Type IV Pili of Streptococcus sanguinis Contribute to Pathogenesis in Experimental Infective Endocarditis

This work provides evidence that type IV pili produced by Streptococcus sanguinis SK36 are critical to the ability of these bacteria to attach to and colonize the aortic heart valve (endocarditis). We found that an S. sanguinis type IV pili mutant strain was defective in causing platelet-dependent aggregation in a 24-h infection assay but not in a 1-h platelet aggregation assay, suggesting that the type IV pili act at later stages of vegetation development.

Barbeya oleoides Leaves Extracts: In Vitro Carbohydrate Digestive Enzymes Inhibition and Phytochemical Characterization

This study investigated the in vitro inhibitory potential of different solvent extracts of leaves of Barbeya oleoides on key enzymes related to type 2 diabetes mellitus (α-glucosidase and α-amylase) in combination with an aggregation assay (using 0.01% Triton X-100 detergent) to assess the specificity of action. The methanol extract was the most active in inhibiting α-glucosidase and α-amylase, with IC50 values of 6.67 ± 0.30 and 25.62 ± 4.12 µg/mL, respectively. However, these activities were significantly attenuated in the presence of 0.01% Triton X-100. The chemical analysis of the methanol extract was conducted utilizing a dereplication approach combing LC-ESI-MS/MS and database searching. The chemical analysis detected 27 major peaks in the negative ion mode, and 24 phenolic compounds, predominantly tannins and flavonol glycosides derivatives, were tentatively identified. Our data indicate that the enzyme inhibitory activity was probably due to aggregation-based inhibition, perhaps linked to polyphenols.

Neutrophil Granule Proteins Inhibit Amyloid Beta Aggregation and Neurotoxicity

Background: A role for neutrophils in the pathogenesis of Alzheimer’s disease (AD) is emerging. We previously showed that the neutrophil granule proteins cationic antimicrobial protein of 37 kDa (CAP37), cathepsin G (CG), and neutrophil elastase (NE) directly bind the amyloid-beta peptide Aβ1-42, a central player in AD pathogenesis. CAP37, CG, and NE are serine proteases that can cleave Aβ1-42 at different sites and with different catalytic activities. Objective: In this study, we compared the effects of these three proteins on Aβ1-42 fibrillation and neurotoxicity. Methods: Using mass spectrometry and in vitro aggregation assay, we found that NE and CG effi- ciently cleave Aβ1-42. This cleavage correlates well with the inhibition of Aβ1-42 aggregation into fi- brils. In contrast, CAP37 did not efficiently cleave Aβ1-42, but was still able to inhibit its fibrillation, most likely through a quenching effect. Inhibition of Aβ1-42 aggregation by NE and CG neutralized its toxicity measured in cultured neurons. In contrast, inhibition of Aβ1-42 aggregation by CAP37 did not inhibit its neurotoxicity. Results: We found that a peptide derived from CAP37 could mimic the quenching and inhibition of Aβ1-42 aggregation effects of the full-length protein. Additionally, this peptide was able to inhibit the neurotoxicity of the most toxic Aβ1-42 aggregate, an effect that was not found with the full-length CAP37. Conclusion: These results shed light on the mechanisms of action of neutrophil granule proteins with regard to inhibition of Aβ1-42 aggregation and neurotoxicity and open up a possible strategy for the discovery of new disease-modifying drugs for AD.

Addressing Compound Reactivity and Aggregation Assay Interferences: Case Studies of Biochemical High-Throughput Screening Campaigns Benefiting from the National Institutes of Health Assay Guidance Manual Guidelines

Compound-dependent assay interferences represent a continued burden in drug and chemical probe discovery. The open-source National Institutes of Health/National Center for Advancing Translational Sciences (NIH/NCATS) Assay Guidance Manual (AGM) established an “Assay Artifacts and Interferences” section to address different sources of artifacts and interferences in biological assays. In addition to the frequent introduction of new chapters in this important topic area, older chapters are periodically updated by experts from academia, industry, and government to include new technologies and practices. Section chapters describe many best practices for mitigating and identifying compound-dependent assay interferences. Using two previously reported biochemical high-throughput screening campaigns for small-molecule inhibitors of the epigenetic targets Rtt109 and NSD2, the authors review best practices and direct readers to high-yield resources in the AGM and elsewhere for the mitigation and identification of compound-dependent reactivity and aggregation assay interferences.

Macrostachyols A-D, oligostilbenes from Gnetum macrostachyum inhibited in vitro human platelet aggregation

Introduction: Gnetum macrostachyum is a known Thai medicinal plant as a source of bioactive oligostilbenes, which possess platelet inhibitory activities. The study aimed to evaluate the in vitro human platelet aggregation inhibitory activities of macrostachyols A-D (compounds 1-4) isolated from the roots of G. macrostachyum. Methods: The in vitro human platelet aggregation assay was assayed with a 96-well microtiter plate format. The well-known aggregating agents were used to investigate the possible mechanism of inhibition, including adenosine diphosphate (ADP), arachidonic acid (AA), thromboxane A2 analog (U-46619), collagen, thrombin, and thrombin receptor-activating peptide-6 (TRAP-6). Results: Compound 1 was more potent than ibuprofen (positive control) on the adenosine diphosphate- induced platelet aggregation assay (P < 0.05). Compound 3 was more potent than 1, 2, and 4 (P < 0.05), but all active oligostilbenes were less potent than the positive control (P < 0.05) on the arachidonic acid-induced platelet aggregation assay. The oligostilbenes 1, 2, 3, and 4 also displayed the inhibitory effects on the U-46619-induced platelet aggregation. The tetrameric stilbenes 1 was the only compound that exhibited inhibitory effects on thrombin-induced platelet aggregation without TRAP-6 mediated platelet aggregation. Conclusion: The findings revealed the inhibitory effects of oligostilbenes on human platelet aggregation through a target-specific experimental design. It suggests that oligostilbenes from this plant might be applied as antiplatelet aggregation agents in platelet hyperreactivity- related diseases.

Antibody-mediated procoagulant platelets in SARS-CoV-2- vaccination associated immune thrombotic thrombocytopenia

The COVID-19 pandemic has resulted in significant morbidity and mortality worldwide. To prevent severe infection, mass COVID-19 vaccination campaigns with several vaccine types are currently underway. We report pathological and immunological findings in 8 patients who developed vaccine-induced immune thrombotic thrombocytopenia (VITT) after administration of SARS-CoV-2 vaccine ChAdOx1 nCoV-19. We analyzed patient material using enzyme immune assays, flow cytometry and heparin-induced platelet aggregation assay and performed autopsies on two fatal cases. Eight patients (5 female, 3 male) with a median age of 41.5 years (range, 24 to 53) were referred to us with suspected thrombotic complications 6 to 20 days after ChAdOx1 nCoV-19 vaccination. All patients had thrombocytopenia at admission. Patients had a median platelet count of 46.5 x109/L (range, 8 to 92). Three had a fatal outcome and 5 were successfully treated. Autopsies showed arterial and venous thromboses in various organs and the occlusion of glomerular capillaries by hyaline thrombi. Sera from VITT patients contain high titer antibodies against platelet factor 4 (PF4) (OD 2.59±0.64). PF4 antibodies in VITT patients induced significant increase in procoagulant markers (P-selectin and phosphatidylserine externalization) compared to healthy volunteers and healthy vaccinated volunteers. The generation of procoagulant platelets was PF4 and heparin dependent. We demonstrate the contribution of antibody-mediated platelet activation in the pathogenesis of VITT.

Response of Escherichia coli minimal ter operon to UVC and auto-aggregation: pilot study

Aim The study of minimal ter operon as a determinant of tellurium resistance (TeR) is important for the purpose of confirming the relationship of these genes to the pathogenicity of microorganisms. The ter operon is widespread among bacterial species and pathogens, implicated also in phage inhibition, oxidative stress and colicin resistance. So far, there is no experimental evidence for the role of the Escherichia coli (E. coli) minimal ter operon in ultraviolet C (UVC) resistance, biofilm formation and auto-aggregation. To identify connection with UVC resistance of the minimal ter operon, matched pairs of Ter-positive and -negative E. coli cells were stressed and differences in survival and whole genome sequence analysis were performed. This study was aimed also to identify differences in phenotype of cells induced by environmental stress. Methods In the current study, a minimal ter operon(terBCDEΔF) originating from the uropathogenic strain E. coli KL53 was used. Clonogenic assay was the method of choice to determine cell reproductive death after treatment with UVC irradiation at certain time intervals. Bacterial suspensions were irradiated with 254 nm UVC-light (germicidal lamp in biological safety cabinet) in vitro. UVC irradiance output was 2.5 mW/cm2 (calculated at the UVC device aperture) and plate-lamp distance of 60 cm. DNA damage analysis was performed using shotgun sequencing on Illumina MiSeq platform. Biofilm formation was measured by a crystal violet retention assay. Auto-aggregation assay was performed according to the Ghane, Babaeekhou & Ketabi (2020). Results A large fraction of Ter-positive E. coli cells survived treatment with 120-s UVC light (300 mJ/cm2) compared to matched Ter-negative cells; ∼5-fold higher resistance of Ter-positive cells to UVC dose (p = 0.0007). Moreover, UVC surviving Ter-positive cells showed smaller mutation rate as Ter-negative cells. The study demonstrated that a 1200-s exposure to UVC (3,000 mJ/cm2) was sufficient for 100% inhibition of growth for all the Ter-positive and -negative E. coli cells. The Ter-positive strain exhibited of 26% higher auto-aggregation activities and was able to inhibit biofilm formation over than Ter- negative strain (**** P < 0.0001). Conclusion Our study shows that Ter-positive cells display lower sensitivity to UVC radiation, corresponding to a presence in minimal ter operon. In addition, our study suggests that also auto-aggregation ability is related to minimal ter operon. The role of the minimal ter operon (terBCDEΔF) in resistance behavior of E. coli under environmental stress is evident.

Probiotic and Metabolic Characterization of Vaginal Lactobacilli for a Potential Use in Functional Foods

The main aim of this work was to verify the metabolic and functional aptitude of 15 vaginal strains belonging to Lactobacillus crispatus, Lactobacillus gasseri, and Limosilactobacillus vaginalis (previously Lactobacillus vaginalis), already characterized for their technological and antimicrobial properties. In order to evaluate the metabolic profile of these vaginal strains, a phenotype microarray analysis was performed on them. Functional parameters such as hydrophobicity, auto-aggregation, deconjugation of bile salts, adhesion to an intestinal cell line (Caco-2), and a simulated digestion process were evaluated for these strains. A good number of these strains showed hydrophobicity values higher than 70%. Regarding the auto-aggregation assay, the most promising strains were L. crispatus BC9 and BC1, L. gasseri BC10 and BC14, and L. vaginalis BC17. Moreover, L. crispatus BC4, BC6, BC7, and BC8 were characterized by strong bile salts hydrolase activity (BHS). In addition, L. crispatus BC8 and L. vaginalis BC17 were characterized by a medium ability to adhere to Caco-2 cells. Data related to digestion process showed a strong ability of vaginal lactobacilli to withstand this stress. In conclusion, the data collected show the metabolic versatility and several exploitable functional properties of the investigated vaginal lactobacilli.

Inhibition of Shear-Induced Platelet Aggregation by Xueshuantong via Targeting Piezo1 Channel-Mediated Ca2+ Signaling Pathway

XueShuanTong (XST) comprising therapeutically active ginsenosides, a lyophilized extract of Panax notoginseng roots, is extensively used in traditional Chinese medicine to treat ischemic heart and cerebrovascular diseases. Our recent study shows that treatment with XST inhibits shear-induced thrombosis formation but the underlying mechanism remained unclear. This study aimed to investigate the hypothesis that XST inhibited shear-induced platelet aggregation via targeting the mechanosensitive Ca2+-permeable Piezo1 channel by performing platelet aggregation assay, Ca2+ imaging and Western blotting analysis. Exposure to shear at physiologically (1,000–2000 s−1) and pathologically related rates (4,000–6,000 s−1) induced platelet aggregation that was inhibited by treatment with GsMTx-4. Exposure to shear evoked robust Ca2+ responses in platelets that were inhibited by treatment with GsMTx-4 and conversely enhanced by treatment with Yoda1. Treatment with XST at a clinically relevant concentration (0.15 g L−1) potently inhibited shear-induced Ca2+ responses and platelet aggregation, without altering vWF-mediated platelet adhesion and rolling. Exposure to shear, while resulting in no effect on the calpain-2 expression in platelets, induced calpain-2-mediated cleavage of talin1 protein, which is known to be critical for platelet activation. Shear-induced activation of calpain-2 and cleavage of talin1 were attenuated by treatment with XST. Taken together, our results suggest that XST inhibits shear-induced platelet aggregation via targeting the Piezo1 channel to prevent Piezo1-mediated Ca2+ signaling and downstream calpain-2 and talin1 signal pathway, thus providing novel insights into the mechanism of the therapeutic action of XST on platelet aggregation and thrombosis formation.

Evaluation of Phyllanthus niruri L. from Malaysia for In-vitro Anti-Urolithiatic Properties by Different Solvent Extraction

The Phyllanthus niruri is traditionally used for curing of kidney disorders and urinary stones in Malaysia. Hence the current work was aimed to evaluate the effect of different solvents extract (n- hexane, ethyl acetate, methanol and water) of P. niruri for in vitro anti-urolithiatic properties in terms of inhibition activity on CaOx by using the rate of CaOx aggregation assay and dissolution of calcium oxalate (CaOx) crystal by using titrimetry method. Cystone was used as positive control. The effects of cystone on slope of nucleation and aggregation as well as growth of CaOx were evaluated spectrophotometrically. The highest yield percentage of P.niruri was occupied by methanol (5.74 %). The maximum inhibition against aggregation of CaOx crystals was also occupied by methanol (66.67 % ± 1.61) and was comprised with alkaloid, steroid, terpenoid and tannin. Dissolution effect on calcium oxalate crystals indicates that the aqueous extracts of P. niruri was found to be more effective in dissolution of CaOx with 63.33 % ± 1.44. P. niruri significantly (P < 0.05) inhibited the slope of nucleation and aggregation of CaOx crystallization, and reduced the crystal density. The results of the present study confirmed that P. niruri leaves can be used as remedial mediator for urolithiasis. However, further studies are required for isolation and identification of active constituents and their in-vivo confirmation.