) production. CBDV also alters the dysregulation of gut microbiota associated to colitis. Finally, CBDV lessens cytokine expression in colonic biopsies from pediatric patients with ulcerative colitis, a condition in which TRPA1 was up-regulated. Our preclinical study shows that CBDV exerts intestinal anti-inflammatory effects in mice via TRPA1, and in children with active UC. Since CBDV has a favorable safety profile in humans, it may be considered for possible clinical trials in patients with UC., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Near-infrared spectroscopy (NIRS) has been widely used to determine various composition traits of many dairy products in the industry. In the last few years, near-infrared (NIR) instruments have become more and more accessible, and now, portable devices can be easily used in the field, allowing the direct measurement of important quality traits. However, the comparison of the predictive performances of different NIR instruments is not simple, and the literature is lacking. These instruments may use different wavelength intervals and calibration procedures, making it difficult to establish whether differences are due to the spectral interval, the chemometric approach, or the instrument's technology. Hence, the aims of this study were (1) to evaluate the prediction accuracy of chemical contents (5 traits), pH, texture (2 traits), and color (5 traits) of 37 categories of cheese; (2) to compare 3 instruments [2 benchtop, working in reflectance (R) and transmittance (T) mode (NIRS-R and NIRS-T, respectively) and 1 portable device (VisNIRS-R)], using their entire spectral ranges (1100-2498, 850-1048, and 350-1830 nm, respectively, for NIRS-R, NIRS-T and VisNIRS-R); (3) to examine different wavelength intervals of the spectrum within instrument, comparing also the common intervals among the 3 instruments; and (4) to determine the presence of bias in predicted traits for specific cheese categories. A Bayesian approach was used to develop 8 calibration models for each of 13 traits. This study confirmed that NIR spectroscopy can be used to predict the chemical composition of a large number of different cheeses, whereas pH and texture traits were poorly predicted. Color showed variable predictability, according to the trait considered, the instrument used, and, within instrument, according to the wavelength intervals. The predictive performance of the VisNIRS-R portable device was generally better than the 2 laboratory NIRS instruments, whether with the entire spectrum or selected intervals. The VisNIRS-R was found suitable for analyzing chemical composition in real time, without the need for sample uptake and processing. Our results also indicated that instrument technology is much more important than the NIR spectral range for accurate prediction equations, but the visible range is useful when predicting color traits, other than lightness. Specifically for certain categories (i.e., caprine, moldy, and fresh cheeses), dedicated calibrations seem to be needed to obtain unbiased and more accurate results., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Background: The long non-coding RNA (lncRNA) growth arrest-specific transcript 5 ( GAS5 ) seems to be involved in the regulation of mediators of tissue injury, in particular matrix metalloproteinases (MMPs), implicated in the pathogenesis of inflammatory bowel disease (IBD). We investigated the role of GAS5 in regulating MMP2 and MMP9 expression in pediatric patients with IBD and in vitro., Methods: In total, 25 IBD patients were enrolled: For each patient paired inflamed and non-inflamed biopsies were collected. RNA was extracted and GAS5 , MMP2 , and MMP9 were quantified by TaqMan assay. The expression of GAS5 and MMPs was also determined in the human monocytic THP1 cells differentiated into macrophages and stimulated with lipopolysaccharide (LPS). The function of GAS5 was assessed by overexpressing the lncRNA and evaluating the MMPs levels., Results: Real-time PCR results demonstrated a downregulation of GAS5 and an upregulation of both MMPs in inflamed tissues. In vitro data confirmed the trend observed in patients for the three genes: The stimulation with LPS promoted a downregulation of GAS5 while an increase of MMPs was observed. Overexpression experiments showed that higher levels of GAS5 lead to a decrease of both enzymes., Conclusion: These results provide new information about the role of GAS5 in IBD: The lncRNA could mediate tissue damage by modulating the expression of MMPs.

Background: Intestinal inflammation and epithelial injury are the leading actors of inflammatory bowel disease (IBD), causing an excessive pro-inflammatory cytokines expression. Tristetraprolin (TTP), an mRNA binding protein, plays a role in regulating the inflammatory factors, recognizing specific sequences on the 3' untranslated region of cytokine mRNAs. TTP activity depends on its phosphorylation state: the unphosphorylated TTP degrades pro-inflammatory cytokine mRNAs; on the contrary, the phosphorylated TTP fails to destabilize mRNAs furthering their expression. The phospho-TTP forms a complex with the chaperone protein 14-3-3. This binding could be one of the factors that promote intestinal inflammation as a cause of disease progression., Aim: To assess if TTP phosphorylation has a role in paediatric IBD., Methods: The study was carried out on a cohort of paediatric IBD patients. For each patient enrolled, a specimen of inflamed and non-inflamed colonic mucosa was collected. Furthermore, the experiments were conducted on macrophages differentiated from blood samples of the same patients. Macrophages from healthy donors' blood were used as controls. Co-immunoprecipitation assay and immunoblotting analyses were performed to observe the formation of the phospho-TTP/14-3-3 complex. In the same samples TNF-α expression was also evaluated as major factor of the pro-inflammatory activity., Results: In this work we studied indirectly the phosphorylation of TTP through the binding with the chaperone protein 14-3-3. In inflamed and non-inflamed colon mucosa of IBD paediatric patients immunoblot assay demonstrated a higher expression of the TTP in inflamed samples respect to the non-inflamed; the co-immunoprecipitated 14-3-3 protein showed the same trend of expression. In the TNF-α gene expression analysis higher levels of the cytokine in inflamed tissues compared to controls were evident. The same experiments were conducted on macrophages from IBD paediatric patients and healthy controls. The immunoblot results demonstrated a high expression of both TTP and co-immunoprecipitated 14-4-3 protein in IBD-derived macrophages in comparison to healthy donors. TNF-α protein levels from macrophages lysates showed the same trend of expression in favour of IBD paediatric patients compared to healthy controls., Conclusion: In this work, for the first time, we describe a relation between phospho-TTP/14-3-3 complex and IBD. Indeed, a higher expression of TTP/14-3-3 was recorded in IBD samples in comparison to controls., Competing Interests: Conflict-of-interest statement: The authors declare no competing financial, personal or professional conflicts of interest., (©The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved.)

In dairy goats, very little is known about the effect of the 2 most important indirect indicators of udder health [somatic cell count (SCC) and total bacterial count (TBC)] on milk composition and cheese yield, and no information is available regarding the effects of lactose levels, pH, and NaCl content on the recovery of nutrients in the curd, cheese yield traits, and daily cheese yields. Because large differences exist among dairy species, conclusions from the most studied species (i.e., bovine) cannot be drawn for all types of dairy-producing animals. The aims of this study were to quantify, using milk samples from 560 dairy goats, the contemporary effects of a pool of udder health indirect indicators (lactose level, pH, SCC, TBC, and NaCl content) on the recovery of nutrients in the curd (%REC), cheese yield (%CY), and daily cheese yields (dCY). Cheese-making traits were analyzed using a mixed model, with parity, days in milk (DIM), lactose level, pH, SCC, TBC, and NaCl content as fixed effects, and farm, breed, glass tube, and animal as random effects. Results indicated that high levels of milk lactose were associated with reduced total solids recovery in the curd and lower cheese yields, because of the lower milk fat and protein contents in samples rich in lactose. Higher pH correlated with higher recovery of nutrients in the curd and higher cheese yield traits. These results may be explained by the positive correlation between pH and milk fat, protein, and casein in goat milk. High SCC were associated with higher recovery of solids and energy in the curd but lower recovery of protein. The higher cheese yield obtained from milk with high SCC was due to both increased recovery of lactose in the curd and water retention. Bacterial count proved to be the least important factor affecting cheese-making traits, but it decreased daily cheese yields, suggesting that, even if below the legal limits, TBC should be considered in order to monitor flock management and avoid economic losses. The effect of NaCl content on milk composition was linked with lower recovery of all nutrients in the curd during cheese-making. In addition, high milk NaCl content led to reductions in fresh cheese yield and cheese solids. The indirect indicators of the present study significantly affected the cheese-making process. Such information should be considered, to adjust the milk-to-cheese economic value and the milk payment system., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Objectives: Therapeutic drug monitoring is becoming increasingly important in clinical decision-making in children with inflammatory bowel disease (IBD). However, enzyme-linked immunosorbent assay (ELISA) assays do not allow results to be provided in real-time. We sought to compare 2 point-of-care (POC) devices for quantification of serum infliximab concentration with 2 validated ELISA assays in children with IBD., Methods: We studied 32 serum samples from 19 children with IBD treated with infliximab. Serum samples were collected immediately before drug infusion (trough level). Infliximab was measured using 2 POC infliximab assays, Quantum Blue (POC IFX/QB) and Rida Quick (POC IFX/RQ), and 2 ELISA assays: Lisa-Tracker (used as primary reference), and Promonitor (used as second control). Intraclass correlation coefficient (ICC) was assessed for quantitative comparison. Qualitative analysis was also performed to evaluate whether POC assays would correctly classify infliximab serum according to a target window (between 3 and 7 μg/mL)., Results: ICC was 0.82 and 0.87 for POC IFX/QB and POC IFX/RQ with the primary reference ELISA assay, respectively; ICC between the 2 ELISA assays was 0.87. Classification of results according to therapeutic intervals showed good agreement between pairs of assays, with kappa of 0.67 and 0.80 for POC IFX/QB and POC IFX/RQ, respectively, with reference ELISA, and 0.81 between the 2 ELISAs. Accuracy of POC assays was better for drug levels <3 μg/mL., Conclusions: POC infliximab assays showed good agreement with traditional ELISA assays. POC devices may represent a viable option for real-time therapeutic drug monitoring in children treated with infliximab.

The assessment of the efficiency of the cheese-making process (ECMP) is crucial for the profitability of cheese-factories. A simple way to estimate the ECMP is the measure of the estimated cheese-making losses (ECL), expressed by the ratio between the concentration of each constituent in the residual whey and in the processed milk. The aim of this research was to evaluate the influence of the season and cheese factory on the efficiency of the cheese-making process in Parmigiano Reggiano cheese manufacture. The study followed the production of 288 Parmigiano Reggiano cheese on 12 batches in three commercial cheese factories. For each batch, samples of the processed milk and whey were collected. Protein, casein, and fat ECL resulted in an average of 27.01%, 0.72%, and 16.93%, respectively. Both milk crude protein and casein contents were negatively correlated with protein ECL, r = -0.141 ( p ≤ 0.05), and r = -0.223 ( p ≤ 0.001), respectively. The same parameters resulted in a negative correlation with casein ECL ( p ≤ 0.001) (r = -0.227 and -0.212, respectively). Moreover, fat ECL was correlated with worse milk coagulation properties and negatively correlated with casein content (r = -0.120; p ≤ 0.05). In conclusion, ECLs depend on both milk characteristics and season.

We investigated whether variation of the sheep Growth Hormone Receptor (GHR), Growth Hormone Releasing Hormone Receptor (GHRHR) and Insulin-Like Growth Factor 1 (IGF1) genes were associated with milk coagulation properties (MCP) in sheep. The GHR, GHRHR and IGF1 genes are part of the GH system, which is known to modulate metabolism, growth and reproduction as well as mammogenesis and galactopoiesis in dairy species. A total of 380 dairy Sarda sheep were genotyped for 36 SNPs mapping to these three genes. Traditional MCP were measured as rennet coagulation time (RCT), curd-firming time (k20) and curd firmness at 30 m (a30). Modeling of curd firming over time (CFt) was based on a 60 m lactodynamographic test, generating a total of 240 records of curd firmness (mm) for each milk sample. The model parameters obtained included: the rennet coagulation time as a result of modeling all data available (RCTeq, min); the asymptotic potential value of curd firmness (CFP, mm) at an infinite time; the CF instant rate constant (kCF, %/min); the syneresis instant rate constant (kSR, %/min); the maximum value of CF (CFmax, mm) and the time at achievement of CFmax (tmax, min). Statistical analysis revealed that variation of the GHR gene was significantly associated with RCT, kSR and CFP (P < 0.05). No other significant associations were detected. These findings may be useful for the dairy industry, as well as for selection programs.

Introduction : Medical treatment of pediatric inflammatory bowel diseases (IBD) has been greatly changed by the introduction of a number of biologic agents that are able to target various players of the immune response. In particular, monoclonal antibodies against the pro-inflammatory cytokine TNF-alpha (TNF) such as infliximab, adalimumab, and golimumab are now in the clinics both in induction and maintenance therapy, and several efforts are currently ongoing to optimize the use of these drugs in children. Areas covered : This review focuses on therapeutic drug monitoring (TDM) of anti-TNF levels and antidrug antibodies (ADAs), in IBD children. A revision of the analytical assays used for assessing anti-TNF plasma levels is also provided. Expert opinion : Although there is a consensus across studies that higher anti-TNF trough levels are associated with a better clinical outcome, and that early anti-TNF serum measurements could be predictive of long-term response, it is still not clear what the best predictive time of sampling is and what the ideal target drug plasma concentration to achieve. Indeed, there are a number of published studies, particularly in pediatric cohorts, limited by the population size analyzed and more prospective large studies are needed to examine the value of these predictive markers.

Polychemotherapeutic protocols for the treatment of pediatric acute lymphoblastic leukemia (ALL) always include thiopurines. Specific approaches vary in terms of drugs, dosages and combinations. Such therapeutic schemes, including risk-adapted intensity, have been extremely successful for children with ALL who have reached an outstanding 5-year survival of greater than 90% in developed countries. Innovative drugs such as the proteasome inhibitor bortezomib and the bi-specific T cell engager blinatumomab are available to further improve therapeutic outcomes. Nevertheless, daily oral thiopurines remain the backbone maintenance or continuation therapy. Pharmacogenetics allows the personalization of thiopurine therapy in pediatric ALL and clinical guidelines to tailor therapy on the basis of genetic variants in TPMT and NUDT15 genes are already available. Other genes of interest, such as ITPA and PACSIN2 , have been implicated in interindividual variability in thiopurines efficacy and adverse effects and need additional research to be implemented in clinical protocols. In this review we will discuss current literature and clinical guidelines available to implement pharmacogenetics for tailoring therapy with thiopurines in pediatric ALL., Competing Interests: All authors declared that there are no conflicts of interest., (© The Author(s) 2019.)

Background: Long non-coding RNA growth arrest-specific 5 ( GAS5 ) is deregulated in many cancers because of its role in cell growth arrest and apoptosis. Additionally, GAS5 interacts with glucocorticoid receptor, making it a potential pharmacotranscription marker of glucocorticoid (GC) therapy. In this study, we aimed at analysing GAS5 expression in the remission induction therapy phase of childhood acute lymphoblastic leukemia (ALL), in which GCs are mandatorily used, and to correlate it with therapy response., Methods: GAS5 expression was measured in peripheral blood mononuclear cells taken from 29 childhood ALL patients at diagnosis, on day 15 and day 33 of remission induction therapy using RT-qPCR methodology., Results: Our results have shown interindividual differences in GAS5 expression at all time points. For each ALL patient, GAS5 expression was higher on day 15 in comparison to its level at diagnosis (p<0.0005). On day 33, the level of GAS5 expression decreased in comparison with day 15 (p<0.0005), but it was still significantly higher than at diagnosis for the majority of patients (p=0.001). Patients whose number of blasts on day 8 was below 100 per μL of peripheral blood had a higher GAS5 expression at diagnosis (p=0.016), and lower ratio day 15/diagnosis (p=0.009)., Conclusions: Our results suggest that the expression level of GAS5 could be a potential marker of therapy response in remission induction therapy of childhood ALL., Competing Interests: Conflict of interest Conflict of interest statement: The authors stated that they have no conflicts of interest regarding the publication of this article.