Release of bifidogenic N-glycans from native bovine colostrum proteins by an endo-ß-N-acetylglucosaminidase.

Milk glycoproteins play various biological roles including antibacterial, antiviral activities, modulating immune responses in living organisms. Released N-glycans from milk glycoproteins act as growth substrates for infant-associated bifidobacteria, which are key members of the breastfed infant's gut. To date, the mechanisms, and contributions of glycans to the biological activities of glycoproteins remain to be elucidated. Only by testing both the released glycans and the deglycosylated protein in their native (i.e., non-denatured) form, can the individual contribution to the biological activity of glycoproteins be elucidated. However, for conventional enzymatic and chemical deglycosylation strategies to work efficiently, glycoprotein denaturation is required, which alters the protein native shape, hindering further investigations of its biological roles. An endo-ß-N-acetylglucosaminidase (EndoBI-1) from Bifidobacterium longum subsp. infantis ATCC 15697 (B. infantis) was characterized as having the ability to release N-glycans from bovine milk glycoproteins efficiently, without the denaturation. In this study, the activity of EndoBI-1 was compared to a commercial enzyme to release N-glycans, the peptide-N-glycosidase F (PNGase F), using dairy glycoproteins as the substrate. The kinetic evaluation showed that EndoBI-1 displayed higher activity on native glycoproteins than PNGase F, with 0.036 mg/mL×min and 0.012 mg/mL×min glycan release, respectively. EndoBI-1 released a broader array of glycan structures compared to PNGase F from native glycoproteins. Thirty-two and fifteen distinct compositions were released from the native glycoproteins by EndoBI-1 and PNGase F, respectively, as characterized by advanced mass spectrometry. EndoBI-1 can be considered a promising enzyme for the release of N-glycans and their protein backbone in the native form, which will enable effective glycan release and will facilitate subsequent investigations to reveal their contribution to glycoproteins' biological roles.

Role of milk glycome in prevention, treatment, and recovery of COVID-19.

Milk contains all essential macro and micro-nutrients for the development of the newborn. Its high therapeutic and antimicrobial content provides an important function for the prevention, treatment, and recovery of certain diseases throughout life. The bioactive components found in milk are mostly decorated with glycans, which provide proper formation and modulate the biological functions of glycosylated compounds. The glycome of milk consists of free glycans, glycolipids, and N- and O- glycosylated proteins. Recent studies have shown that both free glycans and glycan-containing molecules have antiviral characteristics based on different mechanisms such as signaling, microbiome modulation, natural decoy strategy, and immunomodulatory action. In this review, we discuss the recent clinical studies and potential mechanisms of free and conjugated glycans' role in the prevention, treatment, and recovery of COVID-19.

Lactoferrin for COVID-19 prevention, treatment, and recovery.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), a unique beta-coronavirus, has caused the most serious outbreak of the last century at the global level. SARS-CoV-2 infections were firstly reported in the city of Wuhan in China in 2019 and this new disease was named COVID-19 by World Health Organization (WHO). As this novel disease can easily be transmitted from one individual to another via respiratory droplets, many nations around the world have taken several precautions regarding the reduction in social activities and quarantine for the limitation of the COVID-19 transmission. SARS-CoV-2 is known to cause complications that may include pneumonia, acute respiratory distress syndrome (ARDS), multi-organ failure, septic shock, and death. To prevent and treat COVID-19, some significant studies have been conducted since the outbreak. One of the most noticeable therapeutic approaches is related to a multifunctional protein, lactoferrin. Lactoferrin (Lf) is an 80 kDa cationic glycoprotein that has a great range of benefits from improving the immunity to antiviral effects due to its unique characteristics such as the iron-binding ability. This review summarizes the characteristics of SARS-CoV-2 and the potential applications of Lf for the prevention, treatment, and recovery of COVID-19.

Benefits of A2 Milk for Sports Nutrition, Health and Performance.

Bovine milk is one of the best pre-and pro-workout sources for athletes owing to its rich nutritional content. Even though bovine milk consumption significantly benefits athletes' health and performance, many athletes cannot consume bovine milk since they struggle with gastrointestinal problems caused after milk consumption. Especially, the consumption of regular milk, which contains A1 ß-casein, is associated with a variety of diseases ranging from gastrointestinal discomfort to ischemic heart diseases. The main reason behind this is related to ß-casomorphine 7 (BCM-7), which is derived from A1 ß-casein during the digestion of A1 milk. A1 ß-casein is formed as a result of a point mutation in the position of 67th in the amino acid sequence A2 ß-casein by changing proline to histidine. Therefore, this mutated form of ß-casein in regular milk cannot easily be digested by the human-associated digestion enzymes. A2 milk, which includes A2 ß-casein instead of A1 ß-casein, is the best substitute for regular milk with the same nutritional content. This natural form of milk positively affects the athlete's health as well as performance without causing any gastrointestinal discomfort or more serious problems which are seen in the consumption of regular milk. In this review, A2 milk and its potential health effects in comparison to diseases related to A1 milk consumption are discussed.

Immobilization of a Bifidobacterial Endo-ß-N-Acetylglucosaminidase to Generate Bioactive Compounds for Food Industry.

Conjugated N-glycans are considered next-generation bioactive prebiotic compounds due to their selective stimulation of beneficial microbes. These compounds are glycosidically attached to proteins through N-acetylglucosamines via specific asparagine residue (AsN-X-Ser/Thr). Certain bacteria such as Bifidobacterium longum subspecies infantis (B. infantis) have been shown to be capable of utilizing conjugated N-glycans, owing to their specialized genomic abilities. B. infantis possess a unique enzyme, Endo-ß-N-acetylglucosaminidase (EndoBI-1), which cleaves all types of conjugated N-glycans from glycoproteins. In this study, recombinantly cloned EndoBI-1 enzyme activity was investigated using various immobilization methods: 1) adsorption, 2) entrapment-based alginate immobilization, 3) SulfoLink-, and 4) AminoLink-based covalent bonding immobilization techniques were compared to develop the optimum application of EndoBI-1 to food processes. The yield of enzyme immobilization and the activity of each immobilized enzyme by different approaches were investigated. The N-glycans released from lactoperoxidase (LPO) using different immobilized enzyme forms were characterized using MALDI-TOF mass spectrometry (MS). As expected, regardless of the techniques, the enzyme activity decreased after the immobilization methods. The enzyme activity of adsorption and entrapment-based alginate immobilization was found to be 71.55% ± 0.6 and 20.32% ± 3.18, respectively, whereas the activity of AminoLink- and SulfoLink-based covalent bonding immobilization was found to be 58.05 ± 1.98 and 47.49% ± 0.30 compared to the free form of the enzyme, respectively. However, extended incubation time recovery achieved activity similar to that of the free form. More importantly, each immobilization method resulted in the same glycan profile containing 11 different N-glycan structures from a model glycoprotein LPO based on MALDI-TOF MS analysis. The glycan data analysis suggests that immobilization of EndoBI-1 is not affecting the enzyme specificity, which enables full glycan release without a limitation. Hence, different immobilization methods investigated in this study can be chosen for effective enzyme immobilization to obtain bioactive glycans. These findings highlight that further optimization of these methods can be a promising approach for future processing scale-up and commercialization of EndoBI-1 and similar enzymes.

Wet milling of buckwheat cultivars and some quality properties of the fractions.

The hulled buckwheat cultivars (Aktas cv. and Günes cv.) were wet-milled, and then some chemical, yields, colour, functional properties, phenolic compound, antioxidant activity, and pasting, thermal and retrogradation properties of starches were investigated and compared with the wholegrain buckwheat flour (with hull) and buckwheat groat flour (without hull) of the same cultivars. Sodium dodecyl sulphate polyacrylamide gel electrophoresis patterns of flours and protein fractions were examined under reducing and non-reducing conditions. The hull, germ+dietary fibre, protein and starch fractions were collected. The total recovery for Aktas cv. and Günes cv. cultivars were 98.1% and 96.1%; total starch yields were 51.6% and 49.7%; pasting temperatures of the starches were found as 83.7 and 85.7°C; and final viscosities of starches were determined as 3.5 and 3.4 Pa·s, respectively. The resistant starch contents of starch fractions of Aktas cv. ve Günes cv. were found as 3.28% and 3.62%, respectively. The highest total phenolic compound contents were detected with dimethyl sulphoxide extraction in the germ+dietary fibre fractions. The highest 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity and trolox equivalent antioxidant capacity were found in the hull fraction (as 81.7%) and germ+dietary fibre fraction (as 11.8 mmol/kg) of Aktas cv. cultivar.

Biofortified Whey/Deglycosylated Whey and Chickpea Protein Matrices: Functional Enrichment by Black Mulberry Polyphenols.

Morus nigra L. (black mulberry-BM) is a promising nutraceutical fruit containing biologically active polyphenols like anthocyanins, proanthocyanidins, catechins, and stilbenes, with well-established anti-inflammatory, antidiabetic, anti-obesity, and anticancer biofunctions. However, these health-promoting properties in raw fruit are greatly masked due to the presence of soluble and insoluble carbohydrates in excess amounts restricting daily intake of the required dose to achieve targeted effects. In the current study, different protein sources (defatted whey and chickpea flours) were optimized through different conditions to capture polyphenols from BM juice while diminishing its glucose content. To optimize polyphenol-protein interactions, various pHs (3.7, 4.2, and 4.7), matrix concentrations (20, 50, and 80 g protein/L), and incubation times (5, 20, and 45 min) were tested. In the present work, optimized BM polyphenol enriched whey matrix inhibited pro-inflammatory mediators and promoted Nrf-2 dependent cytoprotective enzyme expressions in lipopolysaccharide (LPS) induced macrophages at low doses. In addition, whey proteins were also subjected to an enzymatic deglycosylation process by using recently identified EndoBI-1 enzyme for the specific cleavage of N-glycan core in all glycan types including high mannoses, hybrids as well as complex glycans found on defatted whey proteins. After this process, the polyphenol sorption capacity of deglycosylated whey proteins was found to be significantly higher (37%) than the capacity of non-treated normal whey protein under optimized conditions. In conclusion, deglycosylation of protein matrices could be a novel strategy for efficient sorption/concentration of polyphenols from fruits and vegetables, however, more detailed studies are needed to understand this effect.

Determining Total Protein and Bioactive Protein Concentrations in Bovine Colostrum.

Colostrum is a complex biological fluid produced by mammals immediately after parturition. It meets all the nutritional requirements for neonates as a good source of macro- and micronutrients, bioactive peptides, and growth factors. Bovine colostrum is also a potential source of nutrition and bioactive because of its rich protein content that includes immunoglobulin G (IgG) and lactoferrin. However, the level of lactoferrin and IgG in bovine colostrum changes markedly during the lactation period. Therefore, monitoring the concentration of IgG and lactoferrin for the use of bovine colostrum as a protein source is an important question to study. Methods in this article describe how to determine protein content, as well as specific concentrations of lactoferrin and IgG. These methods include the following steps: Isolation of bovine colostrum proteins, Determination of protein concentration via Bicinchoninic acid assay (BCA), Visualization of proteins via SDS-PAGE, Determination of lactoferrin, and IgG concentration using an ELISA Assay.

Recombinant Production of Bifidobacterial Endoglycosidases for N-glycan Release.

Protein glycosylation is a diverse and common post-translational modification that has been associated with many important roles such as protein function, including protein folding, stability, enzymatic protection, and biological recognition. N-glycans attached to glycoproteins (such as lactoferrin, lactadherin, and immunoglobulins) cannot be digested by the host and reach the large intestine, where they are consumed by certain beneficial microbes. Therefore, they are considered next-generation prebiotic compounds that can selectively stimulate the gut microbiome's beneficial microorganisms. However, the isolation of these new classes of prebiotics requires novel enzymes. Here, we describe the recombinant production of novel glycosidases from different Bifidobacteria strains (isolated from infants, rabbits, chicken, and bumblebee) for improved N-glycan isolation from glycoproteins. The method presented in this study includes the following steps: molecular cloning of Bifidobacterial genes by an in vivo recombinational cloning strategy, control of transformation success, protein induction, and protein purification.

Bovine Colostrum and Its Potential for Human Health and Nutrition.

Colostrum is the first milk produced post-partum by mammals and is compositionally distinct from mature milk. Bovine colostrum has a long history of consumption by humans, and there have been a number of studies investigating its potential for applications in human nutrition and health. Extensive characterization of the constituent fractions has identified a wealth of potentially bioactive molecules, their potential for shaping neonatal development, and the potential for their application beyond the neonatal period. Proteins, fats, glycans, minerals, and vitamins are abundant in colostrum, and advances in dairy processing technologies have enabled the advancement of bovine colostrum from relative limitations of a fresh and unprocessed food to a variety of potential applications. In these forms, clinical studies have examined bovine colostrum as having the substantial potential to improve human health. This review discusses the macro-and micronutrient composition of colostrum as well as describing well-characterized bioactives found in bovine colostrum and their potential for human health. Current gaps in knowledge are also identified and future directions are considered in order to elevate the potential for bovine colostrum as a component of a healthy diet for a variety of relevant human populations.

Potential Applications of Endo-ß-N-Acetylglucosaminidases From Bifidobacterium longum Subspecies infantis in Designing Value-Added, Next-Generation Infant Formulas.

Human milk is the optimal source of infant nutrition. Among many other health benefits, human milk can stimulate the development of a Bifidobacterium-rich microbiome through human milk oligosaccharides (HMOs). In recent years, the development of novel formulas has placed particular focus on incorporating some of the beneficial functional properties of human milk. These include adding specific glycans aimed to selectively stimulate the growth of Bifidobacterium. However, the bifidogenicity of human milk remains unparalleled. Dietary N-glycans are carbohydrate structures conjugated to a wide variety of glycoproteins. These glycans have a remarkable structural similarity to HMOs and, when released, show a strong bifidogenic effect. This review discusses the biocatalytic potential of the endo-ß-N-acetylglucosaminidase enzyme (EndoBI-1) from Bifidobacterium longum subspecies infantis (B. infantis), in releasing N-glycans inherently present in infant formula as means to increase the bifidogenicity of infant formula. Finally, the potential implications for protein deglycosylation with EndoBI-1 in the development of value added, next-generation formulas are discussed from a technical perspective.

Leukemic stem cells shall be searched in the bone marrow before "tyrosine kinase inhibitor-discontinuation" in chronic myeloid leukemia.

BACKGROUND: Leukemic stem cells (LSCs) of chronic myeloid leukemia (CML), persisting in the bone marrow (BM) niche, could be responsible for the relapses within the patients of whom the treatment-free remission (TFR) had been attempted. We assessed the presence of the CML LSCs in the peripheral blood (PB) and concurrently in the BM in the patients with chronic-phase CML (CP CML). PATIENTS AND METHODS: Thirty-eight patients with CP CML were included into the study. CD45+ /CD34+ /CD38- cells with positive CD26 expression were considered as CML LSCs (CD26+ LSC) by using multiparameter flow cytometry (FCM). RESULTS: Mean BCR-ABL, PB LSC, and BM LSC were 58.528 IS (37.405-83.414 IS), 237.5 LSC/µL (16-737.5 LSC/µL), and 805 LSC/106  WBCs (134.6-2470 LSC/106  WBCs), respectively, in newly diagnosed CML patients. In the patients with BCR-ABL positive hematopoiesis, mean BCR-ABL, PB LSCs, and BM LSCs were 30.09 IS (0.024-147.690 IS), 13.5 LSC/µL (0-248.7 LSC/µL) and 143.5 LSC/106  WBCs (9-455.2 LSC/106  WBCs), respectively. No CML LSCs were detected in PB of patients who achieved deep molecular response (DMR). BM LSCs of the patients who were in DMR were 281.1 LSC/106  WBCs (3.1-613.7 LSC/106  WBCs). The amount of PB LSCs was highest in patients with newly diagnosed CML (P < .001). CONCLUSION: LSCs persisted in the BM of the patients with DMR, whereas there was no LSCs in the peripheral blood. The investigation of the CML LSCs in bone marrow before deciding TKI discontinuation could be justified to achieve and maintain stable TFR.

Production of Bovine Colostrum for Human Consumption to Improve Health.

Colostrum contains all essential nutrients for the neonate during the first days of life, with impacts that continue far beyond these first days. Bovine colostrum has been used for human consumption due to the high concentrations of bioactive proteins, vitamins, minerals, growth factors, as well as free and conjugated oligosaccharides. Processes involved in the preparation of bovine colostrum for human consumption play a pivotal role in preserving and maintaining the activity of the bioactive molecules. As bovine colostrum is a multifunctional food that offers a myriad of benefits for human health, assessing the main processes used in preparing it with both advantages and disadvantages is a crucial point to discuss. We discuss major processes effects for colostrum production on the nutritional value, some advanced technologies to preserve processed bovine colostrum and the end-product forms consumed by humans whether as dairy products or dietary supplements.

FGF-2 is a driving force for chromosomal instability and a stromal factor associated with adverse clinico-pathological features in prostate cancer.

BACKGROUND: There is mounting evidence to suggest that stromal cells play an integral role in the progression of prostate cancer (PCa). One of the most frequently altered growth factors in PCa is fibroblast growth factor-2 (FGF-2). It has previously been proposed that early stages of PCa are characterized by a primarily exogenous, that is, stromal cell-derived FGF-2 production, whereas advanced tumors rely more on an autocrine FGF-2 production. Prostate cancer progression is characterized by an increase of genomic instability including aneuploidy and structural chromosomal alterations. Herein, we address 2 problems that have not been comprehensively answered. First, we ask whether exogenous FGF-2 can directly drive genomic instability to promote PCa progression. Second, we investigate whether and to what extent stromal FGF-2 expression is maintained in advanced PCa and whether this influences tumor progression and patient prognosis. METHODS: In vitro experiments to investigate the role of FGF-2 in numerical and structural chromosomal instability were performed using immunofluorescence microscopy, fluorescence in situ hybridization and single cell electrophoresis. A human patient-derived xenograft mouse model recapitulating osteoblastic PCa bone metastasis was used for in vivo validation experiments. The prognostic role of stromal FGF-2 expression was analyzed using immunohistochemical staining of a tissue microarray with primary tumor specimens from 162 predominantly high-risk patients with PCa. RESULTS: Our results show that FGF-2 not only rapidly induces mitotic defects and numerical chromosomal imbalances but also an enhanced DNA breakage to promote chromosomal instability. Using the patient-derived xenograft model, we show that a deregulation of the FGF axis results in an increase of mitotic aberrations as well as DNA damage checkpoint activation in vivo. The FGFR inhibitor dovitinib was found to reduce numerical chromosomal instability as well as DNA breakage, thus underscoring the relevance of the FGF axis in promoting genomic instability. An overexpression of tumor cell-associated FGF-2 was detected in 52 of 162 patients (32.1%), whereas a stromal overexpression was found in 27 of 165 patients (16%). Remarkably, a strong stromal FGF-2 expression was associated with a significantly higher clinical stage and higher biochemical recurrence rate. Patients with strong stromal FGF-2 expression also had a significantly worse biochemical recurrence-free survival. CONCLUSIONS: Our results underscore that exogenous FGF-2 can shape PCa cell genomes and that stromal FGF-2 expression is detectable in a sizeable proportion of advanced PCa where it is associated with adverse clinico-pathological features. Our results highlight the impact of the tumor stroma on malignant progression and provide a rationale for a further exploration of components of the tumor stroma as therapeutic targets in PCa.

Effective downsizing but enhanced intratumoral heterogeneity following neoadjuvant sorafenib in patients with non-metastatic renal cell carcinoma.

OBJECTIVES: To evaluate the safety and feasibility of sorafenib prior to surgery for downsizing tumors in patients with non-metastatic cT1-3 renal tumors together with a characterization of functional intratumoral heterogeneity (ITH). MATERIALS AND METHODS: The effects of 4-week sorafenib prior to curative surgery were assessed in a prospective, single-center, randomized, placebo-controlled, double-blinded, pilot trial in patients with T1-3N0M0 renal cell carcinoma (RCC). Patients received sorafenib or placebo for 28 days prior to surgery. MRI was performed at baseline and prior to surgery to calculate tumor volume. The clinical responses were further characterized on the molecular level by immunohistochemical stainings for Ki-67, cleaved caspase-3, and CD31. RESULTS: After enrolling 20 patients into the study, 14 patients were randomized, of which 12 patients were available for analysis. While no significant change in tumor volume was seen for placebo (range = -24.2-0.2%) a reduction of 29.0% (range = -4.9-61.1%) was detected for sorafenib (p < 0.05). Primary renal tumor diameter changed from 10.6 cm (range = 6.5-10.8) to 10.7 cm (range = 6.7-11.1) in the placebo group, and from 5.4 cm (range = 4.3-7.3) to 4.4 cm (range = 3.5-6.8) for the sorafenib group, at baseline vs. 28 days of treatment. Correlative assessment of proliferation, apoptosis, and microvessel density revealed an enhanced degree of functional ITH in treated patients suggesting adaptive and/or regenerative processes with potential relevance for the development of drug resistance. CONCLUSIONS: Sorafenib in standard dosage, given preoperatively for 28 days, was clinically active in downsizing tumors in patients with locally confined, non-metastatic RCC together but led to an enhanced functional ITH in the residual tumor tissue.

Harnessing the p53-PUMA axis to overcome DNA damage resistance in renal cell carcinoma.

Resistance to DNA damage-induced apoptosis is a hallmark of cancer and a major cause of treatment failure and lethal disease outcome. A tumor entity that is largely resistant to DNA-damaging therapies including chemo- or radiotherapy is renal cell carcinoma (RCC). This study was designed to explore the underlying molecular mechanisms of DNA damage resistance in RCC to develop strategies to resensitize tumor cells to DNA damage-induced apoptosis. Here, we show that apoptosis-resistant RCC cells have a disconnect between activation of p53 and upregulation of the downstream proapoptotic protein p53 upregulated modulator of apoptosis (PUMA). We demonstrate that this disconnect is not caused by gene-specific repression through CCCTC-binding factor (CTCF) but instead by aberrant chromatin compaction. Treatment with an HDAC inhibitor was found to effectively reactivate PUMA expression on the mRNA and protein level and to revert resistance to DNA damage-induced cell death. Ectopic expression of PUMA was found to resensitize a panel of RCC cell lines to four different DNA-damaging agents tested. Remarkably, all RCC cell lines analyzed were wild-type for p53, and a knockdown was likewise able to sensitize RCC cells to acute genotoxic stress. Taken together, our results indicate that DNA damage resistance in RCC is reversible, involves the p53-PUMA axis, and is potentially targetable to improve the oncological outcomes of RCC patients.

Herniation of the liver: an extremely rare entity.

We hereby present the case of a 75 years old female who was complaining of right upper quadrant abdominal pain. She had a history of cystectomy, cholecystectomy and choledochotomy operations for liver hydatid cyst 5 years ago. In addition, multiple endoscopic retrograde cholangiopancreatography sessions had been performed for recurrent biliary duct stones in the last 4 years. Radiological investigations revealed the presence of cirrhosis and the herniation of the left liver lobe through the abdominal incisional hernia defect. Secondary sclerosing cholangitis as a result of the previous operations was suggested to be the probable etiology for cirrhosis. The cirrhotic patient with an advanced age was found to be high risk for surgery. In addition, her symptoms were minimal. Thus, she was managed conservatively. Herniation of the liver is very rare. It is quite difficult to speculate any predisposing risk factors for liver herniation because of the rarity of this condition.