RADIOIMMUNOASSAY FOR DETECTION OF ANTI-RINDERPEST ANTIBODIES IN CATTLES

Liquid Phase Radioimmunoassay (RIA) was developed to detect and measure anti-rinderpest immunoglobulins in field animals sera, within two hours. Rinderpest virus adapted on Vero cell line culture and antigen purified by treatment with Triton-Gentron 13 Butanol, and, labeled with I isotope, using chloramin T iodination method. Comparative studies for detecting anti-rinderpest immunoglobulin in 80 calves sera samples, using the developed assay in parallel with virus neutralization test (VNT). The study showed 58.75 % agreement between the two methods. However, 71 % of seven months old, non-vaccinated calves showed anti rinderpest antibodies in their sera, also 81 % of 10 months old vaccinated calves were developed antibodies in their blood. These results demonstrate the development of sensitive, specific and rapid quantitative / qualitative radioimmunoassay, necessary for screening the development of immunity against rinderpest in cattle.

In Vitro Phytochemical Screening, Cytotoxicity Studies of Curcuma longa Extracts with Isolation and Characterisation of Their Isolated Compounds

The Curcuma longa plant is endowed with multiple traditional and therapeutic utilities and is here explored for its phytochemical constituents and cytotoxic potential. Turmeric rhizomes were extracted from three different solvents and screened for the presence of different phytochemical constituents, observation of which indicated that the polar solvents favoured extraction of greater versatile phytochemical constituents. These extracts were investigated for their cytotoxic potential by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay on three different of cell lines including SCC-29B (oral cancer cell line), DU-145 (prostate cancer cell line) and the Vero cell line (healthy cell line/non-cancerous cell line). This assay was performed by taking three extracts from isolated curcuminoids and a pure bioactive compound bisdemethoxycurcumin (BD). Bisdemethoxycurcumin was isolated from curcuminoids and purified by column and thin-layer chromatography, and its structural characterisation was performed with different spectroscopic techniques such as FTIR, NMR (1H Proton and 13C Carbon-NMR) and LC-MS. Amongst the extracts, the ethanolic extracts exhibited stronger cytotoxic potential against the oral cancer cell line (SCC-29B) with an IC50value of 11.27 μg/mL,and that this was too low of a cytotoxicity against the Vero cell line. Although, curcuminoids have also shown a comparable cytotoxic potential against SCC-29B (IC50 value 16.79 μg/mL), it was not as potent against the ethanolic extract, and it was even found to be cytotoxic against healthy cell lines at a very low dose. While considering the isolated compound, bisdemethoxycurcumin, it also possessed a cytotoxic potential against the prostate cancer cell line (DU-145) (IC50 value of 93.28 μg/mL), but was quite safe for the healthy cell line in comparison to doxorubicin.

Design, Synthesis and Biological Investigation of Some Novel Quinazolin-4(3H)-One Tethered 1, 3, 4-Thiadiazole-Thiol Motifs as Direct Enoyl Acyl Carrier Protein Reductase Inhibitors

Aims: In this study was two noteworthy pharmacophores quinazolin-4(3H)-one and 1,3,4-thiadiazole through methylene bridge were utilized to design, synthesize and characterize some novel 2-methyl quinazolin-4(3H)-one and 6-chloro-2-methyl quinazolin-4(3H)-one tethered S-substituted-1,3,4-thiadiazole-thiol structural analogs respectively as direct Mycobacterium Tuberculosis (MTB) enoyl acyl carrier protein reductase (InhA) inhibitors. Study Design: Design of structural analogs of quinazolin-4(3H)-one tethered 1,3,4-thiadiazole-thiol through methylene bridge by functional group modifications in core scaffold followed by computational studies to select promising compounds. Synthesis of some novel compounds, structural characterization and screening of biological activity of the same. Methodology: The molecular docking of designed compunds was carried out using schrodinger Glide XP into the active site of MTB InhA with protein data bank code (PDB ID: 2H7M). The interactions were evaluated based on the glide G score compared with reference standard isoniazid. Ten new compounds 7(A1-A10) were synthesized, characterized and screened for their in-vitro antitubercular activity by Microplate Almar Blue Assay (MABA) method followed by cytotoxicity evaluation of compounds 7A4 and 7A10 using Vero cell line. Results: All the designed compounds of series 7(A1-A10) had drug-like characteristics and were non-toxic to normal cells. In the molecular docking studies, compounds 7A4, 7A5, and 7A10 demonstrated strong binding affinity in the active region of MTB InhA protein and retained necessary amino acid interaction, similar to co-crystal 2H7M. Synthesized compounds 7(A1-A10) were found to have good antitubercular activity. Out of the series the compounds 7A4 and 7A10 were found to possess excellent antitubercular activity equipotent to reference standard streptomycin with minimum inhibitory concentration (MIC) value of 6.25µg/ml. The cytotoxic potential of compounds 7A4 and 7A10 showed remarkable selectivity index against Vero cell line. Conclusion: The findings of this study highlights the importance of tethering two pharmacophoric motifs in one compound to develop novel antitubercular agents that can be exploited as promising leads as direct InhA inhibitors.

Comparative Transcriptomics Analyses of a Vero Cell Line in Suspension Versus Adherent Culture Conditions

The Vero cell line is the most used continuous cell line for viral vaccine manufacturing. Its anchorage-dependent use renders scaling-up challenging and operations very labor intensive which affects cost effectiveness. Thus, efforts to adapt Vero cells to suspension cultures have been invested but hurdles such as the long doubling time and low cell viability remain to be addressed. In this study, building on the recently published Vero cell line annotated genome, a functional genomics analysis of the Vero cells adapted to suspension is performed to better understand the genetic and phenotypic switches at play during the adaptation of Vero cells from anchorage-dependent to suspension cultures. Results show a downregulation of the epithelial to mesenchymal transition (EMT) pathway, highlighting the dissociation between the adaptation to suspension process and EMT. Surprisingly, an upregulation of cell adhesion components is observed, notably the CDH18 gene, the cytoskeleton pathway, and the extracellular pathway. Moreover, a downregulation of the glycolytic pathway are balanced by an upregulation of the asparagine metabolism pathway, promoting cell adaptation to nutrient deprivation. A downregulation of the adherens junctions and the folate pathways alongside with the FYN gene are possible explanations behind the currently observed low cell viability and long doubling time.

Modular Synthesis and Antiproliferative Activity of New Dihydro-1H-pyrazolo[1,3-b]pyridine Embelin Derivatives

A set of new dihydro-1H-pyrazolo[1,3-b]pyridine and pyrazolo[1,3-b]pyridine embelin derivatives was synthesized through a multicomponent reaction from natural embelin, 3-substituted-5-aminopyrazoles and aldehydes. The synthesized compounds were evaluated against three hematologic tumor cell lines, HEL (acute erythroid leukemia), K-562 (chronic myeloid leukemia) and HL-60 (acute myeloid leukemia), and five breast cancer cell lines (SKBR3, MCF-7, MDA-MB-231, BT-549, HS-578T). The primate non-malignant kidney Vero cell line was used as the control of cytotoxicity. From the obtained results, some structure–activity relationships were outlined. Furthermore, in silico prediction of physicochemical properties and ADME parameters were determined for the derivatives with the best antiproliferative values.

In Vitro Activity of Cysteamine Against SARS-CoV-2 Variants Alpha, Beta,Gamma and Delta

Global COVID-19 pandemic is caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Continuous emergence of new variants and their rapid spread are jeopardizing vaccine countermeasures to a significant extent. While currently available vaccines are effective at preventing illness associated with SARS-CoV-2 infection, these have been shown to be less effective at preventing breakthrough infection and transmission from a vaccinated individual to others. Here we demonstrate broad antiviral activity of cysteamine HCl in vitro against variants of SARS-CoV-2 assayed in a highly permissible Vero cell line. Cysteamine HCl inhibited infection of alpha, beta, gamma and delta variants effectively and the inhibitory activity was shown to manifest during the early stages of viral infection. Cysteamine is a very well-tolerated US FDA-approved drug used chronically as a topical ophthalmic solution to treat ocular cystinosis in patients who receive it hourly or QID lifelong at concentrations 3 to 4 times higher than that required to inhibit SARS CoV-2 in tissue culture. Application of cysteamine as a topical nasal treatment can potentially : 1) mitigate existing infection 2) prevent infection in exposed individuals, and 3) limit the contagion in vulnerable populations..

COMPARATIVE STUDY BETWEEN ROTA VIRUSES ISOLATED FROM DIARRHEATIC INFANT BABIES AND NEWLY BORN CALVES

Rota viruses were isolated in vero continuous cell line from infant babies and newly borne calves affected by diarrhea after treatment in every passage with 10 mg/ml of trypsin and adding 0.5 mg/ml of trypsin in the maintenance media. The isolated viruses induced distinctive and progressive type of cytopatheic effect in infected cells and highest titer was 2x10 6. TCID,50/0.1 for bovine viral isolate. The isolated 50/0.1 for human viral isolate and 2x10 » viruses were identified by indirect fluorescent antibody technique by using reference calf rotavirus antisera. Comparative study were conducted on both human and bovine viral isolate including growth in different cell culture, vero cell line was very sensitive to support growth of both viruses than secondary embryonic calf kidney cell culture, lamb testes and primary embryonic chicken fibroblast cell culture. Both viruses induced morphologically similar kind or plaques in vero cell line but plaqes formed by human isolate were larger in size about 1.5 - 2.5 mm in diameter than those of bovine isolate 1.5 - 2 mm. Cross reactive viral antigens were detected between the isolated viruses in indirect fluorescent antibody technique but not in serum neutralization test by using reference bovine rotavirus antisera.

In vitro antiplasmodial activity, cytotoxicity, antioxidant action and GC-FID analysis of Allanblackia floribunda Oliv

This study evaluated the in vitro antiplasmodial efficacy and cytotoxicity of Allanbackia floribunda stem bark extract, leaf extract and oil. It also assessed the phytochemical compositions and antioxidant action of the stem bark fractions as well as the phytochemical fingerprint of the most active fraction (dichloromethane). Trager and Jensen method was used to culture Plasmodium falciparum, Mark III test developed by WHO was used to assess the antiplasmodial activity of the plant’s crude extract and fractions against the ring stage of P. falciparum strain, Pf3D7. Cytotoxicity was determined against Vero cell line using microculture tetrazolium (MTT) test. Gas chromatography with flame ionization detection (GC-FID) was employed to identify phytochemical fingerprint of the most active fraction. The stem bark extract had better antiplasmodial activity (IC50Pf3D7 of 4.3 ± 0.17 μg/mL) compared with the leaf extract (IC50Pf3D7, 8.0 ± 0.28 μg/mL) and oil (IC50Pf3D7 > 100 μg/mL). Both the leaf and stem bark extracts were found to be non-cytotoxic compared with the standard cytotoxic drug, doxorubicin. The selectivity indices (S.I.) of the extracts against the parasite were 20.06 and 8.85 for the stem bark and leaf respectively. Dichloromethane fraction had the highest inhibition against the P. falciparum parasite with IC50Pf3D7 of 1.51 μg/ mL. GC-FID analysis showed high presence antiplasmodial flavonoids and terpenes. This investigation confirmed that A. floribunda stem bark has potent activity against P. falciparum, and it is relatively safe to normal cell. Article Highlights Allanblackia floribunda methanol stem bark and leaf extracts could inhibit the growth of chloroquine sensitive Plasmodium falciparum (Pf3D7) in vitro. The stem bark infusion of Allanblackia floribunda was found to be nontoxic and safe at moderate doses to normal cell line (Vero cell line). Dichloromethane fraction of the stem bark showed excellent inhibition against chloroquine sensitive malaria parasite.

Active Flavonoids from Colubrina greggii var. greggii S. Watson against Clinical Isolates of Candida spp.

Candida albicans is the most commonly implicated agent in invasive human fungal infections. The disease could be presented as minimal symptomatic candidemia or can be fulminant sepsis. Candidemia is associated with a high rate of mortality and high healthcare and hospitalization costs. The surveillance programs have reported the distribution of other Candida species reflecting the trends and antifungal susceptibilities. Previous studies have demonstrated that C. glabrata more frequently presents fluconazole-resistant strains. Extracts from Mexican plants have been reported with activity against pulmonary mycosis, among them Colubrina greggii. In the present study, extracts from the aerial parts (leaves, flowers, and fruits) of this plant were evaluated against clinical isolates of several species of Candida (C. albicans, C. glabrata, C. parapsilosis, C. krusei, and C. tropicalis) by the broth microdilution assay. Through bioassay-guided fractionation, three antifungal glycosylated flavonoids were isolated and characterized. The isolated compounds showed antifungal activity only against C. glabrata resistant to fluconazole, and were non-toxic toward brine shrimp lethality bioassay and in vitro Vero cell line assay. The ethyl acetate and butanol extracts, as well as the fractions containing the mixture of flavonoids, were more active against Candida spp.