Synthesis and preliminary in vitro activity of mono- and bis-1H-1,2,3-triazole-tethered ß-lactam-isatin conjugates against the human protozoal pathogen Trichomonas vaginalis.

In this study, we describe the synthesis of mono- and bis-1H-1,2,3-triazole-tethered ß-lactam-isatin conjugates using copper-catalysed azide-alkyne cycloaddition reaction between mono- and di-propargylated azetidin-2-ones and N-alkylazido isatins. The synthesized conjugates were evaluated for their preliminary in vitro analysis against Trichomonas vaginalis at 50 µM. The efficacy of synthesized hybrids was observed to depend on the substituent at N-1 position of ß-lactam ring, as well as the presence of single/double 1H-1,2,3-triazole linker. Among the synthesized conjugates, the presence of a p-tolyl substituent at N-1 of ß-lactam ring was preferred for good activity profiles while the increase in spacer length did not influence the efficacy of the compounds. Compounds with high levels of potency were further analysed to determine their IC50 values, as well as cytotoxicity profiles against mammalian cells. The most active compound in the synthesized conjugates displayed an IC50 value of 10.49 µM against cultured G3 strain of T. vaginalis and was non-toxic to cultured mammalian HeLa cells at the same concentration.

Clinical performance evaluation of BD SARS-CoV-2 reagents for BD MAXTM System in asymptomatic individuals.

Transmission by asymptomatic individuals is a persistent hurdle in the effort to control the spread of SARS-CoV-2. Therefore, it is essential to continue developing assays and evaluate their performance for detection of SARS-CoV-2 in individuals without COVID-19 symptoms. In this study, 223 nasopharyngeal swab specimens collected from COVID-19 asymptomatic individuals were tested using the BD SARS-CoV-2 (RT-PCR-based) reagents for the BD MAX™ System and compared with results obtained with the Biomerieux BioFire® Respiratory RT-PCR Panel. Positive and negative percent agreements of 100% (95% CI, 84.5%-100%) and 99.0% (95% CI, 96.5%-99.7%), respectively, were observed for the BD SARS-CoV-2 assay. These results demonstrate the effectiveness of the BD SARS-CoV-2 assay for detecting SARS-CoV-2 in asymptomatic individuals and suggest that this assay can facilitate optimized case surveillance and infection control efforts. Investigations using larger sample sizes of asymptomatic individuals would be beneficial to support the findings in this study.

A closer look at the spectroscopic properties of possible reaction intermediates in wild-type and mutant (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase.

(E)-4-Hydroxy-3-methylbut-2-enyl diphosphate reductase (IspH or LytB) catalyzes the terminal step of the MEP/DOXP pathway where it converts (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) into the two products, isopentenyl diphosphate and dimethylallyl diphosphate. The reaction involves the reductive elimination of the C4 hydroxyl group, using a total of two electrons. Here we show that the active form of IspH contains a [4Fe-4S] cluster and not the [3Fe-4S] form. Our studies show that the cluster is the direct electron source for the reaction and that a reaction intermediate is bound directly to the cluster. This active form has been trapped in a state, dubbed FeS(A), that was detected by electron paramagnetic resonance (EPR) spectroscopy when one-electron-reduced IspH was incubated with HMBPP. In addition, three mutants of IspH have been prepared and studied, His42, His124, and Glu126 (Aquifex aeolicus numbering), with particular attention paid to the effects on the cluster properties and possible reaction intermediates. None of the mutants significantly affected the properties of the [4Fe-4S](+) cluster, but different effects were observed when one-electron-reduced forms were incubated with HMBPP. Replacing His42 led to an increased K(M) value and a much lower catalytic efficiency, confirming the role of this residue in substrate binding. Replacing the His124 also resulted in a lower catalytic efficiency. In this case, however, the enzyme showed the loss of the [4Fe-4S](+) EPR signal upon addition of HMBPP without the subsequent formation of the FeS(A) signal. Instead, a radical-type signal was observed in some of the samples, indicating that this residue plays a role in the correct positioning of the substrate. The incorrect orientation in the mutant leads to the formation of substrate-based radicals instead of the cluster-bound intermediate complex FeS(A). Replacing the Glu126 also resulted in a lower catalytic efficiency, with yet a third type of EPR signal being detected upon incubation with HMBPP. (31)P and (2)H ENDOR measurements of the FeS(A) species incubated with regular and (2)H-C4-labeled HMBPP reveal that the substrate binds to the enzyme in the proximity of the active-site cluster with C4 adjacent to the site of linkage between the FeS cluster and HMBPP. Comparison of the spectroscopic properties of this intermediate to those of intermediates detected in (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase and ferredoxin:thioredoxin reductase suggests that HMBPP binds to the FeS cluster via its hydroxyl group instead of a side-on binding as previously proposed for the species detected in the inactive Glu126 variant. Consequences for the IspH reaction mechanism are discussed.

Single-cell analysis of murine long-term hematopoietic stem cells reveals distinct patterns of gene expression during fetal migration.

BACKGROUND: Long-term hematopoietic stem cells (LT-HSCs) migrate from the fetal liver (FL) to the fetal bone marrow (FBM) during development. Various adhesion and chemotactic receptor genes have been implicated in the migration of adult LT-HSCs. However, their role in the migration of fetal LT-HSCs is not clearly understood due, in part, to the rare number of these cells in fetal tissues, which preclude classical gene expression analysis. The aim of this study is to characterize the expression of migration related genes in fetal LT-HSC across different anatomical locations during development. METHODOLOGY/PRINCIPAL FINDINGS: We isolated fetal LT-HSC from different developmental stages, as well as different anatomical locations, and performed single-cell multiplex RT-qPCR and flow cytometry analysis of eight molecules involved in adult LT-HSC migration. Our results show that the gene expression of the chemokine receptor Cxcr4 in LT-HSC varies across developmental microenvironments and times, while the cadherin Cdh2 (Ncad) and the calcium receptor Casr show higher gene expression and variability only in FBM at 17.5 days post coitum (dpc). The cadherin Cdh5 (Vecad) maintains high expression variability only during fetal development, while the integrin subunit Itga5 (α5) increases its variability after 14.5 dpc. The integrin subunits Itga4 (α4) and Itgal (Lfa1), as well as the selectin ligand Selplg (Psgl1), did not show differences in their expression in single LT-HSCs irrespective of the developmental times or anatomical microenvironments studied. CONCLUSIONS/SIGNIFICANCE: Our data demonstrate that the expression pattern of phenotypically identical, single LT-HSCs fluctuates as a function of developmental stage and anatomical microenvironment. This is the first exhaustive gene expression comparison of migration-related molecules in fetal tissues across developmental times, enhancing the understanding of LT-HSC migration fate decisions during development.