Gelatin zymography is widely used to detect gelatinase activity, which is performed on unfixed tissue because it is assumed that fixation inactivates enzymes. However, using fixed tissues has several advantages over using fresh tissues for such prevention of tissue decay, thereby preserving the proteins as well as the morphology and structure of the specimens. In this study, we investigated the effects of the four commonly used fixatives (ethanol, acetone, zinc-based fixative (ZBF), and paraformaldehyde (PFA)) on the gelatinolytic activity in mouse brain tissue. Multiple protocols were employed to extract proteins from the fixed brain tissue. Western blotting and in-gel zymography (IGZ) were used to detect the gelatinase proteins and gelatinolytic activity of the extractions, respectively. In situ zymography (ISZ) revealed that ethanol, acetone, ZBF, and short-time PFA fixation did not inhibit gelatinolytic activity. Neither 1% Triton + 1 M NaCl nor 10% DMSO + 1 M NaCl was effective in extracting proteins from ethanol-, acetone-, ZBF-, or PFA-fixed brain tissues. However, 8 M urea + 4% CHAPS effectively extracted gelatinase proteins from ethanol- and acetone-fixed tissues while retaining the gelatinolytic activity. 2% SDS effectively extracted gelatinase proteins from ethanol-, acetone-, and ZBF-fixed tissues while retaining the gelatinolytic activity. Although 2% SDS + heating extracted gelatinase proteins from ethanol-, acetone-, ZBF-, and even long-term PFA-fixed tissues, the gelatinolytic activity was not retained. Our findings suggest that both ISZ and IGZ can be performed on fixed brain tissue, which is anticipated to be an improvement over the conventionally used gelatin zymography methods. (J Histochem Cytochem 71: 481-493, 2023).

Recent policies and silvicultural management call for forest regeneration that involve the selection of tree species able to cope with low soil nutrient availability in forest ecosystems. Understanding the impact of different tree species on the rhizosphere processes (e.g., enzyme activities) involved in nutrient mobilisation is critical in selecting suitable species to adapt forests to environmental change. Here, we visualised and investigated the rhizosphere distribution of enzyme activities (cellobiohydrolase, leucine-aminopeptidase, and acid phosphomonoesterase) using zymography. We related the distribution of enzyme activities to the seedling root morphological traits of European beech (Fagus sylvatica) and Norway spruce (Picea abies), the two most cultivated temperate tree species that employ contrasting strategies in soil nutrient acquisition. We found that spruce showed a higher morphological heterogeneity along the roots than beech, resulting in a more robust relationship between rhizoplane-associated enzyme activities and the longitudinal distance from the root apex. The rhizoplane enzyme activities decreased in spruce and increased in beech with the distance from the root apex over a power-law equation. Spruce revealed broader rhizosphere extents of all three enzymes, but only acid phosphomonoesterase activity was higher compared with beech. This latter result was determined by a larger root system found in beech compared with spruce that enhanced cellobiohydrolase and leucine-aminopeptidase activities. The root hair zone and hair lengths were significant variables determining the distribution of enzyme activities in the rhizosphere. Our findings indicate that spruce has a more substantial influence on rhizosphere enzyme production and diffusion than beech, enabling spruce to better mobilise nutrients from organic sources in heterogeneous forest soils.

The aim of the present study was to evaluate the in situ/in vivo effect of quercetin on dentin erosion and abrasion.
Human dentin blocks (2 × 2 × 2 mm) were embedded and assigned to 6 groups: 75 μg/mL, 150 μg/mL and 300 μg/mL quercetin (Q75, Q150, Q300); 120 μg/mL chlorhexidine (CHX, positive control); and deionized water and ethanol (the negative controls). The specimens were treated with the respective solutions for 2 min and then subjected to in situ/in vivo erosive/abrasive challenge for 7 d as follows: in vivo erosion 4 times a day and then in vivo toothbrush abrasion after the first and last erosive challenges of each day. Dentin loss was assessed by profilometry. An additional dentin specimen was used to evaluate the penetration depth of quercetin into dentin by tracking the spatial distribution of its characteristic Raman peak. Moreover, dentin blocks (7 × 1.7 × 0.7 mm) were used to detect the impact of quercetin on dentin-derived matrix metalloproteinase (MMP) inhibition by in situ zymography, and the inhibition percentage (%) was calculated. Additionally, the potential collagen crosslinking interactions with quercetin were detected by Raman spectroscopy, and the crosslinking degree was determined with a ninhydrin assay. Fully demineralized dentin beams (0.5 × 0.5 × 10 mm) were used to evaluate the impact of quercetin on the mechanical properties of dentin collagen fibre by the ultimate micro-tensile strength test (μUTS). The data were analysed by one-way analysis of variance and Tukey\'s test (α = 0.05).
Compared to the negative controls, all treatment solutions significantly reduced dentin loss. The dentin loss of Q150 and Q300 was significantly less than that of CHX (all P < 0.05). The amount of quercetin decreased with increasing dentin depth, and the maximum penetration depth was approximately 25-30 µm. In situ zymography showed that quercetin significantly inhibited the activities of dentin-derived MMPs. The inhibitory percentages of Q75 and Q150 were significantly lower than that of CHX (all P < 0.05), but no significant difference was found between Q300 and CHX (P = 0.58). The collagen crosslinking interactions with quercetin primarily involved hydrogen bonding and the degree of crosslinking increased in a concentration-dependent manner. Statistically significant increases in μUTS values were observed for demineralized dentin beams after quercetin treatment compared with those of the control treatments (all P < 0.05).
This study provides the first direct evidence that quercetin could penetrate approximately 25-30 µm into dentin and further prevent dentin erosion and abrasion by inhibiting dentin-derived MMP activity as well as crosslinking collagen of the demineralized organic matrix.

Paddy soils regularly experience redox oscillations during the wetting and draining stages, yet the effects of short-term presence of oxygen (O2) on in-situ microbial hotspots and enzyme activities in anoxic ecosystems remain unclear. To fill this knowledge gap, we applied soil zymography to localize hotspots and activities of phosphomonoesterase (PME), β-glucosidase (BG), and leucine aminopeptidase (LAP) in three compartments of rice-planted rhizoboxes (top bulk, rooted, and bottom bulk paddy soil) under oxic (+O2) and anoxic (O2) conditions. Short-term (35 min) aeration decreased PME activity by 13-49 %, BG by 4-52 %, and LAP by 12-61 % as compared with O2 in three soil compartments. The percentage of hotspot area was higher by 3-110 % for PME, by 10-60 % for BG, and by 12-158 % for LAP under +O2 vs. O2 conditions depending on a rice growth stage. Irrespective of the aeration conditions, the rhizosphere extent of rice plants for three enzymes was generally greater under higher moisture conditions and at earlier growth stage. Higher O2 sensitivity for the tested enzymes at bottom bulk soil versus other compartments suggested that short-term aeration during conventional zymography may lead to underestimation of nutrient mobilization in subsoil compared to top bulk soil. The intolerance of anaerobic microorganisms against the toxicity of O2 in the cells and the shift of microbial metabolic pathways may explain such a short-term suppression by O2. Our findings, therefore, show that anoxic conditions and soil moisture should be kept during zymography and probably other in-situ soil imaging methods when studying anoxic systems.

Phosphorus (P) availability is closely related to the distributions of pH, O2 and phosphatase activities in the rhizosphere of plants growing in soils and sediments. In this study, the P uptake processes and mechanisms of Vallisneria natans (V. natans) during two vegetation periods (i.e., week three and six) were revealed using three noninvasive 2D imaging techniques: planar optode (PO), diffusive gradients in thin films (DGT) and zymography. The results showed that increased phosphatase activity, O2 concentration and root-induced acidification were observed together in the rhizosphere of root segments and tips. In week three, when V. natans was young, the flux of DGT-labile P accumulated more in the rhizosphere in comparison with the bulk sediment. This was because increased phosphatase activity (of up to 35%) and root-induced acidification (with pH decreasing by up to 0.25) enhanced P acquisition of V. natans by the third week. However, the flux of DGT-labile P turned to depletion during weeks three to six of V. natans growth, after Fe plaque formed at the matured stage. The constant hydrolysis of phosphatase and acidification could not compensate for the P demand of the roots by the sixth week. At this stage, Fe plaque become the P pool, due to P fixation with solid Fe(III) hydroxides. Subsequently, V. natans roots acquired P from Fe plaque via organic acid complexation of Fe(III).

The critical step of Trichinella spiralis infection is that the muscle larvae (ML) are activated to intestinal infective larvae (IIL) which invade the intestinal columnar epithelium to further develop. The IIL excretory/secretory (ES) proteins play an important role in host-parasite interaction. Proteolytic enzymes are able to mediate the tissue invasion, thereby increasing the susceptibility of parasites to their hosts. The aim of the current study was to screen and identify the natural active proteases in T. spiralis IIL ES proteins using Western blot and gel zymography combined with liquid chromatography tandem mass spectrometry (LC-MS/MS). The T. spiralis ML and IIL ES proteins were collected from the in vitro cultures and their enzymatic acitvities were examined by gelatin zymography and azocasein degradation. The protease activities were partially inhibited by PMSF, E-64 and EDTA. Three protein bands (45, 118 and 165 kDa) of T. spiralis IIL ES proteins were identified by shotgun LC-MS/MS because they have hydrolytic activity to gelatin compared to the ML ES proteins. Total of 30 T. spiralis proteins were identified and they are mainly serine proteinases (19), but also metalloproteinases (7) and cysteine proteinases (3). The qPCR results indicated that transcription levels of four T. spiralis protease genes (two serine proteases, a cathepsin B-like cysteine proteinase and a zinc metalloproteinase) at IIL stage were obviously higher than at the ML stage. These proteolytic enzymes are directly exposed to the host intestinal milieu and they may mediate the worm invasion of enteral epithelium and escaping from the host\'s immune responses. The results provide the new insights into understanding of the interaction of T. spiralis with host and the invasion mechanism.

This study evaluated the use of a new collagen-reactive monomer (CRM), isocyanate-terminated urethane methacrylate precursor, which has covalent affinity to dental collagen, in the formation of dentin-resin bonds and compared it with 2 other dental adhesives. Dentin specimens were bonded with either the CRM-based adhesive (CBA), One-Step (OS; Bisco, Inc.), or a negative adhesive (NA) control and subjected to 24-h storage in water, thermocycling to simulate 1-y clinical function, or a matrix metalloproteinase-mediated aging process. We tested the microtensile bond strength (µTBS), characterized the bonding interface with an atomic force microscope, conducted micro-Raman analysis, and performed leakage tests and in situ zymography. CBA and OS exhibited comparable bonding strength after 24 h (P > 0.05); however, there was a sharp decrease in µTBS after aging for all except CBA (P < 0.001). Raman spectra results indicated increased collagen crosslinking and chemical reaction between the adhesive and collagen in the CBA group. CBA achieved high-quality hybridization with collagen, improving mechanical properties and integrity, and decreased the enzyme-mediated degradation of the bonding interface by inhibiting collagenolytic activity. With the promising bonding durability of coapplied CBA, CRM may be the first dental adhesive to provide strong and long-lasting resin-dental collagen bonding without the additional conditioning step. The use of CBA results in high-quality hybrid layers that protect the resin-dentin interface from harmful biological and chemical activities commonly occurring in the oral environment.

This study investigated the effects of cold storage at different temperatures (4, -0.5, -3, and -20 °C) on protein degradation and its relationship to structural changes of black carp muscle. At -0.5 and 4 °C, major structural changes occurred, including the formation of gaps between myofibers and myofibrils, breakage of myofibrils and myofibers, and degradation of sarcoplasmic reticulum. Gel-based proteomic analysis showed that these structural changes were accompanied by degradation of a series of myofibrillar proteins, including titin, nebulin, troponin, myosin, myomesin, myosin-binding protein, and α-actinin. Loss of extractable gelatinolytic and caseinolytic protease activities was also observed. At -3 and -20 °C, formation of ice crystals was the most noticeable change. The major proteins were degraded at different locations in the black carp muscle, and gelatinolytic and caseinolytic proteases appear to contribute to the degradation of those proteins.

The water-associated attributes of resin-dentin interfaces created by contemporary adhesives are important determinants of bond integrity and stability. In the present work, these attributes were estimated from the perspectives of causality, to examine the behavior of the first and most-recently launched versions of universal adhesives when applied in either the etch-and-rinse mode or the self-etch mode.
The immediate cause of interfacial permeability and the time-dependent cause of water sorption were investigated in conjunction with the intermediate effect of interface degradation and the more long-term effect of loss of mechanical strength, before and after thermomechanical cycling. The results were compared with control etch-and-rinse and self-etch adhesives.
Although the introduction of this new class of universal adhesives has brought forth significant changes to the dental adhesion arena, including more application options, reduced bonding armamentarium and increased user friendliness, the water-associated attributes that are critical for making resin-dentin bonds more durable to environmental challenges and less susceptible to degradation have remained unchanged at large, when compared with benchmarks established by former classes of adhesives.
It appears that the current trend of adhesive development has brought forth significant changes but lacks the vigor that demarcates progress and technological sublimity.
The advent of the user friendly universal adhesives has brought forth significant changes to the dental adhesion arena. However, the elements that are critical for making resin-dentin bonds more durable to environmental challenges and less susceptible to degradation have remained unchanged at large.

Zymography, the detection of proteolytic activities on the basis of protein substrate degradation, has been a technique described in the literature for at least in the past 50 years. In this study, we used substrate immersing zymography to analyze proteolysis of proteases. Instead of being directly added into a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, the substrates were added into the immersing solution after electrophoresis. With substrate immersing zymography, some characters of proteases, such as enzyme forms, potential proteolytic activity, molecular weights, presence of complexes, and potentially active enzyme fragments in complex biological samples, can be determined.