Showing papers on "Proteolytic enzymes published in 2020"

Drug Development and Medicinal Chemistry Efforts toward SARS-Coronavirus and Covid-19 Therapeutics.

Abstract: The COVID-19 pandemic caused by SARS-CoV-2 infection is spreading at an alarming rate and has created an unprecedented health emergency around the globe. There is no effective vaccine or approved drug treatment against COVID-19 and other pathogenic coronaviruses. The development of antiviral agents is an urgent priority. Biochemical events critical to the coronavirus replication cycle provided a number of attractive targets for drug development. These include, spike protein for binding to host cell-surface receptors, proteolytic enzymes that are essential for processing polyproteins into mature viruses, and RNA-dependent RNA polymerase for RNA replication. There has been a lot of ground work for drug discovery and development against these targets. Also, high-throughput screening efforts have led to the identification of diverse lead structures, including natural product-derived molecules. This review highlights past and present drug discovery and medicinal-chemistry approaches against SARS-CoV, MERS-CoV and COVID-19 targets. The review hopes to stimulate further research and will be a useful guide to the development of effective therapies against COVID-19 and other pathogenic coronaviruses.

Oral Candidiasis: A Disease of Opportunity.

Abstract: Oral candidiasis, commonly referred to as “thrush,” is an opportunistic fungal infection that commonly affects the oral mucosa. The main causative agent, Candida albicans, is a highly versatile commensal organism that is well adapted to its human host; however, changes in the host microenvironment can promote the transition from one of commensalism to pathogen. This transition is heavily reliant on an impressive repertoire of virulence factors, most notably cell surface adhesins, proteolytic enzymes, morphologic switching, and the development of drug resistance. In the oral cavity, the co-adhesion of C. albicans with bacteria is crucial for its persistence, and a wide range of synergistic interactions with various oral species were described to enhance colonization in the host. As a frequent colonizer of the oral mucosa, the host immune response in the oral cavity is oriented toward a more tolerogenic state and, therefore, local innate immune defenses play a central role in maintaining Candida in its commensal state. Specifically, in addition to preventing Candida adherence to epithelial cells, saliva is enriched with anti-candidal peptides, considered to be part of the host innate immunity. The T helper 17 (Th17)-type adaptive immune response is mainly involved in mucosal host defenses, controlling initial growth of Candida and inhibiting subsequent tissue invasion. Animal models, most notably the mouse model of oropharyngeal candidiasis and the rat model of denture stomatitis, are instrumental in our understanding of Candida virulence factors and the factors leading to host susceptibility to infections. Given the continuing rise in development of resistance to the limited number of traditional antifungal agents, novel therapeutic strategies are directed toward identifying bioactive compounds that target pathogenic mechanisms to prevent C. albicans transition from harmless commensal to pathogen.

Bacteriocins, Potent Antimicrobial Peptides and the Fight against Multi Drug Resistant Species: Resistance Is Futile?

Abstract: Despite highly specialized international interventions and policies in place today, the rapid emergence and dissemination of resistant bacterial species continue to occur globally, threatening the longevity of antibiotics in the medical sector. In particular, problematic nosocomial infections caused by multidrug resistant Gram-negative pathogens present as a major burden to both patients and healthcare systems, with annual mortality rates incrementally rising. Bacteriocins, peptidic toxins produced by bacteria, offer promising potential as substitutes or conjugates to current therapeutic compounds. These non-toxic peptides exhibit significant potency against certain bacteria (including multidrug-resistant species), while producer strains remain insusceptible to the bactericidal peptides. The selectivity and safety profile of bacteriocins have been highlighted as superior advantages over traditional antibiotics; however, many aspects regarding their efficacy are still unknown. Although active at low concentrations, bacteriocins typically have low in vivo stability, being susceptible to degradation by proteolytic enzymes. Another major drawback lies in the feasibility of large-scale production, with these key features collectively limiting their current clinical application. Though such limitations require extensive research, the concept of expanding bacteriocins from food preservation to human health opens many fascinating doors, including novel drug delivery systems and anticancer treatment applications.

Stapled Helical Peptides Bearing Different Anchoring Residues.

Abstract: A large proportion of protein-protein interactions (PPIs) occur between a short peptide and a globular protein domain; the peptides involved in surface interactions play important roles, and there is great promise for using peptide motifs to interfere with protein interactions. Peptide inhibitors show more promise in blocking large surface protein interactions compared to small molecule inhibitors. However, peptides have drawbacks including poor stability against circulating proteolytic enzymes and an intrinsic inability to penetrate cell membranes. Stapled helical peptides, by adopting a preformed, stable α-helical conformation, exhibit improved proteolytic stability and membrane permeability compared to linear bioactive peptides. In this review, we summarize the broad aspects of peptide stapling for chemistry, biophysics, and biological applications and specifically highlight the methodology by providing an inventory of different anchoring residues categorized into two natural amino acids, two nonnatural amino acids, or a combination of natural and nonnatural amino acids. Additional advantages of specific peptide stapling techniques, including but not limited to reversibility, bio-orthogonal reactivity, and photoisomerization, are also discussed individually. This review is expected to provide a broad reference for the rational design of druggable stapled peptides targeting therapeutic proteins, particularly those involved in PPIs, by considering the impact of anchoring residues, functional cross-linkers, physical staple length, staple components, and the staple motif on the biophysical properties of the peptides.

Hepatic Stellate Cells and Hepatocarcinogenesis

Abstract: Hepatic stellate cells (HSCs) are a significant component of the hepatocellular carcinoma (HCC) tumor microenvironment (TME). Activated HSCs transform into myofibroblast-like cells to promote fibrosis in response to liver injury or chronic inflammation, leading to cirrhosis and HCC. The hepatic TME is comprised of cellular components, including activated HSCs, tumor-associated macrophages, endothelial cells, immune cells, and non-cellular components, such as growth factors, proteolytic enzymes and their inhibitors, and other extracellular matrix (ECM) proteins. Interactions between HCC cells and their microenvironment have become topics under active investigation. These interactions within the hepatic TME have the potential to drive carcinogenesis and create challenges in generating effective therapies. Current studies reveal potential mechanisms through which activated HSCs drive hepatocarcinogenesis utilizing matricellular proteins and paracrine crosstalk within the TME. Since activated HSCs are primary secretors of ECM proteins during liver injury and inflammation, they help promote fibrogenesis, infiltrate the HCC stroma, and contribute to HCC development. In this review, we examine several recent studies revealing the roles of HSCs and their clinical implications in the development of fibrosis and cirrhosis within the hepatic TME.

Effect of enzymatic hydrolysis on molecular weight distribution, techno-functional properties and sensory perception of pea protein isolates

Abstract: Pea protein isolate (Pisum sativum “Navarro”) was hydrolyzed with 11 proteolytic enzymes at different hydrolysis times (15, 30, 60, and 120 min) to improve techno-functional and sensory properties. The degree of hydrolysis and changes within the molecular weight distribution were used as indicators for a reduced allergenic potential. The highest degree of hydrolysis was reached by Esperase hydrolysates (9.77%) after 120 min of hydrolysis, whereas Chymotrypsin hydrolysates showed the lowest (1.81%). Hydrolysis with Papain, Trypsin, Bromelain, Esperase, Savinase, and Alcalase suggested an effective degradation of the 72 kDa-convicilin fraction. Papain and Trypsin hydrolysates showed a degradation of the 50 kDa-mature vicilin after 15 min of hydrolysis. Most hydrolysates showed a significant increase in protein solubility at pH 4.5 at all times of hydrolysis. Trypsin hydrolysates showed the highest foaming (2271%) and emulsifying (719 mL/g) capacities. The bitterness of the hydrolysates was strongly correlated (P Industrial relevance Due to their high protein content, peas are becoming an attractive ingredient for the food industry. However, pea protein isolates are often characterized by poor techno-functional and sensory properties. Enzymatic hydrolysis is known to change the molecular weight distribution of proteins. Consequently, the techno-functional and immunogenic properties might be altered selectively. In this study, enzymatic hydrolysis was applied, resulting in highly functional pea protein hydrolysates with a hypothesized reduction of main allergens. The lower bitter perception highlights their high potential as valuable functional food ingredients.

Sustainable extraction of proteins and bioactive substances from pomegranate peel (Punica granatum L.) using pressurized liquids and deep eutectic solvents

Abstract: Pomegranate peel is a source of proteins, bioactive peptides, and phenolic compounds. The simultaneous extraction of these compounds required the use of polluting solvents and reagents that are non-suitable. This work targets the development of green methodologies based on pressurized liquids (PLE) or deep eutectic solvents (DES) for the extraction of these compounds. Extracts were digested with different proteolytic enzymes and different functionalities (antioxidant, hypocholesterolemia, and antihypertensive capacities) were evaluated. Highly antioxidant and hypocholesterolemic extracts and hydrolysates were obtained using PLE while high antihypertensive capacity was observed in the hydrolysates from proteins extracted using DES. Peptides and polyphenols were identified by HPLC-ESI-Q-TOF/MS. Higher amounts of peptides were shown in hydrolysates from DES extracts while hydrolysates from PLE extracts presented higher amounts of phenolic compounds. Some peptides were assigned to proteins from Punica granatum. Both green methods improved the extraction of bioactive compounds from pomegranate peel compared to the non-sustainable method. Industrial relevance The development of green methodologies which employ sustainable solvents such as pressurized liquids (PLE) and deep eutectic solvents (DES) allows extracting proteins and bioactive compounds from pomegranate peel. In addition, these solvents improve the extraction of health beneficial compounds compared to the non-sustainable and polluting solvents. Therefore, they could be used for the development of nutraceuticals and functional foods or even in medicinal, cosmetic, and pharmaceutical preparations.

Ultrafast enzymatic digestion of proteins by microdroplet mass spectrometry.

Abstract: Enzymatic digestion for protein sequencing usually requires much time, and does not always result in high sequence coverage. Here we report the use of aqueous microdroplets to accelerate enzymatic reactions and, in particular, to improve protein sequencing. When a room temperature aqueous solution containing 10 µM myoglobin and 5 µg mL−1 trypsin is electrosonically sprayed (−3 kV) from a homemade setup to produce tiny (∼9 µm) microdroplets, we obtain 100% sequence coverage in less than 1 ms of digestion time, in sharp contrast to 60% coverage achieved by incubating the same solution at 37 °C for 14 h followed by analysis with a commercial electrospray ionization source that produces larger (∼60 µm) droplets. We also confirm the sequence of the therapeutic antibody trastuzumab (∼148 kDa), with a sequence coverage of 100% for light chains and 85% for heavy chains, demonstrating the practical utility of microdroplets in drug development. Mass spectrometry (MS)-based protein sequencing usually relies on in-solution proteolytic digestion, which is time-consuming and inefficient for certain proteins. Here, the authors achieve full protein sequence coverage in less than 1 ms by subjecting protein-protease mixtures to electrosonic spray ionization-MS.

Matrix Metalloproteinases and Glaucoma Treatment.

Abstract: Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that degrade extracellular matrix (ECM) components such as collagen and have important roles in multiple biological processes, including development and tissue remodeling, both in health and disease. The activity of MMPs is influenced by the expression of MMPs and tissue inhibitors of metalloproteinase (TIMPs). In the eye, MMP-mediated ECM turnover in the juxtacanalicular region of the trabecular meshwork (TM) reduces outflow resistance in the conventional outflow pathway and helps maintain intraocular pressure (IOP) homeostasis. An imbalance in the MMP/TIMP ratio may be involved in the elevated IOP often associated with glaucoma. The prostaglandin analog/prostamide (PGA) class of topical ocular hypotensive medications used in glaucoma treatment reduces IOP by increasing outflow through both conventional and unconventional (uveoscleral) outflow pathways. Evidence from in vivo and in vitro studies using animal models and anterior segment explant and cell cultures indicates that the mechanism of IOP lowering by PGAs involves increased MMP expression in the TM and ciliary body, leading to tissue remodeling that enhances conventional and unconventional outflow. PGA effects on MMP expression are dependent on the identity and concentration of the PGA. An intracameral sustained-release PGA implant (Bimatoprost SR) in development for glaucoma treatment can reduce IOP for many months after expected intraocular drug bioavailability. We hypothesize that the higher concentrations of bimatoprost achieved in ocular outflow tissues with the implant produce greater MMP upregulation and more extensive, sustained MMP-mediated target tissue remodeling, providing an extended duration of effect.

A Review of Small Molecule Inhibitors and Functional Probes of Human Cathepsin L.

Abstract: Human cathepsin L belongs to the cathepsin family of proteolytic enzymes with primarily an endopeptidase activity. Although its primary functions were originally thought to be only of a housekeeping enzyme that degraded intracellular and endocytosed proteins in lysosome, numerous recent studies suggest that it plays many critical and specific roles in diverse cellular settings. Not surprisingly, the dysregulated function of cathepsin L has manifested itself in several human diseases, making it an attractive target for drug development. Unfortunately, several redundant and isoform-specific functions have recently emerged, adding complexities to the drug discovery process. To address this, a series of chemical biology tools have been developed that helped define cathepsin L biology with exquisite precision in specific cellular contexts. This review elaborates on the recently developed small molecule inhibitors and probes of human cathepsin L, outlining their mechanisms of action, and describing their potential utilities in dissecting unknown function.

Stapled Peptides Based on Human Angiotensin-Converting Enzyme 2 (ACE2) Potently Inhibit SARS-CoV-2 Infection In Vitro .

Abstract: SARS-CoV-2 uses human angiotensin-converting enzyme 2 (ACE2) as the primary receptor to enter host cells and initiate the infection. The critical binding region of ACE2 is an ∼30-amino-acid (aa)-long helix. Here, we report the design of four stapled peptides based on the ACE2 helix, which is expected to bind to SARS-CoV-2 and prevent the binding of the virus to the ACE2 receptor and disrupt the infection. All stapled peptides showed high helical contents (50 to 94% helicity). In contrast, the linear control peptide NYBSP-C showed no helicity (19%). We have evaluated the peptides in a pseudovirus-based single-cycle assay in HT1080/ACE2 cells and human lung cell line A549/ACE2, overexpressing ACE2. Three of the four stapled peptides showed potent antiviral activity in HT1080/ACE2 (50% inhibitory concentration [IC50]: 1.9 to 4.1 µM) and A549/ACE2 (IC50: 2.2 to 2.8 µM) cells. The linear peptide NYBSP-C and the double-stapled peptide StRIP16, used as controls, showed no antiviral activity. Most significantly, none of the stapled peptides show any cytotoxicity at the highest dose tested. We also evaluated the antiviral activity of the peptides by infecting Vero E6 cells with the replication-competent authentic SARS-CoV-2 (US_WA-1/2020). NYBSP-1 was the most efficient, preventing the complete formation of cytopathic effects (CPEs) at an IC100 of 17.2 µM. NYBSP-2 and NYBSP-4 also prevented the formation of the virus-induced CPE with an IC100 of about 33 µM. We determined the proteolytic stability of one of the most active stapled peptides, NYBSP-4, in human plasma, which showed a half-life (T 1/2) of >289 min.IMPORTANCE SARS-CoV-2 is a novel virus with many unknowns. No vaccine or specific therapy is available yet to prevent and treat this deadly virus. Therefore, there is an urgent need to develop novel therapeutics. Structural studies revealed critical interactions between the binding site helix of the ACE2 receptor and SARS-CoV-2 receptor-binding domain (RBD). Therefore, targeting the entry pathway of SARS-CoV-2 is ideal for both prevention and treatment as it blocks the first step of the viral life cycle. We report the design of four double-stapled peptides, three of which showed potent antiviral activity in HT1080/ACE2 cells and human lung carcinoma cells, A549/ACE2. Most significantly, the active stapled peptides with antiviral activity against SARS-CoV-2 showed high α-helicity (60 to 94%). The most active stapled peptide, NYBSP-4, showed substantial resistance to degradation by proteolytic enzymes in human plasma. The lead stapled peptides are expected to pave the way for further optimization of a clinical candidate.

Detection Methods for Aflatoxin M1 in Dairy Products.

Abstract: Mycotoxins are toxic compounds produced mainly by fungi of the genera Aspergillus, Fusarium and Penicillium. In the food chain, the original mycotoxin may be transformed in other toxic compounds, reaching the consumer. A good example is the occurrence of aflatoxin M1 (AFM1) in dairy products, which is due to the presence of aflatoxin B1 (AFB1) in the animal feed. Thus, milk-based foods, such as cheese and yogurts, may be contaminated with this toxin, which, although less toxic than AFB1, also exhibits hepatotoxic and carcinogenic effects and is relatively stable during pasteurization, storage and processing. For this reason, the establishment of allowed maximum limits in dairy products and the development of methodologies for its detection and quantification are of extreme importance. There are several methods for the detection of AFM1 in dairy products. Usually, the analytical procedures go through the following stages: sampling, extraction, clean-up, determination and quantification. For the extraction stage, the use of organic solvents (as acetonitrile and methanol) is still the most common, but recent advances include the use of the Quick, Easy, Cheap, Effective, Rugged, and Safe method (QuEChERS) and proteolytic enzymes, which have been demonstrated to be good alternatives. For the clean-up stage, the high selectivity of immunoaffinity columns is still a good option, but alternative and cheaper techniques are becoming more competitive. Regarding quantification of the toxin, screening strategies include the use of the enzyme-linked immunosorbent assay (ELISA) to select presumptive positive samples from a wider range of samples, and more reliable methods-high performance liquid chromatography with fluorescence detection or mass spectroscopy-for the separation, identification and quantification of the toxin.

Cationic Arginine-Rich Peptides (CARPs): A Novel Class of Neuroprotective Agents With a Multimodal Mechanism of Action.

Abstract: There are virtually no clinically available neuroprotective drugs for the treatment of acute and chronic neurological disorders, hence there is an urgent need for the development of new neuroprotective molecules. Cationic arginine-rich peptides (CARPs) are an expanding and relatively novel class of compounds, which possess intrinsic neuroprotective properties. Intriguingly, CARPs possess a combination of biological properties unprecedented for a neuroprotective agent including the ability to traverse cell membranes and enter the CNS, antagonize calcium influx, target mitochondria, stabilize proteins, inhibit proteolytic enzymes, induce pro-survival signaling, scavenge toxic molecules, and reduce oxidative stress as well as, having a range of anti-inflammatory, analgesic, anti-microbial, and anti-cancer actions. CARPs have also been used as carrier molecules for the delivery of other putative neuroprotective agents across the blood-brain barrier and blood-spinal cord barrier. However, there is increasing evidence that the neuroprotective efficacy of many, if not all these other agents delivered using a cationic arginine-rich cell-penetrating peptide (CCPPs) carrier (e.g., TAT) may actually be mediated largely by the properties of the carrier molecule, with overall efficacy further enhanced according to the amino acid composition of the cargo peptide, in particular its arginine content. Therefore, in reviewing the neuroprotective mechanisms of action of CARPs we also consider studies using CCPPs fused to a putative neuroprotective peptide. We review the history of CARPs in neuroprotection and discuss in detail the intrinsic biological properties that may contribute to their cytoprotective effects and their usefulness as a broad-acting class of neuroprotective drugs.

NLRP3 Inflammasome and Its Central Role in the Cardiovascular Diseases.

Abstract: Background/Aims. NLRP3 inflammasome, an inflammasome which consists of nucleotide-binding oligomerization domain- (Nod-) like receptor3 (NLRP3) scaffold, apoptosis-associated speck-like protein (ASC) containing a CARD adaptor, and pro-caspase-1, is assembled after the cytoplasmic leucine-rich repeats (LRRs) of NLRP3 sense pathogens or danger signals. In recent years, the role of inflammasome in cardiovascular diseases has attracted mounting attention, and the in-depth study of its mechanism is gradually clear. Materials. The NLRP3 inflammasome controls the activation of the proteolytic enzyme caspase-1. Caspase-1 in turn regulates the maturation of the proinflammasome cytokines IL-1β and IL-18, which leads to an inflammatory response. We made a mini-review on the association of regulatory mechanisms of NLRP3 inflammasome with the development of cardiovascular diseases systematically based on the recent research studies. Discussion. The inflammasome plays an indispensable role in the development of atherosclerosis, coronary heart diseases (CHD), and heart ischemia-reperfusion (I/R) injury, and NLRP3 inflammasome may become a new target for the prevention and treatment of cardiovascular diseases. Effective regulation of NLRP3 may help prevent or even treat cardiovascular diseases. Conclusion. This mini-review focuses on the association of regulatory mechanisms of NLRP3 inflammasome with the development of cardiovascular diseases, which may supply some important clues for future therapies and novel drug targets for cardiovascular diseases.

Metal-Enhanced Fluorescence by Bifunctional Au Nanoparticles for Highly Sensitive and Simple Detection of Proteolytic Enzyme

Abstract: Although fluorescence-based analytical methods have been used in intracellular analyses, their sensitivity is low for the precise analysis of intracellular proteolytic enzymes to observe cell apoptosis related to cancer and neurodegenerative diseases. In this study, a metal-enhanced-fluorescence (MEF)-based highly sensitive biosensor for the detection of proteolytic enzymes is proposed for the first time by using a bifunctional Au nanoparticle (AuNP), which is connected to the fluorophore by both single-stranded DNA (ssDNA) and a peptide. Once caspase-3, a proteolytic enzyme, cuts the peptide specifically, the fluorescence signal is drastically increased because the ssDNA maintains an optimal distance for the MEF. The proposed sensing method shows the highly sensitive detection of caspase-3 based on just a simple enzymatic cleavage reaction within 1 h, and caspase-3-related preapoptotic cell detection was successfully carried out with high sensitivity. The proposed sensing method is a rapid, simple, and one-step technique for the real-time monitoring of intracellular proteolytic enzymes and can be applied to the early diagnosis of cancer and neurodegenerative diseases.

The Practical Potential of Bacilli and Their Enzymes for Industrial Production

Abstract: Bacillus spp. are an affordable source of enzymes due to their wide distribution, safety in work, ease of cultivation, and susceptibility to genetic transformations. Researchers are particularly interested in proteolytic enzymes, which constitute one of the most diverse groups of microbial proteins in terms of properties. Despite the long history of their research, this group of enzymes continue to show great potential for practical application in the biomedical industry, as well as in the agricultural industry. Thus, the unique properties of bacillary proteinases, such as stability in a wide range of temperatures and pH, high specificity, biodegradability of a wide range of substrates, and the high potential of sequenced Bacillus genomes are a powerful foundation for the development of new biotechnologies. The current review aims to discuss recent studies on various enzymes in particular, proteinases produced by bacteria of the genus Bacillus, along with their prospective practical applications. This article also presents an interpretive summary of the recent developments on the usage of probiotic Bacillus strains as potential feed additives.

Role of Matrix Metalloproteinase 9 in Ocular Surface Disorders.

Abstract: Objectives (1) To explore the role and significance of Matrix Metalloproteinase 9 (MMP-9), a proteolytic enzyme, in various ocular surface diseases of inflammatory, infectious, and traumatic etiology (2), to further elucidate the molecular mechanisms responsible for its overexpression in ocular surface disease states, and (3) to discuss possible targets of therapeutic intervention. Methods A literature review was conducted of primary sources from 1995 onward using search results populated from the US National Library of Medicine search database. Results MMP-9 overexpression has been found in in vitro and in vivo models of dry eye disease (DED), corneal ulceration, microbial keratitis, corneal neovascularization, ultraviolet light-induced radiation, and a host of additional surface pathologies. MMP-9 is involved in an intricate signal transduction cascade that includes induction by many proinflammatory molecules including interleukin-1 (IL-1), tumor necrosis factor alpha (TNF-a), nuclear factor kappa light chain enhancer of activated B cells (NF-kB), platelet-activating factor, activator protein 1 (AP-1), and transforming growth factor beta (TGF-B). MMP-9 expression is blunted by a diverse array of molecular factors, such as tissue inhibitors of metalloproteinases, cyclosporine A (CyA), PES_103, epigalloccatechin-3-gallate (EGCG), N-acetylcysteine (NaC), ascorbate, tetracyclines, and corticosteroids. Inhibition of MMP-9 frequently led to improvement of ocular surface disease. Conclusions Novel insights into the mechanistic action of MMP-9 provide potential for new therapeutic modulations of ocular surface diseases mediated by its overexpression.

Review of recently used techniques and materials to improve the efficiency of orally administered proteins/peptides

Abstract: The main objective of present review is to explore and evaluate the effectiveness of recently developed methods to improve the bioavailability of orally administered biopharmaceutical drugs. A systematic search of sciencedirect, tandfonline and Google Scholar databases based on various sets of keywords was performed. All results were evaluated based on their abstracts, and irrelevant studies were neglected during further evaluation. At present, biopharmaceuticals are used as injectable therapies as they are not absorbed adequately from the different routes of drug administration, particularly the oral one. Their insufficient absorption is attributed to their high molecular weight, degradation by proteolytic enzymes, high hydrophilicity and rigidity of the absorptive tissues. From industrial aspect incorporation of enzyme inhibitors (EIs) and permeation enhancers (PEs) and mucoadhesive polymers into conventional dosage forms may be the easiest way of formulation of orally administered macromolecular drugs, but the effectiveness of protection and absorption enhancement here is the most questionable. Conjugation may be problematic from regulatory aspect. Encapsulation into lipid-based vesicles sufficiently protects the incorporated macromolecule and improves intestinal uptake but have considerable stability issues. In contrast, polymeric nanocarriers may provide good stability but provides lower internalization efficacy in comparison with the lipid-based carriers. It can be concluded that the combination of the advantages of mucoadhesive polymeric and lid-based carriers in hybrid lipid/polymer nanoparticles may result in improved absorption and might represent a potential means for the oral administration of therapeutic proteins in the near future.

Accelerated Lysis and Proteolytic Digestion of Biopsy-Level Fresh-Frozen and FFPE Tissue Samples Using Pressure Cycling Technology.

Abstract: Pressure cycling technology (PCT)-assisted tissue lysis and digestion have facilitated reproducible and high-throughput proteomic studies of both fresh-frozen (FF) and formalin-fixed paraffin-embedded (FFPE) tissue of biopsy scale for biomarker discovery. Here, we present an improved PCT method accelerating the conventional procedures by about two-fold without sacrificing peptide yield, digestion efficiency, peptide, and protein identification. The time required for processing 16 tissue samples from tissues to peptides is reduced from about 6 to about 3 h. We analyzed peptides prepared from FFPE hepatocellular carcinoma (HCC) tissue samples by the accelerated PCT method using multiple MS acquisition methods, including short-gradient SWATH-MS, PulseDIA-MS, and 10-plex TMT-based shotgun MS. The data showed that up to 8541 protein groups could be reliably quantified from the thus prepared peptide samples. We applied the accelerated sample preparation method to 25 pairs (tumorous and matched benign) of HCC samples followed by a single-shot, 15 min gradient SWATH-MS analysis. An average of 18 453 peptides from 2822 proteins were quantified in at least 20% samples in this cohort, while 1817 proteins were quantified in at least 50% samples. The data not only identified the previously known dysregulated proteins such as MCM7, MAPRE1, and SSRP1 but also discovered promising novel protein markers, including DRAP1 and PRMT5. In summary, we present an accelerated PCT protocol that effectively doubles the throughput of PCT-assisted sample preparation of biopsy-level FF and FFPE samples without compromising protein digestion efficiency, peptide yield, and protein identification.

Effect of NaCl replacement by other chloride salts on physicochemical parameters, proteolysis and lipolysis of dry-cured foal "cecina".

Abstract: This study was proposed following the strategy of the meat sector to reduce sodium intake through applying different salting processes instead of the traditional method. Therefore, the influence of two salting treatments (with 50% and 55% of NaCl replacement by other chloride salts) on the chemical, physicochemical, proteolysis and lipolysis of foal cecinas was evaluated and compared to those cecinas salted with a traditional procedure. Regarding physicochemical parameters, cecinas treated with CaCl2 and MgCl2 increased the lipid oxidation and luminosity, while decreased the redness. The highest contents of protein and the lowest of moisture were obtained in cecinas salted with 50% KCl, while the NaCl content was dramatically reduced by the experimental batches (4.25 and 3.40 g/100 g) in comparison with control samples (7.73 g/100 g). The values of texture (hardness) did not reflect differences among batches. The content of free amino acids increased with NaCl replacement. In fact, data suggests that NaCl had more inhibitory power on the proteolytic enzymes than the other salts. On the contrary, lipolytic phenomenon showed lower differences among treatments (mainly individual PUFA). However, these variations could be related to the higher oxidation observed in the samples with NaCl replacement. On the other hand, the substitution of NaCl by other salts had an important influence in mineral contents. The main objective, which is the reduction of sodium intake, was achieved. Nevertheless, a sensory study should carry out to observe how aforementioned changes affect the organoleptic quality of the final product and the consumer's acceptability.

Mild Acid-Responsive “Nanoenzyme Capsule” Remodeling of the Tumor Microenvironment to Increase Tumor Penetration

Abstract: Dense extracellular matrix (ECM) severely impedes the spread of drugs in solid tumors and induces hypoxia, reducing chemotherapy efficiency. Different proteolytic enzymes, such as collagenase (Col) or bromelain, can directly attach to the surface of nanoparticles and improve their diffusion, but the method of ligation may also impair the enzymatic activity due to conformational changes or blockage of the active site. Herein, a "nanoenzyme capsule" was constructed by combining collagenase nanocapsules (Col-nc) with heavy-chain ferritin (HFn) nanocages encapsulating the chemotherapy drug doxorubicin (DOX) to enhance tumor penetration of the nanoparticles by hydrolyzing collagen from the ECM. Col-nc could protect the activity of the enzyme before reaching the site of action while being degraded under mildly acidic conditions in tumors, and the released proteolytic enzyme could digest collagen. In addition, HFn as a carrier could effectively load DOX and had a self-targeting ability, enabling the nanoparticles to internalize into cancer cells more effectively. From in vivo and in vitro studies, we found that collagen was effectively degraded by Col-nc/HFn(DOX) to increase the accumulation and penetration of nanoparticles in the solid tumor site and could alleviate hypoxia inside the tumor to enhance the antitumor effects of DOX. Therefore, the strategy of increasing nanoparticle penetration in this system is expected to provide a potential approach for the clinical treatment of solid tumors.

Drugs with new lease of life as quorum sensing inhibitors: for combating MDR Acinetobacter baumannii infections.

Abstract: The emergence of multidrug-resistant (MDR) strains is a major health problem worldwide. There is an urgent need for novel strategies to combat bacterial infections caused by MDR strains like Pseudomonas aeruginosa and Acinetobacter baumannii. Quorum sensing (QS) is a critical communication system in bacterial community controlling survival and virulence. The awareness of the importance of QS in bacterial infections has stimulated research to identify QS inhibitors (QSIs) to defeat microbes. In this study, four FDA-approved drugs (besides azithromycin as positive QSI) were tested for potential QS inhibition against clinical A. baumannii isolates and P. aeruginosa (PAO1) standard strain. The inhibitory effect of these drugs on virulence factors of both microbes has been investigated. The studied virulence factors include biofilm formation, twitching and swarming motilities, proteolytic enzyme production, and resistance to oxidative stress. The four tested drugs (erythromycin, levamisole, chloroquine, and propranolol) inhibited QS in Chromobacterium violaceum by 84, 72, 55.1, and 37.3%, respectively. They also significantly inhibited virulence factors in both PAO1 and A. baumannii at sub-inhibitory concentrations. These findings were confirmed by qRT-PCR and mice mortality test, where tested drugs highly repressed the expression of abaI gene and showed significantly improved mice survival rates. In addition, molecular docking studies against AbaI and AbaR proteins of QS system in A. baumannii revealed the potential inhibition of QS by tested drugs. Beside their known activities, the tested drugs could be given new life as QSIs to combat A. baumannii nosocomial infections (alone or in combination with antimicrobials).