-
Microbial Cell Factories Jun 2024Recombinant peptide production in Escherichia coli provides a sustainable alternative to environmentally harmful and size-limited chemical synthesis. However, in-vivo...
BACKGROUND
Recombinant peptide production in Escherichia coli provides a sustainable alternative to environmentally harmful and size-limited chemical synthesis. However, in-vivo production of disulfide-bonded peptides at high yields remains challenging, due to degradation by host proteases/peptidases and the necessity of translocation into the periplasmic space for disulfide bond formation.
RESULTS
In this study, we established an expression system for efficient and soluble production of disulfide-bonded peptides in the periplasm of E. coli. We chose model peptides with varying complexity (size, structure, number of disulfide bonds), namely parathyroid hormone 1-84, somatostatin 1-28, plectasin, and bovine pancreatic trypsin inhibitor (aprotinin). All peptides were expressed without and with the N-terminal, low molecular weight CASPON™ tag (4.1 kDa), with the expression cassette being integrated into the host genome. During BioLector™ cultivations at microliter scale, we found that most of our model peptides can only be sufficiently expressed in combination with the CASPON™ tag, otherwise expression was only weak or undetectable on SDS-PAGE. Undesired degradation by host proteases/peptidases was evident even with the CASPON™ tag. Therefore, we investigated whether degradation happened before or after translocation by expressing the peptides in combination with either a co- or post-translational signal sequence. Our results suggest that degradation predominantly happened after the translocation, as degradation fragments appeared to be identical independent of the signal sequence, and expression was not enhanced with the co-translational signal sequence. Lastly, we expressed all CASPON™-tagged peptides in two industry-relevant host strains during C-limited fed-batch cultivations in bioreactors. We found that the process performance was highly dependent on the peptide-host-combination. The titers that were reached varied between 0.6-2.6 g L, and exceeded previously published data in E. coli. Moreover, all peptides were shown by mass spectrometry to be expressed to completion, including full formation of disulfide bonds.
CONCLUSION
In this work, we demonstrated the potential of the CASPON™ technology as a highly efficient platform for the production of soluble peptides in the periplasm of E. coli. The titers we show here are unprecedented whenever parathyroid hormone, somatostatin, plectasin or bovine pancreatic trypsin inhibitor were produced in E. coli, thus making our proposed upstream platform favorable over previously published approaches and chemical synthesis.
Topics: Escherichia coli; Periplasm; Disulfides; Peptides; Recombinant Proteins; Aprotinin
PubMed: 38840157
DOI: 10.1186/s12934-024-02446-6 -
Foods (Basel, Switzerland) Apr 2024A novel fibrinolytic enzyme was produced by the liquid fermentation of . The enzyme was purified from the culture supernatant by hydrophobic interactions, gel...
A novel fibrinolytic enzyme was produced by the liquid fermentation of . The enzyme was purified from the culture supernatant by hydrophobic interactions, gel filtration, and ion exchange chromatographies. It was purified by 241.02-fold, with a specific activity of 3619 U/mg and a final yield of 10.02%. SDS-PAGE analysis confirmed the purity of the enzyme, showing a single band with a molecular weight of 19.5 kDa. The first nine amino acids of the N-terminal of the purified enzyme were A-T-Y-T-G-G-S-Q-T. The enzyme exhibited optimal activity at a temperature of 42 °C and pH 7.6. Its activity was significantly improved by Zn, K, Ca, Mn, and Mg while being inhibited by Fe, Fe, Al, and Ba. The activity of the enzyme was completely inhibited by ethylenediamine tetraacetic acid (EDTA), and it was also dose-dependently inhibited by phenylmethylsulfonyl fluoride (PMSF) and soy trypsin inhibitor (SBTI). However, inhibitors such as N-α-tosyl-L-phenylalanine chloromethyl ketone (TPCK), aprotinin, and pepstatin did not significantly affect its activity, suggesting that the enzyme was a serine-like metalloproteinase. The enzyme acted as both a plasmin-like fibrinolytic enzyme and a plasminogen activator, and it also exhibited the capability to hydrolyze fibrinogen and fibrin. In vitro, it demonstrated the ability to dissolve blood clots and exhibit anticoagulant properties. Furthermore, it was found that the enzyme prolonged activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT), and reduced the levels of fibrinogen (FIB) and prothrombin activity (PA). Based on these studies, the enzyme has great potential to be developed as a natural agent for the prevention and treatment of thrombotic diseases.
PubMed: 38731663
DOI: 10.3390/foods13091292 -
Neurosurgical Review Apr 2024Antifibrinolytics have gained increasing attention in minimizing blood loss and mitigating the risks associated with massive transfusions, including infection and... (Meta-Analysis)
Meta-Analysis
Antifibrinolytics have gained increasing attention in minimizing blood loss and mitigating the risks associated with massive transfusions, including infection and coagulopathy in pediatric patients undergoing spine surgery. Nevertheless, the selection of optimal agent is still a matter of debate. We aim to review the utility of these agents and compare the efficacy of antifibrinolytics in pediatric and adolescent spine surgeries. A comprehensive search was performed in Scopus, Web of Science, and MEDLINE databases for relevant works. Studies providing quantitative data on predefined outcomes were included. Primary outcome was perioperative bleeding between the groups. Secondary outcomes included transfusion volume, rate of complications, and operation time. Twenty-eight studies were included in the meta-analysis incorporating 2553 patients. The use of Tranexamic acid (RoM: 0.71, 95%CI: [0.62-0.81], p < 0.001, I = 88%), Aprotinin (RoM: 0.54, 95%CI: [0.46-0.64], p < 0.001, I = 0%), and Epsilon-aminocaproic acid (RoM: 0.71, 95%CI: [0.62-0.81], p < 0.001, I = 60%) led to a 29%, 46%, and 29% reduction in perioperative blood loss, respectively. Network meta-analysis revealed higher probability of efficacy with Tranexamic acid compared to Epsilon-aminocaproic acid (P score: 0.924 vs. 0.571). The rate of complications was not statistically different between each two antifibrinolytic agent or antifibrinolytics compared to placebo or standard of care. Our network meta-analysis suggests a superior efficacy of all antifibrinolytics compared to standard of care/placebo in reducing blood loss and transfusion rate. Further adequately-powered randomized clinical trials are recommended to reach definite conclusion on comparative performance of these agents and to also provide robust objective assessments and standardized outcome data and safety profile on antifibrinolytics in pediatric and adolescent pediatric surgeries.
Topics: Humans; Antifibrinolytic Agents; Child; Blood Loss, Surgical; Network Meta-Analysis; Spine; Tranexamic Acid; Adolescent; Blood Transfusion; Neurosurgical Procedures; Treatment Outcome
PubMed: 38644447
DOI: 10.1007/s10143-024-02424-x -
Transfusion May 2024The role of aprotinin in modern cardiac surgery is not well defined. While licensed for use in isolated coronary artery bypass grafting it is more commonly used for... (Observational Study)
Observational Study
BACKGROUND
The role of aprotinin in modern cardiac surgery is not well defined. While licensed for use in isolated coronary artery bypass grafting it is more commonly used for cases deemed to be at an increased risk of bleeding. The relative efficacy, and safety profile, of aprotinin as compared to other antifibrinolytics in these high-risk cases is uncertain.
STUDY DESIGN AND METHODS
A retrospective observational study with propensity matching to determine whether aprotinin versus tranexamic acid reduced bleeding or transfusion requirements in patients presenting for surgical repair of type A aortic dissection (TAD).
RESULTS
Between 2016 and 2022, 250 patients presented for repair of TAD. A total of 231 patients were included in the final analysis. Bleeding and transfusion were similar between both groups in both propensity matched and unmatched cohorts. Compared to tranexamic acid, aprotinin use did not reduce transfusion requirements for any product. Rates of bleeding in the first 12 h, return to theater and return to intensive care unit with an open packed chest were similar between groups. There was no difference in rates of renal failure, stroke, or death.
CONCLUSION
Aprotinin did not reduce the risk of bleeding or transfusion requirements in patients undergoing repair of type A aortic dissections. Efficacy of aprotinin may vary depending on the type of surgery performed and the underlying pathology.
Topics: Humans; Tranexamic Acid; Aprotinin; Retrospective Studies; Female; Male; Aortic Dissection; Middle Aged; Aged; Antifibrinolytic Agents; Blood Transfusion; Blood Loss, Surgical
PubMed: 38581276
DOI: 10.1111/trf.17819 -
ACS Applied Materials & Interfaces Apr 2024Proteins are promising substances for introducing new drug carriers with efficient blood circulation due to low possibilities of clearance by macrophages. However, such...
A Microfluidics-Assisted Double-Barreled Nanobioconjugate Synthesis Introducing Aprotinin as a New Moonlight Nanocarrier Protein: Tested toward Physiologically Relevant 3D-Spheroid Models.
Proteins are promising substances for introducing new drug carriers with efficient blood circulation due to low possibilities of clearance by macrophages. However, such natural biopolymers have highly sophisticated molecular structures, preventing them from being assembled into nanoplatforms with manipulable payload release profiles. Here, we report a novel anticancer nanodrug carrier moonlighting protein, Aprotinin, to be used as a newly identified carrier for cytotoxic drugs. The Aprotinin-Doxorubicin (Apr-Dox) nanobioconjugate was prepared via a single-step microfluidics coflow mixing technique, a feasible and simple way to synthesize a carrier-based drug design with a double-barreled approach that can release and actuate two therapeutic agents simultaneously, i.e., Apr-Dox in 1:11 ratio (the antimetastatic carrier drug aprotinin and the chemotherapeutic drug DOX). With a significant stimuli-sensitive (i.e., pH) drug release ability, this nanobioconjugate achieves superior bioperformances, including high cellular uptake, efficient tumor penetration, and accumulation into the acidic tumor microenvironment, besides inhibiting further tumor growth by halting the urokinase plasminogen activator (uPA) involved in metastasis and tumor progression. Distinctly, in healthy human umbilical vein endothelial (HUVEC) cells, drastically lower cellular uptake of nanobioconjugates has been observed and validated compared to the anticancer agent Dox. Our findings demonstrate an enhanced cellular internalization of nanobioconjugates toward breast cancer, prostate cancer, and lung cancer both in vitro and in physiologically relevant biological 3D-spheroid models. Consequently, the designed nanobioconjugate shows a high potential for targeted drug delivery via a natural and biocompatible moonlighting protein, thus opening a new avenue for proving aprotinin in cancer therapy as both an antimetastatic and a drug-carrying agent.
Topics: Male; Humans; Aprotinin; Microfluidics; Nanoparticles; Doxorubicin; Antineoplastic Agents; Drug Delivery Systems; Drug Carriers; Breast Neoplasms; Hydrogen-Ion Concentration; Drug Liberation; Tumor Microenvironment
PubMed: 38564228
DOI: 10.1021/acsami.3c16548 -
Scandinavian Cardiovascular Journal :... Dec 2024To describe current on- (isolated coronary arterty bypass grafting, iCABG) and off-label (non-iCABG) use of aprotinin and associated safety endpoints in adult patients...
In Nordic countries 30-day mortality rate is half that estimated with EuroSCORE II in high-risk adult patients given aprotinin and undergoing mainly complex cardiac procedures.
To describe current on- (isolated coronary arterty bypass grafting, iCABG) and off-label (non-iCABG) use of aprotinin and associated safety endpoints in adult patients undergoing high-risk cardiac surgery in Nordic countries. Data come from 10 cardiac surgery centres in Finland, Norway and Sweden participating in the European Nordic aprotinin patient registry (NAPaR). 486 patients were given aprotinin between 2016 and 2020. 59 patients (12.1%) underwent iCABG and 427 (87.9%) non-iCABG, including surgery for aortic dissection (16.7%) and endocarditis (36.0%). 89.9% were administered a full aprotinin dosage and 37.0% were re-sternotomies. Dual antiplatelet treatment affected 72.9% of iCABG and 7.0% of non-iCABG patients. 0.6% of patients had anaphylactic reactions associated with aprotinin. 6.4% (95 CI% 4.2%-8.6%) of patients were reoperated for bleeding. Rate of postoperative thromboembolic events, day 1 rise in creatinine >44μmol/L and new dialysis for any reason was 4.7% (95%CI 2.8%-6.6%), 16.7% (95%CI 13.4%-20.0%) and 14.0% (95%CI 10.9%-17.1%), respectively. In-hospital mortality and 30-day mortality was 4.9% (95%CI 2.8%-6.9%) and 6.3% (95%CI 3.7%-7.8%) in all patients versus mean EuroSCORE II 11.4% (95%CI 8.4%-14.0%, < .01). 30-day mortality in patients undergoing surgery for aortic dissection and endocarditis was 6.2% (95%CI 0.9%-11.4%) and 6.3% (95%CI 2.7%-9.9%) versus mean EuroSCORE II 13.2% (95%CI 6.1%-21.0%, = .11) and 14.5% (95%CI 12.1%-16.8%, = .01), respectively. NAPaR data from Nordic countries suggest a favourable safety profile of aprotinin in adult cardiac surgery.
Topics: Adult; Humans; Aprotinin; Blood Loss, Surgical; Cardiac Surgical Procedures; Endocarditis; Aortic Dissection; Hemostatics
PubMed: 38555873
DOI: 10.1080/14017431.2024.2330347 -
Hematology, Transfusion and Cell Therapy Apr 2024The use of strategies to reduce blood loss and transfusions is essential in the treatment of surgical patients, including in complex cardiac surgeries and those that use...
The use of strategies to reduce blood loss and transfusions is essential in the treatment of surgical patients, including in complex cardiac surgeries and those that use cardiopulmonary bypass. Antifibrinolytics, such as epsilon-aminocaproic acid (EACA) and tranexamic acid (TXA), are widely used in these procedures, as well as in other types of surgeries. These medicines are included in the World Health Organization (WHO) list of 'essential medicines'. Scientific evidence demonstrates the effectiveness of EACA in reducing bleeding and the need for transfusions in heart surgery. EACA is highly recommended for use in heart surgery by the American Society of Anesthesiology Task Force on Perioperative Blood Management. Regarding the safety of EACA, there is no robust evidence of any significant thrombotic potential. TXA has also been shown to be effective in reducing the use of blood transfusions in cardiac and non-cardiac surgeries and is considered safer than other antifibrinolytic agents. There is no evidence of any increased risk of thromboembolic events with TXA, but doses greater than 2 g per day have been associated with an increased risk of seizures. It is also important to adjust the dose in patients with renal impairment. In conclusion, antifibrinolytics, such as EACA and TXA, are effective in reducing blood loss and transfusion use in cardiac and non-cardiac surgeries, without causing serious adverse effects.
PubMed: 38555249
DOI: 10.1016/j.htct.2024.02.011 -
FASEB Journal : Official Publication of... Mar 2024Trypanosoma cruzi is the causative agent of Chagas disease, a chronic pathology that affects the heart and/or digestive system. This parasite invades and multiplies in...
Trypanosoma cruzi is the causative agent of Chagas disease, a chronic pathology that affects the heart and/or digestive system. This parasite invades and multiplies in virtually all nucleated cells, using a variety of host cell receptors for infection. T. cruzi has a gene that encodes an ecotin-like inhibitor of serine peptidases, ISP2. We generated ISP2-null mutants (Δisp2) in T. cruzi Dm28c using CRISPR/Cas9. Epimastigotes of Δisp2 grew normally in vitro but were more susceptible to lysis by human serum compared to parental and ISP2 add-back lines. Tissue culture trypomastigotes of Δisp2 were more infective to human muscle cells in vitro, which was reverted by the serine peptidase inhibitors aprotinin and camostat, suggesting that host cell epitheliasin/TMPRSS2 is the target of ISP2. Pretreatment of host cells with an antagonist to the protease-activated receptor 2 (PAR2) or an inhibitor of Toll-like receptor 4 (TLR4) selectively counteracted the increased cell invasion by Δisp2, but did not affect invasion by parental and add-back lines. The same was observed following targeted gene silencing of PAR2, TLR4 or TMPRSS2 in host cells by siRNA. Furthermore, Δisp2 caused increased tissue edema in a BALB/c mouse footpad infection model after 3 h differently to that observed following infection with parental and add-back lines. We propose that ISP2 contributes to protect T. cruzi from the anti-microbial effects of human serum and to prevent triggering of PAR2 and TLR4 in host cells, resulting in the modulation of host cell invasion and contributing to decrease inflammation during acute infection.
Topics: Animals; Mice; Humans; Trypanosoma cruzi; Toll-Like Receptor 4; Receptor, PAR-2; Chagas Disease; Antiviral Agents; Serine Proteinase Inhibitors; Inflammation; Serine; Serine Endopeptidases
PubMed: 38526868
DOI: 10.1096/fj.202302091RR -
Tissue Engineering. Part A Jun 2024Fiber-reinforcement approaches have been used to replace aligned tissues with engineered constructs after injury or surgical resection, strengthening soft biomaterial...
Fiber-reinforcement approaches have been used to replace aligned tissues with engineered constructs after injury or surgical resection, strengthening soft biomaterial scaffolds and replicating anisotropic, load-bearing properties. However, most studies focus on the macroscale aspects of these scaffolds, rarely considering the cell-biomaterial interactions that govern remodeling and extracellular matrix organization toward aligned neo-tissues. As initial cell-biomaterial responses within fiber-reinforced microenvironments likely influence the long-term efficacy of repair and regeneration strategies, here we elucidate the roles of spatial orientation, substrate stiffness, and matrix remodeling on early cell-fiber interactions. Bovine mesenchymal stromal cells (MSCs) were cultured in soft fibrin gels reinforced with a stiff 100 µm polyglycolide-co-caprolactone fiber. Gel stiffness and remodeling capacity were modulated by fibrinogen concentration and aprotinin treatment, respectively. MSCs were imaged at 3 days and evaluated for morphology, mechanoresponsiveness (nuclear Yes-associated protein [YAP] localization), and spatial features including distance and angle deviation from fiber. Within these constructs, morphological conformity decreased as a function of distance from fiber. However, these correlations were weak ( = 0.01043 for conformity and = 0.05542 for nuclear YAP localization), illustrating cellular heterogeneity within fiber-enforced microenvironments. To better assess cell-fiber interactions, we applied machine-learning strategies to our heterogeneous dataset of cell-shape and mechanoresponsive parameters. Principal component analysis (PCA) was used to project 23 input parameters (not including distance) onto 5 principal components (PCs), followed by agglomerative hierarchical clustering to classify cells into 3 groups. These clusters exhibited distinct levels of morpho-mechanoresponse (combination of morphological conformity and YAP signaling) and were classified as high response (HR), medium response (MR), and low response (LR) clusters. Cluster distribution varied spatially, with most cells (61%) closest to the fiber (0-75 µm) belonging to the HR cluster, and most cells (55%) furthest from the fiber (225-300 µm) belonging to the LR cluster. Modulation of gel stiffness and fibrin remodeling showed differential effects for HR cells, with stiffness influencing the level of mechanoresponse and remodeling capacity influencing the location of responding cells. Together, these novel findings demonstrate early trends in cellular patterning of the fiber-reinforced microenvironment, showing how spatial orientation, substrate biophysical properties, and matrix remodeling may guide the amplitude and localization of cellular mechanoresponses. These trends may guide approaches to optimize the design of microscale scaffold architecture and substrate properties for enhancing organized tissue assembly at the macroscale.
PubMed: 38517095
DOI: 10.1089/ten.TEA.2024.0017 -
The Journal of Physical Chemistry. B Apr 2024In this study, a three-layered multicenter ONIOM approach is implemented to characterize the naive folding pathway of bovine pancreatic trypsin inhibitor (BPTI). Each...
In this study, a three-layered multicenter ONIOM approach is implemented to characterize the naive folding pathway of bovine pancreatic trypsin inhibitor (BPTI). Each layer represents a distinct level of theory, where the initial layer, encompassing the entire protein, is modeled by a general all-atom force-field GFN-FF. An intermediate electronic structure layer consisting of three multicenter fragments is introduced with the state-of-the-art semiempirical tight-binding method GFN2-TB. Higher accuracy, specifically addressing the breaking and formation of the three disulfide bonds, is achieved at the innermost layer using the composite DFT method rSCAN-3c. Our analysis sheds light on the structural stability of BPTI, particularly the significance of interlinking disulfide bonds. The accuracy and efficiency of the multicenter QM/SQM/MM approach are benchmarked using the oxidative formation of cystine. For the folding pathway of BPTI, relative stabilities are investigated through the calculation of free energy contributions for selected intermediates, focusing on the impact of the disulfide bond. Our results highlight the intricate trade-off between accuracy and computational cost, demonstrating that the multicenter ONIOM approach provides a well-balanced and comprehensive solution to describe electronic structure effects in biomolecular systems. We conclude that multiscale energy landscape exploration provides a robust methodology for the study of intriguing biological targets.
Topics: Animals; Cattle; Aprotinin; Cystine; Disulfides; Protein Folding; Proteins
PubMed: 38512062
DOI: 10.1021/acs.jpcb.4c00104