Enzymatic Cross-Linking of Dynamic Thiol-Norbornene Click Hydrogels

Enzyme-mediated in situ forming hydrogels are attractive for many biomedical applications because gelation afforded by enzymatic reactions can be readily controlled not only by tuning macromer compositions, but also by adjusting enzyme kinetics. For example, horseradish peroxidase (HRP) has been used extensively for in situ cross-linking of macromers containing hydroxyl-phenol groups. The use of HRP to initiate thiol-allylether polymerization has also been reported, yet no prior study has demonstrated enzymatic initiation of thiol-norbornene gelation. In this study, we discovered that HRP can generate the thiyl radicals needed for initiating thiol-norbornene hydrogelation, which has only been demonstrated previously using photopolymerization. Enzymatic thiol-norbornene gelation not only overcomes light attenuation issue commonly observed in photopolymerized hydrogels, but also preserves modularity of the cross-linking. In particular, we prepared modular hydrogels from two sets of norbornene-modified macromers, 8-arm poly(ethylene glycol)-norbornene (PEG8NB) and gelatin-norbornene (GelNB). Bis-cysteine-containing peptides or PEG-tetra-thiol (PEG4SH) was used as a cross-linker for forming enzymatically and orthogonally polymerized hydrogel. For HRP-initiated PEG-peptide hydrogel cross-linking, gelation efficiency was significantly improved via adding tyrosine residues on the peptide cross-linkers. Interestingly, these additional tyrosine residues did not form permanent dityrosine cross-links following HRP-induced gelation. As a result, they remained available for tyrosinase-mediated secondary cross-linking, which dynamically increased hydrogel stiffness. In addition to material characterizations, we also found that both PEG- and gelatin-based hydrogels exhibited excellent cytocompatibility for dynamic 3D cell culture. The enzymatic thiol-norbornene gelation scheme presented here offers a new cross-linking mechanism for preparing modularly and dynamically cross-linked hydrogels

Horseradish and soybean peroxidases: comparable tools for alternative niches?

Horseradish and soybean peroxidases (HRP and SBP, respectively) are useful biotechnological tools. HRP is often termed the classical plant heme peroxidase and although it has been studied for decades, our understanding has deepened since its cloning and subsequent expression, enabling numerous mutational and protein engineering studies. SBP, however, has been neglected until recently, despite offering a real alternative to HRP: SBP actually outperforms HRP in terms of stability and is now used in numerous biotechnological applications, including biosensors. Review of both is timely. This article summarizes and discusses the main insights into the structure and mechanism of HRP, with special emphasis on HRP mutagenesis, and outlines its use in a variety of applications. It also reviews the current knowledge and applications to date of SBP, particularly biosensors. The final paragraphs speculate on the future of plant heme-based peroxidases, with probable trends outlined and explored

Intestinal absorption of macromolecules during viral enteritis: an experimental study on rotavirus-infected conventional and germ-free mice.

Epithelial transport and degradation of horseradish peroxidase (HRP), a macromolecular tracer, was studied in conventional and germ-free suckling mice following an experimental infection with rotavirus. Conventional and germ-free mice developed diarrhea from days 2 to 8 postinfection (pi), with growth failure. In mucosal homogenates, infectious virus detected by immunofluorescence on MA 104 cells was present from day 2 through day 8 pi in germ-free mice, but persisted longer (day 13 pi) in conventional mice. Only mild histological lesions were observed during diarrhea, but obvious macrovacuolation of epithelial cells and increased cellular density occurred during the convalescence period (days 9 to 13 pi). Intact and degraded HRP fluxes from mucosa to serosa were measured in vitro on segments of jejunum mounted in Ussing chambers. Both groups of mice developed increased HRP permeability during the experimental period, but at different times after inoculation: during the diarrheal period (days 2 and 3 pi) conventional mouse epithelium absorbed five times more HRP than noninfected controls and during the convalescence period (days 9 to 13 pi) HRP absorption in germ-free mice rose 10-fold as compared to its level before infection. In both cases, this increase in HRP permeability was entirely due to an increase in intact HRP absorption, probably via a transcellular route, and occurred without any alteration in degraded HRP transport. These results indicate that in mice, rotavirus infection causes a transient rise in gut permeability to undegraded proteins. The intestinal microflora seems to affect the timing, magnitude, and duration of this increased permeability

Comparison of Alternative Meat Inspection Regimes for Pigs From Non-Controlled Housing – Considering the Cost of Error

Denmark has not had cases of bovine tuberculosis (bovTB) for more than 30 years but is obliged by trade agreements to undertake traditional meat inspection (TMI) of finisher pigs from non-controlled housing to detect bovTB. TMI is associated with higher probability of detecting bovTB but is also more costly than visual-only inspection (VOI). To identify whether VOI should replace TMI of finisher pigs from non-controlled housing, the cost of error – defined here as probability of overlooking infection and associated economic costs - should be assessed and compared with surveillance costs. First, a scenario tree model was set up to assess the ability of detecting bovTB in an infected herd (HSe) calculated for three within-herd prevalences, WHP (1, 5 and 10%), for four different surveillance scenarios (TMI and VOI with or without serological test, respectively). HSe was calculated for six consecutive 4-week surveillance periods until predicted bovTB detection (considered high-risk periods HRP). 1-HSe was probability of missing all positives by each HRP. Next, probability of spread of infection, Pspread, and number of infected animals moved were calculated for each HRP. Costs caused by overlooking bovTB were calculated taking into account Pspread, 1-HSe, eradication costs, and trade impact. Finally, the average annual costs were calculated by adding surveillance costs and assuming one incursion of bovTB in either 1, 10 or 30 years. Input parameters were based on slaughterhouse statistics, literature and expert opinion. Herd sensitivity increased by high-risk period and within-herd prevalence. Assuming WHP=5%, HSe reached median 90% by 2nd HRP for TMI, whereas for VOI this would happen after 6th HRP. Serology had limited impact on HSe. The higher the probability of infection, the higher the probability of detection and spread. TMI resulted in lowest average annual costs, if one incursion of bovTB was expected every year. However, when assuming one introduction in 10 or 30 years, VOI resulted in lowest average costs. It may be more cost-effective to focus on imported high-risk animals coming into contact with Danish livestock, instead of using TMI as surveillance on all pigs from non-controlled housing

Localization of brain stem motoneurons innervating the laryngeal muscles in the rufous horseshoe bat,rhinolophus rouxi

The motoneurons innervating the laryngeal muscles were localized in the rufous horseshoe bat,Rhinolophus rouxi, using the HRP method. HRP was applied to the cricothyroid muscle and to the cut end of the recurrent laryngeal nerve. Labeled motoneurons were found in two completely separated regions of the nucleus ambiguus. The motoneurons innervating the cricothyroid muscle via the superior laryngeal nerve (SLN) are located withinn the ventrolateral portion of the nucleus reaching the caudal pole of the motor nucleus of the facial nerve. The motoneurons innervating the other intrinsic laryngeal muscles via the recurrent laryngeal nerve (RLN) are situated in the caudal half of the nucleus ambiguus. The innervation is strictly homolateral

Model Pasangan Bersesuaian (Adjacency Pair) Dalam Stuktur Percakapan Bahasa Jerman

Pengertian percakapan berkaitan dengan pemikiran kita tentang bahasa. Bahasa diperlukan sebagai suatu sistem komunikasi verbal. Kaidah-kaidah bahasa dirumuskan dalam bentuk yang mencirikan elemen bahasa. Melalui proses ini struktur suatu bahasa ditemukan. Masalah yang muncul adalah dalam mendistribusikan struktur bahasa itu dalam pemakaian bahasa khususnya percakapan. Oleh karena itu, sebenarnya struktur bahasa itu tidak dapat dipisahkan dengan percakapan. Demikian juga dengan bahasa Jerman, secara umum memiliki struktur percakapan dan kaidahnya. Salah satu cara untuk mengenali struktur percakapan adalah dengan menganalisis model-model pasangan bersesuaian (Adjacency Pair)

Metode Pembelajaran Dan Pengembangan Kemampuan Verbal Bagi Anak Autis

Autis adalah gangguan pervasive pada anak yang ditandai dengan adanya gangguan dan keterlambatan dalam bidang interaksi sosial, komunikasi verbal/non verbal, kognitif, bahasa, prilaku, sensori dan emosi. Gangguan komunikasi pada anak autis ditandai dengan tidak adanya kontak mata, terlambat berbicara atau sama sekali belum dapat bicara dan terkadang huruf vokal saja belum mampu mengucapkannya secara sepontan harus dibantu dengan membuka mulut si anak, sulit untuk memulai percakapan dengan orang lain, mengulang kata-kata atau membeo, berbicara dalam bahasa yang tidak dapat dimengerti atau bahasa planet, serta tidak memahami pembicaraan orang lain. Jadi, salah satu ciri gangguan komunikasi yang muncul pada anak autis adalah terlambat bicara atau sama sekali belum dapat bicara. Adapun sistem pengajaran untuk mengembangkan kemampuan verbal anak adalah dengan permainan tiba-tiba, lomba menamai benda, lagu dan nyanyian, menonton televisi, dan permainan berpura-pura

Functional expression of horseradish peroxidase in Saccharomyces cerevisiae and Pichia pastoris

The ability to engineer proteins by directed evolution requires functional expression of the target polypeptide in a recombinant host suitable for construction and screening libraries of enzyme variants. Bacteria and yeast are preferred, but eukaryotic proteins often fail to express in active form in these cells. We have attempted to resolve this problem by identifying mutations in the target gene that facilitate its functional expression in a given recombinant host. Here we examined expression of HRP in Saccharomyces cerevisiae. Through three rounds of directed evolution by random point mutagenesis and screening, we obtained a 40-fold increase in total HRP activity in the S.cerevisiae culture supernatant compared with wild-type, as measured on ABTS [2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)] (260 units/l/OD_(600)). Genes from wild-type and two high-activity clones were expressed in Pichia pastoris, where the total ABTS activity reached 600 units/l/OD_(600) in shake flasks. The mutants show up to 5.4-fold higher specific activity towards ABTS and 2.3-fold higher specific activity towards guaiacol