In Drosophila Hemolymph, Serine Proteases Are the Major Gelatinases and Caseinases.

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 Native Dentilisin Complex from Treponema denticola by Preparative Continuous Polyacrylamide Gel Electrophoresis and Functional Analysis by Gelatin Zymography.

Periodontal disease is characterized by the destruction of the hard and soft tissues comprising the periodontium. This destruction translates to a degradation of the extracellular matrices (ECM), mediated by bacterial proteases, host-derived matrix metalloproteinases (MMPs), and other proteases released by host tissues and immune cells. Bacterial pathogens interact with host tissue, triggering adverse cellular functions, including a heightened immune response, tissue destruction, and tissue migration. The oral spirochete Treponema denticola is highly associated with periodontal disease. Dentilisin, a T. denticola outer membrane protein complex, contributes to the chronic activation of pro-MMP-2 in periodontal ligament (PDL) cells and triggers increased expression levels of activators and effectors of active MMP-2 in PDL cells. Despite these advances, no mechanism for dentilisin-induced MMP-2 activation or PDL cytopathic behaviors leading to disease is known. Here, we describe a method for purification of large amounts of the dentilisin protease complex from T. denticola and demonstrate its ability to activate MMP-2, a key regulator of periodontal tissue homeostasis. The T. denticola dentilisin and MMP-2 activation model presented here may provide new insights into the dentilisin protein and identify potential therapeutic targets for further research. Key features • This protocol builds upon a method described by Cunningham et al. [1] for selective release of Treponema outer membrane proteins. • We adapted the protocol for the purification of biologically active, detergent-stable outer membrane protein complexes from large batch cultures of T. denticola. • The protocol involves large-scale preparative electrophoresis using a Model 491 Prep Cell. • We then use gelatin zymography to demonstrate the activity of the purified dentilisin complex by its ability to activate matrix metalloproteinase 2 (MMP-2).

Hyperaccumulator extracts promoting the phytoremediation of rare earth elements (REEs) by Phytolacca americana: role of active microbial community in rhizosphere hotspots.

The increased usage of rare earth elements (REEs) leads to the extensive exploitation of rare earth mines, and the REEs pollution in soil caused by the legacy mine tailings has brought great harm to environment and human health. Although Phytolacca americana can remove REEs from contaminated soil to some extent, there is still an urgent problem to improve its efficiency. Hyperaccumulator extract is a new organic material with potential in metal phytoextraction, but its role in REEs phytoremediation and the related underlying processes remain unclear. In this study, hyperaccumulator extracts from P. americana root (PR), stem (PS), leaf (PL) and EDTA were used to improve the phytoremediation efficiency of REEs with P. americana. Soil zymography was applied to assess the enzyme hotspots' spatial distribution in the rhizosphere, and the hotspots' microbial communities were also identified. The results indicated that the application of hyperaccumulator extracts improved the biomass and REEs uptake of P. americana, and the highest REEs content in plant was observed in the treatment of PS, which increased 299% compared to that of the control. Hotspots area of ß-glucosidase, leucine aminopeptidase and acid phosphatase were concentrated in the pant rhizosphere along the roots and increased 2.2, 5.3 and 2.2 times after PS application compared to unamended soils. The PS application increased the relative abundance of Proteobacteria, Cyanobacteria, Bacteroidota and Firmicutes phyla in rhizosphere. Soil fungi have a higher contribution on promoting REEs activation than that of bacteria. Available P and extractable REEs were leading predictors for the plant biomass and REEs concentrations. The co-occurrence network showed that the application of PS creates a more efficient and stable microbial network compared to other treatments. In conclusion, stem-derived hyperaccumulator extract is excellent in stimulating REEs phytoremediation with P. americana by improving hotspots microbial activities and form a healthy rhizosphere microenvironment.

Matrix Metalloproteinase-9 Contributes to Epilepsy Development after Ischemic Stroke in Mice.

Epilepsy, a neurological disorder affecting over 50 million individuals globally, is characterized by an enduring predisposition and diverse consequences, both neurobiological and social. Acquired epilepsy, constituting 30% of cases, often results from brain-damaging injuries like ischemic stroke. With one third of epilepsy cases being resistant to existing drugs and without any preventive therapeutics for epileptogenesis, identifying anti-epileptogenic targets is crucial. Stroke being a leading cause of acquired epilepsy, particularly in the elderly, prompts the need for understanding post-stroke epileptogenesis. Despite the challenges in studying stroke-evoked epilepsy in rodents due to poor long-term survival rates, in this presented study the use of an animal care protocol allowed for comprehensive investigation. We highlight the role of matrix metalloproteinase-9 (MMP-9) in post-stroke epileptogenesis, emphasizing MMP-9 involvement in mouse models and its potential as a therapeutic target. Using a focal Middle Cerebral Artery occlusion model, this study demonstrates MMP-9 activation following ischemia, influencing susceptibility to seizures. MMP-9 knockout reduces epileptic features, while overexpression exacerbates them. The findings show that MMP-9 is a key player in post-stroke epileptogenesis, presenting opportunities for future therapies and expanding our understanding of acquired epilepsy.

Purification and characterization of amylases from three freshwater fish species providing new insight application as enzyme molecular markers for zymography.

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.

Contribution of Biogas Slurry-Derived Colloids to Plant P Uptake and Phosphatase Activities: Spatiotemporal Response.

The bioavailability for varied-size phosphorus (P)-binding colloids (Pcoll) especially from external P sources in soil terrestrial ecosystems remains unclear. This study evaluated the differential contribution of various-sized biogas slurry (BS)-derived colloids to plant available P uptake in the rhizosphere and the corresponding patterns of phosphatase response. Keeping the same content of total P input (15 mg kg-1), we applied different size-fractioned BS-derived colloids including nanosized colloids (NCs, 1-20 nm), fine-sized colloids (FCs, 20-220 nm), and medium-sized colloids (MCs, 220-450 nm) respectively to conduct a 45-day rice (Oryza sativa L.) rhizotron experiment. During the whole cultivation period, the dynamics of chemical characteristics and P fractions in each experimental rhizosphere soil solution were analyzed. The spatial and temporal dynamics examination of P-transforming enzymes (acid phosphatases) in the rice rhizosphere was visualized by a soil zymography technique after 5, 25, and 45 days of rice transplantation. The results indicated that the acid phosphatase activities and its hot spot areas were significantly 1) correlated with the relative bioavailability of colloidal P (RBAcoll), 2) increased with the colloid-free (truly dissolved P) and BS-derived NC addition, and 3) affected by the plant growth stage. With the nanosized BS colloid addition, the RBAcoll and plant biomass were respectively found to be the highest (64% and 1.22 g plant-1), in which the acid phosphatase-catalyzed hydrolysis of organic Pcoll played an important role. All of the above suggested that nanosized BS-derived colloids are an effective alternative to conventional phosphorus fertilizer for promoting plant P uptake and P bioavailability.

Differential impacts of sewage sludge and biochar on phosphorus-related processes: An imaging study of the rhizosphere.

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.

Upregulation of Matrix Metalloproteinases in the Metastatic Cascade of Breast Cancer to the Brain.

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. AIMS: In this study, we sought the MMPs expression in the metastatic cascade of TNBC. METHODS AND 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. CONCLUSION: 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.

Gelatin Zymography Can Be Performed on Fixed Brain Tissue.

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).

Prognostic value of different forms of serum MMP-2 and MMP-9 in dogs with mammary tumors; a longitudinal study.

Gelatinases (MMP-2 and MMP-9) are related to tumor invasion and metastasis. In humans, the diagnostic value of serum levels of gelatinases has been confirmed in breast cancer, but their diagnostic value in canine mammary tumors is still unknown. This study aimed to determine the serum level of gelatinases in dogs with mammary tumors in order to determine their value in the diagnosis of malignancy or benign tumors and also in predicting the possibility of metastasis and recurrence. Frequent measurement of MMP-2 and MMP-9 by gelatin zymography in serum before and after surgical treatment has not previously been studied for monitoring mammary tumors in dogs. Thus, the serum levels of MMP-2 and MMP-9 in 26 dogs with mammary tumors before surgical treatment and then 1, 4 and 12 months after surgery were evaluated by gelatin zymography. Serum samples of 26 healthy dogs with normal conditions were used as control. Dogs with benign and malignant mammary tumors showed bands of pro-MMP-2, pro-MMP-9 and active MMP-9. However, only pro-MMP-2 and pro-MMP-9 bands appeared in the serum of control group. Our results showed that the presence of active MMP-9, regardless of its level, was prognostically important for metastasis and or recurrence (M/R). However, the presence of active MMP-2 band was more important for M/R than active MMP-9, as its presence coincides with definitive M/R. It seems that serum gelatin zymography could possibly be used at regular intervals before and after surgery to evaluate the probability of M/R in dogs with mammary tumors. More research is needed in this regard.

Matrix metalloproteinase- 9 may contribute to collagen structure modification during postmortem aging of beef.

Matrix metalloproteinases (MMPs) are responsible for the turnover of intramuscular connective tissue in live animals. We hypothesize that MMPs may play a role in postmortem aging of beef muscles for the degradation of connective tissues. Four different experiments were performed to: 1) characterize MMP activity during postmortem aging of beef; 2) determine if the native beef MMP can contribute to connective tissue degradation in a simulated standard industry postmortem aging condition; 3) explore approaches to improve the native beef MMP activity and 4) characterize MMP activity in beef from cattle supplemented with supranutritional level of Zn. In experiment 1, MMP was active throughout the entire aging periods (3, 21, 42 and 63 d) for beef muscles Longissimus lumborum, Gluteus medius and Gastrocnemius, and the unknown MMP responsible for the collagen degradation was identified as MMP-9 by Western Blot. In experiment 2 and 3, MMP-9 activity was noticeable in the gels after 42 d of storage in the cooler. Moreover, the addition of ZnCl2 in the model system significantly increased MMP-9 activity when compared to the control (P < 0.01). In experiment 4, Longissimus thoracis from animals supplemented with a supranutritional Zn level had increased Zn availability and MMP-9 activity than those from animals fed with a control diet (P < 0.05). Further research is needed better understand MMP-9 mechanism during postmortem aging of meat. With a better understanding of MMP-9 in the aging process, the beef industry can provide better connective tissue management strategies for lower-quality beef cuts.

A new quantitative insight: Interaction of polyethylene microplastics with soil - microbiome - crop.

In this study, the interaction between primary/secondary PE MPs and soil - microbiome - crop complex system and PE MPs enrichment behavior in crops were studied by using the self-developed quantitative characterization method of Eu-MPs and in situ zymography. The results demonstrated for the first time the enrichment effect of micron-sized PE (> 10 µm) in crops, manifested as roots>leaves>stems. Primary PE MPs significantly increased soil TN, TC, SOM and ß-glu activity and inhibited Phos activity. Age-PE MPs significantly reduced soil TN, TP, ß-glu and Phos activities and also have significant inhibitory effects on plant height, stem diameter, and leaf dry weight of maize. Age-PE MPs significantly affected soil microbial diversity, mainly caused by bacterial genera such as UTCFX1, Sphingomonas, Subgroup-6 and Gemmatimonas. Age-PE MPs also affected some metabolism related to microbial community composition and maize growth, including Glycerolipid, Citrate cycle (TCA cycle), C5-Branched dibasic acid, Arginine and proline, Tyrosine metabolism, pentose phosphate pathway, Valine, leucine and isoleucine biosynthesis. These research results indicated that the PE MPs, which are widely present in farmland soils, can affect crop growth, soil microbial community and metabolic function after aging, thus affecting agroecosystems and terrestrial biodiversity.

Tissue Inhibitor of Matrix Metalloproteinases-1 (TIMP-1) and Pulmonary Involvement in COVID-19 Pneumonia.

Background: The aim of the study was to longitudinally evaluate the association between MMP-2, MMP-9, TIMP-1 and chest radiological findings in COVID-19 patients. Methods: COVID-19 patients were evaluated based on their hospital admission (baseline) and three months after hospital discharge (T post) and were stratified into ARDS and non-ARDS groups. As a control group, healthy donors (HD) were enrolled. Results: At the baseline, compared to HD (n = 53), COVID-19 patients (n = 129) showed higher plasma levels of MMP-9 (p < 0.0001) and TIMP-1 (p < 0.0001) and the higher plasma activity of MMP-2 (p < 0.0001) and MMP-9 (p < 0.0001). In the ARDS group, higher plasma levels of MMP-9 (p = 0.0339) and TIMP-1 (p = 0.0044) and the plasma activity of MMP-2 (p = 0.0258) and MMP-9 (p = 0.0021) compared to non-ARDS was observed. A positive correlation between the plasma levels of TIMP-1 and chest computed tomography (CT) score (ρ = 0.2302, p = 0.0160) was observed. At the T post, a reduction in plasma levels of TIMP-1 (p < 0.0001), whereas an increase in the plasma levels of MMP-9 was observed (p = 0.0088). Conclusions: The positive correlation between TIMP-1 with chest CT scores highlights its potential use as a marker of fibrotic burden. At T post, the increase in plasma levels of MMP-9 and the reduction in plasma levels of TIMP-1 suggested that inflammation and fibrosis resolution were still ongoing.

Protective Effects of a Mixed Medicinal Herb Extract (NUC1) on Collagenase-Induced Osteoarthritis in Rabbits.

NUC1 (Nutraceutical compound 1) is an ethanol extract composed of a formulation based on medicinal herbs traditionally used for the treatment of arthritis in Korea and China. This study investigated the therapeutic effects of NUC1 on osteoarthritis (OA). The protective effect of NUC1 on OA was tested in a rabbit model of collagenase-induced arthritis (CIA) for 4 weeks. Results were compared among four groups (n = 9 per group): the normal group (untreated), the CIA group (vehicle control), the NUC1 group (CIA rabbits treated with 200 mg/kg NUC1), and the JOINS group (positive control, CIA rabbits treated with 200 mg/kg JOINS tablet). NUC1 significantly inhibited NO production (p < 0.05 at 125 µg/ml, p < 0.01 at 250 µg/ml, and p < 0.001 at 500 µg/ml) and iNOS expression in macrophages, in a concentration-dependent manner. NUC1 also inhibited the release and protein expression of MMP-1, 3, and 13, in TNF-α-induced chondrosarcoma cells in a concentration-dependent manner. In vivo, the MMP-1 and MMP-3 levels in synovial fluids were significantly (p < 0.05) lower in NUC1 group (77.50 ± 20.56 and 22.50 ± 7.39 pg/ml, respectively) than in the CIA group (148.33 ± 68.58 and 77.50 ± 20.46 pg/ml, respectively). Also, in histopathological, NUC1 ameliorated articular cartilage damage in OA by increasing the abundance of chondrocytes and proteoglycan in the articular cartilage. Thus, NUC1 showed promise as a potential therapeutic agent, and it can be generalized to a broader study population in different OA animal models.

Glutaraldehyde-based desensitizers' influence on bonding performances and dentin enzymatic activity of universal adhesives.

OBJECTIVES: 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. METHODS: 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). RESULTS: 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). CONCLUSIONS: Glutharldeadeyde-based products increased bond strength and determined a stabilization of the adhesive interface over time apparently not related to the MMPs inhibition. CLINICAL SIGNIFICANCE: 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.

The effect of carbodiimide on push-out bond strength of fiber posts and endogenous enzymatic activity.

BACKGROUND: To investigate the effect of 0.3 M 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) aqueous solution pretreatment on push-out bond strength (PBS) and matrix-metalloproteinases (MMPs) activity within radicular dentin when different post cementation strategies were employed. METHODS: One hundred and twenty monoradicular human teeth were endodontically treated and randomly divided into six groups, depending on the cementation strategy and root dentin pretreatment (n = 20): EAR: cementation with an etch-and-rinse adhesive (LuxaBond Total Etch, DMG) and resin cement (LuxaCore Z Dual, DMG); EAR/EDC: 1 min EDC pretreatment after etching + EAR; SE: cementation with a self-etch primer (Multilink Primer, Ivoclar Vivadent) and corresponding cement (Multilink Automix, Ivoclar Vivadent); SE/EDC: self-etch primer + EDC pretreatment + SE; SA: cementation with a universal self-adhesive cement (RelyX Universal, 3 M); SA/EDC: EDC pretreatment + SA. Slices were submitted to PBS test and interfacial nanoleakage evaluation 24 h after cementation or after thermocycling (40.000 cycles, 5-55 °C). To investigate the effect of EDC on MMPs activity, 4 additional first maxillary premolars per group were processed for in situ zymography analysis. Multivariate ANOVA and post hoc Tukey tests were used to analyze PBS values. The data from in situ zymography were analyzed with Kruskal-Wallis test and Dunn's pairwise multiple comparison procedures (α = 0.05). RESULTS: The variables "EDC pretreatment", "root region" and "thermocycling" significantly influenced PBS (p < 0.05), while the variable "cementation strategy" had no influence (p > 0.05). Thermocycling significantly reduced PBS in SE and SA groups (p < 0.05). EDC was effective in preserving PBS after artificial aging. EDC pretreatment significantly reduced enzymatic activity at baseline in EAR and SE groups, and in SA group after thermocycling (p < 0.05). CONCLUSIONS: The use of EDC prevents the reduction of bond-strength values after artificial aging and silences endogenous enzymatic activity within radicular dentin when different cementation strategies were employed.

Ultradiluted SARS-CoV-2 Spike Protein mitigates hyperinflammation in lung via ferritin and MMP-9 regulation in BALB/c mice.

AIM: This study investigated the prophylactic and therapeutic role of ultradiluted preparation of the Delta variant of SARS-CoV-2 recombinant spike (S) protein during S antigen-induced inflammatory process of disease progression along with the probable mechanism of action. MAIN METHODS: Ultradiluted S protein (UDSP) was prepared and administered orally to adult BALB/c mice before and after administration of S antigen intranasally. After an observation period of 72 h, animals were sacrificed and expression level of ferritin was assayed through ELISA. The genetic expressions of cytokines, IL-6, IL-10, IL-1ß, TNFα, IL-17, MMP-9, TIMP-1, ferritin light and heavy chains, and mitochondrial ferritin from lung tissues were investigated through RT-PCR. Formalin-fixed lung tissue sections were stained with hematoxylin and eosin to observe the degree of pathological changes. The activity of MMP-9 in lung tissues was investigated through gelatin zymography and immunofluorescence of MMP-9 in lung tissue sections was performed to revalidate the finding from gelatin zymography. Systems biology approach was used to elucidate a probable pathway where UDSP attenuated the inflammation through the regulation of pro- and anti-inflammatory cytokines. KEY FINDINGS: UDSP attenuated the S antigen-induced hyperinflammation in the lung by regulating pro- and anti-inflammatory cytokines, calming cytokine storm, reducing ferritin level both in transcriptional and translational levels, and restoring critical ratio of MMP-9: TIMP-1. SIGNIFICANCE: Our findings suggest a probable pathway by which UDSP might have attenuated inflammation through the regulation of cytokines, receptors, and other molecules. This proclaims UDSP as a promising antiviral agent in the treatment of COVID-19-induced immunopathogenesis.

Can Carbodiimide (EDC) and Chitosan Cross-linking Agents Effect the Longevity of Fiberglass Posts Luted with Different Types of Composite Cements to Root Dentin?

PURPOSE: To evaluate the effect of carbodiimide (EDC) and chitosan (CHI) on the enzymatic activity (EA) and bond strength (BS) of different composite cements to root dentin. MATERIALS AND METHODS: Ninety (90) maxillary canines were sectioned, standardizing the length of the roots. The roots were endodontically treated, prepared, divided into 3 groups according to dentin treatment (distilled water [DW], CHI 0.2 wt%, or EDC 0.5M), and further subdivided into 3 subgroups according to composite cement (RelyX ARC [3M Oral Care], Panavia F 2.0 [Kuraray Noritaki], or RelyX U200 [3M Oral Care]). Of the slices obtained by sectioning, the most cervical of each third were subjected to a push-out test and the most apical were subjected to in-situ zymography. Half of the slices were analyzed immediately, and the other half after 6 months. The results were analyzed with ANOVA or the chi-squared test. RESULTS: RelyX ARC showed higher BS associated with CHI, while RelyX U200 showed higher BS associated with EDC (p = 0.044). For Panavia F 2.0, the treatment did not influence BS (p > 0.05). For the cervical and middle thirds, no differences were observed between the cements, while the apical third revealed higher BS for RelyX U200 (p < 0.001). The highest percentage of adhesive-to-dentin failures was observed for Panavia F 2.0. EDC showed the lowest percentage of adhesive-to-dentin failures. According to zymographic analysis, DW and CHI showed greater fluorescence for RelyX ARC, while EDC exhibited the lowest fluorescence of all cements (p > 0.05). CONCLUSION: The different mechanisms of action of solutions for pre-treatment of intraradicular dentin yielded different results depending on the adhesive used. EDC resulted in higher bond strength and higher enzyme inhibition for RelyX U200, while the treatment with chitosan resulted in higher bond strength and lower enzymatic activity for RelyX ARC. Although EDC and chitosan treatments did not influence the bond strength for Panavia F 2.0, both resulted in higher enzyme inhibition for this composite cement.

Screening of MMP-2 Inhibiting Phytoconstituents for the Development of Newer Pancreatic Cancer Treatment Modalities.

BACKGROUND: Pancreatic cancer metastasis is characterized by a higher incidence of morbidity and mortality. The present study attempts to identify phytocomponents with the potential to inhibit the secretion of MMP-2 by pancreatic cancer cells and ascertain the efficacy of individual components. METHODS: Overall survival analysis carried out revealed reduced survival of patients with high MMP-2 expression. Data analysis from TCGA revealed increased MMP-2 expression in pancreatic cancer patients compared to adjacent normal tissues. The expression of MMP-2 was reported at different stages of pancreatic cancer (Stage I-IV). To understand the relevance of phytocomponents in binding to the catalytic site of MMP-2, molecular docking studies were performed to find the effectiveness based on Glide score/energy. To substantiate the in-silico analysis, the eight components were also tested in vitro for reducing the survival in PANC-1 cells at three different time points (24, 48, and 72 hours). Finally, zymography analysis was performed using the eight components in the PANC-1 conditioned media of treated cells to ascertain the enzymatic activity of MMP-2. RESULTS: The obtained results suggest plumbagin, emodin, and EGCG exert potential inhibition in PANC-1 cells, among other phytocomponents tested. Therefore, as assessed using computational studies, the binding ability of plumbagin, emodin, and EGCG can be interpreted as inhibiting effects on MMP-2 activities. CONCLUSION: These compounds could find potential application in preventing the progression, sustenance, and metastasis of pancreatic cancer and need to be explored further using a pre-clinical model system in order to validate the efficacy, bioavailability, and safety.

Effect of adhesive strategy on resin cement bonding to dentin.

OBJECTIVE: The cement bonding strategy and the polymerization mode can influence the prognosis of indirect restorations. The microtensile bond strength (µTBS) and dentin endogenous enzymatic activity of a dual-cure resin cement (PV5) used in combination with two dentin surface conditioners (accelerator-enhancer primer, TP or universal adhesive, UA) were evaluated. MATERIALS AND METHODS: PV5 was used to lute composite overlays after dentin treatment with TP or UA. The resin cement was self-cured, SC (1 h at 37 °C) or dual-cured, DC (20 s light-cure followed by 15 min self-cure at 37°C). The µTBS test, fractographic analysis, and the in situ zymography evaluations were performed after 24 h (T0 ) or 1 yr (T12 ) of artificial storage. Data were statistically analyzed (α = 0.05). RESULTS: TP/DC obtained the highest adhesive strengths (45 ± 9 and 36.6 ± 8), while UA/SC (17 ± 8 and 11 ± 4) the lowest, both at T0 and T12 , respectively. DC resulted in superior bonding values than the SC, independent of the dentin surface treatment (p < 0.05). The type of adhesive, curing mode and aging influenced the gelatinolytic activity (p < 0.05). CONCLUSIONS: The dual-cure resin cement used in combination with its accelerator-enhancer primer showed superior bonding performances with respect to universal adhesive. Dual-curing the resin cement was determinant to enhance bonding capability over time, independent of the adhesive strategy. CLINICAL RELEVANCE: Clinicians must be aware to faithfully follow manufacturer's recommendation regarding the adhesive strategy suggested with the resin cement used.