The Role of Proteases and Serpin Protease Inhibitors in β-Cell Biology and Diabetes

Regulation of the equilibrium between proteases and their inhibitors is fundamental to health maintenance. Consequently, developing a means of targeting protease activity to promote tissue regeneration and inhibit inflammation may offer a new strategy in therapy development for diabetes and other diseases. Specifically, recent efforts have focused on serine protease inhibitors, known as serpins, as potential therapeutic targets. The serpin protein family comprises a broad range of protease inhibitors, which are categorized into 16 clades that are all extracellular, with the exception of Clade B, which controls mostly intracellular proteases, including both serine- and papain-like cysteine proteases. This review discusses the most salient, and sometimes opposing, views that either inhibition or augmentation of protease activity can bring about positive outcomes in pancreatic islet biology and inflammation. These potential discrepancies can be reconciled at the molecular level as specific proteases and serpins regulate distinct signaling pathways, thereby playing equally distinct roles in health and disease development.

Allergen Shedding in Human Milk from Mothers of Preterm Infants: Proteomic and Peptidomic Feasibility and Pilot Analysis

Rationale: There is little information regarding the allergen content of milk feeds in the preterm population. Previous studies have evaluated specific proteins/peptides via ELISA, but no studies have performed a broad analysis of the allergenic peptide content and protease activity of milk feeds in this population. Preterm infants spend a critical window of time for immune development in the Newborn Intensive Care Unit (NICU), and may receive fortified donor milk, maternal milk or formula feeds via nasogastric tube or bottle instead of fresh breastmilk via breastfeeding. Methods: To evaluate feasibility, we initially performed mass spectrometry on four human milk samples (two term and two preterm) from the Mommy’s Milk Human Milk Biorepository (HMB) which included maternal surveys of diet and environmental exposures. We analyzed the results against the University of Nebraska FASTA database and UniProt for a total of 2211 protein sequences. We then further analyzed 5 samples from the Microbiome, Atopy and Prematurity (MAP) pilot study along with formula and human milk fortifier controls and performed not only mass spectrometry, but also peptidomic and protease activity analysis. Results: Each HMB sample had between 806 and 1007 proteins, with 37 to 44 non-human proteins/sample encompassing 26 plant and animal species. Bovine proteins were the most numerous; seven unique Bos taurus proteins were found in all four samples, and three contained Bos d 5. Cat, dog, mosquito, salmon, and crab were detected in all four samples. All donors ingested fish, shellfish and tree nuts, and all had salmon and crab proteins in their milk samples; two almond proteins were detected in three samples. Aeroallergens, including dust mite (Der f 28, Der f 25) and mold (Cla h 4) were identified in all samples. Two samples contained allergens to latex (Hev b 9) and chicken (Gal d 10). One sample contained several unique proteins, including carrot, two molds (including Pen c 19) and Der f 33-like protein. In the preterm MAP samples, 784 digested non-human proteins were identified, 30 were non-bovine in origin. Proteins from 23 different species including aeroallergens, food, and contact allergens were identified. Protease activity was highest in human milk samples without human milk fortifier and lowest in preterm formula. Conclusions: These findings represent the first preterm milk feed mass spectrometry and protease analysis with identification of known allergenic proteins to food, contact and aeroallergens. The varying degree of protein detection may reflect variable individual secretion and augmentation of feeds. This raises questions of whether the composition of milk feeds in the NICU impact the development of atopic disease in the preterm population and whether the complex interaction between allergens, proteases, and other human milk components can serve to induce sensitization or tolerance to allergens in infants.

Culinary Properties of Raw Versus Conventional Soy Sauce during Tuna Preparation

Soy sauce is a traditional Japanese seasoning made from fermented soybeans. As global demand grows, identifying novel soy sauce applications and benefits must become a priority. While conventional soy sauce undergoes heat-sterilization, filter-sterilization produces a lighter-colored (raw) soy sauce with preserved mold enzyme activities. As the impact of raw soy sauce during food (especially seafood) preparation remains unstudied, the present study compared the differential impact of raw and conventional soy sauce on tuna culinary properties. First, soy sauce color and protease activity were assessed. Next, tuna was marinated in soy sauce and non-alcoholic mirin for 0, 10, 35, or 60 min. Finally, marinated tuna properties (mass, salt content, surface salt penetration, color, rupture load, surface wetness, and protein content) were objectively assessed, and subjective sensory evaluation (appearance, aroma, wetness, softness, saltiness, umami, and overall taste) was performed by a blinded panel. Findings confirmed the lighter color of and the preservation of protease activity in raw soy sauce. Raw soy sauce significantly enhanced surface tenderization, salt penetration, and wetness, while both soy sauces increased surface firmness via salt-induced dehydration. Respondents significantly preferred the appearance and saltiness level of raw soy sauce-marinated tuna, and the umami and overall taste of tuna marinated in raw soy sauce for 60 min. The findings of this study, to our knowledge, demonstrate for the first time the potential culinary superiority of raw soy sauce in certain applications, and support future research to further define such applications.

A rare CTSC mutation in Papillon-Lefèvre Syndrome results in abolished serine protease activity and reduced NET formation but otherwise normal neutrophil function

Papillon-Lefèvre Syndrome (PLS) is an autosomal recessive monogenic disease caused by loss-of-function mutations in the CTSC gene, thus preventing the synthesis of the protease Cathepsin C (CTSC) in a proteolytically active form. CTSC is responsible for the activation of the pro-forms of the neutrophil serine proteases (NSPs; Elastase, Proteinase 3 and Cathepsin G), suggesting its involvement in a variety of neutrophil functions. In PLS neutrophils, the lack of CTSC protease activity leads to inactivity of the NSPs. Clinically, PLS is characterized by an early, typically pre-pubertal, onset of severe periodontal pathology and palmoplantar hyperkeratosis. However, PLS is not considered an immune deficiency as patients do not typically suffer from recurrent and severe (bacterial and fungal) infections. In this study we investigated an unusual CTSC mutation in two siblings with PLS, a 503A>G substitution in exon 4 of the CTSC gene, expected to result in an amino acid replacement from tyrosine to cysteine at position 168 of the CTSC protein. Both patients bearing this mutation presented with pronounced periodontal pathology. The characteristics and functions of neutrophils from patients homozygous for the 503A>G CTSC mutation were compared to another previously described PLS mutation (755A>T), and a small cohort of healthy volunteers. Neutrophil lysates from patients with the 503A>G substitution lacked CTSC protein and did not display any CTSC or NSP activity, yet neutrophil counts, morphology, priming, chemotaxis, radical production, and regulation of apoptosis were without any overt signs of alteration. However, NET formation upon PMA-stimulation was found to be severely depressed, but not abolished, in PLS neutrophils.

Effect of Different Agricultural Farming Practices on Microbial Biomass and Enzyme Activities of Celery Growing Field Soil

Soil quality is directly affected by alterations in its microbiological, biological, biochemical, physical, and chemical aspects. The microbiological activities of soil can affect soil fertility and plant growth because it can speed up the cycling of nutrients, enzymes, and hormones that are needed by plants for proper growth and development. The use of different agricultural management practices can influence microbial biomass and enzyme activities by altering soil microclimate, soil microorganism habitat, and nutrient cycling. Based on this, the present work planned to evaluate the impact of conventional, low-input, and organic farming systems in a vegetable field growing celery on microbial biomass and different soil enzyme activities. The present study showed a comparison of the effect of different practices on biological soil quality indicators during two sampling times, i.e., one month after colonization and one month before harvesting. It was observed that the soil microbial biomass in the organic farming system was significantly higher than that found in conventional and low-input practices. Under an organic farming system, the soil microbial biomass in December was significantly higher than that in October. The soil microbial biomass carbon in the 0–20 cm soil layer showed higher variation compared to that in the 20–40 cm layer for all the three of the farming management practices that were used in the study. Additionally, the soil total carbon and total organic carbon were recorded as being higher in the December samples than they were in the October samples. Under all the three of the management practices that were applied, the soil catalase activity was higher in the October samples than it was in the December soil samples that were collected the from 20–40 cm soil layer compared to those that were taken from the 0–20 cm layer. The application of organic fertilizer (chicken and cowmanure compost) resulted inincreases in the soil urease and in the protease activity. The protease activity of the soil samples that were extracted from the 0–20 cm and 20–40 cm soil layers in October was higher in the samples that were taken from farms using conventional practices than it was in the samples that were taken from farms using organic and low-input practices, while the samples that were collected during December from both of the soil layers showed higher protease activity when organic methods had been used. No significant variation in the soil urease activity was observed between the two soil layer samples. Urease activity was the highest when organic management practices were being used, followed by the low-input and the conventional modes. For the conventional and low-input practices, the soil urease activity showed an obvious trend of change that was related to thetime of sampling, i.e., activity in December was significantly higher than activity in October. The novelty of this study was to determine the microbial biomass carbon and enzymatic activity in a six-field crop rotation (tomato, cucumber, celery, fennel, cauliflower, and eggplant) using three management practices: low-input, conventional, and organic systems. The present study showed that the long-term application of organic fertilizers plays a large role in maintaining excellent microbial and enzyme activitythat result in improved soil quality.