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The classic antipsychotic drug haloperidol is known to possess neurotoxic effects in experimental animals. The aim is to investigate neuronal injury, liver damage, and genotoxicity effects of haloperidol and their possible modulation by treatment with α-lactalbumin (Lacprodan® α-10) in the rat. Lacprodan® α-10 (100 or 200 mg/kg) was concurrently given with haloperidol (1.5 mg/kg) for 2 weeks. Compared with the saline control group, rats treated with only haloperidol exhibited significantly decreased reduced glutathione concentration and superoxide dismutase activity along with raised malondialdehyde levels in both brain and liver tissue. Additionally, haloperidol caused significant structural and numerical chromosomal aberrations, increased hepatic DNA fragmentation and DNA damage of peripheral blood lymphocytes. Oral administration of Lacprodan® α-10 in a dose of 200 mg/kg resulted in a significant increase in reduced glutathione concentration and decreased lipid peroxidation (malondialdehyde) in brain and liver tissue. Moreover, Lacprodan® α-10 (100–200 mg/kg) resulted in a significant increase in superoxide dismutase activity compared to the haloperidol control group. Lacprodan® α-10 ameliorated the gentotoxcity and DNA damage caused by haloperidol. The histological assessment showed that haloperidol caused degenerated neurocytes, and pericellular vacuolation. The liver exhibited significant damage with necrosis of hepatocytes, focal area of lymphocyte infiltration and hemorrhage in between hepatocytes. These changes were markedly ameliorated by treatment with Lacprodan® α-10 (200 mg/kg). Lacprodan® α-10 can prevent the neurotoxic and genotoxic/DNA damage caused by the haloperidol. Therefore, Lacprodan® α-10 is likely to prove of value in reducing these adverse effects in patients treated with haloperidol. [ABSTRACT FROM AUTHOR]

Abstract The impact of sodium hexametaphosphate (SHMP) on the aggregation behavior of α-lactalbumin (α-LA) was studied at pH 7.4 and 2.0. Turbidity measurements showed a concentration-dependent aggregation of α-LA at pH 2.0 in the presence of SHMP, while no aggregation was observed at pH 7.4. Light scattering (LS) and Thioflavin-T (ThT) data revealed that the aggregation was rapid, following nucleation-independent pathways. In other kinetics experiments such as turbidity and ThT confirmed that SHMP-induced α-LA aggregation was dependent on SHMP concentration rather than incubation time. Once formed, the aggregates remained unchanged for up to five days. Intrinsic fluorescence studies indicated conformational changes in α-LA upon SHMP addition, and dye-binding assays with ThT and Congo Red demonstrated the formation of amyloid-like aggregates. Far-UV circular dichroism (CD) data suggested a structural transition from α-helical to β-structures in α-LA in the presence of SHMP at pH 2.0. Molecular docking studies confirmed stronger interactions between α-LA and SHMP at pH 2.0 (ΔG = −6.2 kcal/mol) compared to pH 7.4 (ΔG = −5.3 kcal/mol), driven by electrostatic forces and hydrogen bonding. These results suggest that SHMP induces amyloid-like aggregation of α-LA, particularly at acidic pH.

Alpha-lactalbumin (α-LA) is a major whey protein found in breast milk and plays a crucial role in the growth and development of infants. In this study, Bacillus subtilis RIK1285 harboring AprE signal peptide (SP) was selected as the original strain for the production of α-LA. It was found that α-LA was identified in the pellet after ultrasonic disruption and centrifugation instead of in the fermentation supernatant. The original strain most likely only produced α-LA intracellular, but not extracellular. To improve the expression and secretion of α-LA in RIK1285, a library of 173 homologous SPs from the B. subtilis 168 genome was fused with target LALBA gene in the pBE-S vector and expressed extracellularly in RIK1285. SP YjcN was determined to be the best signal peptide. Bands in supernatant were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and purified by nickel column to calculate the highest yield signal peptide. In addition, different promoters (PaprE, P43, and Pglv) were compared and applied. The results indicated that the strain RIK1285-pBE-Pglv-YjcN-LALBA had the highest α-LA yield, reaching 122.04 μg/mL. This study demonstrates successful expression and secretion of human α-LA in B. subtilis and establishes a foundation for simulating breast milk for infant formulas and developing bioengineered milk.

Food processing, which generates both by-products and waste, requires a revision of modern processes in the framework of the development of non-residual, environmentally friendly processes. Dairy by-products require the development of complex zero-waste technologies. Electroactivation is an emerging process to overcome those challenges, which allows for non-residual processing of milk by-products. Electroactivation, managing both the technological regimes and the geometric/technical parameters of the electrolyzers used to process different types of whey, allows both the electrofractionation of the whey proteins recovered in protein mineral concentrates enriched, under certain process conditions, with a certain protein content, but also the simultaneous isomerization of lactose into lactulose following two mechanisms, and creating a closed process cycle. The geometry of the electrolyzers and the content of the secondary liquid (anodic liquid) influence both the extraction and the formation of protein compounds enriched with alpha-lactalbumin, as well as the formation of a complex between calcium and isomerized lactulose.

Purpose of the review: The objective of this review is to provide an analysis of early-phase clinical trials investigating vaccine therapies for triple-negative breast cancer (TNBC). Specifically, the focus is on ongoing trials that are actively recruiting or in progress, while excluding vaccines that target neoantigens or those that have already completed trials. Recent findings: Over the past decade, notable transformations have occurred in the strategy of breast cancer vaccine design. Traditional approaches to identifying tumor antigens, such as SEREX, have been replaced with modern techniques, such as RNA sequencing, HLA typing, and immunoinformatics. These new methods enable the identification and characterization of tumor antigens. Notably, current clinical investigations into tumor targets extend beyond mutated self-proteins or proteins that are overexpressed following neoplastic transformation. Clinical researchers are currently examining protein targets associated with cancer stem cells or non-malignant immune regulatory cell types within the tumor microenvironment. However, the application of up-to-date antigen delivery methods for certain types of breast cancer vaccine therapies still lags behind. Another significant transformation in comparison to previous breast cancer vaccine therapies is the emphasis on stimulating robust T-cell responses against breast cancer cells, independent of any B-cell response directed at the tumor. Summary: In conclusion, we critically assessed the tumor antigens targeted by vaccine immunotherapies in these new clinical trials, the delivery methods used for these antigens, and conclude by discussing potential future directions for the development of new TNBC vaccine therapies. [ABSTRACT FROM AUTHOR]

ObjectivesProtein overfeeding in infants can have negative effects, such as diabetes and childhood obesity; key to reducing protein intake from formula is improving protein quality. The impact of a new infant formula [study formula (SF)] containing alpha-lactalbumin, lactoferrin, partially hydrolyzed whey, and whole milk on growth and tolerance compared to a commercial formula (CF) and a human milk reference arm was evaluated.MethodsThis randomized, double-blind trial included healthy, singleton, term infants, enrollment age ≤14 days. Primary outcome was mean daily weight gain. Secondary outcomes were anthropometrics, formula intake, serum amino acids, adverse events, gastrointestinal characteristics, and general disposition.ResultsNon-inferiority was demonstrated. There were no differences between the formula groups for z scores over time. Formula intake [-0.33 oz/kg/day, 95% confidence interval (CI): -0.66 to -0.01, P = 0.05] and mean protein intake (-0.13 g/kg/day, 95% CI: -0.26 to 0.00, P = 0.05) were lower in the SF infants, with higher serum essential amino acid concentrations (including tryptophan) compared to the CF infants. Energetic efficiency was 14.0% (95% CI: 8.3%, 19.7%), 13.0% (95% CI: 6.0%, 20.0%), and 18.1% (95% CI: 9.4%, 26.8%) higher for weight, length, and head circumference, respectively, in SF infants compared to the CF infants. SF infants had significantly fewer spit-ups and softer stool consistency than CF infants.ConclusionsThe SF resulted in improved parent-reported gastrointestinal tolerance and more efficient growth with less daily formula and protein intake supporting that this novel formula may potentially reduce the metabolic burden of protein overfeeding associated with infant formula.

High protein intake during infancy results in accelerated early weight gain and potentially later obesity. The aim of this follow-up study at 12 months was to evaluate if modified low-protein formulas fed during early infancy have long-term effects on growth and metabolism. In a double-blinded RCT, the ALFoNS study, 245 healthy-term infants received low-protein formulas with either alpha-lactalbumin-enriched whey (α-lac-EW; 1.75 g protein/100 kcal), casein glycomacropeptide-reduced whey (CGMP-RW; 1.76 g protein/100 kcal), or standard infant formula (SF; 2.2 g protein/100 kcal) between 2 and 6 months of age. Breastfed (BF) infants served as a reference. At 12 months, anthropometrics and dietary intake were assessed, and serum was analyzed for insulin, C-peptide, and insulin-like growth factor 1 (IGF-1). Weight gain between 6 and 12 months and BMI at 12 months were higher in the SF than in the BF infants (p = 0.019; p < 0.001, respectively), but were not significantly different between the low-protein formula groups and the BF group. S-insulin and C-peptide were higher in the SF than in the BF group (p < 0.001; p = 0.003, respectively), but more alike in the low-protein formula groups and the BF group. Serum IGF-1 at 12 months was similar in all study groups. Conclusion: Feeding modified low-protein formula during early infancy seems to reduce insulin resistance, resulting in more similar growth, serum insulin, and C-peptide concentrations to BF infants at 6-months post intervention. Feeding modified low-protein formula during early infancy results in more similar growth, serum insulin, and C-peptide concentrations to BF infants 6-months post intervention, probably due to reduced insulin resistance in the low-protein groups. [ABSTRACT FROM AUTHOR]

Milk production by dairy cows is sensitive to increased levels of stress hormones such as glucocorticoids (GC) that also regulate the transcription of several genes required for milk synthesis. Whereas previous studies identified that an exogenous GC such as dexamethasone (DEX) transiently suppresses milk yield in several species without any pronounced effect on milk protein or fat percentage, the mechanism underlying this effect has not been established. In this study we sought to establish changes within the mammary glands of non-pregnant dairy cows in their second lactation (n = 3-4; 648-838 kg) following a single dose of exogenous DEX. Changes in the udder were monitored by serial biopsy of alternating quarters, concurrent with quarter-level monitoring of milk yield and composition. Dexamethasone increased serum glucose levels from 12-36 h (p

This research investigated the antioxidant activities and quality characteristics of functional yogurt supplemented with naturally derived protein, specifically lysozyme (LZ), as well as its thermally modified variants PLZ treated at 63°C for 30 minutes (hold type pasteurization method) and BLZ treated at 100°C for 10 minutes (using boiling temperature). Among these variants, the best antioxidant capacity was reported for PLZ, followed by BLZ and LZ. Subsequently, different yogurt samples (YCtrl, YLZ, YPLZ, and YBLZ) were prepared. Through weekly assessments, YPLZ consistently retained the highest antioxidant capacity. Moreover, YPLZ received the highest sensory evaluation scores with the best microbiological quality until the end of the examination period up to 35 days at 4 ±1°C. It was noted that PLZ displayed increased surface hydrophobicity due to gradual thermal denaturation, leading to structural changes and weak cross-dimerization with native milk alpha-lactalbumin in YPLZ. Gel electrophoresis results suggested that proteolysis by lactic acid bacteria (LAB) could facilitate the formation of low-molecular-weight peptides with enhanced antioxidant stability via weak-force dimerization. In conclusion, yogurt fortified with lysozyme, processed using hold-type pasteurization, presents a promising avenue for developing functional yogurt with antioxidant properties.

α-Lactalbumin (α-LA), which is encoded by the LALBA gene, is a major whey protein that binds to Ca2+ and facilitates lactose synthesis as a regulatory subunit of the synthase enzyme complex. In addition, it has been shown to play central roles in immune modulation, cell-growth regulation, and antimicrobial activity. In this study, a multitechnical approach was used to fully characterize the LALBA gene and its variants in both coding and regulatory regions for domestic camelids (dromedary, Bactrian camel, alpaca, and llama). The gene analysis revealed a conserved structure among the camelids, but a slight difference in size (2,012 bp on average) due to intronic variations. Promoters were characterized for the transcription factor binding sites (11 found in total). Intraspecies sequence comparison showed 36 SNPs in total (2 in the dromedary, none in the Bactrian camel, 22 in the alpaca, and 12 in the llama), whereas interspecies comparison showed 86 additional polymorphic sites. Eight SNPs were identified as trans-specific polymorphisms, and 2 of them (g.112A>G and g.1229A>G) were particularly interesting in the New World camels. The first creates a new binding site for transcription factor SP1. An enhancing effect of the g.112G variant on the expression was demonstrated by 3 independent pGL3 gene reporter assays. The latter is responsible for the p.78Ile>Val AA replacement and represents novel allelic variants (named LALBA A and B). A link to protein variants has been established by isoelectric focusing (IEF), and bioinformatics analysis revealed that carriers of valine (g.1229G) have a higher glycosylation rate. Genotyping methods based on restriction fragment length polymorphism (PCR-RFLP) were set up for both SNPs. Overall, adenine was more frequent (0.54 and 0.76) at both loci. Four haplotypes were found, and the AA and GA were the most common with a frequency of 0.403 and 0.365, respectively. Conversely, a putative biological gain characterizes the haplotype GG. Therefore, opportunities for rapid directional selection can be realized if this haplotype is associated with favorable milk protein properties. This study adds knowledge at the gene and protein level for α-LA (LALBA) in camelids and importantly contributes to a relatively unexplored research area in these species. [ABSTRACT FROM AUTHOR]

The protein composition in goat milk undergoes changes throughout the different lactation periods, displaying distinct characteristics that are influenced by the dynamic nature of protein composition and concentration during the transition from colostrum secretion to mature milk. To evaluate the dynamics of whey proteins of Saanen goats during the colostral phase and the first month of lactation, 110 milk samples from 11 healthy mammary halves of seven Saanen goats were selected through a clinical evaluation. Whey was obtained by rennet coagulation of the mammary secretion. The biuret method determined total protein concentration, and their fractions were identified by 12% dodecyl sulfate-polyacrylamide gel electrophoresis. Maximum concentrations of all protein fractions were observed in the first 12 h of lactation, reducing throughout the study. Modification of the protein predominance was also observed. The transition from colostrum secretion to milk occurred 5 or 7 d postpartum. [ABSTRACT FROM AUTHOR]

Lactose is the primary carbohydrate in the milk of most mammals and is unique in that it is only synthesized by epithelial cells in the mammary glands. Lactose is also essential for the development and nutrition of infants. Across species, the concentration of lactose in milk holds a strong positive correlation with overall milk volume. Additionally, there is a range of examples where the onset of lactose synthesis as well as the content of lactose in milk varies between species and throughout a lactation. Despite this diversity, the precursors, genes, proteins and ions that regulate lactose synthesis have not received the depth of study they likely deserve relative to the significance of this simple and abundant molecule. Through this review, our objective is to highlight the requirements for lactose synthesis at the biochemical, cellular and temporal levels through a comparative approach. This overview also serves as the prelude to a companion review describing the dietary, hormonal, molecular, and genetic factors that regulate lactose synthesis.

Milk is critical for the survival of all mammalian offspring, where its production by a mammary gland is also positively associated with its lactose concentration. A clearer understanding of the factors that regulate lactose synthesis stands to direct strategies for improving neonatal health while also highlighting opportunities to manipulate and improve milk production and composition. In this review we draw a cross-species comparison of the extra- and intramammary factors that regulate lactose synthesis, with a special focus on humans, dairy animals, and rodents. We outline the various factors known to influence lactose synthesis including diet, hormones, and substrate supply, as well as the intracellular molecular and genetic mechanisms. We also discuss the strengths and limitations of various in vivo and in vitro systems for the study of lactose synthesis, which remains an important research gap.

Background: In Spain, IgE-mediated cow's milk protein allergy (CMPA) affects approximately 0.69% of infants. Molecular diagnosis may be useful for monitoring natural spontaneous tolerance development in CMPA. The aim of this study was to retrospectively analyse a cohort of paediatric patients with IgE-mediated CMPA who were avoiding milk products awaiting natural tolerance and determine the relationship between disease persistence and major cow's milk allergens. Methods: A retrospective chart review of 200 patients diagnosed with IgE-mediated CMPA between 2011 and 2020 was conducted. Patients strictly avoided milk products until an oral food challenge was performed. The main outcome was the introduction of liquid milk following a negative oral food challenge and its correlation with IgE and SPT measurements of milk components at diagnosis. Secondary outcomes included the rate of allergic reactions and anaphylaxis during the treatment period and its correlation with IgE and SPT measurements. Results: Of the 200 charts analysed, 122 patients had a negative oral food challenge to milk (61.0%) (95% confidence interval (CI): 54.1–67.5) following a period of strict avoidance of milk. Higher levels of component-specific IgE, especially casein, were associated with failure in the oral food challenge (p = 0.02). Allergic reactions were experienced by 106 children (53%), of which 34 (17%; 95% CI: 12.4–22.8) had anaphylactic reactions. The risk of anaphylaxis was not predicted by raised IgE levels. Conclusions: While a large proportion of children acquired natural tolerance to cow's milk following a period of strict avoidance, IgE-mediated CMPA persisted in many children. Casein IgE levels at diagnosis were raised in those who failed to achieve natural tolerance. Allergic reactions to milk, including anaphylaxis, occurred commonly, but this was not predicted by raised IgE levels or SPT measurements. [ABSTRACT FROM AUTHOR]

Cow’s milk allergy has been studied extensively in infants and young children and has public health importance around the globe. We describe the clinical and demographic characteristics of 3 cases of a rare presentation of adult-onset IgE-mediated cows' milk allergy.

Objective: Cases of cow's milk allergy (CMA) who reacted to measles or measles, mumps, and rubella (MMR) vaccines containing alpha-lactalbumin have been reported. The purpose of this study was to assess patients with CMA who received measles or MMR vaccines containing alpha-lactalbumin, as well as the characteristics of those who developed reactions to these vaccines. Study Design: Patients followed up in the allergy clinic for CMA and who received measles or MMR vaccines containing alpha-lactalbumin at 9 or 12 months of age were included in the study, and their characteristics were analyzed retrospectively from the hospital registry system. Results: Forty-nine patients were included in the study. Six patients received the measles vaccine, whereas 43 patients received the MMR vaccine containing alpha-lactalbumin. Vaccine skin tests were performed on these 6 patients. One patient had a positive intradermal test, so an alternative vaccine not containing alpha-lactalbumin was administered. The other 5 patients were vaccinated, and no reaction was observed. Anaphylaxis was observed in 3 of 43 patients who received the MMR vaccine containing alpha-lactalbumin. In all of these patients, the first reaction to dairy products was anaphylaxis. In 2 of those patients, cow's milk-specific IgE (spIgE) levels were >100 kU/L, and alpha-lactalbumin-spIgE levels were also high at 97 and 90 kU/L. The third patient's cow's milk-spIgE level was 15.9 kU/L, whereas the alpha-lactalbumin-spIgE level was 0.04 kU/L. Conclusion: Especially in patients with an initial reaction of anaphylaxis to dairy products and high cow's milk-spIgE levels, the risk of reaction is high with the MMR vaccine. [ABSTRACT FROM AUTHOR]

Background: Secretory carcinoma is a rare histological type of breast neoplasm in humans and dogs that is characterized by the presence of intracellular and extracellular eosinophilic secretions. Case Description: In this case report, we describe the cytological, histological, and immunohistochemical characteristics of secretory mammary carcinoma in a 10-year-old mixed-breed female dog with nodal and bone metastases. The bitch had a history of claudication and a mass in the left humeral scapular region, which revealed osteolysis of the proximal humerus on radiography. Fine-needle aspiration cytology revealed numerous neoplastic cells arranged mostly in cohesive groups, but sometimes isolated, that contained cytoplasmic vacuoles and had a moderate to high nucleus: cytoplasm ratio with frequent karyomegaly and evident nucleoli. Histologically, the neoplasm was organized in solid, tubular structures with luminal spaces filled with eosinophilic secretions and was composed of cells with clear cytoplasm and prominent vacuoles that pushed the nuclei to the periphery, resembling signet ring cells. The extracellular and intracytoplasmic material of the epithelial cells was positive for periodic acid-Schiff (PAS) staining and immunoreactive for alpha-lactalbumin. Two chemotherapy sessions were performed, but one month after surgery, the clinical condition worsened, and euthanasia was elected, accounting for 133 days of survival after surgical removal of the tumor. Conclusion: The bitch presented with secretory mammary carcinoma with nodal and bone metastases, and histological and immunohistochemical characteristics were important for diagnosis. The morphological and immunohistochemical characteristics of this carcinoma were similar to those observed in humans. Mammary gland secretory carcinoma with bone metastasis must be included as a differential diagnosis among canine mammary gland carcinomas showing cellular morphological characteristics of intracytoplasmic vacuolization and eosinophilic secretion.

Protein intake is higher in formula-fed than in breast-fed infants during infancy, which may lead to an increased risk of being overweight. Applying alpha-lactalbumin (α-lac)-enriched whey or casein glycomacropeptide (CGMP)-reduced whey to infant formula may enable further reduction of formula protein by improving the amino acid profile. Growth, nutrient intake, and protein metabolites were evaluated in a randomized, prospective, double-blinded intervention trial where term infants received standard formula (SF:2.2 g protein/100 kcal; n = 83) or low-protein formulas with α-lac-enriched whey (α-lac-EW;1.75 g protein/100 kcal; n = 82) or CGMP-reduced whey (CGMP-RW;1.76 g protein/100 kcal; n = 80) from 2 to 6 months. Breast-fed infants (BF; n = 83) served as reference. Except between 4 and 6 months, when weight gain did not differ between α-lac-EW and BF (p = 0.16), weight gain was higher in all formula groups compared to BF. Blood urea nitrogen did not differ between low-protein formula groups and BF during intervention, but was lower than in SF. Essential amino acids were similar or higher in α-lac-EW and CGMP-RW compared to BF. Conclusion: Low-protein formulas enriched with α-lac-enriched or CGMP-reduced whey supports adequate growth, with more similar weight gain in α-lac-enriched formula group and BF, and with metabolic profiles closer to that of BF infants. [ABSTRACT FROM AUTHOR]

Alpha-lactalbumin (α-La) is a major milk protein essential for the biosynthesis of lactose at the level of mammary glands. α-La directly influences the quality and the volume of the milk since it is directly involved in the lactose synthesis (Ashwell et al., 1997). The PCR-RFLP test was performed to distinguish the different alleles in a population of Romanian Black Spotted cattle, a dairy breed. Genetic polymorphism was detected by digestion with the endonuclease MnlI, followed by electrophoresis in high resolution agarose gel stained with ethidium bromide. Sixty DNA samples from Romanian Black Spotted breed were analyzed for A and B variants. The PCRRFLP test makes feasible the inclusion of α-La genotypes in breeding plans and cattle selection.

Background: Secretory carcinoma is a rare histological type of breast neoplasm in humans and dogs that is characterized by the presence of intracellular and extracellular eosinophilic secretions. Case Description: In this case report, we describe the cytological, histological, and immunohistochemical characteristics of secretory mammary carcinoma in a 10-year-old mixed-breed female dog with nodal and bone metastases. The bitch had a history of claudication and a mass in the left humeral scapular region, which revealed osteolysis of the proximal humerus on radiography. Fine-needle aspiration cytology revealed numerous neoplastic cells arranged mostly in cohesive groups but sometimes isolated, that contained cytoplasmic vacuoles and had a moderate-to-high nucleus: cytoplasm ratio with frequent karyomegaly and evident nucleoli. Histologically, the neoplasm was organized in solid, tubular structures with luminal spaces filled with eosinophilic secretions and was composed of cells with clear cytoplasm and prominent vacuoles that pushed the nuclei to the periphery, resembling signet ring cells. The extracellular and intracytoplasmic material of the epithelial cells was positive for periodic acid-Schiff staining and immunoreactive for alpha-lactalbumin. Two chemotherapy sessions were performed, but 1 month after surgery, the clinical condition worsened, and euthanasia was elected, accounting for 133 days of survival after surgical removal of the tumor. Conclusion: The bitch presented with secretory mammary carcinoma with nodal and bone metastases, and histological and immunohistochemical characteristics were important for diagnosis. The morphological and immunohistochemical characteristics of this carcinoma were similar to those observed in humans. Mammary gland secretory carcinoma with bone metastasis must be included as a differential diagnosis among canine mammary gland carcinomas showing cellular morphological characteristics of intracytoplasmic vacuolization and eosinophilic secretion. [ABSTRACT FROM AUTHOR]

Milk production by dairy cows is sensitive to increased levels of stress hormones such as glucocorticoids (GC) that also regulate the transcription of several genes required for milk synthesis. Whereas previous studies identified that an exogenous GC such as dexamethasone (DEX) transiently suppresses milk yield in several species without any pronounced effect on milk protein or fat percentage, the mechanism underlying this effect has not been established. In this study we sought to establish changes within the mammary glands of non-pregnant dairy cows in their second lactation (n = 3–4; 648–838 kg) following a single dose of exogenous DEX. Changes in the udder were monitored by serial biopsy of alternating quarters, concurrent with quarter-level monitoring of milk yield and composition. Dexamethasone increased serum glucose levels from 12–36 h (p

The interaction of Nilotinib (NIL) with alpha lactalbumin (α-LA) were studied by spectrofluorimetry, UV-Vis spectroscopy, circular dichroism (CD) and molecular modeling methods. Static quenching procedure fluorescence spectroscopy revealed that the fluorescence quenching of α-LA by NIL was the reason of formation of complex) NIL–α-LA. Fluorescence, UV-Vis, and CD spectra of NIL–α-LA complex showed that the conformation of α-LA has been changed in the presence of NIL. Based on the fluorescence quenching analyses the binding constant is calculated which is (467.73 × 104 M–1 at 298 K) and number of ligand-binding site is equal to one then the thermodynamic parameters of this complex are (ΔG = –33.011 kJ mol−1, ΔS = –80.005 J mol−1 K−1; ΔH = −157.70 kJ mol−1) at 298 K which were computed by using van't Hoff equation and revealed that the reaction between ligand and protein is spontaneous and hydrogen bonding and van der Waals forces played a key role in the binding of NIL to α-LA. The results of molecular docking investigations have good agreement with the results of fluorescence spectroscopy studies. The molecular dynamic simulation (MDS) showed that free α-LA and the NIL–α-LA complex reached equilibration after 20 ns according to monitoring their root mean square deviation (RMSD). Finally, analyzing the root mean square fluctuations (RMSF) show that interaction of NIL with α-LA did not cause a significant conformational change in α-LA during the simulation. [ABSTRACT FROM AUTHOR]

Myo-inositol and D-chiro-inositol are insulin sensitising agents. In the ovary, myo-inositol acts as second messenger of Follicle Stimulating Hormone (FSH). Both molecules were administered to Polycystic Ovary Syndrome (PCOS) women. The gynaecologist Vittorio Unfer was the first to give specific value to myo-inositol for the treatment of PCOS: this important innovation opened new ways of research to identify efficient therapies based on myo-inositol alone or with low doses of D-chiro-inositol. Significant successes were also gained using myo-inositol in treating male and female infertility. Unfer's researches allowed to identify "the D-Chiro-Inositol Paradox in the Ovary" and the best myo-inositol/D-chiro-inositol ratio (40:1) for the treatment of PCOS. Furthermore, his studies allowed to improve the inositol's efficacy using alpha-lactalbumin. As shown in this review, the main stages of Unfer's scientific career have been closely intertwined with important phases of the recent pharmacological research about the topic. [ABSTRACT FROM AUTHOR]

Glycated protein is a kind of promising material that can improve the bioavailability of bioactive compounds and achieve sustained release under digestion. In this study, the α-lactalbumin (ALA)-dextran conjugates synthesized by Maillard reaction were fabricated to load and protect quercetin. Quercetin-loaded micelles stabilized by the ALA-dextran conjugates 1:4 showed the smallest size (428.57 ± 5.64 nm) with highest encapsulation efficiency (94.38% ± 0.50%) of quercetin. Compared to ALA/dextran mixture complex, the conjugates-based micelles had better pH, ionic strength and photothermal stability. Furthermore, the micelles composed of the conjugates 1:2 and 1:4 showed the best controlled release effect during the simulated digestion, releasing 62.41% and 66.15% of quercetin from the total encapsulated contents, respectively, which was mainly related to the resistance of glycated ALA to the enzymes. The findings indicated that ALA-dextran conjugates could be effectively designed for the ideal delivery system of hydrophobic bioactive compounds in food industry.

Milk proteins are known for their high nutritional quality, based on their essential amino acid composition, and they exhibit a wide range of bioactivities, including satiety, antimicrobial, mineral-binding, and anti-lipidemic properties. Because of their unique water solubility, milk proteins are readily separated into casein and whey fractions, which can be further fractionated into many individual proteins, including alpha-S1- and alpha-S2-caseins, beta-casein, and kappa-casein, and the whey proteins alpha-lactalbumin, lactoferrin, beta-lactoglobulin, and glycomacropeptide. Many of these proteins have unique bioactivities. Further, over the past 30 years, peptides that are encrypted in the primary amino acid sequences of proteins and released along with amino acids during digestion are increasingly recognized as biologically active protein metabolites that may have beneficial effects on human health. This review examines the current state of the science on the contribution of dairy proteins and their unique peptides and amino acids to human health. [ABSTRACT FROM AUTHOR]

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder of hematopoietic stem cells. Although there have been advancements in treatment, there is still a need to develop a biotherapeutic agent. A new variant of the human alpha-lactalbumin-oleic acid (HALOA) complex has been synthesized, which showed similarities with SNARE. The native α-LA was treated with EDTA to remove Ca2+ ions confirmed by ICP-OES and Arsenazo III to unfold and attain apo structure. The apo LA was mixed with OA in a specific ratio, leading to HALOA complex formation. The conformational state from native to complex was elucidated by circular dichroism (far; 190-260 nm and near; 260-340 nm UV-CD), which confirmed that the complex consists of a majority of turns and β-sheet structure. SDS-PAGE result showed the masking effect of OA on apo α-LA. In the lane of the complex, there was no band detected. However, 1-anilino-8-naphthalene sulfonate (ANS) dye has shown maximum fluorescence intensity with the complex because of the availability of hydrophobic patches, which was further validated by NMR spectroscopy indicating the masking effect of OA on the apo α-LA. In the lane of the complex, there was no band detected. However, 1-anilino-8-naphthalene sulfonate (ANS) dye has shown maximum fluorescence intensity with the complex because of the availability of hydrophobic patches, which was further validated by NMR spectroscopy indicating the masking effect of OA on the apo a-LA. The SNARE behavior of the complex (500 nm) has been confirmed by TEM. This new structural variant complex shows anti-tumor activity on chronic myeloid leukemia by targeting the IL-8, survivin, and induces apoptosis through DNA fragmentation, but not against normal cells. Overall, the formulated complex shows that SNARE-like behavior can be used as a promising anti-tumor agent with lower toxicity and maximum bioavailability. [ABSTRACT FROM AUTHOR]

In this work binding properties and conformational changes in alpha lactalbumin (α-LA) upon interaction with sorafenib were investigated by spectrofluorimetry, UV-Vis spectroscopy, Fourier transform infrared spectroscopy and molecular modeling methods. The fluorescence spectroscopic results revealed that sorafenib could effectively quench the intrinsic fluorescence of alpha lactalbumin through a static quenching mechanism. Evaluation of the thermodynamic parameters (ΔH0 = –120.167 kJ mol–1, ΔS0 = –309.507 J mol–1 K–1) suggested that the binding process was spontaneous while hydrogen bonds and van der Waals forces played a major role in this interaction. The fluorescence, UV-absorption and FT-IR spectra showed that conformational changes occurred in alpha lactalbumin structure after interaction with sorafenib. The molecular docking studies showed one binding site in alpha lactalbumin which most of its interactions are hydrophobic. The value of calculated docking ΔG0 (–27.11232 kJ mol–1) (is in agreement with those obtained from fluorescence spectroscopy measurement. Finally, molecular dynamics simulation was performed on the best docked complex by considering the permanence and flexibility of α-LA–sorafenib complex in the binding site. [ABSTRACT FROM AUTHOR]

OBJECTIVE: This open-label non-randomized clinical study aimed at evaluating the effects of myo-inositol plus alpha-lactalbumin in two groups of PCOS women, treated in Mexico and Italy. Alpha-lactalbumin was used being effective in increasing myo-inositol intestinal absorption. This effect is very useful in greatly reducing the therapeutic failure of myo-inositol in some patients (inositol resistant subjects). PATIENTS AND METHODS: The study involved 34 normal weight or overweight patients (14 in Mexico and 20 in Italy), aged 18 to 40 years, with anovulation and infertility > 1 year and insulin resistance diagnosed by HOMA-Index. Patients were administered orally with 2 g myo-inositol, 50 mg alpha-lactalbumin, and 200 μg of folic acid twice a day for 6 months. Controls were the same patients at t0 (baseline). The primary outcome was HOMA-index decrease after 3 and 6 months of treatment. Other parameters monitored were BMI, progesterone, LH, FSH, total testosterone, free testosterone, androstenedione, total cholesterol, HDL, LDL, triglycerides. RESULTS: Recovery was general, and its relevance was higher when the starting point was further away from the normal range. The most important results were obtained with insulin, HOMA-index, LH, and androstenedione. No significant adverse effects were detected in both groups of patients. CONCLUSIONS: This clinical trial demonstrated for the first time that myo-inositol and alpha- lactalbumin improve important parameters in PCOS patients characterized by different metabolic profiles. [ABSTRACT FROM AUTHOR]

HAMLET is a protein‐lipid complex with a specific and broad bactericidal and tumoricidal activity, that lacks cytotoxic activity against healthy cells. In this study, we show that HAMLET also has general immune‐stimulatory effects on primary human monocyte‐derived dendritic cells and macrophages (Mo‐DC and Mo‐M) and murine RAW264.7 macrophages. HAMLET, but not its components alpha‐lactalbumin or oleic acid, induces mature CD14low/–CD83+ Mo‐DC and M1‐like CD14+CD86++ Mo‐M surface phenotypes. Concomitantly, inflammatory mediators, including IL‐2, IL‐6, IL‐10, IL‐12 and MIP‐1α, were released in the supernatant of HAMLET‐stimulated cells, indicating a mainly pro‐inflammatory phenotype. The HAMLET‐induced phenotype was mediated by calcium, NFκB and p38 MAPK signaling in Mo‐DCs and calcium, NFκB and ERK signaling in Mo‐M as inhibitors of these pathways almost completely blocked the induction of mature Mo‐DCs and M1‐like Mo‐M. Compared to unstimulated Mo‐DCs, HAMLET‐stimulated Mo‐DCs were more potent in inducing T cell proliferation and HAMLET‐stimulated macrophages were more efficient in phagocytosis of Streptococcus pneumoniae in vitro. This indicates a functionally activated phenotype of HAMLET‐stimulated DCs and macrophages. Combined, we propose that HAMLET has a two‐fold anti‐bacterial activity; one inducing direct cytotoxic activity, the other indirectly mediating elimination of bacteria by activation of immune cells of the myeloid lineage. [ABSTRACT FROM AUTHOR]

Abstract Background Myo-inositol (MI), successfully used in polycystic ovary syndrome (PCOS), was administered with α-LA to exploit its action of favouring the passage of other molecules through biological barriers, and also considering its anti-inflammatory effect. Methods PCOS patients, according to the Rotterdam ESHRE–ASRM criteria, with anovulation and infertility > 1 year, were included in this open and prospective study. The preliminary phase was aimed at determining a set of MI-resistant PCOS patients. This treatment involved 2 g MI, taken twice per day by oral route, for three months. The Homeostasis Model Assessment (HOMA) index and MI plasma levels were measured. In the main phase, previously selected MI-resistant patients received the same daily amount of MI plus 50 mg α-LA twice a day, for a further three months. Ovulation was assessed using ultrasound examination on days 12, 14 and 20 of the cycle. The HOMA index, lipid, hormone and MI plasma levels were detected at baseline and at the end of this phase. Results Thirty-seven anovulatory PCOS subjects were included in the study. Following MI treatment, 23 of the 37 women (62%) ovulated, while 14 (38%) were resistant and did not ovulate. In the latter group, MI plasma levels did not increase. These MI-resistant patients underwent treatment in the main phase of the study, receiving MI and α-LA. After this combined treatment, 12 (86%) of them ovulated. Their MI plasma levels were found to be significantly higher than at baseline; also, a hormone and lipid profile improvement was recorded. Conclusion The combination of MI with α-LA allowed us to obtain significant progress in the treatment of PCOS MI-resistant patients. Therefore, this new formulation was able to re-establish ovulation, greatly increasing the chances of desired pregnancy. Trial Registration Clinical trial registration number: NCT03422289 (ClinicalTrials.gov registry).

The separation of α-lactalbumin (α-LAC) and β-lactoglobulin (β-LG) by ultrafiltration (UF) is a technological challenge due to their similar molecular size. As high hydrostatic pressure (HHP) treatment generates β-LG complexes of a larger molecular size than native β-LG, it was suggested that pressurization of skim milk could improve the separation of α-LAC by UF. The strategy was effective, but the pressurization affected UF performance, α-LAC recovery and purity. Hence, the combination of HHP and UF might not be the most efficient strategy for producing an enriched α-LAC fraction, but this study generated significant scientific knowledge about HHP in separation technology. [ABSTRACT FROM AUTHOR]

This review details the physiologic roles of two insulin sensitizers, myo -inositol (MI) and d -chiro-inositol (DCI). In the human ovary, MI is a second messenger of follicle-stimulating hormone (FSH) and DCI is an aromatase inhibitor. These activities allow a treatment for polycystic ovary syndrome (PCOS) to be defined based on the combined administration of MI and DCI, where the best MI:DCI ratio is 40:1. Moreover, MI enhances the effect of metformin and clomiphene on the fertility of PCOS women seeking pregnancy. As impaired intestinal transport may lead to unsuccessful inositol treatment, we also discuss new data on the use of alpha-lactalbumin to boost inositol absorption. Overall, the physiological activities of MI and DCI dictate the dosages and timing of inositol supplementation in the treatment of PCOS. Myo -inositol (MI) and d -chiro-inositol (DCI) are two stereoisomers of inositol. These natural molecules are safe and well-tolerable and have insulin-sensitizing activity. In addition, MI mediates FSH signaling. MI and DCI are dietary supplements with proven therapeutic activity in polycystic ovary syndrome (PCOS). New preclinical and clinical studies support the importance of administering a combination of MI and DCI in the 40:1 ratio. Of note, this is the physiologic ratio in blood. Inositol absorption at intestinal level and its therapeutic effect in PCOS are significantly improved with the coadministration of alpha-lactalbumin. In conclusion, inositols represent an important therapeutic advance for PCOS treatment. [ABSTRACT FROM AUTHOR]

The protein‐nanoparticles conjugate has been developed as a targeted drug delivery system which selectively and preferentially delivers the therapeutic agents. Here, the alpha‐lactalbumin (ALA) was purified by size exclusion chromatography and confirmed by gel electrophoresis and mass spectrometry (MS). Functionalization of gold nanoparticles (AuNP, ∼29 nm) with ALA was optimized by light scattering and zeta potential measurements. The nanoconjugates (AuNP‐ALA) and ALA showed strong binding and affinity with curcumin and gemcitabine studied using biophysical analysis. The presence of native form of curcumin with ALA was investigated by MS. 8‐anilino‐1‐naphthalenesulfonic acid binding assay showed 95 % surface hydrophilicity of ALA. Circular dichroism and Fourier transform infrared spectroscopy analysis shows increased alpha‐helicity due to the binding of drugs, revealed large increase in non‐covalent interaction. Vesicular membrane interaction with ALA and nanoconjugate exhibited strong interaction at the vesicles surface. The alpha‐lactalbumin‐nanoparticle conjugate in combination with drugs, could be exploited to establish a targeted drug delivery system. [ABSTRACT FROM AUTHOR]

Lactoferrin (Lf) and other whey proteins have been isolated from red deer milk for the first time using a three-step anion and cation exchange chromatography protocol. The separated deer Lf was subject to in vitro gastric and duodenal digestions to generate peptides. The purity of the deer Lf and its hydrolysis products were analyzed by SDS-PAGE. The antibacterial activity of the deer Lf and its hydrolysates were investigated and was compared to cow counterpart. Gastric and duodenal digested deer Lf had strong bactericidal activity against E. coli ATCC 25922 with minimum inhibition concentration (MIC) of 280 µM and 402 µM, respectively. These results suggest that deer milk contains bioactive whey proteins and can generate bioactive peptides, which can benefit human health by inhibiting food-borne pathogenic bacteria.

The bovine whey consists of more than 200 different types of proteins, of which β-lactoglobulin, α-lactalbumin, serum albumin, immunoglobulins and lactoferrin predominate. However, their concentrations are not stable due to the existence of protein dynamics during a transition from colostrum secretion to mature milk. To evaluate the dynamics of whey proteins of Jersey cows during a colostral phase and first month of lactation and an influence of the number of lactations, 268 milk samples from 135 Jersey cows were selected through a clinical evaluation. Whey was obtained by rennet coagulation of the mammary secretion. The concentration of total proteins was determined by the biuret method and their fractions were identified by 12% dodecyl sulfate-polyacrylamide gel electrophoresis (12% SDS-PAGE). Maximum concentrations of all protein fractions were observed in the first 12 h of lactation, reducing over the course of the study. Modification of the protein predominance was also observed. The transition from colostrum secretion to milk occurred between 24 and 72 h postpartum. There was an influence of the number of lactations on the dynamics of whey proteins, indicating that multiparous cows had better immunological and nutritional quality when compared to primiparous cows. [ABSTRACT FROM AUTHOR]

To gain knowledge on the molecular basis of diversity of several clans of Saudi camel (Camelus dromedarius) characterization of these animals was conducted at both genetic and protein levels. To this end, blood and milk samples were collected from several camel breeds at different Saudi Arabia locations (northern Jeddah, Riyadh, and Alwagh governorates). Genomic DNA was extracted from blood of four Saudi camel breeds (Majahem, Safra, Wadha, and Hamara), and DNA fragments of the casein and α-lactalbumin genes were amplified. The retrieved DNA sequences were analyzed for genetic variability. The inter-simple sequence repeat technique was used for confirming the relationships among the analyzed camel breeds, and the PCR-RFLP with two restriction enzymes was utilized for exploring their molecular variations. The number of haplotypes, gene diversity, nucleotide diversity, average number of nucleotide differences, and sequence conservation were calculated for all the analyzed DNA sequences. These analyses revealed the presence of several single nucleotide polymorphisms in the analyzed DNA sequences. A group of neighbor joining trees was built for inferring the evolutionary variations among the studied animals. Protein profiling of milk from different camel clans was also conducted, and differences between and within the Saudi camel clans were easily found based on the isoelectric focusing (IEF) profiles using ampholytes with different IEF range. This study revealed that analyzed camel breeds show low levels of genetic differences. This may be a reflection of the evolutionary history of C. dromedarius that was domesticated based on a highly homogeneous ancestor ecotype. [ABSTRACT FROM AUTHOR]

[Display omitted] • The isomer transition did not change the quenching type of Azo to α-La. • The unique structure of aromatic cluster 2 effected the binding of Azo with α-La. • The cis -isomer of Azo binds to α-La more strongly than the trans -isomer. • The binding of Azo will lead to a slight discrete trend in the structure of α-La. The combination of light and photoresponsive compounds provides a peculiar way of regulating biological systems. Azobenzene is a classical organic compound with photoisomerization properties. Exploring the interactions between azobenzene and proteins can deepen the biochemical applications of the azobenzene compounds. In this paper, the interaction of 4-[(2,6-dimethylphenyl)diazenyl]-3,5-dimethylphenol with alpha-lactalbumin was investigated by UV–Vis absorption spectra, multiple fluorescence spectra, computer simulations, and circular dichroism spectra. Most critically, the interaction differences between proteins and the trans - and cis -isomer of ligands have been analyzed and compared. Results showed that both isomers of ligands were bound to alpha-lactalbumin to form ground state complexes and statically quenched the steady-state fluorescence of alpha-lactalbumin. The van der Waals forces and hydrogen bonding dominated the binding; the difference is that the binding of the cis -isomer to alpha-lactalbumin is more rapidly stabilized, and the binding strength is greater than the trans -isomer. These binding differences were modeled and analyzed by molecular docking and kinetic simulations, and we found that both isomers bind through the hydrophobic aromatic cluster 2 of alpha-lactalbumin. However, the bent structure of the cis -isomer is more closely aligned with the construction of the aromatic cluster and may have contributed to the above differences. [ABSTRACT FROM AUTHOR]

The fluorescence quenching measurements and docking studies were used for investigating the binding of kaempferol and genistein as functional food ingredients to bovine α-lactalbumin (BLA) as a natural vehicle for bioactive ions and molecules. The closeness of the tryptophan residues to the bound ligands than to the tyrosine residues and the considerable change in microregion of the tryptophan residues in BLA is observed upon the binding of the genistein and kaempferol to BLA by synchronous fluorescence. The essential parameters which describe the characteristics of the binding such as the thermodynamic parameters, the binding constants and the binding stoichiometry were obtained from the fluorescence quenching measurements. The extent of vicinity of the bound ligands to the tryptophan residues of BLA was estimated by Förster's distance. The major role of hydrogen bonds in stabilizing the final ligand–protein complexes was concluded from the thermodynamic parameters which were calculated in different temperatures. The results of docking studies indicated that kaempferol and gnistein come closer to the Trp-60 than to other tryptophans and bind to BLA by three and five hydrogen bonds, respectively. The calculated distances between bound ligands and tryptophans obtained by docking studies were in agreement with fluorescence resonance energy transfer results. These findings can reveal that BLA can not only carry the metal ions, but also has binding affinity for natural polyphenolic compounds.

Breast cancer immunotherapy is a potent treatment option, with antibody therapies such as trastuzumab increasing 2-year survival rates by 50%. However, active immunotherapy through vaccination has generally been clinically ineffective. One potential means of improving vaccine therapy is by delivering breast cancer antigens to dendritic cells (DCs) for enhanced antigen presentation. To accomplish this in vivo, we pseudotyped lentiviral vector (LV) vaccines with a modified Sindbis Virus glycoprotein so that they could deliver genes encoding the breast cancer antigen alpha-lactalbumin (Lalba) or erb-b2 receptor tyrosine kinase 2 (ERBB2 or HER2) directly to resident DCs. We hypothesized that utilizing these DC-targeting lentiviral vectors as a breast cancer vaccine could lead to an improved immune response against self-antigens found in breast cancer tumors. Indeed, single injections of the vaccine vectors were able to amplify antigen-specific CD8 T cells 4–6-fold over naïve mice, similar to the best published vaccine regimens. Immunization of these mice completely inhibited tumor growth in a foreign antigen environment (LV-ERBB2 in wildtype mice), and it reduced the rate of tumor growth in a self-antigen environment (LV-Lalba in wildtype or LV-ERBB2 in MMTV-huHER2 transgenic). These results show that a single injection with targeted lentiviral vectors can be an effective immunotherapy for breast cancer. Furthermore, they could be combined with other immunotherapeutic regimens to improve outcomes for patients with breast cancer. [ABSTRACT FROM AUTHOR]

Vaccination is a promising strategy to trigger and boost immune responses against cancer or infectious disease. We have designed, synthesized and characterized aliphatic-polyester (poly(lactic- co -glycolic acid) (PLGA) nanoparticles (NP) to investigate how the nature of protein association (adsorbed versus entrapped) and polymer/surfactant concentrations impact on the generation and modulation of antigen-specific immune responses. The ability of the NP formulations to target dendritic cells (DC), be internalized and activate the T cells was characterized and optimized in vitro and in vivo using markers of DC activation and co-stimulatory molecules. Ovalbumin (OVA) was used as a model antigen in combination with the engraftment of CD4 + and CD8 + T cells, carrying a transgenic OVA-responding T cell receptor (TCR), to trace and characterize the activation of antigen-specific CD4 + and CD8 + lymph node T cells upon NP vaccination. Accordingly, the phenotype and frequency of immune cell stimulation induced by the NP loaded with OVA, isolated or in combination with synthetic unmethylated cytosine-phosphate-guanine (CpG) oligodeoxynucleotide (ODN) motifs, were characterized. DC-NP interactions increased with incubation time, presenting internalization values between 50 and 60% and 30–40%, in vitro and in vivo , respectively. Interestingly, animal immunization with antigen-adsorbed NP up-regulated major histocompatibility complex (MHC) class II (MHCII), while NP entrapping the antigen up-regulated MHCI, suggesting a more efficient cross-presentation. On the other hand, rather surprisingly, the surfactant used in the NP formulation had a major impact on the activation of antigen presenting cells (APC). In fact, DC collected from lymph nodes of animals immunized with NP prepared using poly(vinil alcohol) (PVA), as a surfactant, expressed significantly higher levels of CD86, MHCI and MHCII. In addition, those NP prepared with PVA and co-entrapping OVA and the toll-like receptor (TLR) ligand CpG, induced the most profound antigen-specific T cell response, by both CD4 + and CD8 + T cells, in vivo . Overall, our data reveal the impact of NP composition and surface properties on the type and extension of induced immune responses. Deeper understanding on the NP-immune cell crosstalk can guide the rational development of nano-immunotherapeutic systems with improved and specific therapeutic efficacy and avoiding off-target effects. [ABSTRACT FROM AUTHOR]