A novel proteomic approach for the identification and relative quantification of disulfide-bridges in the human milk proteome.

This study explores the disulfide bridges present in the human milk proteome by a novel approach permitting both positional identification and relative quantification of the disulfide bridges. Human milk from six donors was subjected to trypsin digestion without reduction. The digested human milk proteins were analyzed by nanoLC-timsTOF Pro combined with data analysis using xiSEARCH. A total of 85 unique disulfide bridges were identified in 25 different human milk proteins. The total relative abundance of disulfide bridge-containing peptides constituted approximately 5% of the total amount of tryptic-peptides. Seven inter-molecular disulfide bridges were identified between either α-lactalbumin and lactotransferrin (5) or αS1-casein and κ-casein (2). All cysteines involved in the observed disulfide bridges of α-lactalbumin and lactotransferrin were mapped onto protein models using AlphaFold2 Multimer to estimate the length of the observed disulfide bridges. The lengths of the disulfide bridges of lactotransferrin indicate a potential for multi- or poly-merization of lactotransferrin. The high number of intramolecular lactotransferrin disulfide bridges identified, suggests that these are more heterogeneous than previously presumed. SIGNIFICANCE: Disulfide-bridges in the human milk proteome are an often overseen post-transaltional modification. Thus, mapping the disulfide-bridges, their positions and relative abundance, are valuable new knowledge needed for an improved understanding of human milk protein behaviour. Although glycosylation and phosphorylation have been described, even less information is available on the disulfide bridges and the disulfide-bridge derived protein complexes. This is important for future work in precision fermentation for recombinant production of human milk proteins, as this will highlight which disulfide-bridges are naturally occouring in human milk proteins. Further, this knowledge would be of value for the infant formula industry as it provides more information on how to humanize bovine-milk based infant formula. The novel method developed here can be broadly applied in other biological systems as the disulfid-brigdes are important for the structure and functionality of proteins.

Frequency of misdiagnosis in hypertrophic cardiomyopathy.

BACKGROUND: Hypertrophic Cardiomyopathy (HCM) is characterized by unexplained left ventricle hypertrophy (LVH) ≥15 mm. The condition is often hereditary and family screening is recommended to reduce the risk of adverse disease complications and premature death among relatives. Correct diagnosis of index patients is important to ensure that only relatives at risk of disease development are invited for family screening. PURPOSE: To investigate if patients with ICD-10 codes for HCM (DI421) or hypertrophic obstructive cardiomyopathy (DI422) fulfilled recognised diagnostic criteria. METHODS: All patients with ICD-10 codes for HCM or HOCM at a Department of Cardiology were identified and had their diagnosis validated by a cardiac investigation or a review of their medical records and previous investigations. RESULTS: Two hundred and forty patients had ICD-10 codes for HCM/HOCM, of whom 202 (84%, 202/240) underwent re-examination, while 38 (16%, 38/240) had their hospital notes reviewed. Seventy-six patients (32%, n = 76/240) did not fulfil diagnostic criteria, of whom 39, (51%, n = 39/76) had normal (10 mm) or modest LV wall thickness (11-14 mm). The remaining 37 patients (49%, n = 37/76) had LVH ≥15 mm, which was well-explained by uncontrolled hypertension, (32%, n = 24/76), aortic valve stenosis (19%, n = 7/76) or wild-type amyloidosis (16%, 6/76). CONCLUSION: One-third of patients with ICD-10 codes for HCM or HOCM did not fulfil recognised diagnostic criteria. Incorrect diagnosis of HCM may cause unnecessary family investigations which may be associated with anxiety, and a waste of health care resources. This highlights the need for specialised cardiomyopathy services to ensure correct diagnosis and management of HCM.

Educational attainment in eating disorders: What can we learn from visualising data.

BACKGROUND: Educational attainment is an understudied outcome in eating disorders (ED). We compared the educational attainment of individuals with and without ED. METHODS: This study is a nationwide, register-based, observational epidemiological study using record linkage. The studied cohorts were (1) all persons treated psychiatrically for ED from 1970 to 2014, and (2) a control population matched for sex, age, and place of residence. The International Standard Classification of Education 2011 was used to classify educational attainment. We employed ineqord, a series of graphical and analytical tools that are appropriate for comparing the distributions of ordinal data (Jenkins, 2020). RESULTS: Females with ED attained higher educational levels than males with ED. Males with ED had lower average educational levels than controls. On average, female controls attained higher educational levels than patients with ED in the eating disorders not otherwise specified or overeating groups. Females with anorexia nervosa, differed from matched controls: While their median was the same, too many participants were in the lower and higher levels of educational attainment. Females with bulimia nervosa had higher educational levels than matched controls on average. CONCLUSIONS: Educational attainment differs between individuals with and without out ED for all ED diagnoses and in both sexes.

Fingerprinting of Proteases, Protease Inhibitors and Indigenous Peptides in Human Milk.

The presence of proteases and their resulting level of activity on human milk (HM) proteins may aid in the generation of indigenous peptides as part of a pre-digestion process, of which some have potential bioactivity for the infant. The present study investigated the relative abundance of indigenous peptides and their cleavage products in relation to the abundance of observed proteases and protease inhibitors. The proteomes and peptidomes in twelve HM samples, representing six donors at lactation months 1 and 3, were profiled. In the proteome, 39 proteases and 29 protease inhibitors were identified in 2/3 of the samples. Cathepsin D was found to be present in higher abundance in the proteome compared with plasmin, while peptides originating from plasmin cleavage were more abundant than peptides from cathepsin D cleavage. As both proteases are present as a system of pro- and active- forms, their activation indexes were calculated. Plasmin was more active in lactation month 3 than month 1, which correlated with the total relative abundance of the cleavage product ascribed to plasmin. By searching the identified indigenous peptides in the milk bioactive peptide database, 283 peptides were ascribed to 10 groups of bioactivities. Antimicrobial peptides were significantly more abundant in month 1 than month 3; this group comprised 103 peptides, originating from the ß-CN C-terminal region.

Discovery of NSD2-Degraders from Novel and Selective DEL Hits.

NSD2 is a histone methyltransferase predominantly catalyzing di-methylation of histone H3 on lysine K36. Increased NSD2 activity due to mutations or fusion-events affecting the gene encoding NSD2 is considered an oncogenic event and a driver in various cancers, including multiple myelomas carrying t(4;14) chromosomal translocations and acute lymphoblastic leukemia's expressing the hyperactive NSD2 mutant E1099 K. Using DNA-encoded libraries, we have identified small molecule ligands that selectively and potently bind to the PWWP1 domain of NSD2, inhibit NSD2 binding to H3K36me2-bearing nucleosomes, but do not inhibit the methyltransferase activity. The ligands were subsequently converted to selective VHL1-recruiting NSD2 degraders and by using one of the most efficacious degraders in cell lines, we show that it leads to NSD2 degradation, decrease in K3 K36me2 levels and inhibition of cell proliferation.

The obesity-linked human lncRNA AATBC stimulates mitochondrial function in adipocytes.

Adipocytes are critical regulators of metabolism and energy balance. While white adipocyte dysfunction is a hallmark of obesity-associated disorders, thermogenic adipocytes are linked to cardiometabolic health. As adipocytes dynamically adapt to environmental cues by functionally switching between white and thermogenic phenotypes, a molecular understanding of this plasticity could help improving metabolism. Here, we show that the lncRNA Apoptosis associated transcript in bladder cancer (AATBC) is a human-specific regulator of adipocyte plasticity. Comparing transcriptional profiles of human adipose tissues and cultured adipocytes we discovered that AATBC was enriched in thermogenic conditions. Using primary and immortalized human adipocytes we found that AATBC enhanced the thermogenic phenotype, which was linked to increased respiration and a more fragmented mitochondrial network. Expression of AATBC in adipose tissue of mice led to lower plasma leptin levels. Interestingly, this association was also present in human subjects, as AATBC in adipose tissue was inversely correlated with plasma leptin levels, BMI, and other measures of metabolic health. In conclusion, AATBC is a novel obesity-linked regulator of adipocyte plasticity and mitochondrial function in humans.

Clinical outcomes using a 3D printed tandem-needle-template and the EMBRACE-II planning aims for image guided adaptive brachytherapy in locally advanced cervical cancer.

BACKGROUND: Extensive local disease or narrow vagina may compromise brachytherapy (BT) in patients with cervical cancer. This is the first study to analyze long-term outcomes of using 3D printed vaginal tandem-needle templates (3DP TNT) for transvaginal insertion of needles in parallel (P) or parallel and oblique (P&O) direction to the tandem. MATERIAL AND METHODS: All patients treated with BT using 3DP TNT from 2015-2020 were included. Decision to use a 3DP TNT and preplanning were made after 4-5 weeks of external beam radiotherapy, based on gynecological examination and MRI with a tandem-ring applicator in situ. The TNT was 3D-printed in house consisting of a circular template with P&O holes for guidance of plastic needles and a shaft fitting the uterine tandem. Thus, the radioactive source was never in direct contact with the 3DP TNT. The TNT was 3D printed in a standard or personalized configuration. Planning aims were based on the Embrace II protocol. RESULTS: 101 patients (median age of 63 years) were included: 49 with P needles only and 52 with P&O needles. Personalized TNT was used in 19 patients in the P&O group. Performance status (WHO) was > 0 in 48%. FIGO2018 stage III-IV was present in 77%. T-score at diagnosis and BT was 9.1 and 6.3 respectively, with a significantly higher T-score in the P&O compared to P group. The mean high-risk CTV D90 was 93 Gy with no significant difference between the two groups. Three-year local control rates were 85%, 95%, 75% for the overall, P- and P&O group respectively and 68%, 80% and 56% for cancer specific survival. Grade ≥3 treatment related complications were observed in 10 (10%) patients. CONCLUSIONS: 3DP TNT for BT in cervical cancer provides successful management of very extensive local disease and/or unfavorable anatomy with the possibility for treatment individualization.

Bioactive milk peptides: an updated comprehensive overview and database.

Partial digestion of milk proteins leads to the formation of numerous bioactive peptides. Previously, our research team thoroughly examined the decades of existing literature on milk bioactive peptides across species to construct the milk bioactive peptide database (MBPDB). Herein, we provide a comprehensive update to the data within the MBPDB and a review of the current state of research for each functional category from in vitro to animal and clinical studies, including angiotensin-converting enzyme (ACE)-inhibitory, antimicrobial, antioxidant, dipeptidyl peptidase (DPP)-IV inhibitory, opioid, anti-inflammatory, immunomodulatory, calcium absorption and bone health and anticancer activity. This information will help drive future research on the bioactivities of milk peptides.

Exercise increases myocardial free fatty acid oxidation in subjects with metabolic dysfunction-associated fatty liver disease.

BACKGROUND AND AIMS: Metabolic dysfunction-associated fatty liver disease (MAFLD) is associated with dyslipidemia and may promote cardiac lipotoxicity. Myocardial free fatty acids (FFA) oxidation (MOFFA) is normal in pre-diabetes, but reduced in heart failure. We hypothesized that during exercise MOFFA, very low-density lipoprotein triglycerides (VLDL-TG) secretion, hepatic FFA utilization, and lactate production differ among obese subjects with and without MAFLD. METHODS: Nine obese subjects with MAFLD and 8 matched subjects without MAFLD (Control) without a history of heart failure and cardiovascular disease were compared before and after 90-min exercise at 50% Peak oxygen consumption. Basal and exercise induced cardiac and hepatic FFA oxidation, uptake and re-esterification and VLDL-TG secretion were measured using [11C]palmitate positron-emission tomography and [1-14C]VLDL-TG. RESULTS: In the heart, increased MOFFA was observed after exercise in MAFLD, whereas MOFFA decreased in Control (basal vs exercise, MAFLD: 4.1 (0.8) vs 4.8 (0.8) µmol·100 ml-1 min-1; Control: 4.9 (1.8) vs 4.0 (1.1); µmol·100 ml-1 min-1, mean (SD), p < 0.048). Hepatic FFA fluxes were significantly lower in MAFLD than Control and increased ≈ two-fold in both groups. VLDL-TG secretion was 50% greater in MAFLD at rest and similarly suppressed during exercise. Plasma lactate increased significantly less in MAFLD than Control during exercise. CONCLUSIONS: Using robust tracer-techniques we found that obese subjects with MAFLD do not downregulate MOFFA during exercise compared to Control, possibly due to diminished lactate supply. Hepatic FFA fluxes are significantly lower in MAFLD than Control, but increase similarly with exercise. VLDL-TG export remains greater in MAFLD compared to Control. Basal and post-exercise myocardial and hepatic FFA, VLDL-TG and lactate metabolism is abnormal in subjects with MAFLD compared to Control.

Assessing welfare risks in unowned unsocialised domestic cats in Denmark based on associations with low body condition score.

BACKGROUND: Populations of unowned unsocialised cats are present worldwide. Generally, there is concern about their welfare. Low body condition score (BCS) is a potentially relevant indicator that is relatively easy to assess: emaciated cats are likely to have welfare problems while thin cats may be at risk of becoming emaciated. The objective of this study was to assess the association of low BSC with a selection of factors relating to the host, disease, and infection in unowned unsocialised domestic cats. We necropsied 598 euthanised unowned unsocialised cats from Denmark. We recorded each cat's age-group, sex, and neuter status, together with its pregnancy status, the season and location of trapping, as well as gross lesions at necropsy. We also tested for feline immunodeficiency virus and feline leukaemia virus, recorded presence of ectoparasites, and a subsample of the cats were also tested for endoparasites. Cats with no or sparse adipose deposits were categorised as having low BCS, and logistic regression was used to determine the factors associated with low BCS. RESULTS: Of the cats, 11.4% had low BCS. Season, age-group and sex were associated with low BCS and confounded potential associations with other variables. Intact adult males and females in spring and early summer were at highest risk of low BCS. When these factors were taken into account, cats with biting lice had 2.8 (95% confidence interval (CI) 1.4-5.4) times higher odds of low BCS, and cats with gastro-intestinal findings (i.e., enlarged mesenteric lymph nodes, abdominal hernia, diarrhoea, obstructive foreign bodies, or diaphragmatic hernia) had 50 (95% CI 10-417) times higher odds of low BCS, than cats with no such findings. Cats with low BCS were primarily adult intact cats with tooth lesions and skin lesions, ear mite infection, and positive test result for feline immunodeficiency virus. CONCLUSIONS: The results highlight associations that can be used to define a risk profile: low BCS, notably in summer-autumn, in an unowned unsocialised cat was associated with underlying, less visible problems. Thus, low BCS can be more than a step towards being emaciated; it can also be an indicator of other underlying welfare problems.

Lessons Learned, Challenges Taken, and Actions Made for "Precision" Immunohistochemistry. Analysis and Perspectives From the NordiQC Proficiency Testing Program.

Immunohistochemistry (IHC) has for decades been an integrated method within pathology applied to gain diagnostic, prognostic, and predictive information. However, the multimodality of the analytical phase of IHC is a challenge to ensure the reproducibility of IHC, which has been documented by external quality assessment (EQA) programs for many biomarkers. More than 600 laboratories participate in the Nordic immunohistochemical Quality Control EQA program for IHC. In the period, 2017-2021, 65 different biomarkers were assessed and a total of 31,967 results were evaluated. An overall pass rate of 79% was obtained being an improvement compared with 71% for the period, 2003-2015. The pass rates for established predictive biomarkers (estrogen receptor, progesterone receptor, and HER2) for breast carcinoma were most successful showing mean pass rates of 89% to 92%. Diagnostic IHC biomarkers as PAX8, SOX10, and different cytokeratins showed a wide spectrum of pass rates ranging from 37% to 95%, mean level of 75%, and attributed to central parameters as access to sensitive and specific antibodies but also related to purpose of the IHC test and validation performed accordingly to this. Seven new diagnostic biomarkers were introduced, and all showed inferior pass rates compared with the average level for diagnostic biomarkers emphasizing the challenge to optimize, validate, and implement new IHC biomarkers. Nordic immunohistochemical Quality Control operates by "Fit-For-Purpose" EQA principles and for programmed death-ligand 1, 2 segments are offered aligned to the "3-dimensional" approach-bridging diagnostic tests, drugs to be offered, and diseases addressed. Mean pass rates of 65% and 79% was obtained in the 2 segments for programmed death-ligand 1.

Human visceral and subcutaneous adipose stem and progenitor cells retain depot-specific adipogenic properties during obesity.

Abdominal obesity associates with cardiometabolic disease and an accumulation of lipids in the visceral adipose depot, whereas lipid accumulation in the subcutaneous depot is more benign. We aimed to further investigate whether the adipogenic properties where cell-intrinsic, or dependent on a depot-specific or obesity-produced microenvironment. We obtained visceral and subcutaneous biopsies from non-obese women (n = 14) or women living with morbid obesity (n = 14) and isolated adipose stem and progenitor cells (ASPCs) from the stromal vascular fraction of non-obese (n = 13) and obese (n = 13). Following in vitro differentiation into mature adipocytes, we observed a contrasting pattern with a lower gene expression of adipogenic markers and a higher gene expression of immunogenic markers in the visceral compared to the subcutaneous adipocytes. We identified the immunogenic factor BST2 as a marker for visceral ASPCs. The effect of obesity and insulin resistance on adipogenic and immunogenic markers in the in vitro differentiated cells was minor. In contrast, differentiation with exogenous Tumor necrosis factor resulted in increased immunogenic signatures, including increased expression of BST2, and decreased adipogenic signatures in cells from both depots. Our data, from 26 women, underscore the intrinsic differences between human visceral and subcutaneous adipose stem and progenitor cells, suggest that dysregulation of adipocytes in obesity mainly occurs at a post-progenitor stage, and highlight an inflammatory microenvironment as a major constraint of human adipogenesis.

Access and quality of biomarker testing for precision oncology in Europe.

BACKGROUND: Predictive biomarkers are essential for selecting the best therapeutic strategy in patients with cancer. The International Quality Network for Pathology, the European Cancer Patient Coalition and the European Federation of Pharmaceuticals Industries and Associations evaluated the access to and quality of biomarker testing across Europe. METHODS: Data sources included surveys of 141 laboratory managers and 1.665 patients, and 58 in-depth interviews with laboratory managers, physicians and payers. Four access metrics (laboratory access, test availability, test reimbursement, test order rate) and three quality metrics (quality scheme participation, laboratory accreditation, test turnaround time) were applied to rank the results. RESULTS: The access to precision medicines is higher in countries with public national reimbursement processes in place. Lack of diagnostic laboratory infrastructure, inefficient organization and/or insufficient public reimbursement narrow the access to single biomarker tests in many European countries. In countries with limited public reimbursement, pharma and patients' out of pocket were the primary funding sources for testing. Uptake of multi-biomarker next generation sequencing (NGS) is highly varied, ranging from 0% to >50%. Financial constraints, a lack of NGS testing capabilities and the failure to include NGS testing in the guidelines represent the main barriers to NGS implementation. The quality of biomarker testing is highest in Western and Northern Europe, with more than 90% of laboratories participating in quality assurance schemes. CONCLUSIONS: Our data clearly indicate the need for a call to action to ensure the clinical implementation of precision medicine in Europe.

A Consortium for Analytic Standardization in Immunohistochemistry.

CONTEXT.­: The authors announce the launch of the Consortium for Analytic Standardization in Immunohistochemistry, funded with a grant from the National Cancer Institute. As with other laboratory testing, analytic standards are important for many different stakeholders: commercial vendors of instruments and reagents, biopharmaceutical firms, pathologists, scientists, clinical laboratories, external quality assurance organizations, and regulatory bodies. Analytic standards are customarily central to assay development, validation, and method transfer into routine assays and are critical quality assurance tools. OBJECTIVE.­: To improve immunohistochemistry (IHC) test accuracy and reproducibility by integrating analytic standards into routine practice. To accomplish this mission, the consortium has 2 mandates: (1) to experimentally determine analytic sensitivity thresholds (lower and upper limits of detection) for selected IHC assays, and (2) to inform IHC stakeholders of what analytic standards are, why they are important, and how and for what purpose they are used. The consortium will then publish the data and offer analytic sensitivity recommendations where appropriate. These mandates will be conducted in collaboration and coordination with clinical laboratories, external quality assurance programs, and pathology organizations. DATA SOURCES.­: Literature review and published external quality assurance data. CONCLUSIONS.­: Integration of analytic standards is expected to (1) harmonize and standardize IHC assays; (2) improve IHC test accuracy and reproducibility, both within and between laboratories; and (3) dramatically simplify and improve methodology transfer for new IHC protocols from published literature or clinical trials to clinical IHC laboratories.

Cysteine residues are responsible for the sulfurous off-flavor formed in heated whey protein solutions.

Odor-active volatile sulfur compounds are formed in heated food protein systems. In the present study, hydrogen sulfide (H2S) was found to be the most abundant sulfur volatile in whey protein solutions (whey protein isolate [WPI], a whey model system and single whey proteins) by gas chromatography-flame photometric detector (GC-FPD) analysis after heat treatments (60-90 °C for 10 min, 90 °C for 120 min and UHT-like treatment). H2S was detected in WPI after heating at 90 °C for 10 min, and was significantly increased at higher heat load (90 °C for 120 min and the UHT-like treatment). Site-specific LC-MS/MS-based proteomic analysis was conducted, monitoring desulfurization reactions in these protein systems to investigate the mechanism of H2S formation in heated WPI. Cysteine residues from beta-lactoglobulin were found to be responsible for the formation of H2S in heated WPI, presumably via beta-elimination.

Cost-Effectiveness of PD-L1 Testing in Non-Small Cell Lung Cancer (NSCLC) Using In Vitro Diagnostic (IVD) Versus Laboratory-Developed Test (LDT).

INTRODUCTION: Accurate PD-L1 testing for non-small cell lung cancer (NSCLC) maximizes the benefits of immune checkpoint inhibitor (ICI) drugs like pembrolizumab. False negative test results deny ICI treatments to eligible patients, worsening clinical and economic outcomes, while false positives increase costs by using ICI treatments without their benefits. This study evaluates the cost-effectiveness of PD-L1 testing with an in vitro diagnostic (IVD) compared to a laboratory-developed test (LDT) for allocating patients with NSCLC to treatment with either pembrolizumab or chemotherapy using the German healthcare system as a model. METHODS: We developed a decision analytical model to evaluate the cost-effectiveness of PD-L1 testing with a regulatory body approved IVD compared to an LDT from the national German healthcare payer (statutory health insurance system) perspective. Accuracy of PD-L1 testing was based on data from two independent proficiency testing programs. The 1-year model was based on outcomes data from the KEYNOTE-024 clinical trial and treatment patterns reflecting current German practices. RESULTS: IVDs produced accurate PD-L1 testing results in 93% (752/811) of tested cases compared to 73% (492/672) with LDTs. Most misclassifications concerned false negatives, occurring in 21% of LDTs vs 7% of IVDs. Total costs of the IVD group (48,878 €) were 196 € higher than the LDT group (48,682 €). These costs incorporate testing, first- and second-line therapy, managing treatment-related grade 3+ adverse events (AEs), and end-of-life costs for those who died within the year. Total effectiveness (percentage of patients successfully diagnosed and prescribed the correct therapy per German treatment guidelines) was 19 percentage points higher for the IVD group (88%) compared to the LDT group (69%). These differences in costs and effects lead to an incremental cost-effectiveness ratio (ICER) of 1057 €. CONCLUSION: Compared to LDT technology, on-label IVD use for PD-L1 testing is only slightly more costly and substantially more effective for aligning patients with PD-L1-positive NSCLC with ICI therapy according to German practice guidelines. Given these findings, changes to testing and reimbursement policies may be considered to maximize patient outcomes in NSCLC.

Comparison of Antibodies to Detect Uroplakin in Urothelial Carcinomas.

Immunohistochemistry for Uroplakin (UP) II and III is used to determine urothelial origin of carcinomas of unknown primary site and are especially valuable to differentiate urothelial carcinomas (UCs) from lung squamous cell carcinomas and prostate carcinomas. In the Nordic immunohistochemical Quality Control assessment scheme, only 45% of the participants obtained a sufficient staining result for UP. Primary antibodies (Abs) against UPII were most successful with a pass rate of 86%. No Abs against UPIII provided sufficient staining results. A comparative study was carried out on a larger cohort of tissue samples with optimized methods for the UPII mouse monoclonal antibody (mmAb) clone BC21, UPIII mmAb clone AU-1, and rabbit monoclonal antibody (rmAb) clone SP73 to evaluate the performance in a standardized way. Tissue microarrays containing 58 UCs, 111 non-UCs, and 20 normal tissues were included. The UP stains were evaluated by using H-score. Based on H-scores, samples were categorized as high-expressor (150 to 300), moderate-expressor (10 to 149), low-expressor (1 to 9), and negative (<1). The UPII mmAb clone BC21 obtained a significant higher analytical sensitivity of 69% for UCs compared with the UPIII Abs mmAb clone AU-1 and rmAb clone SP73 with 19% and 29%, respectively. No high-expressor UCs were seen for the UPIII Abs, whereas 13% of the positive UCs obtained an H-score >150 for the UPII Ab. The 2 UPIII Abs gave an analytical specificity of 100% compared with 97% for the UPII Ab being positive in 2 ovarian carcinomas and 1 cervical squamous cell carcinoma.

Oxidation of Whey Proteins during Thermal Treatment Characterized by a Site-Specific LC-MS/MS-Based Proteomic Approach.

Thermal treatment is often employed in food processing to tailor product properties by manipulating the ingredient functionality, but these elevated temperatures may accelerate oxidation and nutrient loss. Here, oxidation of different whey protein systems [α-lactalbumin (α-LA), ß-lactoglobulin (ß-LG), a mix of α-LA and ß-LG (whey model), and a commercial whey protein isolate (WPI)] was investigated during heat treatment at 60-90 °C and a UHT-like treatment by LC-MS-based proteomic analysis. The relative modification levels of each oxidation site were calculated and compared among different heat treatments and sample systems. Oxidation increased significantly in protein systems after heating at ≥90 °C but decreased in systems with higher complexity [pure protein (α-LA > ß-LG) > whey model > WPI]. In α-LA, Cys, Met, and Trp residues were found to be most prone to oxidation. In ß-LG-containing protein systems, Cys residues were suggested to scavenge most of the reactive oxidants and undergo an oxidation-mediated disulfide rearrangement. The rearranged disulfide bonds contributed to protein aggregation, which was suggested to provide physical protection against oxidation. Overall, limited loss of amino acid residues was detected after acidic hydrolysis followed by UHPLC analysis, which showed only a minor effect of heat treatment on protein oxidation in these protein systems.

Influence of Lactose on the Maillard Reaction and Dehydroalanine-Mediated Protein Cross-Linking in Casein and Whey.

A liquid chromatography-mass spectrometry method based on multiple reaction monitoring (MRM) was developed for the simultaneous quantification of markers representing two potentially competing pathways, the Maillard reaction and the dehydroalanine pathway. The two pathways involve the same residues in the proteins to some extent, namely, the essential amino acid lysine, as well as free-amino terminals available on proteins and polypeptides, competition between the two pathways in food systems may occur. The developed method comprises the following markers of the Maillard reaction: furosine, N-ε-(carboxyethyl)lysine (CEL) and N-ε-(carboxymethyl)lysine (CML), together with the dehydroalanine reaction pathway markers; lanthionine (LAN) and lysinoalanine (LAL), as well as lysine itself. The validated method was then used for the absolute quantification of heat-induced protein modifications in model systems of micellar casein and whey protein isolates (MCI and WPI, respectively) in the presence or absence of lactose. As expected, the Maillard reaction markers furosine, CEL and CML increased during the applied heat treatment in the presence of lactose, whereas the dehydroalanine markers, LAN and LAL increased with heating in both MCI and WPI, both in the presence and absence of lactose, although at lower levels in the presence of lactose, confirming the competing state of the two pathways.