Concise Synthesis of Alternariol and Alternariol-9-monomethyl ether as well as their D3-Isotopologues

Alternariol (AOH) and alternariol-9-monomethyl ether (AME) are two secondary metabolites of Alternaria fungi which can be found in various foodstuff like tomatoes, nuts, and grains. Due to their toxicity and potential mutagenic activity the need for the development of high-throughput methods for the supervision of AOH- and AME-levels is of increasing interest. As the availability of both native and labeled AOH and AME analytical standards is very limited we herein wish to present a novel concise approach towards their synthesis employing a ruthenium-catalyzed ortho-arylation as the key step. Finally, we demonstrate their suitability as internal standards in stable-isotope dilution assay (SIDA)-HPLC-MS/MS analysis commonly used for the quantification of the natural products in food and feed.

Time to revisit the endpoint dilution assay and to replace the TCID50 as a measure of a virus sample’s infection concentration

The endpoint dilution assay’s output, the 50% infectious dose (ID50), is calculated using the Reed-Muench or Spearman-Kärber mathematical approximations, which are biased and often miscalculated. We introduce a replacement for the ID50 that we call Specific INfection (SIN) along with a free and open-source web-application, midSIN (https://midsin.physics.ryerson.ca) to calculate it. midSIN computes a virus sample’s SIN concentration using Bayesian inference based on the results of a standard endpoint dilution assay, and requires no changes to current experimental protocols. We analyzed influenza and respiratory syncytial virus samples using midSIN and demonstrated that the SIN/mL reliably corresponds to the number of infections a sample will cause per mL. It can therefore be used directly to achieve a desired multiplicity of infection, similarly to how plaque or focus forming units (PFU, FFU) are used. midSIN’s estimates are shown to be more accurate and robust than the Reed-Muench and Spearman-Kärber approximations. The impact of endpoint dilution plate design choices (dilution factor, replicates per dilution) on measurement accuracy is also explored. The simplicity of SIN as a measure and the greater accuracy provided by midSIN make them an easy and superior replacement for the TCID50 and other in vitro culture ID50 measures. We hope to see their universal adoption to measure the infectivity of virus samples.

Phytochemical Traits and Biological Activity of Eryngium amethystinum and E. alpinum (Apiaceae)

Eryngium amethystinum L. is a wild vegetable used in Croatia. Both E. amethystinum and E. alpinum L. are decorative plants that can also be used in horticulture. To find out the hidden qualities of these two species, the following biological activities were studied: essential oil (EO) composition, analyzed by gas chromatography and gas chromatography with mass spectrometry (GC, GC–MS); phenolic compound content (PC), analyzed by high performance liquid chromatography (HPLC); total phenols as well as total flavonoids, analyzed by ultraviolet–visible spectrophotometry (UV/Vis); antioxidants, analyzed by 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity (DPPH), b-carotene-linoleic acid assay, chelating activity and reducing power of the extracts; and antimicrobial evaluation by micro-dilution assay and inhibition of Candida albicans blastospore germination. The major constituents of EO were β-caryophyllene (15.2%), α-pinene (10.2%) and 2,3,6-trimethylbenzaldehyde (9.3%) for E. amethystinum and caryophyllene oxide (27.9%), bicyclogermacrene (13.2%) and germacrene D (8.2%) for E. alpinum. The methanol extracts of both species showed a broad spectrum of antibacterial and antifungal activity with minimum inhibitory concentrations (MIC) less than or equal to 1.944 and 1.11 mg/mL, respectively.

An Improved Tat/Rev Induced Limiting Dilution Assay With Enhanced Sensitivity and Breadth of Detection

Tat/Rev Induced Limiting Dilution Assay (TILDA) is instrumental in estimating the size of latent reservoirs of HIV-1. Here, we report an optimized TILDA containing a broader detection range compared to the reported methods and high sensitivity. Giving priority to sequence conservation, we positioned the two forward primers and the probe in exon-1 of HIV-1. The reverse primers are positioned in highly conserved regions of exon-7. The optimized TILDA detected eight molecular clones belonging to five major genetic subtypes of HIV-1 with a comparable detection sensitivity. Using the optimized assay, we show that only a minor proportion of CD4+ T cells of primary clinical samples can spontaneously generate multiply spliced viral transcripts. A significantly larger proportion of the cells produced viral transcripts following activation. The optimized TILDA is suitable to characterize HIV-1 latent reservoirs and the therapeutic strategies intended to target the reservoir size.

ATI-2307 Exhibits Equivalent Antifungal Activity in Cryptococcus neoformans Clinical Isolates With High and Low Fluconazole IC50

Half maximal inhibitory concentrations (IC50) to the experimental drug ATI-2307 and complete inhibition (IC90) of the common clinically used antifungal drug amphotericin B were determined by microbroth dilution assay for a collection of 69 clinical isolates of Cryptococcus neoformans from Uganda that had high fluconazole IC50 values. The majority of the clinical isolates tested had fluconazole IC50 at or above 8 µg/mL, but were susceptible to both amphotericin B (IC90 ≤1 μg/mL) and ATI-2307 (IC50 ≤0.0312 µg/mL). No correlation between increased fluconazole minimum inhibitory concentration (MIC) and ATI-2307 or amphotericin B MIC was observed, suggesting that the cellular changes impacting fluconazole susceptibility did not impact the effectiveness of ATI-2307. Our results suggest that ATI-2307 is a promising new antifungal drug for use in the context of high fluconazole or other antifungal drug MICs and/or in combination drug therapy regimens.

Inducible HIV-1 Reservoir Quantification: Clinical Relevance, Applications and Advancements of TILDA

The presence of a stable HIV-1 reservoir persisting over time despite effective antiretroviral suppression therapy precludes a cure for HIV-1. Characterizing and quantifying this residual reservoir is considered an essential prerequisite to develop and validate curative strategies. However, a sensitive, reproducible, cost-effective, and easily executable test is still needed. The quantitative viral outgrowth assay is considered the gold standard approach to quantify the reservoir in HIV-1-infected patients on suppressive ART, but it has several limitations. An alternative method to quantify the viral reservoir following the reactivation of latent HIV-1 provirus detects multiply-spliced tat/rev RNA (msRNA) molecules by real-time PCR [tat/rev induced limiting dilution assay (TILDA)]. This article provides a perspective overview of the clinical relevance, various applications, recent advancements of TILDA, and how the assay has contributed to our understanding of the HIV-1 reservoir.

Exraction of Secondary Metabolites Using Bacillus endopyticus and Its Applications

Bacillus Species was isolated from a soil sample, the isolate was cultivated, identified and its culture medium was extracted. Specific media called Gibbon’s media used for the cultivation and confirmation of the bacteria. 16S rRNA study was done for the morphological identification of the isolate. The crude which was collected from Bacillus endopyticus was purified by column chromatography. The antibacterial activity of the crude extract was identified by agar diffusion test by measuring the minimum inhibitory concentration by dilution assay. The purified extract was active against Staphylococcus aureus, Bacillus cerues, Escherichia coli. The study suggest that Bacillus Sps have the potential to produce antibiotics and can be used to control the growth in future. The further work was able to bring the microbial strains under control. The percentage of cell death was calculated the cell death was 97.95%.