in Vitro Effect of Methamphetamine on Proliferation, Differentiation and Apoptosis of Adipose Tissue Stem Cells

Purpose: Among abused substances, methamphetamine is a psychostimulant drug widely used recreationally with public health importance. This study investigated the effect of methamphetamine on proliferation, differentiation, and apoptosis of human adipose tissue stem cells (AdSCs). Methods: AdSCs were isolated from human abdominal adipose tissue and were characterized for mesenchymal properties and growth kinetics. MTT assay was undertaken to assess methamphetamine toxicity on proliferation and differentiation properties and apoptosis of hAdSCs. Results: Isolated cells were shown to have mesenchymal properties and a population doubling time (PDT) of 40.1 h. Following methamphetamine treatment, expressions of KI-67 and TPX2 as proliferation genes and Col1A1 and PPARg as differentiation genes decreased. Methamphetamine administration increased the expression of Bax and decreased Bcl-2 genes responsible for apoptosis. Conclusions: Our data suggested when AdSCs were exposed to methamphetamine, it decreased proliferation and differentiation properties of stem cells together with an increase in apoptosis. These findings can be added to the literature, especially when methamphetamine is used recreationally for weight loss purposes.

Remdesivir, Molnupiravir and Nirmatrelvir remain active against SARS-CoV-2 Omicron and other variants of concern.

The in vitro effect of GS-441524, remdesivir, EIDD-1931, molnupiravir and nirmatrelvir against the various SARS-CoV-2 VOCs, including Omicron, was determined. VeroE6-GFP cells were pre-treated overnight with serial dilutions of the compounds before infection. The number of fluorescent pixels of GFP signal, determined by high-content imaging on day 4 post-infection, was used as read-out, and the EC50 of each compound on a viral isolate of each VOC was calculated. These experiments were performed in the presence of the Pgp-inhibitor CP-100356 in order to limit compound efflux. A SARS-CoV-2 strain grown from the first Belgian patient sample was used as ancestral strain. All the other isolates were obtained from patients in Belgium as well. Our results indicate that GS-441524, remdesivir, EIDD-1931, molnupiravir and nirmatrelvir retain their activity against the VOCs Alpha, Beta, Gamma, Delta and Omicron. This is in accordance with the observation that the target proteins of these antivirals are highly conserved.

Evaluation of biological activities of highly diluted nucleotide sequences by using cellular models

Background: highly diluted specific nucleic acids (SNA®), designed to modulate viral and cytokine genes expression, are currently used in Micro-Immunotherapy to treat viral infections and immune disorders. Although some preliminary studies have showed clinical benefit of these homeopathic preparations [1], no experimental data are available to explain their mechanism of action. Aims: to investigate the in vitro effect of two sets of highly diluted (HD) SNA targeting i) latent/lytic Epstein-Barr virus (SNA EBV) and ii) TNF-α and its receptor p55 involved in rheumatoid arthritis (SNA RA) on cellular models. Methodology: serial homeopathic dilutions of SNA EBV and SNA RA (15cH-18cH) were tested on a EBV-positive B-lymphoblastoid (B95-8) and on a LPS-stimulated macrophage (THP1) cell lines respectively, in comparison with agitated/diluted water and scramble DNA sequences prepared in the same conditions (negative controls). For B95-8 proliferative model, high mobility group box 1 protein (HMGB1) was used as reference. Analyzed biological parameters on B95-8 were i) cell proliferation measured after 24 and 48h of incubation with HD SNA and ii) expression of the EBV ZEBRA protein in response to TGF-β by Western-blotting (T+24h). For THP1 model, TNF-α synthesis and release were determined by RT-qPCR and ELISA (protein), after stimulation by LPS (1µg/ml) and HD SNA co-administration. Results: we demonstrated that HD SNA RA significantly down-regulated TNF-α synthesis and release. This biological activity was showed to be specific (no effect of HD scramble SNA) and related to the level of dilution (maximal effect with higher dilutions). Unexpectedly, a biological effect of agitated/diluted water was also detected in both cellular models. For B95-8 model, this effect resulted in a significant decrease of B95-8 proliferation (comparable to the HMGB1 reference) and an inhibition of ZEBRA expression. Similarly, a reproducible stimulation effect of HD water was obtained in the LPS-stimulated THP1 model. Conclusions: these findings indicate that highly diluted SNA RA can regulate TNF-α synthesis and release by LPS-stimulated THP1 and support the hypothesis that these homeopathic preparations may act in modulating mRNA expression of the targeted genes. This in vitro work underlines the potential effect of agitated water in context of cellular models for testing biological properties of HD. [1] Jenaer M, Henry MF, Garcia A, Marichal B. Evaluation of 2LHERP in preventing recurrences of genital herpes. Br Homeopath J. 2000 Oct;89(4):174-7.

In vitro effect of salinity and pH on Fusarium sp., the causal agent of sweet-potato root rot

Fusarium root rot in a common pathogen of sweet potato, with a wide range of host plants. In the current study six new isolates of Fusarium sp., collected from infected sweet potato plants, along with a reference strain of Fusarium oxysporum, had their growth behavior studied in various pH and saline conditions. In vitro studies showed that salinity higher than 6% NaCl in the PDA substrate significantly reduces the fungal growth. At 12% NaCl, four of seven strains revealed complete mycelia inhibition. However, for the other two isolates, and for the reference strain, 12% salinity only reduced the growth with 77.4%. Regarding the fungal growth at different pH values, it was noticed that tested fusaria were not perturbed at up to 8.5 alkalinity. However, at a pH of 4.5, the growth rate was reduced, although the growth differences were diminished during prolonged incubation time. Considering the in vitro results, saline water should be tested as preventive immersion treatment on the sweet potato sprouts, before their planting, in order to reduce the incidence of Fusarium infection.

Efficacy of silver and gold nanoparticles obtained from vermiwash: In vitro study on antimicrobial and antidiabetic activities

Emerging nanobiotechnology has provided innovative techniques to synthesize nanoparticles through biological methods to explore the potentialities of biological sources like phytoextracts, microbes, animal secretions and excretion. This research studies the potential of vermiwash to synthesize the silver and gold nanoparticles and evaluate its in vitro effect of antimicrobial and antidiabetic activities. The characterization of the nanoparticles was analyzed through various techniques. Ultraviolet (UV)-Visible spectroscopy showed the maximum absorption spectrum at 413 nm for silver and 541 nm for gold nanoparticles. Fourier transform infrared spectroscopy (FTIR) revealed the reducing agent involved in nanoparticles synthesis. Scanning electron microscope (SEM) images revealed the size of the silver and gold nanoparticles as 24 nm and 50 nm, respectively. Energy dispersive X-ray (EDAX) analysis revealed the elemental composition of the synthesized nanoparticles. X-ray diffraction (XRD) analysis confirmed the crystalline nature of the nanoparticles that displayed the preferential orientation of the crystals toward the (111) plane. Antimicrobial activity was assessed using the resazurin assay method. A minimum inhibitory concentration (MIC) of less than 7.8 µg was observed in Staphylococcus aureus and Klebsiella pneumoniae. In the antifungal activity, MIC at 250 µg was noted in Mucor sp. and Candida albicans. Antidiabetic activity was assessed by α-amylase and α-glucosidase inhibitory assay. IC50 of α-amylase and α-glucosidase activity of the silver nanoparticles was noted as 218 and 221 µg/mL, respectively. IC 50 value for the enzymatic assay dose-dependently confirmed the effect. Conclusively biosynthesized nanoparticles from vermiwash showed potential efficiency of antibacterial, antifungal and antidiabetic activities.

Lactobacillus iners Cell-Free Supernatant Enhances Biofilm Formation and Hyphal/Pseudohyphal Growth by Candida albicans Vaginal Isolates

Candida albicans is a commensal fungus of the vaginal mucosa and the principal etiological agent of vaginal candidiasis. Vaginal dysbiosis has been reported during vulvovaginal candidiasis (VVC), with a progressive decrease in Lactobacillus crispatus population and an increase in L. iners population. To date, the role of L. iners in VVC pathogenesis remains scarcely explored. Herein we investigated the in vitro effect of L. iners cell-free supernatant (CFS) on the ability of C. albicans to form biofilms. Biomass and metabolic activity were measured by crystal violet and XTT assays. Further, light microscopy was performed to determine the effect of L. iners CFS on biofilm cellular morphology. We found that L. iners CFS induced a significant increase in biofilm formation by C. albicans clinical isolates which were categorized as moderate or weak biofilm producers. This effect was associated with an enhancement of hyphal/pseudohyphal growth, and the expression levels of HWP1 and ECE1, which are typical hyphae-associated genes, were upregulated. Overall, these results suggest that L. iners contributes to the pathogenesis of VVC and highlight the complexity of the interaction between C. albicans and vaginal lactobacilli. Understanding these interactions could prove essential for the development of new strategies for treating VVC.

​In-vitro Effect of Different Abiotic Stresses on the Growth and Sporulation of Colletotrichum gloeosporioides causing Anthracnose of Mango

Background: Mango productivity was very much affected due to a major fungal pathogen, Colletotrichum gloeosporioides causing anthracnose mango rot. The present study was carried out to investigate the influence of abiotic factors for the support of superficial growth of isolated fungus and finding a minimum inhibitory concentration of different fungicides. Methods: Among four different culture media tested, the highest radial growth and sporulation of the fungus were recorded in Oatmeal agar (OMA) (84 mm) followed by Conn’s agar (CA), Czapek Dox agar (CDA) and Potato dextrose agar (PDA). Among the different pH tested, pH 7.0 was found to be the best in supporting the good radial growth (69 mm) followed by pH 6.0 (56 mm), pH 5.5 (49 mm), pH 7.5 (43 mm) and pH 8.0 (37 mm). Among the various temperature tested, 25oC (69.32) was found to be the best followed by 20oC (52.53 mm), 30oC (65.23 mm) and 35oC. Result: Among the fungicides tested, Zineb 68% + Hexaconazole 4% WP (avtar) was found best as the radial growth was observed to be 45, 41, 36, 32, 25 mm at 5, 10, 25, 50 and 100 ppm, respectively as compared to 80 mm in control. The fungicide Tricyclazole 18% + Mancozeb 62% WP (Merger) was found to be the least effective in checking the radial growth of C. gloeosporioides even at 100 ppm concentration.

Novel Bioactive Adhesive Monomer CMET Promotes Odontogenic Differentiation and Dentin Regeneration

This study aimed to evaluate the in vitro effect of the novel bioactive adhesive monomer CMET, a calcium salt of 4-methacryloxyethyl trimellitate acid (4-MET), on human dental pulp stem cells (hDPSCs) and its capacity to induce tertiary dentin formation in a rat pulp injury model. Aqueous solutions of four tested materials [4-MET, CMET, Ca(OH)2, and mineral trioxide aggregate (MTA)] were added to the culture medium upon confluence, and solvent (dH2O) was used as a control. Cell proliferation was assessed using the Cell Counting Kit-8 assay, and cell differentiation was evaluated by real-time quantitative reverse transcription-polymerase chain reaction. The mineralization-inducing capacity was evaluated using alizarin red S staining and an alkaline phosphatase activity assay. For an in vivo experiment, a mechanical pulp exposure model was prepared on Wistar rats; damaged pulp was capped with Ca(OH)2 or CMET. Cavities were sealed with composite resin, and specimens were assessed after 14 and 28 days. The in vitro results showed that CMET exhibited the lowest cytotoxicity and highest odontogenic differentiation capacity among all tested materials. The favorable outcome on cell mineralization after treatment with CMET involved p38 and c-Jun N-terminal kinases signaling. The nuclear factor kappa B pathway was involved in the CMET-induced mRNA expression of odontogenic markers. Similar to Ca(OH)2, CMET produced a continuous hard tissue bridge at the pulp exposure site, but treatment with only CMET produced a regular dentinal tubule pattern. The findings suggest that (1) the evaluated novel bioactive adhesive monomer provides favorable biocompatibility and odontogenic induction capacity and that (2) CMET might be a very promising adjunctive for pulp-capping materials.