Mannheimia haemolytica is the main etiological bacterial agent in ruminant respiratory disease. M. haemolytica secretes leukotoxin, lipopolysaccharides, and proteases, which may be targeted to treat infections. We recently reported the purification and in vivo detection of a 110 kDa Zn metalloprotease with collagenase activity (110-Mh metalloprotease) in a sheep with mannheimiosis, and this protease may be an important virulence factor. Due to the increase in the number of multidrug-resistant strains of M. haemolytica, new alternatives to antibiotics are being explored; one option is lactoferrin (Lf), which is a multifunctional iron-binding glycoprotein from the innate immune system of mammals. Bovine apo-lactoferrin (apo-bLf) possesses many properties, and its bactericidal and bacteriostatic effects have been highlighted. The present study was conducted to investigate whether apo-bLf inhibits the secretion and proteolytic activity of the 110-Mh metalloprotease. This enzyme was purified and sublethal doses of apo-bLf were added to cultures of M. haemolytica or co-incubated with the 110-Mh metalloprotease. The collagenase activity was evaluated using zymography and azocoll assays. Our results showed that apo-bLf inhibited the secretion and activity of the 110-Mh metalloprotease. Molecular docking and overlay assays showed that apo-bLf bound near the active site of the 110-Mh metalloprotease, which affected its enzymatic activity.

Thrombosis is the formation of abnormal blood clots in the blood vessels that obstruct blood flow and lead to thrombosis. Current treatments for thrombosis are associated with serious side effects. Therefore there is a need for alternative natural therapy. A fibrinolytic protease was isolated from fresh leaves of Moringa oleifera Lam. and characterized for its potential to solubilize blood clots and hydrolyse fibrin under in-vitro conditions. The isolated protease showed a single protein band on native-PAGE. It showed optimum fibrinolytic activity at pH 8.0, 37 oC with 50 µg protein. The fibrinolytic activity of isolated protease was also confirmed by fibrin zymography. Km and Vmax of isolated protease were determined by the Lineweaver Burk plot. The isolated protease could solubilize 96.41% of blood clots by 96 h under in-vitro conditions. In-vitro fibrin hydrolysis and blood clot solubilization activities shown by an isolated protease from leaves of Moringa oleifera Lam. suggest its fibrinolytic potential to dissolve blood clots. Being a natural molecule and from a dietary plant it can be explored as an alternative natural therapy against thrombosis.

OBJECTIVE: To evaluate the effect of proanthocyanidin-functionalized hydroxyapatite nanoparticles (nHAp_PA) used as pretreatment at different concentrations on the microtensile bond strength (µTBS) and endogenous enzymatic activity (MMPs) on pH-cycled dentin after 24 h and 6 months of artificial aging.
METHODS: Fifty human sound dentin blocks were randomly assigned to 5 groups (n = 10): (i) negative control (no treatment); (ii) positive control (pH-cycling); (iii) pH-cycling + 2% nHAp_PA for 60s; (iv) pH-cycling + 6.5% nHAp_PA for 60s; (v) pH-cycling + 15% nHAp_PA for 60s. A self-etch adhesive was used for bonding procedures before resin composite build-ups. Specimens were tested with the µTBS test after 24 h and 6 months of laboratory storage. The proteolytic activity in each group was evaluated with gelatin zymography and in situ zymography. Data were statistically analyzed (p < 0.05).
RESULTS: At 24 h, the µTBS of the experimental groups were significantly higher than the controls (p ≤ 0.001), and no differences were observed between different concentrations (p > 0.05). Artificial aging significantly decreased bond strength in all groups (p ≤ 0.008); however, nHAp_PA 2% still yielded higher bonding values than controls (p ≤ 0.007). The groups pretreated with nHAp_PA exhibited lower MMP-9 and MMP-2 activities compared to the positive control group and almost the same enzymatic activity as the negative control group. In situ zymography showed that after 6 months of aging, nHAp_PA 2% and nHAp_PA 6,5% decreased enzymatic activity as well as the negative control.
CONCLUSIONS: Dentin pretreatment with nHAp_PA increased the bonding performance of a self-etch adhesive and decreased MMP-2 and MMP-9 activities after 6 months.

Polyamide 4 (PA4) is expected to solve the issue of marine plastic pollution due to its excellent mechanical properties and biodegradability. In this study, to reveal the mechanism of PA4 biodegradation in the marine environment, we isolated 5 strains of PA4-degrading bacteria belonging to Aliiglaciecola, Dasania, and Pseudophaeobacter from a marine environment. The isolated 5 strains are novel PA4-degrading bacteria that are phylogenetically distinct from those isolated in previous studies. In addition, we compared the PA4-degrading activities and structures of the PA4-degrading enzymes secreted by the 5 strains and PA4-degrading strains isolated in our previous study. The PA4-degrading activity in the supernatant of the cultivation solutions differed among the strains. Native-PAGE and zymography using a polyacrylamide gel containing a PA4 emulsion demonstrated that PA4-degrading enzymes are classified into no less than three types of structures. These results suggested that marine PA4-degrading bacteria have multiple PA4-degrading enzymes. Our findings will contribute to a better understanding of the microbial degradation of PA4 in the marine environment.

After separation on gel zymography, Drosophila melanogaster hemolymph displays gelatinase and caseinase bands of varying sizes, ranging from over 140 to 25 kDa. Qualitative and quantitative variations in these bands were observed during larval development and between different D. melanogaster strains and Drosophila species. The activities of these Drosophila hemolymph gelatinase and caseinase were strongly inhibited by serine protease inhibitors, but not by EDTA. Mass spectrometry identified over 60 serine proteases (SPs) in gel bands corresponding to the major D. melanogaster gelatinases and caseinases, but no matrix metalloproteinases (MMPs) were found. The most abundant proteases were tequila and members of the Jonah and trypsin families. However, the gelatinase bands did not show any change in the tequila null mutant. Additionally, no clear changes could be observed in D. melanogaster gel bands 24 h after injection of bacterial lipopolysaccharides (LPS) or after oviposition by Leptopilina boulardi endoparasitoid wasps. It can be concluded that the primary gelatinases and caseinases in Drosophila larval hemolymph are serine proteases (SPs) rather than matrix metalloproteinases (MMPs). Furthermore, the gelatinase pattern remains relatively stable even after short-term exposure to pathogenic challenges.

Purification of amylases from digestive tracts of three freshwater fish species with Q-Sepharose Fast Flow and Sephacryl S-200 columns displayed two isoforms of amylases from Osteochilus hasselti (O1, O2) and three isoforms of those from both Hampala dispar (UB, H1, H2) and Puntioplites proctozystron (P1, P2, P3). The optimum pH values displayed at 7.0 and 8.0, while the optimum temperatures revealed at 40 and 50 °C. Almost isoenzyme activities were activated by NaCl and CaCl2, whereas EDTA and SDS strongly inhibited all enzymatic activities. Verification with an atomic absorption spectrophotometry exhibited the presence of Ca2+ ions in the range of 0.02-13.53 ppm per mg protein indicating that amylases are Ca2+ dependent. Molecular weight analysis revealed 12 to 147 kDa. The UB, O1, and H2 amylases with appropriate molecular masses of 64, 49, and 25 kDa validated with LC-MS/MS were selected. Three certain enzymes revealed high stability in a sample buffer after five cycles of freeze-thawing process upon storage at - 20 °C for 12 weeks. No protein degradation was observed on polyacrylamide gel, and the enzymes still displayed sharp and clear bands on zymograms. The result suggested that the purified fish amylases, which expressed high activities and stabilities, were potentially used as enzyme molecular weight markers for zymography.

BACKGROUND: In patients with triple-negative breast cancer (TNBC), brain metastasis is a fatal consequence. Matrix metalloproteinases (MMPs), especially MMP-2 and MMP-9 as the major members of the MMP family, are involved in many different facets of breast cancer metastasis.
OBJECTIVE: In this study, we sought the MMPs expression in the metastatic cascade of TNBC.
RESULTS: Primary breast cancer cells known as 4T1T were extracted from the tumor mass following the creation of an animal model of TNBC. The brain metastasis lesions of malignant mice were used to extract highly brain metastatic tumor cells known as 4T1B. Gelatinase zymography and real-time polymerase chain reaction (RT-PCR) were used to examine the expression of MMPs at the proteomic and transcriptomic levels in 4T1T and 4T1B. Our results indicated; brain metastatic tumor cells greatly increased their expression of MMPs. In 4T1B, MMP-2 and MMP-9 gene expression were upregulated by 4 and 3.4 folds respectively. Zymographic analysis found MMP activity only in 4T1B.
CONCLUSIONS: These results offer significant information about the massive alteration of MMPs expression in the brain metastasis of TNBC. By analyzing the molecular characteristics of brain metastatic tumor cells, we can understand the molecular and genetic features of brain metastasis and develop tailored therapeutic strategies to combat TNBC brain metastasis.

Recycling of phosphorus (P) from waste streams in agriculture is essential to reduce the negative environmental effects of surplus P and the unsustainable mining of geological P resources. Sewage sludge (SS) is an important P source; however, several issues are associated with the handling and application of SS in agriculture. Thus, post-treatments such as pyrolysis of SS into biochar (BC) could address some of these issues. Here we elucidate how patches of SS in soil interact with the living roots of wheat and affect important P-related rhizosphere processes compared to their BC counterparts. Wheat plants were grown in rhizoboxes with sandy loam soil, and 1 cm Ø patches with either SS or BC placed 10 cm below the seed. A negative control (CK) was included. Planar optode pH sensors were used to visualize spatiotemporal pH changes during 40 days of plant growth, diffusive gradients in thin films (DGT) were applied to map labile P, and zymography was used to visualize the spatial distribution of acid (ACP) and alkaline (ALP) phosphatase activity. In addition, bulk soil measurements of available P, pH, and ACP activity were conducted. Finally, the relative abundance of bacterial P-cycling genes (phoD, phoX, phnK) was determined in the patch area rhizosphere. Labile P was only observed in the area of the SS patches, and SS further triggered root proliferation and increased the activity of ACP and ALP in interaction with the roots. In contrast, BC seemed to be inert, had no visible effect on root growth, and even reduced ACP and ALP activity in the patch area. Furthermore, there was a lower relative abundance of phoD and phnK genes in the BC rhizosphere compared to the CK. Hence, optimization of BC properties is needed to increase the short-term efficiency of BC from SS as a P fertilizer.

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).

To evaluate the influence of two glutaraldehyde-based desensitizers (L: GLUMA Desensitizer, Heraeus Kulzer and G: GLUMA Desensitizer PowerGel) prior to the adhesive procedures on microtensile bond strength (µTBS) to dentin and endogenous enzymatic activity.
Noncarious human third molars (N = 48) were cut to expose middle coronal dentin. Six experimental groups were formed according to the dentin pre-treatment (L or G) and the universal adhesives (IBU - iBond universal, Kulzer or AU - Adhese Universal, Ivoclar Vivadent) used in the self-etch mode (n = 8): 1) L/IBU; 2) G/IBU; 3) IBU; 4) L/AU; 5) G/AU; 6) AU. Specimens were cut into sticks and stressed until failure after 24 h (T0) or 1 yr of aging (T12). Additional 4 teeth were used for in situ zymography evaluation and data were statistically analyzed (α = 0.05).
Dentin pre-treatment, adhesive and aging statistically influenced bond strength and enzymatic activity (P<0.001). AU demonstrated higher bond strength values than IBU (P<0.001). The L resulted in higher bond strength compared to the G and control groups (P<0.001). aging statistically influenced bonding performance, especially when no dentin pre-treatment was performed (P<0.001). In situ zymography revealed that at baseline the control groups exhibited lower interfacial fluorescence compared to the experimental groups, irrespective of the adhesive used (P<0,001). However, after 1 yr of artificial storage, no differences were found among the groups (P>0.05).
Glutharldeadeyde-based products increased bond strength and determined a stabilization of the adhesive interface over time apparently not related to the MMPs inhibition.
The results of this in vitro study suggest that the application of glutaraldehyde-based desensitizers prior to the adhesive procedures when associated with universal adhesives could result in increased bond strength and stabilization of the adhesive interface over time.