Effect of using mycotoxin-detoxifying agents in dairy cattle feed on natural whey starter biodiversity.

Natural whey cultures (NWC) are undefined multiple-strain bacterial starter communities that can be affected by even small changes along the entire dairy chain. We applied a multidisciplinary approach to investigate how the addition of 2 mycotoxin-detoxifying agents [sodium smectite and lignocellulose-based material (B1); leonardite and betaine (B2)] to cow diets modified the microbiota of the NWC in manufacture of a Grana-like cheese. Microbiological and flow cytometry analyses showed that the content and viability of lactic acid bacteria (LAB) and the total whey microbiota were not affected by the detoxifying agents, and Streptococcus thermophilus, Lactobacillus helveticus, and Limosilactobacillus fermentum were the dominant taxa. Random amplified polymorphic DNA-PCR fingerprinting and metagenomic analysis highlighted differences in the bacterial community of the NWC and in the relative abundance of Bacteroidetes that increased when B1 and B2 were included in the diet. Two of 6 St. thermophilus biotypes were detected only in control samples; conversely, none of the Lb. helveticus biotypes found in control samples were isolated from B1 and B2. In vitro tests showed that the 2 binders did not significantly affect the development of St. thermophilus, but they stimulated the growth of Lb. helveticus strains recovered only from B1 and B2 NWC. The addition of binders in cow feed can affect the LAB biotypes present in NWC.

Understanding thiopurine methyltransferase polymorphisms for the targeted treatment of hematologic malignancies.

INTRODUCTION: Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of thiopurines (mercaptopurine (MP) and tioguanine (TG)), chemotherapeutic agents used in the treatment of acute lymphoblastic leukemia (ALL). Polymorphisms in TPMT gene encode diminished activity enzyme, enhancing accumulation of active metabolites, and partially explaining the inter-individual differences in patients' clinical response. AREAS COVERED: This review gives an overview on TPMT gene and function, and discusses the pharmacogenomic implications of TPMT variants in the prevention of severe thiopurine-induced hematological toxicities and the less known implication on TG-induced sinusoidal obstruction syndrome. Additional genetic and non-genetic factors impairing TPMT activity are considered. Literature search was done in PubMed for English articles published since1990, and on PharmGKB. EXPERT OPINION: To titrate thiopurines safely and effectively, achieve the right degree of lymphotoxic effect and avoid excessive myelosuppression, the optimal management will combine a preemptive TPMT genotyping to establish a safe initial dose with a close phenotypic monitoring of TPMT activity and/or of active metabolites during long-term treatment. Compared to current ALL protocols, replacement of TG by MP during reinduction phase in TPMT heterozygotes and novel individualized TG regimens in maintenance for TPMT wild-type subjects could be investigated to improve outcomes while avoiding risk of severe hepatotoxicity.

The non-euphoric phytocannabinoid cannabidivarin counteracts intestinal inflammation in mice and cytokine expression in biopsies from UC pediatric patients.

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-1ß, 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.

Detailed macro- and micromineral profile of milk: Effects of herd productivity, parity, and stage of lactation of cows of 6 dairy and dual-purpose breeds.

The aim of this study was to quantify the major sources of variation in the levels of 15 minerals in individual milk samples collected from cows raised in multibreed dairy herds. The herds (n = 27) were classified into 2 categories, according to milk productivity. Milk productivity was based on the net energy of lactating cows' average daily milk yield. Milk samples were collected from 240 cows of 6 different breeds: 3 specialized dairy (Holstein-Friesian, Brown Swiss, and Jersey) and 3 dual-purpose (Simmental, Rendena, and Alpine Grey). The samples were analyzed for macro-elements (Na, Mg, P, S, K, and Ca), essential micro-elements (Mn, Fe, Cu, Zn, and Se), and environmental micro-elements (B, Si, Sr, and Sn), using inductively coupled plasma-optical emission spectrometry. Data were analyzed using a linear mixed model that included fixed effects of days in milk (DIM), parity, breed, and herd productivity, and a random effect of herd-date within productivity level. Results showed that the effect of herd-date varied across minerals. It was especially large for environmental minerals (ranging from 47 to 91% of total variance) and ranged from 11 to 61% for macrominerals and essential microminerals. Milk samples collected from farms with a high level of herd productivity had a richer mineral profile than samples from low-productivity herds. Parity only influenced macrominerals, with the exception of S and Ca, while DIM influenced almost all minerals, with a few exceptions among the environmental elements. Differences in mineral profile were small between specialized and dual-purpose breeds, but they were large within the group of the specialized cows. These breed differences were reduced after adjusting for milk quality and yield, particularly in the case of milk Mg, S, Ca, Mn, and Zn levels. Milk samples from the Jersey and Brown Swiss cows had higher mineral levels (Sn excluded) than milk from the Holstein-Friesian cows; the other breeds of Alpine origin produced milk of intermediate quality. Our findings suggest that breed has a stronger effect on macrominerals and some of the essential microminerals than herd productivity, parity, and DIM. The modification of the mineral profile in milk seems possible for many minerals, but it likely depends on genetics (e.g., breed, selection) and on environmental and management factors in variable proportions according to the mineral considered.

SERS of cells: What can we learn from cell lysates?

Surface-enhanced Raman spectroscopy (SERS) is a promising and emerging technique to analyze the cellular environment. We developed an alternative, rapid and label-free SERS-based method to get information about the cellular environment by analyzing cells lysates, thus avoiding the need to incorporate nanoparticles into cells. Upon sonicating and filtrating cells, we obtained lysates which, mixed with Au or Ag nanoparticles, yield stable and repeatable SERS spectra, whose overall profile depends on the metal used as substrate, but not on the buffer used for the lysis process. Bands appearing in these spectra were shown to arise mostly from the cytosol and were assigned to adenine, guanine, adenosine and reduced glutathione (GSH). Spectral differences among various cell types also demonstrated that this approach is suitable for cell type identification.

Breed of cow and herd productivity affect milk nutrient recovery in curd, and cheese yield, efficiency and daily production.

Little is known about cheese-making efficiency at the individual cow level, so our objective was to study the effects of herd productivity, individual herd within productivity class and breed of cow within herd by producing, then analyzing, 508 model cheeses from the milk of 508 cows of six different breeds reared in 41 multi-breed herds classified into two productivity classes (high v. low). For each cow we obtained six milk composition traits; four milk nutrient (fat, protein, solids and energy) recovery traits (REC) in curd; three actual % cheese yield traits (%CY); two theoretical %CYs (fresh cheese and cheese solids) calculated from milk composition; two overall cheese-making efficiencies (% ratio of actual to theoretical %CYs); daily milk yield (dMY); and three actual daily cheese yield traits (dCY). The aforementioned phenotypes were analyzed using a mixed model which included the fixed effects of herd productivity, parity, days in milk (DIM) and breed; the random effects were the water bath, vat, herd and residual. Cows reared in high-productivity herds yielded more milk with higher nutrient contents and more cheese per day, had greater theoretical %CY, and lower cheese-making efficiency than low-productivity herds, but there were no differences between them in terms of REC traits. Individual herd within productivity class was an intermediate source of total variation in REC, %CY and efficiency traits (10.0% to 17.2%), and a major source of variation in milk yield and dCY traits (43.1% to 46.3%). Parity of cows was an important source of variation for productivity traits, whereas DIM affected almost all traits. Breed within herd greatly affected all traits. Holsteins produced more milk, but Brown Swiss cows produced milk with higher actual and theoretical %CYs and cheese-making efficiency, so that the two large-framed breeds had the same dCY. Compared with the two large-framed breeds, the small Jersey cows produced much less milk, but with greater actual and theoretical %CYs, similar efficiencies and a slightly lower dCY. Compared with the average of the specialized dairy breeds, the three dual-purpose breeds (Simmental and the local Rendena and Alpine Grey) had, on average, similar dMY, lower actual and theoretical %CY, similar fat and protein REC, and slightly greater cheese-making efficiency.

Associations between pathogen-specific cases of subclinical mastitis and milk yield, quality, protein composition, and cheese-making traits in dairy cows.

The aim of this study was to investigate associations between pathogen-specific cases of subclinical mastitis and milk yield, quality, protein composition, and cheese-making traits. Forty-one multibreed herds were selected for the study, and composite milk samples were collected from 1,508 cows belonging to 3 specialized dairy breeds (Holstein Friesian, Brown Swiss, and Jersey) and 3 dual-purpose breeds of Alpine origin (Simmental, Rendena, and Grey Alpine). Milk composition [i.e., fat, protein, casein, lactose, pH, urea, and somatic cell count (SCC)] was analyzed, and separation of protein fractions was performed by reversed-phase high performance liquid chromatography. Eleven coagulation traits were measured: 5 traditional milk coagulation properties [time from rennet addition to milk gelation (RCT, min), curd-firming rate as the time to a curd firmness (CF) of 20 mm (k20, min), and CF at 30, 45, and 60 min from rennet addition (a30, a45, and a60, mm)], and 6 new curd firming and syneresis traits [potential asymptotical CF at an infinite time (CFP, mm), curd-firming instant rate constant (kCF, % × min-1), curd syneresis instant rate constant (kSR, % × min-1), modeled RCT (RCTeq, min), maximum CF value (CFmax, mm), and time at CFmax (tmax, min)]. We also measured 3 cheese yield traits, expressing the weights of total fresh curd (%CYCURD), dry matter (%CYSOLIDS), and water (%CYWATER) in the curd as percentages of the weight of the processed milk, and 4 nutrient recovery traits (RECPROTEIN, RECFAT, RECSOLIDS, and RECENERGY), representing the percentage ratio between each nutrient in the curd and milk. Milk samples with SCC > 100,000 cells/mL were subjected to bacteriological examination. All samples were divided into 7 clusters of udder health (UH) status: healthy (cows with milk SCC < 100,000 cells/mL and uncultured); culture-negative samples with low, medium, or high SCC; and culture-positive samples divided into contagious, environmental, and opportunistic intramammary infection (IMI). Data were analyzed using a linear mixed model. Significant variations in the casein to protein ratio and lactose content were observed in all culture-positive samples and in culture-negative samples with medium to high SCC compared to normal milk. No differences were observed among contagious, environmental, and opportunistic pathogens, suggesting an effect of inflammation rather than infection. The greatest impairment in milk quantity and composition, clotting ability, and cheese production was observed in the 2 UH status groups with the highest milk SCC (i.e., contagious IMI and culture-negative samples with high SCC), revealing a discrepancy between the bacteriological results and inflammatory status, and thus confirming the importance of SCC as an indicator of udder health and milk quality.

Factors affecting variations in the detailed fatty acid profile of Mediterranean buffalo milk determined by 2-dimensional gas chromatography.

Buffalo milk is the world's second most widely produced milk, and increasing attention is being paid to its composition, particularly the fatty acid profile. The objectives of the present study were (1) to characterize the fatty acid composition of Mediterranean buffalo milk, and (2) to investigate potential sources of variation in the buffalo milk fatty acid profile. We determined the profile of 69 fatty acid traits in 272 individual samples of Mediterranean buffalo milk using gas chromatography. In total, 51 individual fatty acids were identified: 24 saturated fatty acids, 13 monounsaturated fatty acids, and 14 polyunsaturated fatty acids. The major individual fatty acids in buffalo milk were in the order 16:0, 18:1 cis-9, 14:0, and 18:0. Saturated fatty acids were the predominant fraction in buffalo milk fat (70.49%); monounsaturated and polyunsaturated fatty acids were at 25.95 and 3.54%, respectively. Adopting a classification based on carbon-chain length, we found that medium-chain fatty acids (11-16 carbons) represented the greater part (53.7%) of the fatty acid fraction of buffalo milk, whereas long-chain fatty acids (17-24 carbons) and short-chain fatty acids (4-10 carbons) accounted for 32.73 and 9.72%, respectively. The n-3 and n-6 fatty acids were 0.46 and 1.77%, respectively. The main conjugated linoleic acid, rumenic acid, represented 0.45% of total milk fatty acids. Herd/test date and stage of lactation were confirmed as important sources of variation in the fatty acid profile of buffalo milk. The percentages of short-chain and medium-chain fatty acids in buffalo milk increased in early lactation (+0.6 and +3.5%, respectively), whereas long-chain fatty acids decreased (-4.2%). The only exception to this pattern was butyric acid, which linearly decreased from the beginning of lactation, confirmation that its synthesis is independent of malonyl-CoA. These results seem to suggest that in early lactation the mobilization of energy reserves may have less influence on the fatty acid profile of buffalo milk than that of cow milk, probably due to a shorter and less severe period of negative energy balance. Parity affected the profiles of a few traits and had the most significant effects on branched-chain fatty acids. This work provided a detailed overview of the fatty acid profile in buffalo milk including also those fatty acids present in small concentrations, which may have beneficial effects for human health. Our results contributed also to increase the knowledge about the effects of some of the major factors affecting buffalo production traits and fatty acid concentrations in milk, and consequently its technological and nutritional properties.

Role of inosine triphosphate pyrophosphatase gene variant on fever incidence during zidovudine antiretroviral therapy.

Zidovudine, the antiretroviral drug used to treat HIV infection, commonly causes adverse effects, such as systemic fever and gastrointestinal alterations. In the present study, the potential role of inosine triphosphate pyrophosphatase (ITPA) gene variant on the incidence of adverse events during antiretroviral therapy (ART) of HIV with zidovudine was discussed. Individuals from Northeastern Brazil (N = 204) receiving treatment for HIV-1 infection were recruited. Zidovudine-related adverse effects developed during the treatment were registered. The rs1127354 polymorphism in the ITPA gene was genotyped using real-time PCR to assess whether this single nucleotide polymorphism was associated with the occurrence of zidovudine-related adverse effects. We observed a significant association between the ITPA variant genotype and the reported systemic fever (odds ratio = 7.17, 95% confidence interval = 1.19-43.15; P = 0.032). Zidovudine use could indirectly lead to an increase in the levels of inosine monophosphate in an antimetabolite-like manner, which is converted to inosine triphosphate (ITP). The rs1127354 variant caused a decrease in ITPA activity, thereby leading to ITP accumulation. This in turn resulted in cytotoxicity, which was manifested by neutropenia and fever. Therefore, we hypothesized a pharmacogenetic model involving the ITPA variant genotype in multifactorial components that act together to determine the onset of zidovudine-related adverse effects.

Breed of cow and herd productivity affect milk composition and modeling of coagulation, curd firming, and syneresis.

Milk coagulation properties (MCP) have been widely investigated in the past using milk collected from different cattle breeds and herds. However, to our knowledge, no previous studies have assessed MCP in individual milk samples from several multi-breed herds characterized by either high or low milk productivity, thereby allowing the effects of herd and cow breed to be evaluated independently. Multi-breed herds (n=41) were classified into 2 categories based on milk productivity (high vs. low), defined according to the average milk net energy yielded daily by lactating cows. Milk samples were taken from 1,508 cows of 6 different breeds: 3 specialized dairy (Holstein-Friesian, Brown Swiss, Jersey) and 3 dual-purpose (Simmental, Rendena, Alpine Grey) breeds, and analyzed in duplicate (3,016 tests) using 2 lactodynamographs to obtain 240 curd firming (CF) measurements over 60min (1 every 15 s) for each duplicate. The 5 traditional single-point MCP (RCT, k20, a30, a45, and a60) were yielded directly by the instrument from the available CF measures. All 240 CF measures of each replicate were also used to estimate 4 individual equation parameters: RCT estimated according to curd firm change over time modeling (RCTeq), asymptotic potential curd firmness (CFP), curd firming instant rate constant (kCF), and syneresis instant rate constant (kSR) and 2 derived traits: maximum curd firmness achieved within 45min (CFmax) and time at achievement of CFmax (tmax) by curvilinear regression using a nonlinear procedure. Results showed that the effect of herd-date on traditional and modeled MCP was modest, ranging from 6.1% of total variance for k20 to 10.7% for RCT, whereas individual animal variance was the highest, ranging from 32.0% for tmax to 82.5% for RCTeq. The repeatability of MCP was high (>80%) for all traits except those associated with the last part of the lactodynamographic curve (i.e., a60, kSR, kCF, and tmax: 57 to 71%). Reproducibility, taking into account the effect of instrument, was equal to or slightly lower than repeatability. Milk samples collected in farms characterized by high productivity exhibited delayed coagulation (RCTeq: 18.6 vs. 16.3min) but greater potential curd firmness (CFP: 76.8 vs. 71.9mm) compared with milk samples collected from low-productivity herds. Parity and days in milk influenced almost all MCP. Large differences in all MCP traits were observed among breeds, both between specialized and dual-purpose breeds and within these 2 groups of breeds, even after adjusting for milk quality and yield. Milk quality and MCP of samples from Jersey cows, and coagulation time of samples from Rendena cows were better than in milk from Holstein-Friesian cows, and intermediate results were found with the other breeds of Alpine origin. The results of this study, taking into account the intrinsic limitation of this technique, show that the effects of breed on traditional and modeled MCP are much greater than the effects of herd productivity class, parity, and DIM. Moreover, the variance in individual animals is much greater than the variance in individual herds within herd productivity class. It seems that improvement in MCP depends more on genetics (e.g., breed, selection) than on environmental and management factors.

Cheesemaking in highland pastures: Milk technological properties, cream, cheese and ricotta yields, milk nutrients recovery, and products composition.

Summer transhumance of dairy cows to high Alpine pastures is still practiced in many mountainous areas. It is important for many permanent dairy farms because the use of highland pastures increases milk production and high-priced typical local dairy products often boost farm income. As traditional cheese- and ricotta-making procedures in Alpine pastures are central to this dairy system, the objective of this study was to characterize the quality and efficiency of products and their relationships with the quality and availability of grass during the grazing season. The milk from 148 cows from 12 permanent farms reared on a temporary farm located in Alpine pastures was processed every 2wk during the summer (7 cheesemakings from late June to early September). During each processing, 11 dairy products (4 types of milk, 2 by-products, 3 fresh products, and 2 ripened cheeses) were sampled and analyzed. In addition, 8 samples of fresh forage from the pasture used by the cows were collected and analyzed. At the beginning of the pasture season the cows were at 233±90d in milk, 2.4±1.7 parities, and produced 23.6±5.7kg/d of milk. The milk yield decreased with the move from permanent to temporary farms and during the entire summer transhumance, but partly recovered after the cows returned to the permanent farms. Similar trends were observed for the daily yields of fat, protein, casein, lactose, and energy, as we found no large variations in the quality of the milk, with the exception of the first period of Alpine pasture. The somatic cell counts of milk increased during transhumance, but this resulted from a concentration of cells in a lower quantity of milk rather than an increase in the total number of cells ejected daily from the udder. We noted a quadratic trend in availability of forage (fresh and dry matter weight per hectare), with a maximum in late July. The quality of forage also varied during the summer with a worsening of chemical composition. The evening milk (before and after natural creaming), the whole morning milk, and the mixed vat milk had different chemical compositions, traditional coagulation properties, and curd-firming modeling parameters. These variations over the pasture season were similar to the residual variations with respect to chemical composition, and much lower with respect to coagulation and curd-firming traits. Much larger variations were noted in cream, cheese, and ricotta yields, as well as in nutrient recoveries in curd during the pasture season. The protein content of forage was correlated with some of the coagulation and curd-firming traits, the ether extract of forage was positively correlated with milk fat content and cheese yields, and fiber fractions of forage were unfavorably correlated with some of the chemical and technological traits. Traditional cheese- and ricotta-making procedures showed average cream, cheese, and ricotta yields of 6.3, 14.2, and 4.9%, respectively, and an overall recovery of almost 100% of milk fat, 88% of milk protein, and 60% of total milk solids.

Short communication: Variations in major mineral contents of Mediterranean buffalo milk and application of Fourier-transform infrared spectroscopy for their prediction.

The aims of this study were (1) to assess variability in the major mineral components of buffalo milk, (2) to estimate the effect of certain environmental sources of variation on the major minerals during lactation, and (3) to investigate the possibility of using Fourier-transform infrared (FTIR) spectroscopy as an indirect, noninvasive tool for routine prediction of the mineral content of buffalo milk. A total of 173 buffaloes reared in 5 herds were sampled once during the morning milking. Milk samples were analyzed for Ca, P, K, and Mg contents within 3h of sample collection using inductively coupled plasma optical emission spectrometry. A Milkoscan FT2 (Foss, Hillerød, Denmark) was used to acquire milk spectra over the spectral range from 5,000 to 900 wavenumber/cm. Prediction models were built using a partial least square approach, and cross-validation was used to assess the prediction accuracy of FTIR. Prediction models were validated using a 4-fold random cross-validation, thus dividing the calibration-test set in 4 folds, using one of them to check the results (prediction models) and the remaining 3 to develop the calibration models. Buffalo milk minerals averaged 162, 117, 86, and 14.4mg/dL of milk for Ca, P, K, and Mg, respectively. Herd and days in milk were the most important sources of variation in the traits investigated. Parity slightly affected only Ca content. Coefficients of determination of cross-validation between the FTIR-predicted and the measured values were 0.71, 0.70, and 0.72 for Ca, Mg, and P, respectively, whereas prediction accuracy was lower for K (0.55). Our findings reveal FTIR to be an unsuitable tool when milk mineral content needs to be predicted with high accuracy. Predictions may play a role as indicator traits in selective breeding (if the additive genetic correlation between FTIR predictions and measures of milk minerals is high enough) or in monitoring the milk of buffalo populations for dairy industry purposes.

In vitro sensitivity to methyl-prednisolone is associated with clinical response in pediatric idiopathic nephrotic syndrome.

The aim of this study was to evaluate the in vitro steroid sensitivity as a predictor of clinical response to glucocorticoids in childhood idiopathic nephrotic syndrome (INS). Seventy-four patients (median age 4.33, interquartile range [IQR] 2.82-7.23; 63.5% male) were enrolled in a prospective multicenter study: in vitro steroid inhibition of patients' peripheral blood mononuclear cell proliferation was evaluated by [methyl-(3) H] thymidine incorporation assay at disease onset (T0) and after 4 weeks (T4) of treatment. Steroid dependence was associated with increased in vitro sensitivity at T4 assessed both as drug concentration inducing 50% of inhibition (IC50 ; odds ratio [OR] = 0.48, 95% confidence interval [CI] = 0.24-0.85; P = 0.0094) and maximum inhibition at the highest drug concentration (Imax ; OR = 1.13, 95% CI = 1.02-1.31; P = 0.017). IC50 > 4.4 nM and Imax < 92% at T4 were good predictors for optimal clinical response. These results suggest that this test may be useful for predicting the response to glucocorticoid therapy in pediatric INS.

The 9-MilCA method as a rapid, partly automated protocol for simultaneously recording milk coagulation, curd firming, syneresis, cheese yield, and curd nutrients recovery or whey loss.

The aim of this study was to propose and test a new laboratory cheesemaking procedure [9-mL milk cheesemaking assessment (9-MilCA)], which records 15 traits related to milk coagulation, curd firming, syneresis, cheese yield, and curd nutrients recovery or whey loss. This procedure involves instruments found in many laboratories (i.e., heaters and lacto-dynamographs), with an easy modification of the sample rack for the insertion of 10-mL glass tubes. Four trials were carried out to test the 9-MilCA procedure. The first trial compared 8 coagulation and curd firming traits obtained using regular or modified sample racks to process milk samples from 60 cows belonging to 5 breeds and 3 farms (480 tests). The obtained patterns exhibited significant but irrelevant between-procedure differences, with better repeatability seen for 9-MilCA. The second trial tested the reproducibility and repeatability of the 7 cheesemaking traits obtained using the 9-MilCA procedure on individual samples from 60 cows tested in duplicate in 2 instruments (232 tests). The method yielded very repeatable outcomes for all 7 tested cheese yield and nutrient recovery traits (repeatability >98%), with the exception of the fresh cheese yield (84%), which was affected by the lower repeatability (67%) of the water retained in the curd. In the third trial (96 tests), we found that using centrifugation in place of curd cooking and draining (as adopted in several published studies) reduced the efficiency of whey separation, overestimated all traits, and worsened the repeatability. The fourth trial compared 9-MilCA with a more complex model cheese-manufacturing process that mimics industry practices, using 1,500-mL milk samples (72 cows, 216 tests). The average results obtained from 9-MilCA were similar to those obtained from the model cheeses, with between-method correlations ranging from 78 to 99%, except for the water retained in the curd (r=54%). Our results indicate that new 9-MilCA method is a powerful research tool that allows the rapid, inexpensive, and partly automated analysis processing 40 samples per day with 2 replicates each, using 1 lacto-dynamograph, 2 heaters, and 3 modified sample racks, and yields a complete picture of the cheesemaking process (e.g., milk gelation, curd firming, syneresis, and whey expulsion) as well as the cheese yield and the efficiency of energy or nutrients retention in the cheese or loss in the whey.

Milk skimming, heating, acidification, lysozyme, and rennet affect the pattern, repeatability, and predictability of milk coagulation properties and of curd-firming model parameters: A case study of Grana Padano.

Milk coagulation properties are used to evaluate the cheesemaking aptitude of milk samples. No international standard procedure exists, although laboratories often mimic the production of a full-fat fresh cheese for milk coagulation properties. Questions have arisen about the predictability of such a procedure for different types of cheese production. The aim of this study was to establish a procedure mimicking the production conditions of a long-ripened hard cheese, taking Protected Designation of Origin Grana Padano as a case study. With respect to the traditional conditions (standard procedure; SP), the Grana Padano procedure (GP) modifications were the use of standardized milk, coagulation lower temperature, previous milk acidification, lysozyme addition, and rennet type. Each modification was tested in turn versus the SP and also all together in the GP. Another 3 tests were carried out: SP on naturally creamed milk, SP with double the quantity of rennet, and a simplified GP on a full-fat milk sample. The 10 procedures were tested on 2 subsamples with 2 replicates each and were repeated using individual milk samples from 15 dual-purpose Simmental cows in 4 sessions for a total of 600 tests. Two Formagraph instruments (Foss Electric A/S, Hillerød, Denmark) measuring curd firmness every 15 s were used, prolonging test duration to 60min to obtain 5 traditional single-point milk coagulation properties and 3 parameters of the curd firming model using all 240 points recorded for each replicate. The 8 traits of each replicate were analyzed according to a mixed model with fixed effects of 4 sessions, 10 treatments, 2 instruments, and 16microvats, and random effects of 15 animals and 300 subsamples. Compared with the SP, the coagulation and curd firming was slowed by low temperature and was accelerated by acidification and by adding a double amount of rennet; natural creaming, fat standardization, and rennet with 5% pepsin affected only some traits, whereas lysozyme addition affected none. Combination of all modifications tended to compensate for each of their effects, resulting in similar average patterns between GP and SP. Modifications to repeatability were found for all traits with the exception of 2. The ability of the SP to predict GP, tested through correlations between procedures, was not very high. Whereas SP is used for both research and in the dairy industry, better results for Grana Padano cheesemaking can only be achieved by adopting specific, more complex, and labor-intensive procedures at the research level or, possibly, by specific calibrations through Fourier-transform infrared spectroscopy at the industry level.

Pharmacogenomic approaches for tailored anti-leukemic therapy in children.

Several lympholytic and cytotoxic agents are used in acute lymphoblastic leukemia (ALL) polychemotherapy. Genetic variants for cellular components involved in the pharmacokinetics and pharmacodynamics of these drugs can influence the pharmacological response, and molecular characterization of these genetic variants could be helpful for the comprehension of the mechanisms of resistance or increased sensitivity. The purpose of this review is to carry out an update of recent publications on genes that might influence ALL treatment in terms of outcome and/or toxicity and to underlie the role of genetic variants, particularly single nucleotide polymorphisms (SNP), in predicting clinical response, with particular reference to the current protocol for ALL therapy used in Italy, AIEOP-BFM ALL 2009.

A genome-wide approach identifies that the aspartate metabolism pathway contributes to asparaginase sensitivity.

Asparaginase is an important component for treatment of childhood acute lymphoblastic leukemia (ALL). The basis for interindividual differences in asparaginase sensitivity remains unclear. To comprehensively identify genetic variants important in the cytotoxicity of asparaginase, we used a genome-wide association approach using the HapMap lymphoblastoid cell lines (87 CEU trio members) and 54 primary ALL leukemic blast samples at diagnosis. Asparaginase sensitivity was assessed as the drug concentration necessary to inhibit 50% of growth (inhibitory concentration (IC)(50)). In CEU lines, we tested 2,390,203 single-nucleotide polymorphism (SNP) genotypes at the individual SNP (P<0.001) and gene level (P<0.05), and identified 329 SNPs representing 94 genes that were associated with asparaginase IC(50). The aspartate metabolism pathway was the most overrepresented among 199 pathways evaluated (P=8.1 × 10(-3)), with primary involvement of adenylosuccinate lyase and aspartyl-tRNA synthetase genes. We validated that SNPs in the aspartate metabolism pathway were also associated with asparaginase sensitivity in primary ALL leukemic blast samples (P=5.5 × 10(-5)). Our genome-wide interrogation of CEU cell lines and primary ALL blasts revealed that inherited genomic interindividual variation in a plausible candidate pathway can contribute to asparaginase sensitivity.