Endogenous Proteases Research Articles

Yersinia pestis escapes entrapment in thrombi by targeting platelet function

BackgroundPlatelets are classically recognized for their role in hemostasis and thrombosis. Recent work has demonstrated that platelets can also execute a variety of immune functions. The dual prothrombotic and immunological roles of platelets suggest that they may pose a barrier to the replication or dissemination of extracellular bacteria. However, some bloodborne pathogens, such as the plague bacterium Yersinia pestis, routinely achieve high vascular titers that are necessary for pathogen transmission. ObjectivesIt is not currently known how or if pathogens circumvent platelet barriers to bacterial dissemination and replication. We sought to determine whether extracellular bloodborne bacterial pathogens actively interfere with platelet function, using Y pestis as a model system. MethodsThe interactions and morphological changes of human platelets with various genetically modified Y pestis strains were examined using aggregation assays, immunofluorescence, and scanning electron microscopy. ResultsYersinia pestis directly destabilized platelet thrombi, preventing bacterial entrapment in fibrin/platelet clots. This activity was dependent on two well‐characterized bacterial virulence factors: the Y pestis plasminogen activator Pla, which stimulates host‐mediated fibrinolysis, and the bacterial type III secretion system (T3SS), which delivers bacterial proteins into the cytoplasm of targeted host cells to reduce or prevent effective immunological responses. Platelets intoxicated by the Y pestis T3SS were unable to respond to prothrombotic stimuli, and T3SS expression decreased the formation of neutrophil extracellular traps in platelet thrombi. ConclusionsThese findings are the first demonstration of a bacterial pathogen using its T3SS and an endogenous protease to manipulate platelet function and to escape entrapment in platelet thrombi.

Biotechnological Potential of Araucaria angustifolia Pine Nuts Extract and the Cysteine Protease Inhibitor AaCI-2S.

Protease inhibitors are involved in the regulation of endogenous cysteine proteases during seed development and play a defensive role because of their ability to inhibit exogenous proteases such as those present in the digestive tracts of insects. Araucaria angustifolia seeds, which can be used in human and animal feed, were investigated for their potential for the development of agricultural biotechnology and in the field of human health. In the pine nuts extract, which blocked the activities of cysteine proteases, it was detected potent insecticidal activity against termites (Nasutitermes corniger) belonging to the most abundant termite genus in tropical regions. The cysteine inhibitor (AaCI-2S) was purified by ion-exchange, size exclusion, and reversed-phase chromatography. Its functional and structural stability was confirmed by spectroscopic and circular dichroism studies, and by detection of inhibitory activity at different temperatures and pH values. Besides having activity on cysteine proteases from C. maculatus digestive tract, AaCI-2S inhibited papain, bromelain, ficin, and cathepsin L and impaired cell proliferation in gastric and prostate cancer cell lines. These properties qualify A. angustifolia seeds as a protein source with value properties of natural insecticide and to contain a protease inhibitor with the potential to be a bioactive molecule on different cancer cells.

Bowman-Birk Inhibitors: Insights into Family of Multifunctional Proteins and Peptides with Potential Therapeutical Applications.

Bowman-Birk inhibitors (BBIs) are found primarily in seeds of legumes and in cereal grains. These canonical inhibitors share a highly conserved nine-amino acids binding loop motif CTP1SXPPXC (where P1 is the inhibitory active site, while X stands for various amino acids). They are natural controllers of plants’ endogenous proteases, but they are also inhibitors of exogenous proteases present in microbials and insects. They are considered as plants’ protective agents, as their elevated levels are observed during injury, presence of pathogens, or abiotic stress, i.a. Similar properties are observed for peptides isolated from amphibians’ skin containing 11-amino acids disulfide-bridged loop CWTP1SXPPXPC. They are classified as Bowman-Birk like trypsin inhibitors (BBLTIs). These inhibitors are resistant to proteolysis and not toxic, and they are reported to be beneficial in the treatment of various pathological states. In this review, we summarize up-to-date research results regarding BBIs’ and BBLTIs’ inhibitory activity, immunomodulatory and anti-inflammatory activity, antimicrobial and insecticidal strength, as well as chemopreventive properties.

Tryptic Stability and Antimicrobial Activity of the Derivatives of Polybia-CP with Fine-Tuning Modification in the Side Chain of Lysine

Antimicrobial peptides (AMPs) were believed to be a class of promising antimicrobials to combat the increasing resistant microbes. However, AMPs could be easily degraded by endogenous proteases, which limited their clinic use. Considering that lysine is the only one cationic amino acid residue in polybia-CP which were related to the cleavage of trypsin like serine protease’s digestion, we introduced lysine’s mimics which kept the key function group side chain –NH2 and only varied the methylene chains in the sequence of polybia-CP to investigate the effect of the introduction of non-proteinogenic amino acids on the activity and stability of antimicrobial peptides. In addition, two analogs in which lysine were substituted by the other two proteinogenic cationic amino acids arginine and histidine also were synthesized to evaluate the effect of fine tuning the function group in lysine on its antimicrobial activity and stability. We found that the introduction of amino acids with shortened side chain length of lysine could enhance the trypsin resistance of the derivatives of polybia-CP, while maintain the same or comparable antimicrobial activity with the parent peptide. In addition, while lysine was substituted by histidine, His-CP demonstrated comparable antimicrobial activity and dramatically improved trypsin resistance. Although the analog exhibited excellent antimicrobial activity while lysine was substituted by Arginine residue (Arg-CP), its enzymatic stability was almost the same as its parent peptide. These results suggest that the fine-tuning of side chain of lysine may offer a promising strategy to improve the tryptic stability and retain the antimicrobial activity of antimicrobial peptide.

Antimicrobial antidegradative dental adhesive preserves restoration-tooth bond

ObjectiveAssess the ability of an antimicrobial drug-releasing resin adhesive, containing octenidine dihydrochloride (OCT)-silica co-assembled particles (DSPs), to enhance the biostability and preserve the interfacial fracture toughness (FT) of composite restorations bonded to dentin. Enzyme-catalyzed degradation compromises the dental restoration-tooth interface, increasing cariogenic bacterial infiltration. In addition to bacterial ingress inhibition, antimicrobial-releasing adhesives may exhibit direct interfacial biodegradation inhibition as an additional benefit. MethodsMini short-rod restoration bonding specimens with total-etch adhesive with/without 10% wt. DSPs were made. Interfacial fracture toughness (FT) was measured as-manufactured or post-incubation in simulated human salivary esterase (SHSE) for up to 6-months. Effect of OCT on SHSE and whole saliva/bacterial enzyme activity was assessed. Release of OCT outside the restoration interface was assessed. ResultsNo deleterious effect of DSPs on initial bonding capacity was observed. Aging specimens in SHSE reduced FT of control but not DSP-adhesive-bonded specimens. OCT inhibited SHSE degradation of adhesive monomer and may inhibit endogenous proteases. OCT inhibited bacterial esterase and collagenase. No endogenous collagen breakdown was detected in the present study. OCT increased human saliva degradative esterase activity below its minimum inhibitory concentration towards S. mutans (MIC), but inhibited degradation above MIC. OCT release outside restoration margins was below detection. SignificanceDSP-adhesive preserves the restoration bond through a secondary enzyme-inhibitory effect of released OCT, which is virtually confined to the restoration interface microgap. Enzyme activity modulation may produce a positive-to-negative feedback switch, by increasing OCT concentration via biodegradation-triggered release to an effective dose, then subsequently slowing degradation and degradation-triggered release.

Activation of matrix-bound endogenous proteases by self-etch adhesives.

The study evaluated changes in total enzymatic activity and degradation of demineralized dentin following the application of universal or self-etch adhesives. The universal adhesives -Scotchbond Universal (SU) and All-Bond Universal (ABU) and self-etch adhesives -Adper Easy Bond (EB) and G-aenial Bond (GB) were used for 2 min pretreatment of the dentin beams. Phosphoric acid (PA) treatment as well as no treatment served as controls. Total enzymatic activity was analyzed before and after treatment, collagen degradation was assessed using mass loss, C-terminal telopeptide (CTX) and C-terminal-telopeptide of type I collagen (ICTP) release (24 h, 3-day, 3-week). Over three weeks of incubation, ICTP release of ABU treated beams was significantly higher than other groups (p<0.05), except for SU treated beams (p>0.05) and CTX release of GB treated beams was the highest among the groups with statistically significant difference (p<0.05). The results confirm that the universal adhesives tested have also potential to increase the enzymatic activity in dentin.

Improvement of Pseudoalteromonas haloplanktis TAC125 as a Cell Factory: IPTG-Inducible Plasmid Construction and Strain Engineering

Our group has used the marine bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) as a platform for the successful recombinant production of “difficult” proteins, including eukaryotic proteins, at low temperatures. However, there is still room for improvement both in the refinement of PhTAC125 expression plasmids and in the bacterium’s intrinsic ability to accumulate and handle heterologous products. Here, we present an integrated approach of plasmid design and strain engineering finalized to increment the recombinant expression and optimize the inducer uptake in PhTAC125. To this aim, we developed the IPTG-inducible plasmid pP79 and an engineered PhTAC125 strain called KrPL LacY+. This mutant was designed to express the E. coli lactose permease and to produce only a truncated version of the endogenous Lon protease through an integration-deletion strategy. In the wild-type strain, pP79 assured a significantly better production of two reporters in comparison to the most recent expression vector employed in PhTAC125. Nevertheless, the use of KrPL LacY+ was crucial to achieving satisfying production levels using reasonable IPTG concentrations, even at 0 °C. Both the wild-type and the mutant recombinant strains are characterized by an average graded response upon IPTG induction and they will find different future applications depending on the desired levels of expression.

Distinct alpha-Synuclein species induced by seeding are selectively cleared by the Lysosome or the Proteasome in neuronally differentiated SH-SY5Y cells.

A major pathological feature of Parkinson's disease (PD) is the aberrant accumulation of misfolded assemblies of alpha-synuclein (α-Syn). Protein clearance appears as a regulator of the 'α-Syn burden' underlying PD pathogenesis. The picture emerging is that a combination of pathways with complementary roles, including the Proteasome System and the Autophagy-Lysosome Pathway, contributes to the intracellular degradation of α-Syn. This study addresses the mechanisms governing the degradation of α-Syn species seeded by exogenous fibrils in neuronally differentiated SH-SY5Y neuroblastoma cells with inducible expression of α-Syn. Using human α-Syn recombinant fibrils (pre-formed fibrils, PFFs), seeding and aggregation of endogenous Proteinase K (PK)-resistant α-Syn species occurs within a time frame of 6days, and is still prominent after 12days of PFF addition. Clearance of α-Syn assemblies in this inducible model was enhanced after switching off α-Syn expression with doxycycline. Lysosomal inhibition led to accumulation of SDS-soluble α-Syn aggregates 6days after PFF-addition or when switching off α-Syn expression. Additionally, the autophagic enhancer, rapamycin, induced the clearance of α-Syn aggregates 13days post-PFF addition, indicating that autophagy is the major pathway for aggregated α-Syn clearance. SDS-soluble phosphorylated α-Syn at S129 was only apparent at 7days of incubation with a higher amount of PFFs.Proteasomal inhibition resulted in further accumulation of SDS-soluble phosphorylated α-Syn at S129, with limited PK resistance. Our data suggest that in this inducible model autophagy is mainly responsible for the degradation of fibrillar α-Syn, whereas the proteasome system is responsible, at least in part, for the selective clearance of phosphorylated α-Syn oligomers.

Recombinant neutral protease rNpI as fish feed additive to improve protein digestion and growth

The addition of heavy protein to aquafeed causes low digestibility, high cost and significant nitrogen pollution in the water. The addition of protease is an effective way of improving protein absorption, but few proteases have been developed specifically for aquafeeds. The hydrolysis efficiency of protease additives for plant protein in the neutral gut environment and their synergy with endogenous proteases has not been sufficiently investigated. In this study, the performance of the recombinant neutral protease rNpI as an aquafeed additive to reduce the raw protein materials in the diet of Carassius carassius during 56 days of growth was evaluated. rNpI effectively hydrolysed soya bean protein isolates (SPI) with a high degree of hydrolysation and a high small-peptide yield. The rNpI-treated SPI exhibited a notable antioxidant activity, which benefited fish health. In vitro digestibility indicated that rNpI complemented trypsin in SPI digestion better than papain or Alcalase. The growth trial showed that the addition of rNpI could warrant a reduction in the crude protein and gross energy of feed while producing a lower feed-conversion rate (FCR) and better growth performance. Crucian that were fed an RM-wheat-E diet (5.5% rapeseed meal and 5% wheat reduction) showed the best performance, with the lowest FCR and highest weight gain. These results indicate that rNpI could be an effective additive for the aquafeed industry and could provide economic benefits by reducing aquafeed protein requirements and nitrogen emissions. This study provides novel information and strategies for additive protease use and development in aquafeed.

Protease-Activated Receptor-2 Regulates Neuro-Epidermal Communication in Atopic Dermatitis.

Background: Activation of protease-activated receptor-2 (PAR2) has been implicated in inflammation, pruritus, and skin barrier regulation, all characteristics of atopic dermatitis (AD), as well as Netherton syndrome which has similar characteristics. However, understanding the precise role of PAR2 on neuro-immune communication in AD has been hampered by the lack of appropriate animal models.Methods: We used a recently established mouse model with epidermal overexpression of PAR2 (PAR2OE) and littermate WT mice to study the impact of increased PAR2 expression in epidermal cells on spontaneous and house dust mite (HDM)-induced skin inflammation, itch, and barrier dysfunction in AD, in vivo and ex vivo.Results: PAR2OE newborns displayed no overt abnormalities, but spontaneously developed dry skin, severe pruritus, and eczema. Dermatological, neurophysiological, and immunological analyses revealed the hallmarks of AD-like skin disease. Skin barrier defects were observed before onset of skin lesions. Application of HDM onto PAR2OE mice triggered pruritus and the skin phenotype. PAR2OE mice displayed an increased density of nerve fibers, increased nerve growth factor and endothelin-1 expression levels, alloknesis, enhanced scratching (hyperknesis), and responses of dorsal root ganglion cells to non-histaminergic pruritogens.Conclusion: PAR2 in keratinocytes, activated by exogenous and endogenous proteases, is sufficient to drive barrier dysfunction, inflammation, and pruritus and sensitize skin to the effects of HDM in a mouse model that mimics human AD. PAR2 signaling in keratinocytes appears to be sufficient to drive several levels of neuro-epidermal communication, another feature of human AD.

1,4-Disubstituted 1,2,3-Triazoles as Amide Bond Surrogates for the Stabilisation of Linear Peptides with Biological Activity.

Peptides represent an important class of biologically active molecules with high potential for the development of diagnostic and therapeutic agents due to their structural diversity, favourable pharmacokinetic properties, and synthetic availability. However, the widespread use of peptides and conjugates thereof in clinical applications can be hampered by their low stability in vivo due to rapid degradation by endogenous proteases. A promising approach to circumvent this potential limitation includes the substitution of metabolically labile amide bonds in the peptide backbone by stable isosteric amide bond mimetics. In this review, we focus on the incorporation of 1,4-disubstituted 1,2,3-triazoles as amide bond surrogates in linear peptides with the aim to increase their stability without impacting their biological function(s). We highlight the properties of this heterocycle as a trans-amide bond surrogate and summarise approaches for the synthesis of triazole-containing peptidomimetics via the Cu(I)-catalysed azide-alkyne cycloaddition (CuAAC). The impacts of the incorporation of triazoles in the backbone of diverse peptides on their biological properties such as, e.g., blood serum stability and affinity as well as selectivity towards their respective molecular target(s) are discussed.

Sialorphin Potentiates Effects of [Met5]Enkephalin without Toxicity by Action other than Peptidase Inhibition.

This dose-response study investigated the effects of sialorphin on [Met5]enkephalin (ME)-induced inhibition of contractions in mouse vas deferens and antinociception in male rats. Differences were compared among combinations of three chemical peptidase inhibitors: amastatin, captopril, and phosphoramidon. The ratio of potencies of ME in mouse vas deferens pretreated with both sialorphin (100 µM) and a mixture of the three peptidase inhibitors (1 µM each) was higher than that with the mixture of peptidase inhibitors alone at any dose. Intrathecal administration of sialorphin (100-400 nmol) significantly and dose dependently increased ME (3 nmol)-induced antinociception with the mixture of three peptidase inhibitors (10 nmol each). The degree of antinociception with a combination of any two of the peptidase inhibitors (10 nmol each) in the absence of sialorphin was less than that in the presence of sialorphin (200 nmol). Pretreatment with both sialorphin (200 nmol) and the mixture of three peptidase inhibitors (10 nmol each) produced an approximately 100-fold augmentation in ME (10 nmol)-induced antinociception, but without signs of toxicity such as motor dysfunction in rats. Radioligand receptor binding assay revealed that sialorphin did not affect either binding affinity or maximal binding capacity of [d-Ala2,N-MePhe4,Gly-ol5]enkephalin. These results indicate that sialorphin potentiates the effects of ME without toxicity by a mechanism other than peptidase inhibition and with no effect on its affinity to µ-opioid receptors. SIGNIFICANCE STATEMENT: Sialorphin is regarded as an endogenous peptidase inhibitor that interacts with enkephalin-degrading enzymes. The results of these in vitro and in vivo studies confirm that sialorphin potentiates the effects of [Met5]enkephalin without toxicity by an action other than peptidase inhibition. This suggests that sialorphin offers the advantage of reducing or negating the side effects of opioid drugs and endogenous opioid peptides.

Profiling selectivity of chagasin mutants towards cysteine proteases cruzain or cathepsin L through molecular dynamics simulations

Chagasin, an endogenous cysteine protease inhibitor from Trypanosoma cruzi, can control the activity of the parasitic cruzain and its homologous human cathepsin L. While chagasin inhibits both enzymes with similar potency, mutations have different effects on binding to these enzymes. Mutants T31A and T31A/T32A bind well to cathepsin L, but their affinity for cruzain drops ∼40 to 140–fold. On the other hand, the mutant W93A binds well to cruzain, but it loses potency against cathepsin L. Here, we employed molecular dynamics simulations to understand the selectivity in inhibition of cruzain or cathepsin L by chagasin mutants W93A, T31A, and T31A/T32A. Our results allowed profiling the nonbonded interactions in the interfaces of each mutant with these cysteine proteases. Additionally, we observed differences in the binding conformation of the chagasin loops L2 and L6 of the W93A mutant, favoring interactions with cruzain and reducing interactions with cathepsin L. These differences are associated with a partial dissociation of the W93A-cathepsin L complex, providing a likely cause for the selectivity of the mutant W93A towards cruzain. Communicated by Ramaswamy H. Sarma.

Inhibiting fusion with cellular membrane system: therapeutic options to prevent severe acute respiratory syndrome coronavirus-2 infection.

Severe acute respiratory syndrome coronavirus (SARS-CoV), an enveloped virus with a positive-sense single-stranded RNA genome, facilitates the host cell entry through intricate interactions with proteins and lipids of the cell membrane. The detailed molecular mechanism involves binding to the host cell receptor and fusion at the plasma membrane or after being trafficked to late endosomes under favorable environmental conditions. A crucial event in the process is the proteolytic cleavage of the viral spike protein by the host’s endogenous proteases that releases the fusion peptide enabling fusion with the host cellular membrane system. The present review details the mechanism of viral fusion with the host and highlights the therapeutic options that prevent SARS-CoV-2 entry in humans.

Influence of endodontic irrigants on bond strength between glass-fibre posts and dentin: A systematic review of in vitro studies

Irrigant solutions used during and after endodontic treatment on the dentinal surface can influence the bond strength of glass-fibre posts. The aim of this systematic review was to provide an overview of available scientific evidence on the influence of endodontic irrigants on glass-fibre post bond strength. This review followed the Preferred Reporting Items of Systematic Reviews and Meta-Analyses (PRISMA) guidelines. To identify relevant papers, an electronic search was conducted in PubMed, Cochrane, Elsevier, Scopus and EBSCO databases. There were no language restrictions and the last search was conducted to include studies published up to and including July 1, 2019. We searched for in vitro or ex vivo studies that included human or bovine specimens and reported on dentin-fibre post bond strength with the use of different irrigants for root canals. Common variables were identified to consolidate the data, such as bond strength values, interface failure mode and microstructural changes in the dentin after treatments. From 79 potentially relevant papers identified from the database searches, 19 were included in our study for full-text analysis. Authors of this review independently screened search results, extracted data and assessed the risk of bias. Data heterogeneity was present, with differences in dentin type, bond strength tests and irrigation and luting protocols. Current findings suggest that dentin bonding is adversely affected by 5.25% sodium hypochlorite (NaOCl). Chelators adversely affect bonding to root dentin. Antioxidant agents could recover the bond strength between the glass-fibre post and the dentin. Conditioning with phosphoric acid (H3PO4) and NaOCl/ethylenediaminetetraacetic acid (EDTA) with ultrasonic agitation might be recommended in order to remove the smear layer and produce an adequate resin-tag formation. Ageing affects resin cement-dentin bond strength by degradation of collagen fibrils resulting from the activation of endogenous proteases. Therefore, ethanol (EtOH), chlorhexidine (CHX), or cross-linkers can be used to inhibit the activity of matrix metalloproteinases (MMPs) and maintain bond strength over time to achieve greater and longer-term stability of the bonding interface. There are difficulties in achieving an effective resin-root dentin bond. Therefore, this review is clinically significant because recognising the effects of root canal irrigating substances on the retention of glass-fibre posts is essential to improve the prognosis of endodontically treated tooth restorations.

Role of the activation peptide in the mechanism of protein C activation

Protein C is a natural anticoagulant activated by thrombin in a reaction accelerated by the cofactor thrombomodulin. The zymogen to protease conversion of protein C involves removal of a short activation peptide that, relative to the analogous sequence present in other vitamin K-dependent proteins, contains a disproportionately high number of acidic residues. Through a combination of bioinformatic, mutagenesis and kinetic approaches we demonstrate that the peculiar clustering of acidic residues increases the intrinsic disorder propensity of the activation peptide and adversely affects the rate of activation. Charge neutralization of the acidic residues in the activation peptide through Ala mutagenesis results in a mutant activated by thrombin significantly faster than wild type. Importantly, the mutant is also activated effectively by other coagulation factors, suggesting that the acidic cluster serves a protective role against unwanted proteolysis by endogenous proteases. We have also identified an important H-bond between residues T176 and Y226 that is critical to transduce the inhibitory effect of Ca2+ and the stimulatory effect of thrombomodulin on the rate of zymogen activation. These findings offer new insights on the role of the activation peptide in the function of protein C.

New antimicrobial and collagen crosslinking formulated dentin adhesive with improved bond durability

ObjectiveHere we describe a novel formulation, based on quaternary ammonium (QA) and riboflavin (RF), which combines antimicrobial activities and protease inhibitory properties with collagen crosslinking without interference to bonding capabilities, was investigated. MethodsExperimental adhesives modified with different fractions of dioctadecyldimethyl ammonium bromide quaternary ammonium and riboflavin (QARF) were formulated. Dentine specimens were bonded to resincomposites with control or the experimental adhesives to be evaluated for bond strength, interfacial morphology, micro-Raman analysis, nano-CT and nano-leakage expression. In addition, the antibacterial and biocompatibilities of the experimental adhesives were investigated. The endogenous proteases activities and their molecular binding-sites were studied. ResultsModifying the experimental adhesives with QARF did not adversely affect micro-tensile bond strength or the degree of conversion along with the demonstration of anti-proteases and antibacterial abilities with acceptable biocompatibilities. In general, all experimental adhesives demonstrated favourable bond strength with increased and improved values in 1% QARF adhesive at 24 h (39.2 ± 3.0 MPa) and following thermocycling (34.8 ± 4.3 MPa). SignificanceIt is possible to conclude that the use of QARF with defined concentration can maintain bond strength values when an appropriate protocol is used and have contributed in ensuring a significant decrease in microbial growth of biofilms. Incorporation of 1% QARF in the experimental adhesive lead to simultaneous antimicrobial and anti-proteolytic effects with low cytotoxic effects, acceptable bond strength and interfacial morphology.

While We Wait for a Vaccine Against SARS-CoV-2, Why Not Think About Available Drugs?

At the time of reception of this article (April 2, 2020), efforts to develop a specific vaccine against SARS-Cov-2, the causative agent of the coronavirus disease 2019 (COVID-19), had just begun trial phase 1, but full validation of this and other current developments is likely to take many more months to reach completion. The ongoing pandemic constitutes a major health burden of world proportions that is also having a devastating impact on whole economies worldwide, the knock-on effects of which could be catastrophic especially in poorer countries. Alternative measures to ameliorate the impact and hamper or minimally slow down disease progression are urgently called for. This review discusses past and currently evolving data on the etiological agent of the current pandemic, SARS-CoV-2, and its host cell receptors with a view to disclosing alternative drugs for palliative or therapeutic approaches. Firstly, SARS-CoV-2 exhibits marked tropism for cells that harbor the membrane-bound metalloprotease angiotensin-converting enzyme 2 (ACE2) at their plasmalemma, predominantly in cells lining the oral cavity, upper respiratory tract, and bronchoalveolar cells, making these epithelial mucosae the most likely viral receptor cell targets and entry routes. Secondly, the crystal structures of several coronavirus spike proteins in complex with their cell host target receptors, and of SARS-Cov-2 in complex with an inhibitor, are now available at atomic resolution through X-ray diffraction and cryo-electron microscopy studies. Thirdly, viral entry of other viruses has been successfully blocked by inhibiting viral endogenous proteases or clathrin/dynamin-dependent endocytosis, the same internalization pathway followed by ACE2 and some viruses. Fourthly, the target cell-surface receptor molecules and SARS-CoV-2 possess other putative sites for drugs potentially modulating receptor activity or virus processing. A multi-pronged pharmacological approach attacking more than one flank of the viral-receptor interactions is worth considering as a front-line strategy.

Alpha-2-Macroglobulin Not Significantly Better Than Regular PRP For Knee Arthritis Symptoms

Objectives:Osteoarthritis, a degenerative and debilitating joint disease, is one of the most prevalent diseases in the United States. Despite the significant burden osteoarthritis can place on an individual and society, nonoperative treatment options are still very limited. Little clinical data exists on the use of injectable macromolecules for the treatment of osteoarthritis of the knee. Alpha-2-macroglobulin (A2M), a naturally occurring macromolecule, inhibits the many endogenous and exogenous proteinases presenting in the pathogenesis of osteoarthritis. To date, no studies exist evaluating the effectiveness of A2M with other intra-articular injectables. The purpose of this study is to compare the clinical effectiveness of intra-articular injection of A2M against both platelet-rich plasma (PRP) and corticosteroids.Methods:This prospective randomized control trial was conducted at a single medical center. Seventy-five patients with symptomatic knee osteoarthritis with Kellgren-Lawrence grade 2 or 3 were randomized into one of three cohorts receiving intra-articular injection. One group was given PRP, one group was given A2M, and one group was given corticosteroids. All groups had blood drawn to simulate A2M preparation. Patient reported outcomes were collected prior to injection, 6 weeks and 12 weeks following injection. Several measures were used for clinical assessment including the visual analog scale (VAS), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Knee Injury and Osteoarthritis Outcome Score (KOOS), Lysholm, and Tegner scores.Results:Seventy-five patients with a mean age of 61.08 (± 8.48) were enrolled between June 2018 and February 2019. At six weeks, the A2M group had the greatest decrease in VAS (-1.60) and WOMAC (-10.9), however, the differences were not statistically significant. Between the injection and the twelve week visit, the A2M group had a statistically significant difference in WOMAC score compared with PRP (-18.43 vs -5.70, p <0.02). Overall, both corticosteroid (-1.57) and A2M (-1.70) had a similar improvement in VAS when compared with PRP (-0.61). These differences were also not statistically significant.Conclusions:Our study demonstrates Alpha 2 Macroglobulin shows similar effectiveness to corticosteroids in the treatment of knee osteoarthritis. Both A2M and corticosteroids appear to show marginally better effectiveness than PRP injection, however the differences are small and did not reach statistical significance in most outcome measures.

Neisseria gonorrhoeae NGO2105 Is an Autotransporter Protein Involved in Adhesion to Human Cervical Epithelial Cells and in vivo Colonization

Autotransporters are important virulence factors in the outer membrane of gram-negative bacteria. Although several autotransporters have been identified in Neisseria meningitidis, only IgA1 protease has been identified in Neisseria gonorrhoeae. A sequence analysis showed a marked difference in the distribution of autotransporters between the two strains. It has been speculated that only two autotransporters, the IgA1 protease and the NGO2105 protein, might be encoded by N. gonorrhoeae. Here, we describe the identification of NGO2105, a new autotransporter in N. gonorrhoeae. A sequence alignment showed that NGO2105 is highly similar to the adhesion and penetration protein (App) in N. meningitidis. We found that NGO2105 is exported to the outer membrane, cleaved and released into the culture supernatant by endogenous serine protease activity in N. gonorrhoeae and E. coli. The site-directed mutagenesis of S267A in the predicted enzyme catalytic triad abolished autoproteolytic cleavage to allow secretion. The NGO2105 β-barrel shows the ability to translocate the heterologous Hbp passenger domain. NGO2105 is involved in gonococcal adherence to and invasion into human cervical epithelial cells. Furthermore, antibodies raised against NGO2105 are able to block gonococcal adherence to human cervical epithelial cells. The Δngo2105 mutant and anti-NGO2105 antiserum significantly attenuated the colonization of N. gonorrhoeae in mice. Collectively, our results suggest that the newly identified serine protease autotransporter NGO2105 represents a novel virulence factor of gonococcus and a potential vaccine target.