A Simple and Rapid Microscale Method for Isolating Bacterial Lipopolysaccharides.

Bacterial endotoxins (lipopolysaccharides (LPSs)) are important mediators of inflammatory processes induced by Gram-negative microorganisms. LPSs are the key inducers of septic shock due to a Gram-negative bacterial infection; thus, the structure and functions of LPSs are of specific interest. Often, highly purified bacterial endotoxins must be isolated from small amounts of biological material. Each of the currently available methods for LPS extraction has certain limitations. Herein, we describe a rapid and simple microscale method for extracting LPSs. The method consists of the following steps: ultrasonic destruction of the bacterial material, LPS extraction via heating, LPS purification with organic solvents, and treatment with proteinase K. LPSs that were extracted by using this method contained less than 2-3% protein and 1% total nucleic acid. We also demonstrated the structural integrity of the O-antigen and lipid A via the sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) methods, respectively. We demonstrated the ability of the extracted LPSs to induce typical secretion of cytokines and chemokines by primary macrophages. Overall, this method may be used to isolate purified LPSs with preserved structures of both the O-antigen and lipid A and unchanged functional activity from small amounts of bacterial biomass.

Extracellular components in enteroaggregative Escherichia coli biofilm and impact of treatment with proteinase K, DNase or sodium metaperiodate.

Enteroaggregative E. coli (EAEC) is a major cause of diarrhea worldwide. EAEC are highly adherent to cultured epithelial cells and make biofilms. Both adherence and biofilm formation rely on the presence of aggregative adherence fimbriae (AAF). We compared biofilm formation from two EAEC strains of each of the five AAF types. We found that AAF type did not correlate with the level of biofilm produced. Because the composition of the EAEC biofilm has not been fully described, we stained EAEC biofilms to determine if they contained protein, carbohydrate glycoproteins, and/or eDNA and found that EAEC biofilms contained all three extracellular components. Next, we assessed the changes to the growing or mature EAEC biofilm mediated by treatment with proteinase K, DNase, or a carbohydrate cleavage agent to target the different components of the matrix. Growing biofilms treated with proteinase K had decreased biofilm staining for more than half of the strains tested. In contrast, although sodium metaperiodate only altered the biofilm in a quantitative way for two strains, images of biofilms treated with sodium metaperiodate showed that the EAEC were more spread out. Overall, we found variability in the response of the EAEC strains to the treatments, with no one treatment producing a biofilm change for all strains. Finally, once formed, mature EAEC biofilms were more resistant to treatment than biofilms grown in the presence of those same treatments.

Influence of Chemical Modifications of the Crystallophore on Protein Nucleating Properties and Supramolecular Interactions Network.

Crystallophores are lanthanide complexes that have demonstrated outstanding induction of crystallization for various proteins. This article explores the effect of tailored modifications of the crystallophore first generation and their impact on the nucleating properties and protein crystal structures. Through high-throughput crystallization experiments and dataset analysis, we evaluated the effectiveness of these variants, in comparison to the first crystallophore generation G1. In particular, the V1 variant, featuring a propanol pendant arm, demonstrated the ability to produce new crystallization conditions for the proteins tested (hen-egg white lysozyme, proteinase K and thaumatin). Structural analysis performed in the case of hen egg-white lysozyme along with Molecular Dynamics simulations, highlights V1's unique behavior, taking advantage of the flexibility of its propanol arm to explore different protein surfaces and form versatile supramolecular interactions.

Computer-assisted semi-rational design enhanced the enzymatic activity and protein stability of Proteinase K in calcium-free conditions.

Wild-type Proteinase K binds to two Ca2+ ions, which play an important role in regulating enzymaticactivity and maintaining protein stability. Therefore, a predetermined concentration of Ca2+ must be added during the use of Proteinase K, which increases its commercial cost. Herein, we addressed this challenge using a computational strategy to engineer a Proteinase K mutant that does not require Ca2+ and exhibits high enzymatic activity and protein stability. In the absence of Ca2+, the best mutant, MT24 (S17W-S176N-D260F), displayed an activity approximately 9.2-fold higher than that of wild-type Proteinase K. It also exhibited excellent protein stability, retaining 56.2 % of its enzymatic activity after storage at 4 °C for 5 days. The residual enzymatic activity was 65-fold higher than that of the wild-type Proteinase K under the same storage conditions. Structural analysis and molecular dynamics simulations suggest that the introduction of new hydrogen bond and π-π stacking at the Ca2+ binding sites due to the mutation may be the reasons for the increased enzymatic activity and stability of MT24.

Novel method for classification of prion diseases by detecting PrPres signal patterns from formalin-fixed paraffin-embedded samples.

Prion disease is an infectious and fatal neurodegenerative disease. Western blotting (WB)-based identification of proteinase K (PK)-resistant prion protein (PrPres) is considered a definitive diagnosis of prion diseases. In this study, we aimed to detect PrPres using formalin-fixed paraffin-embedded (FFPE) specimens from cases of sporadic Creutzfeldt-Jakob disease (sCJD), Gerstmann-Sträussler-Scheinker disease (GSS), glycosylphosphatidylinositol-anchorless prion disease (GPIALP), and V180I CJD. FFPE samples were prepared after formic acid treatment to inactivate infectivity. After deparaffinization, PK digestion was performed, and the protein was extracted. In sCJD, a pronounced PrPres signal was observed, with antibodies specific for type 1 and type 2 PrPres exhibited a strong or weak signals depending on the case. Histological examination of serial sections revealed that the histological changes were compatible with the biochemical characteristics. In GSS and GPIALP, prion protein core-specific antibodies presented as PrPres bands at 8-9 kDa and smear bands, respectively. However, an antibody specific for the C-terminus presented as smears in GSS, with no PrPres detected in GPIALP. It was difficult to detect PrPres in V180I CJD. Collectively, our findings demonstrate the possibility of detecting PrPres in FFPE and classifying the prion disease types. This approach facilitates histopathological and biochemical evaluation in the same sample and is safe owing to the inactivation of infectivity. Therefore, it may be valuable for the diagnosis and research of prion diseases.

Accelerating Proteomics Using Broad Specificity Proteases.

Trypsin is the gold-standard protease in bottom-up proteomics, but many sequence stretches of the proteome are inaccessible to trypsin and standard LC-MS approaches. Thus, multienzyme strategies are used to maximize sequence coverage in post-translational modification profiling. We present fast and robust SP3- and STRAP-based protocols for the broad-specificity proteases subtilisin, proteinase K, and thermolysin. All three enzymes are remarkably fast, producing near-complete digests in 1-5 min, and cost 200-1000× less than proteomics-grade trypsin. Using FragPipe resolved a major challenge by drastically reducing the duration of the required "unspecific" searches. In-depth analyses of proteinase K, subtilisin, and thermolysin Jurkat digests identified 7374, 8178, and 8753 unique proteins with average sequence coverages of 21, 29, and 37%, including 10,000s of amino acids not reported in PeptideAtlas' >2400 experiments. While we could not identify distinct cleavage patterns, machine learning could distinguish true protease products from random cleavages, potentially enabling the prediction of cleavage products. Finally, proteinase K, subtilisin, and thermolysin enabled label-free quantitation of 3111, 3659, and 4196 unique Jurkat proteins, which in our hands is comparable to trypsin. Our data demonstrate that broad-specificity proteases enable quantitative proteomics of uncharted areas of the proteome. Their fast kinetics may allow "on-the-fly" digestion of samples in the future.

Advances in Understanding the Antioxidant and Antigenic Properties of Egg-Derived Peptides.

Pepsin, trypsin and proteinase K were used in the present study to hydrolyse the proteins from whole eggs, yolks or whites, and the resulting hydrolysates were characterised in terms of antioxidant and IgE-binding properties, using a combination of in vitro and in silico methods. Based on the degree of hydrolysis (DH) results, the egg yolk proteins are better substrates for all the tested enzymes (DH of 6.2-20.1%) compared to those from egg whites (DH of 2.0-4.4%). The SDS-PAGE analysis indicated that pepsin and proteinase K were more efficient compared to trypsin in breaking the intramolecular peptide bonds of the high molecular weight egg proteins. For all the tested substrates, enzyme-assisted hydrolysis resulted in a significant increase in antioxidant activity, suggesting that many bioactive peptides are encrypted in inactive forms in the parent proteins. The hydrolysates obtained with proteinase K exhibited the highest DPPH radical scavenging activity (124-311 µM Trolox/g protein) and the lowest residual IgE-binding capacity. The bioinformatics tools revealed that proteinase K is able to break the integrity of the main linear IgE-binding epitopes from ovalbumin and ovomucoid. It can be concluded that proteinase K is a promising tool for modulating the intrinsic properties of egg proteins.

Silkworm pupae protein co-degradation by magnetic nanoparticles immobilized proteinase K and Mucor circinelloides aspartic protease for further utilization of sericulture by-products.

Silkworm pupae, by-product of sericulture industry, is massively discarded. The degradation rate of silkworm pupae protein is critical to further employment, which reduces the impact of waste on the environment. Herein, magnetic Janus mesoporous silica nanoparticles immobilized proteinase K mutant T206M and Mucor circinelloides aspartic protease were employed in the co-degradation. The thermostability of T206M improved by enhancing structural rigidity (t1/2 by 30 min and T50 by 5 °C), prompting the degradation efficiency. At 65 °C and pH 7, degradation rate reached the highest of 61.7%, which improved by 26% compared with single free protease degradation. Besides, the immobilized protease is easy to separate and reuse, which maintains 50% activity after 10 recycles. Therefore, immobilized protease co-degradation was first applied to the development and utilization of silkworm pupae resulting in the release of promising antioxidant properties and reduces the environmental impact by utilizing a natural and renewable resource.

Enhanced molecular release from elderly bone samples using collagenase I: insights into fatty acid metabolism alterations.

BACKGROUND: Bone is a metabolically active tissue containing different cell types acting as endocrine targets and effectors. Further, bone is a dynamic depot for calcium, phosphorous and other essential minerals. The tissue matrix is subjected to a constant turnover in response to mechanical/endocrine stimuli. Bone turnover demands high energy levels, making fatty acids a crucial source for the bone cells. However, the current understanding of bone cell metabolism is poor. This is partly due to bone matrix complexity and difficulty in small molecules extraction from bone samples. This study aimed to evaluate the effect of metabolite sequestering from a protein-dominated matrix to increase the quality and amount of metabolomics data in discovering small molecule patterns in pathological conditions. METHODS: Human bone samples were collected from 65 to 85 years old (the elderly age span) patients who underwent hip replacement surgery. Separated cortical and trabecular bone powders were treated with decalcifying, enzymatic (collagenase I and proteinase K) and solvent-based metabolite extraction protocols. The extracted mixtures were analyzed with the high-resolution mass spectrometry (HRMS). Data analysis was performed with XCMS and MetaboAnalystR packages. RESULTS: Fast enzymatic treatment of bone samples before solvent addition led to a significantly higher yield of metabolite extraction. Collagenase I and proteinase K rapid digestion showed more effectiveness in cortical and trabecular bone samples, with a significantly higher rate (2.2 folds) for collagenase I. Further analysis showed significant enrichment in pathways like de novo fatty acid biosynthesis, glycosphingolipid metabolism and fatty acid oxidation-peroxisome. CONCLUSION: This work presents a novel approach for bone sample preparation for HRMS metabolomics. The disruption of bone matrix conformation at the molecular level helps the molecular release into the extracting solvent and, therefore, can lead to higher quality results and trustable biomarker discovery. Our results showed ß-oxidation alteration in the aged bone sample. Future work covering more patients is worthy to identify the effective therapeutics to achieve healthy aging.

From a Droplet to a Fibril and from a Fibril to a Droplet: Intertwined Transition Pathways in Highly Dynamic Enzyme-Modulated Peptide-Adenosine Triphosphate Systems.

Many cellular coassemblies of proteins and polynucleotides facilitate liquid-liquid phase separation (LLPS) and the subsequent self-assembly of disease-associated amyloid fibrils within the liquid droplets. Here, we explore the dynamics of coupled phase and conformational transitions of model adenosine triphosphate (ATP)-binding peptides, ACC1-13Kn, consisting of the potent amyloidogenic fragment of insulin's A-chain (ACC1-13) merged with oligolysine segments of various lengths (Kn, n = 16, 24, 40). The self-assembly of ATP-stabilized amyloid fibrils is preceded by LLPS for peptides with sufficiently long oligolysine segments. The two-component droplets and fibrils are in dynamic equilibria with free ATP and monomeric peptides, which makes them susceptible to ATP-hydrolyzing apyrase and ACC1-13Kn-digesting proteinase K. Both enzymes are capable of rapid disassembly of amyloid fibrils, producing either monomers of the peptide (apyrase) or free ATP released together with cleaved-off oligolysine segments (proteinase K). In the latter case, the enzyme-sequestered Kn segments form subsequent droplets with the co-released ATP, resulting in an unusual fibril-to-droplet transition. In support of the highly dynamic nature of the aggregate-monomer equilibria, addition of superstoichiometric amounts of free peptide to the ACC1-13Kn-ATP coaggregate causes its disassembly. Our results show that the droplet state is not merely an intermediate phase on the pathway to the amyloid aggregate but may also constitute the final phase of a complex amyloidogenic protein misfolding scenario rich in highly degraded protein fragments incompetent to transition again into fibrils.

Comparison of Three Methods to Extract Plasmodium falciparum DNA from Whole Blood and Dried Blood Spots.

This study aimed to compare the effectiveness of three DNA extraction methods: the GF-1 Blood DNA Extraction Kit (GF-1 BD Kit), which employs a spin column along with lysing and washing buffers; the tris-ethylenediaminetetraacetic acid and proteinase K (TE-pK) method, which utilizes a combination of TE buffer and proteinase K for cell lysis; and DNAzol® Direct (DN 131), a single reagent combined with heating for the extraction process. Plasmodium falciparum DNA was extracted from both whole blood and dried blook spots (DBSs), with consideration of DNA concentration, purity, cost, time requirement, and limit of parasite detection (LOD) for each method. The target gene in this study was 18S rRNA, resulting in a 395-bp product using specific primers. In the comparative analysis, the DN 131 method yielded significantly higher DNA quantities from whole blood and DBSs than the GF-1 BD Kit and TE-pK methods. In addition, the DNA purity obtained from whole blood and DBSs using the GF-1 BD Kit significantly exceeded that obtained using the TE-pK and DN 131 methods. For LOD, the whole blood extracted using the DN 131, GF-1 BD Kit, and TE-pK methods revealed 0.012, 0.012, and 1.6 parasites/µL, respectively. In the case of DBSs, the LODs for the DN 131, GF-1 BD Kit, and TE-pK methods were 1.6, 8, and 200 parasites/µL, respectively. The results revealed that the TE-pK method was the most cost-effective, whereas the DN 131 method showed the simplest protocol. These findings offer alternative approaches for extracting Plasmodium DNA that are particularly well-suited for large-scale studies conducted in resource-limited settings.

Characteristics of inhibitory active substances produced by Pseudoalteromonas SW-1.

To confirm the antagonistic activity characterization of the strain Pseudoalteromonas SW-1 (P. SW-1), its cell-free supernatant (CFS) was studied against a clam pathogenic strain of Vibrio Alginolyticu MP-1 (V.MP-1). The CFS of P. SW-1 exhibited evident antagonistic activities against the pathogens, and the absorbance value (600 nm) of V. MP-1 remained at a lower level at 24 h when compared with the control. The results showed that the inhibitory activities of strain P. SW-1 CFS showed differences after treatment with heat, acid and alkali, and proteinase K. The CFS of P. SW-1 inhibitory activities decreased after treatment with heat, but the inhibitory activities of strain P. SW-1 CFS were still effective after treatment with proteinase K for 24 h. The acid and alkali treatments could increase the inhibitory activities of strain P. SW-1 CFS. Therefore, the ammonium sulfate precipitation test also indicated that P. SW-1 could produce some active protein compounds to antagonize pathogenic V. MP-1.

Saliva-based Proteinase K method: A rapid and reliable diagnostic tool for the detection of SARS-COV-2 in children.

Early and accurate detection of viruses in children might help prevent transmission and severe diseases. In this study, the severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) detection in children was evaluated using saliva specimens with a Proteinase K (PTK)-based RNA preparation, as saliva collection is a simple and noninvasive procedure, even in young children, with fewer concerns about sample contamination. The saliva-based PTK and the conventional paired nasopharyngeal aspiration (NPA)-based detection methods were compared between COVID-19-positive and -negative children. In addition, the detection rate for SARS-COV-2 and the difference between admission and discharge by the saliva-based PTK method was tested in COVID-19 patients. The diagnostic accuracy of the saliva-based PTK method was 98.8% compared to NP swab-based reverse transcriptase polymerase chain reaction. Saliva samples showed high sensitivity (94.1%) and specificity (100%) when using the PTK method. Furthermore, the saliva-based PTK method significantly reduced the test processing time by 2 h. Notably, Ct values at discharge increased in saliva samples compared with those at admission, which might indicate patients' clinical conditions or virus activity. In conclusion, the saliva-based PTK implemented in this study streamlines RNA extraction, making the process faster, safer, and more cost-effective, demonstrating that this method is a rapid and reliable diagnostic tool for SARS-CoV-2 detection in children.

Distribution of proteinase K-resistant anti-α-synuclein immunoreactive axons in the cardiac plexus is unbiased to the left ventricular anterior wall.

Lewy body disease (LBD) is characterized by the appearance of Lewy neurites and Lewy bodies, which are predominantly composed of α-synuclein. Notably, the cardiac plexus (CP) is one of the main targets of LBD research. Although previous studies have reported obvious differences in the frequency of Lewy body pathology (LBP) in the CP, none of them have confirmed whether LBP preferably appears in any part of the CP. Thus, we aimed to clarify the emergence and/or propagation of LBP in the CP. In this study, 263 consecutive autopsy cases of patients aged ≥50 years were included, with one region per case selected from three myocardial perfusion areas (MPAs) and subjected to proteinase K and then immunohistochemically stained with anti-α-synuclein antibodies to assess LBP. We stained all three MPAs in 17 cases with low-density LBP and observed the actual distribution of LBP. LBP were identified in the CP in 20.2% (53/263) of patients. Moreover, we found that LBP may appear in only one region of MPAs, mainly in the young-old group (35.3% (6/17) of patients). These findings suggest that it is possible to underestimate LBP in the CP, especially in the young-old group, by restricting the search to only one of the three MPAs.

Genotyping Protocols for Genetically Engineered Mice.

Historically, the laboratory mouse has been the mammalian species of choice for studying gene function and for modeling diseases in humans. This was mainly due to their availability from mouse fanciers. In addition, their short generation time, small size, and minimal food consumption compared to that of larger mammals were definite advantages. This led to the establishment of large hubs for the development of genetically modified mouse models, such as the Jackson Laboratory. Initial research into inbred mouse strains in the early 1900s revolved around coat color genetics and cancer studies, but gene targeting in embryonic stem cells and the introduction of transgenes through pronuclear injection of a mouse zygote, along with current clustered regularly interspaced short palindromic repeat (CRISPR) RNA gene editing, have allowed easy manipulation of the mouse genome. Originally, to distribute a mouse model to other facilities, standard methods had to be developed to ensure that each modified mouse trait could be consistently identified no matter which laboratory requested it. The task of establishing uniform protocols became easier with the development of the polymerase chain reaction (PCR). This chapter will provide guidelines for identifying genetically modified mouse models, mainly using endpoint PCR. In addition, we will discuss strategies to identify genetically modified mouse models that have been established using newer gene-editing technology such as CRISPR. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Digestion with proteinase K followed by purification of genomic DNA using phenol/chloroform Alternate Protocol: Digestion with proteinase K followed by crude isopropanol extraction of genomic DNA for tail biopsy and ear punch samples Basic Protocol 2: Purification of genomic DNA using a semi-automated system Basic Protocol 3: Purification of genomic DNA from semen, blood, or buccal swabs Basic Protocol 4: Purification of genomic DNA from mouse blastocysts to assess CRISPR gene editing Basic Protocol 5: Routine endpoint-PCR-based genotyping using DNA polymerase and thermal cycler Basic Protocol 6: T7E1/Surveyor assays to detect insertion or deletions following CRISPR editing Basic Protocol 7: Detecting off-target mutations following CRISPR editing Basic Protocol 8: Detecting genomic sequence deletion after CRISPR editing using a pair of guide RNAs Basic Protocol 9: Detecting gene knock-in events following CRISPR editing Basic Protocol 10: Screening of conditional knockout floxed mice.

Optimization of extraction-free protocols for SARS-CoV-2 detection using a commercial rRT-PCR assay.

In the ongoing global fight against coronavirus disease 2019 (COVID-19), the sample preparation process for real-time reverse transcription polymerase chain reaction (rRT-PCR) faces challenges due to time-consuming steps, labor-intensive procedures, contamination risks, resource demands, and environmental implications. However, optimized strategies for sample preparation have been poorly investigated, and the combination of RNase inhibitors and Proteinase K has been rarely considered. Hence, we investigated combinations of several extraction-free protocols incorporating heat treatment, sample dilution, and Proteinase K and RNase inhibitors, and validated the effectiveness using 120 SARS-CoV-2 positive and 62 negative clinical samples. Combining sample dilution and heat treatment with Proteinase K and RNase inhibitors addition exhibited the highest sensitivity (84.26%) with a mean increase in cycle threshold (Ct) value of + 3.8. Meanwhile, combined sample dilution and heat treatment exhibited a sensitivity of 79.63%, accounting for a 38% increase compared to heat treatment alone. Our findings highlight that the incorporation of Proteinase K and RNase inhibitors with sample dilution and heat treatment contributed only marginally to the improvement without yielding statistically significant differences. Sample dilution significantly impacts SARS-CoV-2 detection, and sample conditions play a crucial role in the efficiency of extraction-free methods. Our findings may provide insights for streamlining diagnostic testing, enhancing its accessibility, cost-effectiveness, and sustainability.

Optimization of degradation behavior and conditions for the protease K of polylactic acid films by simulation.

With global enforcement of plastic bans and restrictions, the biodegradable plastic, e.g., polylactic acid (PLA), has been extensively employed as a primary substitute for traditional petroleum-based plastics. However, the growing problem associated with PLA waste accumulation is posing grand environmental challenges. In addition, although PLA has the degrading property under natural conditions, the degradation process takes too long and the degradation products cannot be recycled. In this context, enzymatic degradation of PLA arouses great attention in scientific communities. This study aims at selecting the most cost-effective protease from various enzymes and optimizing the enzymolysis conditions towards the degradation of PLA. We will demonstrate that under an optimal temperature of 45 °C, a pH vale of 11, and an enzyme concentration of 0.6 mg mL-1, the protease K would achieve a remarkable degradation efficiency (> 99 %) for PLA films within just 50 min. Finally, molecular dynamics (MD) simulation and molecular docking studies reveal the mechanism behind the protease-induced PLA degradation, providing a promising direction for waste treatment and resource utilization for future biodegradable plastics.

Antimicrobial activity of Lactiplantibacillus plantarum against shiga toxin-producing Escherichia coli.

AIMS: The aim of the present work was to characterize the Lactiplantibacillus sp. LP5 strain, isolated from pork production, and identify bacteriocin-like inhibitory substances produced by this strain. METHODS AND RESULTS: In this study, LP5 was identified by species-specific PCR and 16S rRNA sequencing. Additionally, bacterial growth kinetics, antimicrobial activity, the detection of genes related to plantaricin production, and the genetic expression of plantaricins were determined. Lactiplantibacillus sp. LP5 was identified as Lactiplantibacillus plantarum. The well-diffusion test using cell-free supernatants (CFS), neutralized CFS, CFS treated with catalase, and CFS treated with proteinase K showed that inhibitory effects on a Shiga toxin-producing Escherichia coli (STEC) strain were produced by bacteriocins. The PCR technique allowed the detection of genes encoding E/F plantaricins, as well as J/K and whole genome sequencing, and bacteriocin mining analysis allowed us to confirm the presence of these plantaricins. CONCLUSIONS: We can conclude that the inhibitory effect of L. plantarum LP5 isolated from pigs against the STEC EDL933 strain could be associated with the bacteriocins production and represents a potential use as a probiotic strain.

Plasma untargeted metabolomics with proteinase K discloses phospholipid signature associated with pulmonary arterial hypertension.

Pulmonary arterial hypertension is a rare but life-threatening and clinically heterogeneous disease. The diagnostic schedule of this disorder is complex, and no specific indicator of the arterial etiology has been explored. In this study, untargeted plasma metabolomics was applied to evaluate the metabolic fingerprints of pulmonary arterial hypertension patients. Plasma samples were prepared using a new approach, which applies proteinase K during the sample preparation procedure to increase the metabolite coverage. The metabolic fingerprints were determined via LC-MS and subsequently analyzed with the use of both uni- and multivariate statistics. A total of 21 metabolites were discovered to be significantly altered in pulmonary arterial hypertensive patients. The metabolites were mainly related to the phospholipid metabolic pathways. In this study, decreases were found in the phosphatidylcholines (PCs) [PC(32:0), PC(40:7), PC(42:7)], phosphatidylethanolamine PE(18:0/18:2), lysophosphatidylethanolamines (LPEs) [LPE(22:6), LPE(18:2), LPE(18:0), LPE(20:4), LPE(20:1), LPE(20:0)], lysophosphatidylcholine LPC(20:4) and lysophosphatidylserine LPS(19:0), as well as increase of sphingomyelin SM(36:2), in the plasma samples of pulmonary arterial hypertensive patients in comparison to the control group. Besides their function as components of the biological membranes, these metabolites are also involved in the intracellular signaling pathways that are related to cell proliferation and apoptosis. The results obtained during this study confirm the potential of (untargeted) metabolomics to identify the molecular characteristics of the pathophysiology of pulmonary arterial hypertension. The clinical relevance of this study constitutes the selection of a metabolic panel that can potentially detect and properly diagnose the disease.

Enzymatic Degradation of Polylactic Acid Fibers Supported on a Hydrogel for Sustained Release of Lactate.

The incorporation of exogenous lactate into cardiac tissues is a regenerative strategy that is rapidly gaining attention. In this work, two polymeric platforms were designed to achieve a sustained release of lactate, combining immediate and prolonged release profiles. Both platforms contained electrospun poly(lactic acid) (PLA) fibers and an alginate (Alg) hydrogel. In the first platform, named L/K(x)/Alg-PLA, lactate and proteinase K (x mg of enzyme per 1 g of PLA) were directly loaded into the Alg hydrogel, into which PLA fibers were assembled. In the second platform, L/Alg-K(x)/PLA, fibers were produced by electrospinning a proteinase K:PLA solution and, subsequently, assembled within the lactate-loaded hydrogel. After characterizing the chemical, morphological, and mechanical properties of the systems, as well as their cytotoxicity, the release profiles of the two platforms were determined considering different amounts of proteinase K (x = 5.2, 26, and 52 mg of proteinase K per 1 g of PLA), which is known to exhibit a broad cleavage activity. The profiles obtained using L/Alg-K(x)/PLA platforms with x = 26 and 52 were the closest to the criteria that must be met for cardiac tissue regeneration. Finally, the amount of lactate directly loaded in the Alg hydrogel for immediate release and the amount of protein in the electrospinning solution were adapted to achieve a constant lactate release of around 6 mM per day over 1 or 2 weeks. In the optimized bioplatform, in which 6 mM lactate was loaded in the hydrogel, the amount of fibers was increased by a factor of ×3, the amount of enzyme was adjusted to 40 mg per 1 g of PLA, and a daily lactate release of 5.9 ± 2.7 mM over a period of 11 days was achieved. Accordingly, the engineered device fully satisfied the characteristics and requirements for heart tissue regeneration.