PSI-6 Characterization of in vitro stability for two multi-functional processive endoglucanases as exogenous biocatalysts in pig nutrition

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 278-278
Author(s):  
Laurence Cheng ◽  
Weijun Wang ◽  
Ming Z Z Fan
Keyword(s):  
Three Dimensional ◽  
Enzyme Preparations ◽  
Pork Production ◽  
Profit Margins ◽  
Natural Cellulose ◽  
Gas Purging ◽  
Three Dimensional Models ◽  
In Vitro Stability

Abstract Poor efficiency of dietary fibre utilization limits global pork production profit margins and mitigation of footprint on environment. The objective of this study was to characterize in vitro stability for two unique processive endoglucanases of tCel5A1 and p4818Cel5_2A that are reported to hydrolyze natural cellulose and have multi-functionality towards hemicelluloses (Basit and Akhtar, Biotechnology and Bioengineering, 2018, 115:1675; Wang et al., Scientific Reports 2019, 9:13630). Both tCel5A1 and p4818Cel5_2A were modelled with the SWISS-MODEL online server and analyzed and visualized by PyMOL2.4.1. These two cellulases were overexpressed in ClearColi®BL21(DE3). Their endoglucanase activities were determined using 0.70 % sodium carboxymethyl cellulose and 5 mM dithiothreitol (DTT) with or without N2 gas purging; and incubated at pH 6.5–7.0 and 37 °C for 15 min. The three-dimensional models showed 1 and 4 Cys residues on the surfaces of tCel5A1 and p4818Cel5_2A, respectively, suggesting their susceptibility to auto-oxidation by air-borne O2. This prediction was tested by comparing the activities of N2-purged and non-purged enzyme preparations as well as their processing and incubation handling. Both tCel5A1 and p4818Cel5_2A activities were enhanced (P < 0.01) by 50% when 5-mM DTT and N2-purging were adopted. Furthermore, after incubating both tCel5A1 and p4818Cel5_2A enzyme preparations under the porcine gastric pH (3.5) and pepsin (274 U/mL) as well as intestinal trypsin (78 U/mL) and chymotrypsin (20 U/mL) activities at pH 6.5 during 0–5 h, Eadie-Hofstee inhibition kinetic analyses showed that tCel5A1 and p4818Cel5_2A respectively lost 18 and 68% (P < 0.01) of their initial activities after 2 h under the gastric conditions and more than 90% (P < 0.01) of their initial activities after 2–3 h under the intestinal conditions. Therefore, further enzyme protein engineering and/or post-fermentation treatments, such as coating for by-passing the gastric-intestinal environment, will be required to enable these two processive endoglucanases as efficacious exogenous fibre enzymes.

In Silico Identification and Molecular Characterization of Extracellular Cathepsin L Proteases from Giardia duodenalis

2020 ◽  
Vol 17 (4) ◽  
pp. 342-351
Author(s):  
Sergio A. Durán-Pérez ◽  
José G. Rendón-Maldonado ◽  
Lucio de Jesús Hernandez-Diaz ◽  
Annete I. Apodaca-Medina ◽  
Maribel Jiménez-Edeza ◽  
...  
Keyword(s):  
In Silico ◽  
Cathepsin L ◽  
Three Dimensional ◽  
Giardia Duodenalis ◽  
Evolutionary Genetics ◽  
Extracellular Proteins ◽  
In Silico Identification ◽  
Dimensional Models ◽  
Three Dimensional Models

Background: The protozoan Giardia duodenalis, which causes giardiasis, is an intestinal parasite that commonly affects humans, mainly pre-school children. Although there are asymptomatic cases, the main clinical features are chronic and acute diarrhea, nausea, abdominal pain, and malabsorption syndrome. Little is currently known about the virulence of the parasite, but some cases of chronic gastrointestinal alterations post-infection have been reported even when the infection was asymptomatic, suggesting that the cathepsin L proteases of the parasite may be involved in the damage at the level of the gastrointestinal mucosa. Objective: The aim of this study was the in silico identification and characterization of extracellular cathepsin L proteases in the proteome of G. duodenalis. Methods: The NP_001903 sequence of cathepsin L protease from Homo sapienswas searched against the Giardia duodenalisproteome. The subcellular localization of Giardia duodenaliscathepsin L proteases was performed in the DeepLoc-1.0 server. The construction of a phylogenetic tree of the extracellular proteins was carried out using the Molecular Evolutionary Genetics Analysis software (MEGA X). The Robetta server was used for the construction of the three-dimensional models. The search for possible inhibitors of the extracellular cathepsin L proteases of Giardia duodenaliswas performed by entering the three-dimensional structures in the FINDSITEcomb drug discovery tool. Results: Based on the amino acid sequence of cathepsin L from Homo sapiens, 8 protein sequences were identified that have in their modular structure the Pept_C1A domain characteristic of cathepsins and two of these proteins (XP_001704423 and XP_001704424) are located extracellularly. Threedimensional models were designed for both extracellular proteins and several inhibitory ligands with a score greater than 0.9 were identified. In vitrostudies are required to corroborate if these two extracellular proteins play a role in the virulence of Giardia duodenalisand to discover ligands that may be useful as therapeutic targets that interfere in the mechanism of pathogenesis generated by the parasite. Conclusion: In silicoanalysis identified two proteins in the Giardia duodenalisprotein repertoire whose characteristics allowed them to be classified as cathepsin L proteases, which may be secreted into the extracellular medium to act as virulence factors. Three-dimensional models of both proteins allowed the identification of inhibitory ligands with a high score. The results suggest that administration of those compounds might be used to block the endopeptidase activity of the extracellular cathepsin L proteases, interfering with the mechanisms of pathogenesis of the protozoan parasite Giardia duodenalis.

scholarly journals In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor

2003 ◽  
Vol 77 (6) ◽  
pp. 3669-3679 ◽  
Author(s):  
Caterina Trozzi ◽  
Linda Bartholomew ◽  
Alessandra Ceccacci ◽  
Gabriella Biasiol ◽  
Laura Pacini ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Serine Protease ◽  
In Vitro Selection ◽  
Three Dimensional ◽  
Host Cells ◽  
Dimensional Structure ◽  
In Vitro Systems

ABSTRACT The hepatitis C virus (HCV) serine protease is necessary for viral replication and represents a valid target for developing new therapies for HCV infection. Potent and selective inhibitors of this enzyme have been identified and shown to inhibit HCV replication in tissue culture. The optimization of these inhibitors for clinical development would greatly benefit from in vitro systems for the identification and the study of resistant variants. We report the use HCV subgenomic replicons to isolate and characterize mutants resistant to a protease inhibitor. Taking advantage of the replicons' ability to transduce resistance to neomycin, we selected replicons with decreased sensitivity to the inhibitor by culturing the host cells in the presence of the inhibitor and neomycin. The selected replicons replicated to the same extent as those in parental cells. Sequence analysis followed by transfection of replicons containing isolated mutations revealed that resistance was mediated by amino acid substitutions in the protease. These results were confirmed by in vitro experiments with mutant enzymes and by modeling the inhibitor in the three-dimensional structure of the protease.

ANCD thrombectomy device: in vitro evaluation

2019 ◽  
Vol 12 (1) ◽  
pp. 77-81 ◽  
Author(s):  
Sonia Sanchez ◽  
Ignacio Cortiñas ◽  
Helena Villanova ◽  
Anna Rios ◽  
Iñaki Galve ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Treatment Strategies ◽  
Local Flow ◽  
Guide Catheter ◽  
First Pass ◽  
Thrombectomy Device ◽  
The Mean ◽  
Three Dimensional Models ◽  
Number Of Passes

IntroductionEndovascular treatment of stroke, although highly effective, may fail to reach complete recanalization in around 20% of cases. The Advanced Thrombectomy System (ANCD) is a novel stroke thrombectomy device designed to reduce clot fragmentation and facilitate retrieval by inducing local flow arrest and allowing distal aspiration in combination with a stent retriever. We aimed to assess the preclinical efficacy of ANCD.MethodsSoft red blood cell (RBC)-rich (n=20/group) and sticky fibrin-rich (n=30/group) clots were used to create middle cerebral artery (MCA) occlusions in two vascular phantoms. Three different treatment strategies were tested: (1) balloon guide catheter + Solitaire (BGC+SR); (2) distal access catheter + SR (DAC+SR); and (3) ANCD+SR, until complete recanalization was achieved or to a maximum of three passes. The recanalization rate was determined after each pass.ResultsAfter one pass, ANCD+SR resulted in an increased recanalization rate (94%) for all clots together compared with BGC+SR (66%; p<0.01) or DAC+SR (80%; p=0.04). After the final pass the recanalization rate increased in all three groups but remained higher with ANCD+SR (100%) than with BGC+SR (74%; p<0.01) or DAC+SR (90%; p=0.02). The mean number of passes was lower with ANCD+SR (1.06) than with BGC+SR (1.46) or DAC+SR (1.25) (p=0.01). A logistic regression model adjusted for treatment arm, clot type, and model used showed that both RBC-rich clots (OR 8.1, 95% CI 1.6 to 13.5) and ANCD+SR (OR 3.9, 95% CI 1.01 to 15.8) were independent predictors of first-pass recanalization.ConclusionIn in vitro three-dimensional models replicating MCA-M1 occlusion, ANCD+SR showed significantly better recanalization rates in fewer passes than other commonly used combinations of devices.

The effect of temperature on in vitro germination and germ tube growth of urediniospores of Tranzschelia discolor

1990 ◽  
Vol 41 (3) ◽  
pp. 479 ◽  
Author(s):  
PJ Ellison ◽  
BR Cullis ◽  
RW Bambach ◽  
PF Kable
Keyword(s):  
Germ Tube ◽  
Three Dimensional ◽  
Tube Growth ◽  
Effect Of Temperature ◽  
In Vitro Germination ◽  
Three Dimensional Models ◽  
C 30 ◽  
Tranzschelia Discolor ◽  
Germ Tube Growth

The effect of temperature on in vitro germination and germ tube growth of urediniospores of Tranzschelia discolor was studied over time under constant temperature conditions. Studies were carried out on 1% water agar in the dark at 3�C, 5�C, 8�C, 10�C, 15�C, 20�C, 25�C, 28�C, 30�C and 32�C. Germination was observed at all temperatures between 5 and 30'C, and occurred rapidly over most of this range. At 2 h, germination exceeded 80% at temperatures between 10 and 28�C, and this level was reached at 3 h at 8�C. Germination at 5 and 30�C was much reduced and at 7 h reached only 44% and 38% respectively. Germ tube growth occurred most vigorously at 15 and 20�C, reaching lengths in excess of 500 8m at 9 h. The optimum range was narrower than that for germination, and growth was reduced or poor at 8�C, 10�C, 25�C and 28�C, which were favourable temperatures for germination. Average germ tube lengths at 9 h at these temperatures were 55, 245, 273 and 62 8m, respectively. Three-dimensional models were derived relating germination and germ tube growth to time and temperature.

scholarly journals LKB1 kinase-dependent and -independent defects disrupt polarity and adhesion signaling to drive collagen remodeling during invasion

2016 ◽  
Vol 27 (7) ◽  
pp. 1069-1084 ◽  
Author(s):  
Jessica Konen ◽  
Scott Wilkinson ◽  
Byoungkoo Lee ◽  
Haian Fu ◽  
Wei Zhou ◽  
...  
Keyword(s):  
Cell Polarity ◽  
Cancer Metastasis ◽  
Kinase Activity ◽  
Rho Gtpase ◽  
Three Dimensional ◽  
Serine Threonine Kinase ◽  
Collagen Remodeling ◽  
Threonine Kinase ◽  
Three Dimensional Models

LKB1 is a serine/threonine kinase and a commonly mutated gene in lung adenocarcinoma. The majority of LKB1 mutations are truncations that disrupt its kinase activity and remove its C-terminal domain (CTD). Because LKB1 inactivation drives cancer metastasis in mice and leads to aberrant cell invasion in vitro, we sought to determine how compromised LKB1 function affects lung cancer cell polarity and invasion. Using three-dimensional models, we show that LKB1 kinase activity is essential for focal adhesion kinase–mediated cell adhesion and subsequent collagen remodeling but not cell polarity. Instead, cell polarity is overseen by the kinase-independent function of its CTD and more specifically its farnesylation. This occurs through a mesenchymal-amoeboid morphological switch that signals through the Rho-GTPase RhoA. These data suggest that a combination of kinase-dependent and -independent defects by LKB1 inactivation creates a uniquely invasive cell with aberrant polarity and adhesion signaling that drives invasion into the microenvironment.

scholarly journals The domain architecture of JBP1 suggests synergy between J-base DNA binding and thymidine hydroxylase activity

2018 ◽  
Author(s):  
Athanassios Adamopoulos ◽  
Tatjana Heidebrecht ◽  
Jeroen Roosendaal ◽  
Wouter G Touw ◽  
Isabelle Phan ◽  
...  
Keyword(s):  
Dna Binding ◽  
Domain Architecture ◽  
Three Dimensional ◽  
Specific Sequence ◽  
X Ray ◽  
Tet Proteins ◽  
X Ray Scattering ◽  
Sequence Patterns ◽  
Three Dimensional Models

JBP1 (J-DNA Binding Protein 1) contributes to biosynthesis and maintenance of base J (β-D-glucosyl-hydroxymethyluracil), a modification of thymidine (T) confined to pathogenic protozoa. JBP1 has two known functional domains: an N-terminal thymidine hydroxylase (TH) homologous to the 5-methylcytosine hydroxylase domain in TET proteins; and a J-DNA binding domain (JDBD) that resides in the middle of JBP1. Here we show that removing JDBD from JBP1 results in a soluble protein (Δ-JDBD) with the N- and C-terminal regions tightly associated together in a well-ordered domain. This Δ-JDBD domain retains thymidine hydroxylation activity in vitro, but displays a fifteen-fold lower apparent rate of hydroxylation compared to JBP1. Small Angle X-ray Scattering (SAXS) experiments on JBP1 and JDBD in the presence and absence of J-DNA, and on Δ-JDBD, allowed us to generate low-resolution three-dimensional models. We conclude that Δ-JDBD, and not the N-terminal region of JBP1 alone, is a distinct folding unit. Our SAXS-based model supports the notion that binding of JDBD specifically to J-DNA can facilitate hydroxylation a T 12-14 bp downstream on the complementary strand of the J-recognition site. We postulate that insertion of the JDBD module in the Δ-JDBD scaffold during evolution provided a mechanism to synergize between J recognition and T hydroxylation, ensuring inheritance of J in specific sequence patterns following DNA replication.

scholarly journals Shear bond strength of ceramic laminate veneers to finishing surfaces with different percentages of preserved enamel under a digital guided method

2022 ◽  
Vol 22 (1) ◽  
Author(s):  
Jiakang Zhu ◽  
Jing Gao ◽  
Luming Jia ◽  
Xin Tan ◽  
Chenyang Xie ◽  
...  
Keyword(s):  
Bond Strength ◽  
Shear Bond Strength ◽  
Three Dimensional ◽  
In Vitro Study ◽  
Significant Difference ◽  
Dimensional Models ◽  
Three Dimensional Models ◽  
Laminate Veneers ◽  
Bonding Surface

Abstract Background The purpose of this in vitro study was to evaluate the effect of the percentages of preserved enamel on ceramic laminate veneers’ (CLVs) shear bond strength (SBS). Methods Seventy extracted human maxillary central incisors were scanned and reconstructed into three-dimensional models. The extracted teeth were then embedded and randomly divided into seven groups (n = 10 per group). Based on digital analyses of the three-dimensional models, guided tooth preparation and bonding procedures were performed individually to form seven different percentages (100%, 80%, 60% 50%, 40%, 20% and 0%) of remaining enamel thickness on the bonding surface. Finally, the SBS test was performed, and the data were statistically analysed by one-way ANOVA with LSD post hoc test (α = 0.05). Results The complete enamel surface exhibited the highest SBS (19.93 ± 4.55 MPa), followed by 80% enamel (19.03 ± 3.66 MPa), 60% enamel (18.44 ± 3.65 MPa), 50% enamel (18.18 ± 3.41 MPa), 40% enamel (17.83 ± 3.01 MPa) and 20% enamel (11.32 ± 3.42 MPa) group. The lowest SBS (9.63 ± 3.46 MPa) was detected in 0% enamel group. No significant difference was observed among the 40–100% enamel groups, while the 20% or 0% enamel group demonstrated a significantly lower mean SBS than the 40% enamel group (p < 0.05). Conclusion The SBS value of CLVs bonded to 100% enamel on the finishing surfaces (nearly 20 MPa) was twice that which bonded to 0% enamel (nearly 10 MPa). Bonding to 100% enamel is the most reliable treatment. When dentin exposure is inevitable, enamel should be preserved as much as possible to maintain good bonding. In addition, 40% of preserved enamel on the bonding surface was the minimal acceptable value to fulfil the requirements of good bonding strength.

Sign in / Sign up

Export Citation Format

Share Document