Whey Acidic Protein Research Articles

Screening for Antifungal Indigenous Lactobacilli Strains Isolated from Local Fermented Milk for Developing Bioprotective Fermentates and Coatings Based on Acid Whey Protein Concentrate for Fresh Cheese Quality Maintenance.

The demand for healthy foods without artificial food additives is constantly increasing. Hence, natural food preservation methods using bioprotective cultures could be an alternative to chemical preservatives. Thus, the main purpose of this work was to screen the indigenous lactobacilli isolated from fermented cow milk for their safety and antifungal activity to select the safe strain with the strongest fungicidal properties for the development of bioprotective acid whey protein concentrate (AWPC) based fermentates and their coatings intended for fresh cheese quality maintenance. Therefore, 12 lactobacilli strains were isolated and identified from raw fermented cow milk as protective cultures. The safety of the stains was determined by applying antibiotic susceptibility, haemolytic and enzymatic evaluation. Only one strain, Lacticaseibacillus paracasei A11, met all safety requirements and demonstrated a broad spectrum of antifungal activity in vitro. The strain was cultivated in AWPC for 48 h and grew well (biomass yield 8 log10 cfu mL-1). L. paracasei A11 AWPC fermentate was used as a vehicle for protective culture in the development of pectin-AWPC-based edible coating. Both the fermentate and coating were tested for their antimicrobial properties on fresh acid-curd cheese. Coating with L. paracasei A11 strain reduced yeast and mould counts by 1.0-1.5 log10 cfu mL-1 (p ≤ 0.001) during cheese storage (14 days), simultaneously preserving its flavour and prolonging the shelf life for six days.

Exploring the Potential of Sustainable Acid Whey Cheese Supplemented with Apple Pomace and GABA-Producing Indigenous Lactococcus lactis Strain.

This study aimed to utilize two by-products, acid whey and apple pomace, as well as an indigenous Lactococcus lactis LL16 strain with the probiotic potential to produce a sustainable cheese with functional properties. Acid whey protein cheese was made by thermocoagulation of fresh acid whey and enhancing the final product by adding apple pomace, L. lactis LL16 strain, or a mixture of both. The sensory, the physicochemical, the proteolytic, and the microbiological parameters were evaluated during 14 days of refrigerated storage. The supplementation of the cheese with apple pomace affected (p ≤ 0.05) the cheese composition (moisture, protein, fat, carbohydrate, and fiber), the texture, the color (lightness, redness, and yellowness), and the overall sensory acceptability. The addition of the presumptive probiotic L. lactis LL16 strain decreased (p ≤ 0.05) the concentration of glutamic acid, thus increasing γ-aminobutyric acid (GABA) significantly in the acid whey cheese. The supplementation with apple pomace resulted in slightly (p < 0.05) higher counts of L. lactis LL16 on day seven, suggesting a positive effect of apple pomace components on strain survival. The symbiotic effect of apple pomace and LL16 was noted on proteolysis (pH 4.6-soluble nitrogen and free amino acids) in the cheese on day one, which may have positively influenced the overall sensory acceptance.

Identification of waprin and its microbicidal activity: A novel protein component of honeybee (Apis mellifera) venom

Bee venom is a rich source of biologically and pharmacologically active proteins. Waprin is a protein component of venoms; however, waprin has yet to be identified in bee venom. Moreover, the biological functions of waprin in venoms remain poorly characterized. Thus, in this study, we have identified and characterized waprin: a novel protein component from the venom of honeybees (Apis mellifera). The waprin in A. mellifera venom (Amwaprin) was found to consist of an 80-amino acid mature peptide, in which the whey acidic protein domain contains four conserved disulfide bonds. We discovered the presence of the Amwaprin protein in secreted venom by using an antibody against recombinant Amwaprin produced in baculovirus-infected insect cells. Recombinant Amwaprin exhibited inhibitory activity against microbial serine proteases and elastases but not thrombin or plasmin. It recognized carbohydrates in the microbial cell wall molecules and bound to the live microbial surfaces. The binding action of Amwaprin produced its microbicidal activity by inducing structural damage to bacterial and fungal cell walls. In addition, recombinant Amwaprin is heat-stable and contains no hemolytic activity. These findings demonstrate that Amwaprin acts as a microbicidal and anti-elastolytic agent.

Molecular characterization of two newly identified glycine rich crustin genes (Mn-Gly-Cru3, Mn-Gly-Cru4) and their function in Macrobrachium nipponense innate immunity

Crustin, as an important antibacterial peptide in crustacean, plays a role in the defense against the invasion of foreign pathogens in the innate immune system. In this study, two glycine rich crustin genes (named as Mn-Gly-Cru3 and Mn-Gly-Cru4) were cloned in Macrobrachium nipponense. The full-length cDNA of Mn-Gly-Cru3 and Mn-Gly-Cru4 were 740 and 781 bp with open reading frames of 525 and 576 bp that encode 174 and 191 amino acids, respectively. The genome structure of Mn-Gly-Cru3 and Mn-Gly-Cru4 shows that both contain two exons and one intron. Both Mn-Gly-Cru3 and Mn-Gly-Cru4 protein contains a signal peptide, a cysteine-rich region, a glycine-rich region, and a whey acidic protein domain. Evolutionary analysis showed that Mn-Gly-Cru1, Mn-Gly-Cru2, Mn-Gly-Cru3 and Mn-Gly-Cru4 were clustered together. Mn-Gly-Cru3 and Mn-Gly-Cru4 were widely distributed in multiple tissues. After white spot syndrome virus (WSSV), Staphylococcus aureus or Vibrio parahaemolyticus challenge, the expression levels of Mn-Gly-Cru3 and Mn-Gly-Cru4 in the hemocytes, gills, and stomach were significantly changed. Mn-Gly-Cru3 and Mn-Gly-Cru4 knockdown remarkably increased the copy number of WSSV and the number of S. aureus and V. parahaemolyticus colonies in vivo. Purified rMn-Gly-Cru3 and rMn-Gly-Cru4 proteins could bind to a variety of bacteria and they also had binding activity toward LPS or PGN. Furthermore, rMn-Gly-Cru4 had a stronger binding activity to polysaccharides than rMn-Gly-Cru3. Our study indicates that Mn-Gly-Cru3 and Mn-Gly-Cru4 in M. nipponense are involved in the innate immune defense against bacteria or viruses.

Exploring the immunomodulatory effects and mechanisms of Xinjiang fermented camel milk-derived bioactive peptides based on network pharmacology and molecular docking.

Purpose: Fermented camel milk from Xinjiang is rich in probiotics and has immunomodulatory effects as an important source of bioactive peptides. However, it is not clear whether it is the probiotic or the bioactive peptide that acts. The present study aimed to extract and identify bioactive peptides from fermented camel milk in Xinjiang and investigate their immunomodulatory effects and mechanisms based on network pharmacology and molecular docking. Methods: Four probiotic bacteria were used to ferment the fresh camel milk and the bioactive peptides were extracted and isolated by ultrafiltration and column chromatography. Network pharmacology predicts targets and pathways of action. GeneCards and OMIM-GENE-MAP database were used in order to search disease target genes and screen common target genes. Then we used STRING web to construct a protein-protein interaction (PPI) interaction network of the common target protein. The key targets were analyzed by GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis through the David database. The "drug (bioactive peptide)-disease-targets-pathway" network was established and molecular docking was used for prediction. Results: Two fractions were obtained by UV spectrophotometer; whey acidic protein, α-lactalbumin, and peptidoglycan recognition protein 1 were the main protein-like components of Xinjiang fermented camel milk-derived bioactive peptides. The repeat sequence of peptidoglycan recognition protein 1 was selected and then seven bioactive peptides were obtained. Bioactive peptides had 222 gene targets, anti-inflammatory diseases had 2598 gene targets, and immune regulation had 866 gene targets, the intersection of which was 66 in common gene targets. Gene ontology and KEGG analysis reveals that bioactive peptides mainly play a vital role in the signaling pathways of lipid and atherosclerosis, pathways in cancer. The molecular docking results showed that the seven bioactive peptides bound well to the top four scoring proteins. Conclusion: The immunomodulatory and anti-inflammatory effects and mechanisms of Xinjiang fermented camel milk-derived bioactive peptides were initially investigated by network pharmacology and molecular docking, providing a scientific basis for future studies.

Trichuris WAP and CAP proteins: Potential whipworm vaccine candidates?

Trichuris trichiura and T. suis are gastrointestinal dwelling roundworms that infect humans and pigs, respectively. Heavy infections cause gastrointestinal symptoms and impaired growth and development. Vaccination has the potential to reduce the disease burden of whipworm infection; however, there are currently no commercially available vaccines against these parasites and very few against other gastrointestinal-dwelling nematodes of medical and agricultural importance. The naturally occurring mouse whipworm, T. muris, has been used for decades to model human trichuriasis, and the immunogenic potential of the excretory/secretory material (E/S, which can be collected following ex vivo culture of worms) has been studied in the context of vaccine candidate identification. Despite this, researchers are yet to progress an effective vaccine candidate to clinical trials. The T. muris, T. trichiura, and T. suis genomes each encode between 10 and 27 whey acidic protein (WAP) domain-containing proteins and 15 to 34 cysteine-rich secretory protein/antigen 5/pathogenesis related-1 (CAP) family members. WAP and CAP proteins have been postulated to play key roles in host-parasite interactions and may possess immunomodulatory functions. In addition, both protein families have been explored in the context of helminth vaccines. Here, we use phylogenetic and functional analysis to investigate the evolutionary relationship between WAP and CAP proteins encoded by T. muris, T. trichiura, and T. suis. We highlight several WAP and CAP proteins that warrant further study to understand their biological function and as possible vaccine candidates against T. trichiura and/or T. suis, based on the close evolutionary relationship with WAP or CAP proteins identified within T. muris E/S products.

Genome-wide identification and immune response analysis of serine protease inhibitor genes in the blood clam Tegillarca granosa

Serine protease inhibitors (SPIs) are the main regulators of serine protease activities. In this study, we present a genome-wide identification of SPI genes in T. granosa（TgSPI genes）and their expression characteristics in respond to Vibrio stress. A total of 102 TgSPI genes belonging to eight families, including Serpin, TIL (trypsin inhibitor like cysteine rich domain), Kunitz, Kazal, I84, Pacifastin, WAP (whey acidic protein) and A2M (Alpha-2-macroglobulin) were identified, while no genes belonging to Bowman-Birk, amfpi and Antistasin families were identified. The Kazal family has the most TgSPI genes with 38, and 11 TgSPI genes belong to the mollusc-specific I84 family. The TgSPI genes were found to be randomly distributed on 17 chromosomes with 12 tandem duplicate gene pairs. Expression profiles showed that most TgSPI genes were mainly expressed in immune-related tissues such as hepatopancreas, gill and mantle. In the hepatopancreas, most of TgSPI genes were sensitive to Vibrio stress, 28 and 29 TgSPI genes were up-regulated and down-regulated, respectively. Some up-regulated genes with signal peptides, such as the TgSPIs of I84 family, may act as a mechanism to directly prevent Vibrio from invasion. Six Kazal-type TgSPIs (TgSPI29, 45, 49, 50, 51 and 52) were intracellular proteins and their expression was down-regulated in hemocytes after Vibrio stress. This may have boosted protease activity in hemocytes to the point that more hemoglobin derived peptides were produced and secreted into the hemolymph to exert their anti-Vibrio effects. These findings may provide valuable information for further clarifying the roles of SPIs in the immune defense and will benefit future exploration of the immune function of SPIs in molluscs.

Inhibitory and bactericidal activities analysis of a type IIa crustin from the swimming crab, Portunus trituberculatus

Crustin is an important antibacterial peptide in crustaceans. In this study, a type IIa crustin was cloned from Portunus trituberculatus and named as PtCrustin5. The PtCrustin5 contained the typical domains of type II crustin such as a signal peptide, a cysteine-rich domain (CRD), a whey acidic protein (WAP) domain and a glycine rich region (GRR). The PtCrustin5 showed the highest transcription in hepatopancreas, and its expression level in hepatopancreas, gills and hemocytes increased after challenging with Vibrio alginolyticus for 3 h and 6 h. The recombinant protein of PtCrustin5 (rPtCrustin5) was obtained and the GRR of PtCrustin5 (PtCrustin5-GRR) was synthesized. The bacterial binding assay showed that PtCrustin5 preferred to bind to the three tested gram-positive bacteria including Staphylococcus aureus, Corynebacteri umglutamicum and Micrococcus lysodeikticus. Moreover, rPtCrustin5 and PtCrustin5-GRR displayed inhibitory activity against the three tested gram-positive bacteria. The inhibitory and bactericidal activities of rPtCrustin5 and PtCrustin5-GRR under different conditions were investigated. The rPtCrustin5 and PtCrustin5-GRR displayed inhibitory activity against S. aureus and C. umglutamicum at relatively low concentration under all the tested conditions, while the inhibitory activity of rPtCrustin5 and PtCrustin5-GRR against M. lysodeikticus was affected by pH and salinity. This study enriched our understanding of the immune functions of type II crustin in defending against bacteria and provided theoretical basis for the application of PtCrustin5 and PtCrustin5-GRR in preventing and treating bacteriosis.

Application of Edible Coating Based on Liquid Acid Whey Protein Concentrate with Indigenous Lactobacillus helveticus for Acid-Curd Cheese Quality Improvement.

Edible coatings as carriers for protective lactic acid bacteria (LAB) can enhance hygienic quality to dairy products. Thus, the aim of this study was to improve the quality of artisanal acid-curd cheese by applying liquid acid whey protein concentrate based edible coating with entrapped indigenous antimicrobial Lactobacillus helveticus MI-LH13. The edible fresh acid-curd cheese coating was composed of 100% (w/w) liquid acid whey protein concentrate (LAWPC), apple pectin, sunflower oil, and glycerol containing 6 log10 CFU/mL of strain biomass applied on cheese by dipping. The cheese samples were examined over 21 days of storage for changes of microbiological criteria (LAB, yeast and mould, coliform, enterobacteria, and lipolytic microorganism), physicochemical (pH, lactic acid, protein, fat, moisture content, and colour), rheological, and sensory properties. The coating significantly improved appearance and slowed down discolouration of cheese by preserving moisture during prolonged storage. The immobilisation of L. helveticus cells into the coating had no negative effect on their viability throughout 14 days of storage at 4 °C and 23 °C. The application of coating with immobilised cells on cheeses significantly decreased the counts of yeast up to 1 log10 CFU/g during 14 days (p &lt; 0.05) of storage and suppressed growth of mould for 21 days resulting in improved flavour of curd cheese at the end of storage. These findings indicate that LAWPC-pectin formulation provided an excellent matrix to support L. helveticus cell viability. Acting as protective antimicrobial barrier in fresh cheeses, this bioactive coating can reduce microbial contamination after processing enabling the producers to extend the shelf life of this perishable product.

Effect of casein-whey ingredient blends on the protein stability of model infant formulas

Milk protein concentrate (MPC 80), skim milk powder, acid whey protein concentrate (aWPC 35), cheese whey protein concentrate and isolate (cWPC 35 and cWPI) were assessed for suitability in infant formula (IF) applications with total solids similar to commercial ready-to-drink applications. Serum-phase compounds including phosphate, citrate and chloride in casein-rich streams, especially aWPC 35, played an important role in controlling whey protein thermal aggregation. Replacing cWPC 35 with cWPI improved protein stability at UHT temperatures, indicating that removal of minerals from whey protein streams could increase thermal stability of formulations. At HTST temperatures (95 °C), lactose appeared to have a protective effect against protein denaturation. At 140 °C, however, lactose contributed to cascading Maillard reactions triggering complete protein destabilisation; this occurred most rapidly in MPC 80 formulations that were comparatively low in serum-phase calcium-binding species (phosphate and citrate). These findings highlight how innate minerals and lactose impact protein stability during IF manufacture.

Fascin is essential for mammary gland lactogenesis

Fascin expression has commonly been observed in certain subtypes of breast cancer, where its expression is associated with poor clinical outcome. However, its role in normal mammary gland development has not been elucidated. Here, we used a fascin knockout mouse model to assess its role in normal mammary gland morphogenesis and lactation. Fascin knockout was not embryonically lethal, and its effect on the litter size or condition at birth was minimal. However, litter survival until the weaning stage significantly depended on fascin expression solely in the nursing dams. Accordingly, pups that nursed from fascin−/− dams had smaller milk spots in their abdomen, suggesting a lactation defect in the nursing dams. Mammary gland whole-mounts of pregnant and lactating fascin−/− mice showed significantly reduced side branching and alveologenesis. Despite a typical composition of basal, luminal, and stromal subsets of mammary cells and normal ductal architecture of myoepithelial and luminal layers, the percentage of alveolar progenitors (ALDH+) in fascin−/− epithelial fraction was significantly reduced. Further in-depth analyses of fascin−/− mammary glands showed a significant reduction in the expression of Elf5, the master regulator of alveologenesis, and a decrease in the activity of its downstream target p-STAT5. In agreement, there was a significant reduction in the expression of the milk proteins, whey acidic protein (WAP), and β-casein in fascin−/− mammary glands. Collectively, our data demonstrate, for the first time, the physiological role of fascin in normal mammary gland lactogenesis, an addition that could reveal its contribution to breast cancer initiation and progression.

A role for whey acidic protein four-disulfide-core 12 (WFDC12) in the pathogenesis and development of psoriasis disease.

Whey acidic protein four-disulfide core domain protein 12 (WFDC12) has been implicated in the pathogenesis of psoriasis but the specific molecular mechanism is not clearly defined. In this study, we found the expression of WFDC12 protein closely correlated with psoriasis. WFDC12 in keratinocyte might increase infiltration of Langerhans cells (LCs) and monocyte-derived dendritic cells (moDDCs), up-regulating the co-stimulation molecular CD40/CD86. Th1 cells in lymph nodes were higher in K14-WFDC12 transgenic psoiasis-like mice. Meanwhile, the mRNA of IL-12 and IFN-γ in the lesion skin was significantly increased in transgenic mice. Moreover, we found that the expression of the proteins that participated in the retinoic acid–related pathway and immune signaling pathway was more changed in the lesion skin of K14-WFDC12 transgenic psoriasis-like mice. Collectively, the results implied that WFDC12 might affect the activation of the retinoic acid signaling pathway and regulate the infiltration of DC cells in the skin lesions and lymph nodes, thereby inducing Th1 cells differentiation and increasing the secretion of IFN-γ to exacerbate psoriasis in mice.

Identification and functional characterization of C-type lectins and crustins provide new insights into the immune response of Portunus trituberculatus

A meticulous understanding of the immune characteristics of aquaculture animals is the basis for developing precise disease prevention and control strategies. In this study, four novel C-type lectins (PtCTL-5, PtCTL-6, PtCTL-7 and PtCTL-8) including a single carbohydrate-recognition domain (CRD), and four novel crustins (Ptcrustin-1, Ptcrustin-2, Ptcrustin-3 and Ptcrustin-4) with a single whey acidic protein (WAP) domain were identified from the swimming crab Portunus trituberculatus. Tissue distribution analysis indicated that most of the target genes were predominantly expressed in the hepatopancreas in all examined tissues, except for Ptcrustin-1 which were mainly expressed in the gills. Our results showed that the eight genes displayed various transcriptional profiles across different tissues. In hemocytes, the PtCTL-7 responded quickly to Vibrio alginolyticus and exhibited much more strongly up-regulation than other three PtCTLs. The Ptcrustin-1 rapidly responded to V. alginolyticus within 3 h in all the three tested tissues. Furthermore, recombinant proteins of PtCTL-5 and PtCTL-8 were successfully obtained, and both of them displayed bacterial binding activities toward V. alginolyticus, V. harveyi and Staphylococcus aureus, and only showed antibacterial activity against V. harveyi. These findings provided new insights into the diverse immune response of P. trituberculatus and laid theoretical foundations for the development of precise disease prevention and control strategies in P. trituberculatus farming. Moreover, the specific anti-V. harveyi activities exhibited by rPtCTL-5 and rPtCTL-8 suggested their promising application prospects for controlling diseases caused by V. harveyi.

Effect of Delta-9-tetrahydrocannabinol and cannabidiol on milk proteins and lipid levels in HC11 cells.

Pregnant and lactating women have been discouraged from using cannabis by Health Canada. However, the increasing rate of cannabis use among pregnant women has presented an urgent need to investigate its physiological effects during the perinatal period. During pregnancy, the mammary gland (MG) undergoes remodeling, which involves alveolar differentiation of mammary epithelial cells (MECs), which is essential for breast milk production and secretion. Limited evidence has been reported on the impact of cannabis or its components, delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD), on MG development or MEC differentiation. In this study, we investigated the effects of THC and CBD on the differentiation of MECs by assessing changes in cellular viability, lipid accumulation, and gene and protein expression of major milk protein and lipid synthesizing markers. using the HC11 cells as a model. We hypothesized that THC and CBD will negatively impact the synthesis of milk proteins and lipids, as well as lipid markers in HC11 cells. Our results demonstrated that THC and CBD reduced cellular viability at concentrations above 30μM and 20μM, respectively. Relative to control, 10μM THC and 10μM CBD reduced mRNA levels of milk proteins (CSN2 and WAP), lipid synthesizing and glucose transport markers (GLUT 1, HK2, FASN, FABP4, PLIN2 and LPL), as well as whey acidic protein and lipid levels. In addition, co-treatment of a CB2 antagonist with THC, and a CB2 agonist with CBD, reversed the impact of THC and CBD on the mRNA levels of key markers, respectively. In conclusion, 10μM THC and CBD altered the differentiation of HC11 cells, in part via the CB2 receptor.

Progress in the effect of human epididymis protein 4 on sperm maturation

Human epididymis protein 4(HE4) is a secretory glycoprotein found in human distal epididymis epithelial cells. It is often used in the early diagnosis, efficacy evaluation and monitoring of ovarian cancer, and also has been considered as an effective serum marker for many other types of cancer. However, its function in the process of sperm maturation is not fully unknown. The maturation of sperm in epididymis is characterized by the acquisition of motility and fertilization. As a member of the whey acid protein (WAP) family, several studies proposed the importance of HE4 in the maturity of sperm in epididymis. This article reviews the effect of HE4 on spermatozoa maturation in epididymis, which provides basis for the evaluation of male reproductive ability, early detection, early diagnosis and pathogenesis of male infertility.

Gelation of nanofiltrated acid whey by κ‐carrageenan and xanthan gum

The high nutritional value of whey proteins explains the widespread use of their dry forms in the composition of various food products. Liquid whey protein concentrates obtained by membrane methods can serve as an alternative. The purpose of the work was the thickening of a liquid concentrate of acid whey proteins obtained by nanofiltration (nanofiltrated concentrate – NF concentrate). A positive result in terms of gelation was obtained, first by increasing the total solid content in the experimental samples to 34% compared with 18% in the NF concentrate and second by increasing the mass fraction of κ‐carrageenan to 0.5% or by combining κ‐carrageenan with xanthan gum. The total solids of the samples was increased by the introduction of dry whey protein concentrate and maltodextrin. Organoleptical, rheological and microstructural studies have demonstrated the positive effect of a combination of milk whey proteins, maltodextrin, κ‐carrageenan and xanthan gum on the gelation of NF concentrate.

Anosmin 1 N-terminal domains modulate prokineticin receptor 2 activation by prokineticin 2

The X-linked form of Kallmann syndrome (KS), characterized by hypogonadotropic hypogonadism and anosmia, is due to mutations in the ANOS1 gene that encodes for the extracellular matrix (ECM) protein anosmin 1. Prokineticins (PKs) exert their biological functions through the activation of the G protein-coupled receptors (GPCRs) prokineticin receptor 1 and 2 (PKR1, 2), and mutations in the PK2 and PKR2 genes are involved in the pathogenesis of KS. We have previously shown interaction between PKR2 and anosmin 1 in vitro. In the current report we present evidence of the modulation of PK2/PKR2 activity by anosmin 1, since this protein is able to enhance the activation of the ERK1/2 (extracellular signal-regulated kinase 1/2) pathway elicited by PK2 through PKR2. We also show that the N-terminal region of anosmin 1, capable of binding to the PK2-binding domain of PKR2, seems to be responsible for this effect. The whey acidic protein domain (WAP) is necessary for this modulatory activity, although data from GST pull-down (glutathione-S-transferase) and analysis of the N267K mutation in the fibronectin type III domain 1 (FnIII.1) suggest the cysteine-rich (CR) and the FnIII.1 domains could assist the WAP domain both in the binding to PKR2 and in the modulation of the activation of the receptor by PK2. Our data support the idea of a modulatory role of anosmin 1 in the biological effects controlled by the PK2/PKR2 system.

Widespread alterations upon exposure to the estrogenic endocrine disruptor ethinyl estradiol in the liver proteome of the marine male fish Cyprinodon variegatus.

Quantitative proteomic changes in the liver of adult males of Sheepshead minnow (Cyprinodon variegatus) upon exposure to ethinyl estradiol (EE2) were assessed to provide an advanced understanding of the metabolic pathways affected by estrogenic endocrine disruption in marine fish, and to identify potential novel molecular biomarkers for the environmental exposure to estrogens. From a total of 3188 identified protein groups (hereafter proteins), 463 showed a statistically significant difference in their abundance between EE2 treatment and solvent control samples. The most affected biological processes upon EE2 exposure were related to ribosomal biogenesis, protein synthesis and transport of nascent proteins to endoplasmic reticulum, and nuclear mRNA catabolism. Within the group of upregulated proteins, a subset of 14 proteins, involved in egg production (Vitellogenin, Zona Pellucida), peptidase activity (Cathepsine E, peptidase S1, Serine/threonine-protein kinase PRP4 homolog, Isoaspartyl peptidase and Whey acidic protein), and nucleic acid binding (Poly [ADP-ribose] polymerase 14) were significantly upregulated with fold-change values higher than 3. In contrast, Collagen alpha-2, involved in the process of response to steroid hormones, among others, was significantly downregulated (fold change=0.2). This pattern of alterations in the liver proteome of adult males of C. variegatus can be used to identify promising novel biomarkers for the characterization of exposure of marine fish to estrogens. The Whey acidic protein-like showed the highest upregulation in EE2-exposed individuals (21-fold over controls), suggesting the utility of abundance levels of this protein in male liver as a novel biomarker of xenoestrogen exposure.

Abstract 6018: Expansion of mouse mammary epithelial stem cells in serum-free organoid growth medium

Abstract Growing mammary epithelial cells as organoids in a three-dimensional (3D) cell culture environment represents a more physiological model system than conventional 2D adherent cell culture systems for studying many different aspects of mammary epithelial cell biology. We have developed MammoCult™ Organoid Growth Medium (Mouse), a serum- and phenol red-free medium for the long-term propagation of organoids from mouse mammary tissue. To initiate the cultures, mouse mammary glands are resected and enzymatically dissociated sequentially in Gentle Collagenase/Hyaluronidase, trypsin, and dispase/DNase I to generate a single-cell suspension, and 4.5 x 103 of the liberated cells are then embedded in Corning® Matrigel® and maintained in MammoCult™ Organoid Growth Medium (Mouse). Alternatively, freshly dissociated mammary cells can be expanded in adherent culture in EpiCult™ Plus Medium prior to seeding into Matrigel®. Approximately 8.4 ± 0.6% (mean ± SEM; n=10) of the freshly dissociated cells seeded in the Corning® Matrigel® will proliferate and generate organoids that are typically &amp;gt; 200 µm in diameter and have a highly branched morphology, although organoids with a cystic morphology are also observed. Immunostaining of these branched organoids reveals that they are composed of a polarized epithelium made up of keratin (K) 8-expressing luminal cells and K5/14-expressing basal cells (n=7). Organoids upregulate transcripts for caseins (Csn1s1, Csn2), whey acidic protein (Wap), and lactoferrin (Ltf) upon stimulation with prolactin, hydrocortisone, and insulin (n=4). These multilineage organoids can be established from multiple inbred mouse strains, including C57Bl6/J, FVB, BALB/c, and CD-1. Organoids can be passaged every 10 - 14 days as small fragments at a 1:3 to 1:4 split ratio, and can be maintained for a minimum of 10 passages while still maintaining multilineage potential and a normal karyotype. These results demonstrate that MammoCult™ Organoid Growth Medium (Mouse) efficiently generates and expands mammary epithelial cell-derived organoids and offers researchers a novel tool for in vitro mammary studies. Citation Format: David Rowbotham, Samantha Rothwell, Allen C. Eaves, Sharon A. Louis, John Stingl. Expansion of mouse mammary epithelial stem cells in serum-free organoid growth medium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6018.

A Type Ib Crustin from Deep-Sea Shrimp Possesses Antimicrobial and Immunomodulatory Activity.

Crustins are small antimicrobial proteins produced by crustaceans. Of the many reported crustins, very few are from deep sea environments. Crustins are categorized into several types. Recently, the Type I crustin has been further classified into three subtypes, one of which is Type Ib, whose function is unknown. Here, we studied the function of a Type Ib crustin (designated Crus2) identified from a deep-sea crustacean. Crus2 has a whey acidic protein (WAP) domain and a long C-terminal region (named P58). Recombinant Crus2 bound to peptidoglycan (PGN), lipoteichoic acid (LTA), and lipopolysaccharide (LPS), and killed Gram-positive and Gram-negative bacteria by permeabilizing the bacterial cytomembrane. Consistently, Crus2 dramatically attenuated the inflammatory response induced by LPS and LTA. Disruption of the disulfide bonds in the WAP domain abolished the bactericidal ability of Crus2, but had no effect on the bacterial binding ability of Crus2. Deletion of the C-terminal P58 region moderately affected the antimicrobial activity of Crus2 against some bacteria. P58 as a synthesized peptide could bind bacteria and inhibit the bactericidal activity of Crus2. Taken together, these results revealed different roles played by the WAP domain and the P58 region in Type Ib crustin, and provided new insights into the antimicrobial and immunomodulatory functions of crustins.