SPRY4 acts as an indicator of osteoarthritis severity and regulates chondrocyte hypertrophy and ECM protease expression

Osteoarthritis (OA) causes serious changes in the metabolic and signaling pathways of chondrocytes, including the mitogen-activated protein kinase (MAPK) pathway. However, the role of sprouty RTK signaling antagonist 4 (SPRY4), an inhibitor of MAPK, in the human cartilage tissues and chondrocytes remains to be understood. Here, using SPRY4 gene delivery into healthy and degenerated chondrocytes, we elucidated the role of SPRY4 in preventing chondrocyte hypertrophy. In addition to using the human cartilage tissues with the destabilization of the medial meniscus (DMM) model in Sprague-Dawley (SD) rats, the role of SPRY4 in cartilage tissues and chondrocytes was explored through their molecular and histological analyses. In order to determine the effects of SPRY4 on healthy human chondrocyte hypertrophy, small interfering RNA (siRNA) was used to knock down SPRY4. Lentiviral transduction of SPRY4 into degenerated human chondrocytes allowed us to investigate its ability to prevent hypertrophy. SPRY4 expression levels were higher in healthy human cartilage tissue and chondrocytes than in degenerated human cartilage tissues and hypertrophy-induced chondrocytes. The knockdown of SPRY4 in healthy chondrocytes caused an increase in hypertrophy, senescence, reactive oxygen species (ROS) production, and extracellular matrix (ECM) protease expression. However, all these factors decreased upon overexpression of SPRY4 in degenerated chondrocytes via regulation of the MAPK signaling pathway. We conclude that SPRY4 is a crucial indicator of osteoarthritis (OA) severity and could play an important role in preventing OA in the cartilage by inhibiting chondrocyte hypertrophy.

Natural Products as A Promising Therapy for SARS COV-2; An Overview

Recently emerging pandemic SARS CoV-2 conquered our world since December 2019. Continuous efforts have been done to find out effective immunization and precise treatment stetratigies A way from therapeutic options that were tried in SARS CoV-2, an increased attention is directed to predict natural products and mainly phytochemicals as collaborative measures for this crisis. In this review, most of the mentioned compounds specially flavonoids (biacalin, hesperidin, quercetin, luteolin,, and phenolic (resveratrol, curcumin, and theaflavin) exert their effect through interfering with the action of one or more of this proteins (spike protein, papain like protease, 3 chymotrypsin like cysteine protease, and RNA dependent RNA polymerase) that are involved in viral life cycle beside the anti-inflammatory effect of these compounds. The triterpenoids (celastrol, escin and glycyrrhizin) and the alkaloids (lycorine and cepharanthine) mediated their effect mainly through anti-inflammatory activity. Glycyrrhetinic acid (glycyrrhizin metabolite) dawn regulates ACE 2, and reduces protease expression, thus reduce viral entry. This review may be representing an initial step in long path for designing the successful and effective treatment or vaccine for this pandemic.

Protease-activatable biosensors of SARS-CoV-2 infection for cell-based drug, neutralisation and virological assays

The world is in the grip of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, and there is an urgent unmet clinical need for effective antiviral therapies. Many inhibitors of viral enzymes identified in vitro have limited efficacy against viral replication in cells, but conventional plaque assays are impractical for high-throughput screens. In this study, we therefore engineer cell-based biosensors of SARS-CoV-2 infection. Our assays exploit the cleavage of specific oligopeptide linkers by SARS-CoV-2 Main or Papain-like proteases, leading to the activation of green fluorescent protein (GFP) or firefly luciferase-based reporters. First, we characterise these biosensors in cells using recombinant viral proteases. Next, we confirm their ability to detect endogenous viral protease expression during infection with wildtype SARS-CoV-2. Finally, we develop a sensitive luminescent reporter cell line, confirm that it accurately quantitates infectious SARS-CoV-2 virus, and demonstrate its utility for drug screening and titration of neutralising antibodies.

Membrane protein of SARS-CoV-2 plays a pivotal role in the availability of active testosterone through its interaction with AKR1C2 enzyme leading to the upregulation of TMPRSS2 protease expression

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease (COVID-19) and ongoing pandemic that has devastated humankind. During the COVID-19 pandemic, it was noticed that the mortality rate in men is higher than that in women. The membrane (M) protein of SARS-CoV-2 plays a pivotal role in the viral life cycle regulating intracellular trafficking and processing of spike (S) protein. In infected individuals, M protein inhibits the conversion of active testosterone to its inactive form through its interaction with Aldo-keto reductase family 1 member C2 (AKR1C2) protein. This leads to the high availability of active testosterone and boosts the formation of its complex with an androgen receptor that in turn promotes the transcription of the transmembrane protease serine 2 (TMPRSS2) gene. TMPRSS2 is known to play a pivotal role in the priming of S protein that is necessary for the SARS-CoV-2 entry into the host cell. Therefore, the interaction of the M protein of SARSCoV-2 with AKR1C2 eventually leads to the upregulation of the transcription of the TMPRSS2 gene that results in an enhanced viral infection and in turn higher mortality in men. The interaction of M protein with AKR1C2 could be a possible target for SARSCoV-2 antiviral drug design.

Targeting TMPRSS2 and Cathepsin B/L together may be synergistic against SARS-CoV-2 infection

The entry of SARS-CoV-2 into target cells requires the activation of its surface spike protein, S, by host proteases. The host serine protease TMPRSS2 and cysteine proteases Cathepsin B/L can activate S, making two independent entry pathways accessible to SARS-CoV-2. Blocking the proteases prevents SARS-CoV-2 entry in vitro. This blockade may be achieved in vivo through ‘repurposing’ drugs, a potential treatment option for COVID-19 that is now in clinical trials. Here, we found, surprisingly, that drugs targeting the two pathways, although independent, could display strong synergy in blocking virus entry. We predicted this synergy first using a mathematical model of SARS-CoV-2 entry and dynamics in vitro. The model considered the two pathways explicitly, let the entry efficiency through a pathway depend on the corresponding protease expression level, which varied across cells, and let inhibitors compromise the efficiency in a dose-dependent manner. The synergy predicted was novel and arose from effects of the drugs at both the single cell and the cell population levels. Validating our predictions, available in vitro data on SARS-CoV-2 and SARS-CoV entry displayed this synergy. Further, analysing the data using our model, we estimated the relative usage of the two pathways and found it to vary widely across cell lines, suggesting that targeting both pathways in vivo may be important and synergistic given the broad tissue tropism of SARS-CoV-2. Our findings provide insights into SARS-CoV-2 entry into target cells and may help improve the deployability of drug combinations targeting host proteases required for the entry.

Effects of resveratrol on the growth and enzyme production of Stenotrophomonas maltophilia: a burn wound pathogen

Objective: The purpose of this study was to identify the potential of resveratrol in inhibiting the growth and production of two enzymes, hyaluronidase and protease, in Stenotrophomonas maltophilia, which has become a burn wound pathogen of great significance. Method: Stenotrophomonas maltophilia (ATCC 17666) was cultured in nutrient broth and the microbial load was standardised to 0.5 McFarland standard at 600nm. The study included antimicrobial assays (well diffusion and resazurin dye binding method), hyaluronidase expression regulation assay (hyaluronic acid hydrolysis assay and turbidity assay) and protease expression regulation assay (casein hydrolysis assay and determination of specific activity of protease using tyrosine standard). Results: The minimum inhibitory concentration (MIC) of resveratrol against Stenotrophomonas maltophilia was found to be 125µg/ml. Hyaluronidase production in the organism treated with resveratrol was found to be half that in the untreated organism. The specific activity of protease produced by the organism treated with resveratrol was found to be one-quarter that in the untreated organism, as analysed by the tyrosine standard estimation protocol. Conclusion: Resveratrol was found to be a potent compound to treat Stenotrophomonas maltophilia infections. In addition to the antimicrobial and enzyme-regulatory properties of resveratrol, it also shows anti-oxidant and anti-inflammatory properties. This finding has great scope clinically as resveratrol may prove to be an ideal drug to treat burn wound infections.

Novel histochemical approach for evaluation of tryptase expression in the mast cell population

The paper presents a novel histochemical approach for evaluation of tryptase expression in the mast cell population. To study the selective effect of mast cells (MC) on the parameters of a specific tissue microenvironment, it is necessary to detail the molecular composition of their secretome and analyze the pathways of degranulation. The developed method for combined immunomorphological and histochemical tryptase staining protocols contributes to a more objective determination of the integral level of specific protease expression in the skin MC population. Extra visualization potentials of cytological features and specific aspects of tryptase processing expand efficiency of morphological analysis in both normal and pathological conditions.

Novel nanobiosensor based protease biomarkers for early detection and prognosis of pancreatic cancers.

e16787 Background: Identifying novel biomarkers for early detection and prognosis would allow for improved survival outcomes in pancreatic cancer (PC). Arginase has been implicated in tumorigenesis through its production of L-ornithine. Its aberrant expression provides metabolites for tumor proliferation. Methods: In our study, 47 patients with metastatic PC, 29 with localized PC, and 50 healthy controls were enrolled at KU Cancer Center from 2000 to 2019. Overall survival (OS) was retrospectively assessed and correlated to serum protease levels at diagnosis. Protease expression was analyzed using nanobiosensors, which utilized fluorescent nanoparticles read by Spectra Scan to measure arginase, matrix metalloproteinases (MMPs), urokinase plasminogen activator (uPA), neutrophil elastase (NE), cathepsin B (CTSB), and cathepsin E (CTSE). Survival analysis was completed by Kaplan-Meier method. Results: Protease expression in localized vs. healthy cases was significant for (CTSB [p = 1.33E-10], MMP1 [p = 6.66E-21], MMP3 [p = 1.39E-13], MMP9 [p = 2.77E-08], NE [p = 0.00015], uPA [p = 2.55E-11]) and borderline significant for arginase [p = 0.05082]. Protease expression in metastatic vs. healthy cases was significant for (CTSB [p = 7.06E-06], MMP1 [p = 3.08E-12], MMP3 [p = 2.51E-14], MMP9 [p = 1.73E-08], NE [p = 0.01430], uPA [p = 0.00024]). Further, metastatic vs. localized cancer was significant for (arginase [p = 0.00034], CTSB [p = 0.00584], MMP1 [p = 2.03E-13], NE [p = 2.29E-10], and uPA [p = 6.58E-07]. Characteristics of 21 localized PC patients are illustrated in Table. Median OS was 25m in localized PC with low Arginase expression (mean < 215) vs 17.5m in high Arginase expression (p = 0.0477). Conclusions: Expression pattern of CTSB, MMP, NE, arginase, uPA proteases showed statistical significant difference for detection of localized PC vs metastatic PC vs healthy cases. In localized PC, lower arginase expression showed a statistically significant improvement in survival vs high arginase expression. Arginase expression was borderline significant for detection of localized PC vs healthy controls, likely due to small sample. Arginase as a potential biomarker for early detection and prognosis of PC warrants validation in a larger cohort. [Table: see text]

Targeting TMPRSS2 and Cathepsin B/L Together May Be Synergistic Against SARS-CoV-2 Infection

<p>The entry of SARS-CoV-2 into target cells requires the activation of its surface spike protein, S, by host proteases. The host serine protease TMPRSS2 and cysteine proteases Cathepsin B/L can activate S, making two independent entry pathways accessible to SARS-CoV-2. Blocking the proteases prevents SARS-CoV-2 entry <i>in vitro</i>. This blockade may be achieved <i>in vivo</i> through ‘repurposing’ drugs, a potential treatment option for COVID-19 that is now in clinical trials. Here, we found, surprisingly, that drugs targeting the two pathways, although independent, could display strong synergy in blocking virus entry. We predicted this synergy first using a mathematical model of SARS-CoV-2 entry and dynamics <i>in vitro</i>. The model considered the two pathways explicitly, let the entry efficiency through a pathway depend on the corresponding protease expression level, which varied across cells, and let inhibitors compromise the efficiency in a dose-dependent manner. The synergy predicted was novel and arose from effects of the drugs at both the single cell and the cell population levels. Validating our predictions, available <i>in vitro</i> data on SARS-CoV-2 and SARS-CoV entry displayed this synergy. Further, analysing the data using our model, we estimated the relative usage of the two pathways and found it to vary widely across cell lines, suggesting that targeting both pathways <i>in vivo</i> may be important and synergistic given the broad tissue tropism of SARS-CoV-2. Our findings provide insights into SARS-CoV-2 entry into target cells and may help improve the deployability of drug combinations targeting host proteases required for the entry. <br></p>

Targeting TMPRSS2 and Cathepsin B/L Together May Be Synergistic Against SARS-CoV-2 Infection

<p>The entry of SARS-CoV-2 into target cells requires the activation of its surface spike protein, S, by host proteases. The host serine protease TMPRSS2 and cysteine proteases Cathepsin B/L can activate S, making two independent entry pathways accessible to SARS-CoV-2. Blocking the proteases prevents SARS-CoV-2 entry <i>in vitro</i>. This blockade may be achieved <i>in vivo</i> through ‘repurposing’ drugs, a potential treatment option for COVID-19 that is now in clinical trials. Here, we found, surprisingly, that drugs targeting the two pathways, although independent, could display strong synergy in blocking virus entry. We predicted this synergy first using a mathematical model of SARS-CoV-2 entry and dynamics <i>in vitro</i>. The model considered the two pathways explicitly, let the entry efficiency through a pathway depend on the corresponding protease expression level, which varied across cells, and let inhibitors compromise the efficiency in a dose-dependent manner. The synergy predicted was novel and arose from effects of the drugs at both the single cell and the cell population levels. Validating our predictions, available <i>in vitro</i> data on SARS-CoV-2 and SARS-CoV entry displayed this synergy. Further, analysing the data using our model, we estimated the relative usage of the two pathways and found it to vary widely across cell lines, suggesting that targeting both pathways <i>in vivo</i> may be important and synergistic given the broad tissue tropism of SARS-CoV-2. Our findings provide insights into SARS-CoV-2 entry into target cells and may help improve the deployability of drug combinations targeting host proteases required for the entry. <br></p>