Group A streptococcus (GAS) is a Gram-positive human pathogen that causes invasive infections with mild to life-threatening severity, like toxic shock syndrome, rheumatic heart disease, and necrotizing fasciitis (NF). NF is characterized by a clinical presentation of widespread tissue destruction due to the rapid spread of GAS infection into fascial planes. Despite quick medical interventions, mortality from NF is high. The early onset of the disease is difficult to diagnose because of non-specific clinical symptoms. Moreover, the unavailability of an effective vaccine against GAS warrants a genuine need for alternative treatments against GAS NF. One endoplasmic reticulum stress signaling pathway (PERK pathway) gets triggered in the host upon GAS infection. Bacteria utilize asparagine release as an output of this pathway for its pathogenesis. We reported that the combination of sub-cutaneous (SC) and intraperitoneal (IP) administration of PERK pathway inhibitors (GSK2656157 and ISRIB) cures local as well as systemic GAS infection in a NF murine model, by reducing asparagine release at the infection site. This protocol's methodology is detailed below.

Plasmodium falciparum is a unicellular eukaryotic parasite that causes malaria in humans. The parasite is spread by Anopheles mosquitoes after ingestion of sexual stage parasites known as gametocytes. Malaria transmission depends on parasites switching from the disease-causing asexual blood forms to male and female gametocytes. The current protocol allows the simultaneous isolation of male and female parasites from the same population to study this critical lifecycle stage in a sex-specific manner. We have generated a transgenic P. falciparum cell line that expresses a GFP-tagged parasite protein in female, but not male, parasites. Gametocyte production is stress induced and, through a series of steps, sexual stage parasites are enriched relative to uninfected red blood cells or red blood cells infected with asexual stage parasites. Finally, male and female gametocytes are separated by fluorescence-activated cell sorting. This protocol allows for the separation of up to 12 million live male and female parasites from the same population, which are amenable to further analysis.

Gene expression within the mitochondria of African trypanosomes and other protozoan organisms relies on a nucleotide-specific RNA-editing reaction. In the process exclusively uridine (U)-nucleotides are site-specifically inserted into and deleted from sequence-deficient primary transcripts to convert them into translatable mRNAs. The reaction is catalyzed by a 0.8 MDa multiprotein complex termed the editosome. Here we describe an improved in vitro test to quantitatively explore the catalytic activity of the editosome. The assay uses synthetic, fluorophore-derivatized oligoribonucleotides as editing substrates, which enable the automated electrophoretic separation of the reaction products by capillary electrophoresis (CE) coupled to laser-induced fluorescence (LIF) detection systems. The assay is robust, it requires only nanogram amounts of materials and by using multicapillary CE/LIF-instruments it can be executed in a highly parallel layout. Further improvements include the usage of phosphorothioate-modified and thus RNase-resistant substrate RNAs as well as multiplex-type fluorophore labeling strategies to monitor the U-insertion and U-deletion reaction simultaneously. The assay is useful for investigating the mechanism and enzymology of the editosome. However, it can also be executed in high-throughput to screen for RNA editing-specific inhibitors.

Graphic abstract:

Characteristics of the fluorescence-based in vitro U-insertion/U-deletion RNA-editing (FIDE) assay

There is a pressing need to develop sustainable and efficient methods to protect and stabilize iron objects. To develop a conservation-restoration method for corroded iron objects, this bio-protocol presents the steps to investigate reductive dissolution of ferric iron and biogenic production of stabilizing ferrous iron minerals in the strict anaerobe Desulfitobacterium hafniense (strains TCE1 and LBE). We investigated iron reduction using three different Fe(III) sources: Fe(III)-citrate (a soluble phase), akaganeite (solid iron phase), and corroded coupons. This protocol describes a method that combines spectrophotometric quantification of the complex Fe(II)-Ferrozine^® with mineral characterization by scanning electron microscopy and Raman spectroscopy. These three methods allow assessing reductive dissolution of ferric iron and biogenic mineral production as a promising alternative for the development of an innovative sustainable method for the stabilization of corroded iron.