International Consensus Based Review and Recommendations for Minimum Reporting Standards in Research on Transcutaneous Vagus Nerve Stimulation (Version 2020)

Given its non-invasive nature, there is increasing interest in the use of transcutaneous vagus nerve stimulation (tVNS) across basic, translational and clinical research. Contemporaneously, tVNS can be achieved by stimulating either the auricular branch or the cervical bundle of the vagus nerve, referred to as transcutaneous auricular vagus nerve stimulation(VNS) and transcutaneous cervical VNS, respectively. In order to advance the field in a systematic manner, studies using these technologies need to adequately report sufficient methodological detail to enable comparison of results between studies, replication of studies, as well as enhancing study participant safety. We systematically reviewed the existing tVNS literature to evaluate current reporting practices. Based on this review, and consensus among participating authors, we propose a set of minimal reporting items to guide future tVNS studies. The suggested items address specific technical aspects of the device and stimulation parameters. We also cover general recommendations including inclusion and exclusion criteria for participants, outcome parameters and the detailed reporting of side effects. Furthermore, we review strategies used to identify the optimal stimulation parameters for a given research setting and summarize ongoing developments in animal research with potential implications for the application of tVNS in humans. Finally, we discuss the potential of tVNS in future research as well as the associated challenges across several disciplines in research and clinical practice

DYSPROSIUM DOPED CALCIUM GERMANATE (CA2GеO4) AS A CANDIDATE FOR LED APPLICATION

Powder samples of Ca2GeO4 doped with 0.2, 0.5, 1, 2 and 3 at% Dy3+ were prepared using conventional solid state synthesis technique. XRD analyses show obtaining of the pure phase at all dopant concentrations. Emission and excitation spectra contain the characteristic peaks of Dy3+ ion. The Dy3+ excitation spectrum in the range from 300 to 500 nm show characteristic transitions of Dy3+, attributed to the f–f transitions. The strongest peak is located at 348 nm corresponding to the 6H15/2 →6P7/2 transition. In the luminescent spectra the two emission peaks of Dy3+ appears at 483 nm (4F5/2 →6H15/2 transition) corresponding to blue color and at 575 nm (4F5/2→6H13/2 transition) corresponding to yellow one. CIE coordinates of the samples show different emission colors in the yellow region depending on the active ion concentration. The obtained results confirm that as prepared dysprosium doped materials could be used as yellow phosphors

Identification and Expression Analysis of the <i>C-TERMINALLY ENCODED PEPTIDE</i> Family in <i>Pisum sativum</i> L.

The C-TERMINALLY ENCODED PEPTIDE(CEP) peptides play crucial roles in plant growth and response to environmental factors. These peptides were characterized as positive regulators of symbiotic nodule development in legume plants. However, little is known about the CEP peptide family in pea. Here, we discovered in pea genome 21 CEP genes (PsCEPs), among which three genes contained additional conserved motifs corresponding to the PIP (PAMP-induced secreted peptides) consensus sequences. We characterized the expression patterns of pea PsCEP genes based on transcriptomic data, and for six PsCEP genes with high expression levels in the root and symbiotic nodules the detailed expression analysis at different stages of symbiosis and in response to nitrate treatment was performed. We suggest that at least three PsCEP genes, PsCEP1, PsCEP7 and PsCEP2, could play a role in symbiotic nodule development, whereas the PsCEP1 and PsCEP13 genes, downregulated by nitrate addition, could be involved in regulation of nitrate-dependent processes in pea. Further functional studies are required to elucidate the functions of these PsCEP genes

Sulfonylpiperazine compounds prevent Plasmodium falciparum invasion of red blood cells through interference with actin-1/profilin dynamics.

With emerging resistance to frontline treatments, it is vital that new antimalarial drugs are identified to target Plasmodium falciparum. We have recently described a compound, MMV020291, as a specific inhibitor of red blood cell (RBC) invasion, and have generated analogues with improved potency. Here, we generated resistance to MMV020291 and performed whole genome sequencing of 3 MMV020291-resistant populations. This revealed 3 nonsynonymous single nucleotide polymorphisms in 2 genes; 2 in profilin (N154Y, K124N) and a third one in actin-1 (M356L). Using CRISPR-Cas9, we engineered these mutations into wild-type parasites, which rendered them resistant to MMV020291. We demonstrate that MMV020291 reduces actin polymerisation that is required by the merozoite stage parasites to invade RBCs. Additionally, the series inhibits the actin-1-dependent process of apicoplast segregation, leading to a delayed death phenotype. In vitro cosedimentation experiments using recombinant P. falciparum proteins indicate that potent MMV020291 analogues disrupt the formation of filamentous actin in the presence of profilin. Altogether, this study identifies the first compound series interfering with the actin-1/profilin interaction in P. falciparum and paves the way for future antimalarial development against the highly dynamic process of actin polymerisation

Sulfonylpiperazine compounds prevent Plasmodium falciparum invasion of red blood cells through interference with actin-1/profilin dynamics

Abstract With emerging resistance to frontline treatments, it is vital that new antimalarial drugs are identified to target Plasmodium falciparum. We have recently described a compound, MMV020291, as a specific inhibitor of red blood cell (RBC) invasion, and have generated analogues with improved potency. Here, we generated resistance to MMV020291 and performed whole genome sequencing of 3 MMV020291-resistant populations. This revealed 3 nonsynonymous single nucleotide polymorphisms in 2 genes; 2 in profilin (N154Y, K124N) and a third one in actin-1 (M356L). Using CRISPR-Cas9, we engineered these mutations into wild-type parasites, which rendered them resistant to MMV020291. We demonstrate that MMV020291 reduces actin polymerisation that is required by the merozoite stage parasites to invade RBCs. Additionally, the series inhibits the actin-1-dependent process of apicoplast segregation, leading to a delayed death phenotype. In vitro cosedimentation experiments using recombinant P. falciparum proteins indicate that potent MMV020291 analogues disrupt the formation of filamentous actin in the presence of profilin. Altogether, this study identifies the first compound series interfering with the actin-1/profilin interaction in P. falciparum and paves the way for future antimalarial development against the highly dynamic process of actin polymerisation

Proposed model for MMV291 interference in profilin-mediated filamentous actin polymerisation.

(A) Treadmilling model of profilin’s role in sequestering G-actin and stimulating the exchange of ADP for ATP before delivering the subunits to the barbed end of the growing filament. Here, formin initiates the polymerisation process to form F-actin. Hydrolysis of the G-actin-ATP occurs at this end to produce G-actin-ADP and inorganic phosphate (Pi), to stabilise the filament. The slow release of Pi at the pointed end induces filament instability and proteins such as ADF1 bind to G-actin-ADP to aid in the release of the subunits, thereby severing the filaments. (B) A potential mechanism for MMV291’s inhibitory activity could be through the stabilisation of the G-actin/profilin dimer therefore inhibiting the formation of F-actin and preventing the generation of force required for invasion. ADF1, actin depolymerising factor 1; F-actin, filamentous actin; G-actin, globular actin.</p

MMV291 does not affect actin filaments in HeLa cells.

HeLa cells labelled with SiR-Actin imaged by lattice light-sheet microscopy upon stimulation with DMSO control (A), 5 μM Latrunculin B (B), 200 nM Cytochalasin D (CytD) (C), 2.5 μM MMV291 (D), 5 μM MMV291 (E), 10 μM MMV291 (F), and 20 μM MMV291 (G). Images represent a 100 × 100 μm subregion of a larger 250 × 250 μm field of view. Images are presented as maximum intensity projections with the contrast scaled between 100–400 counts. The images show the same region of cells imaged across multiple time points. Time is presented as HH:MM, and the scale bar represents 20 μm. (TIF)</p

Introduction of the SNPs in <i>profilin</i> and <i>actin-1</i> into 3D7 parasites mediates resistance to MMV291.

(A) i Strategy to create the donor plasmid to introduce PFN(N154Y), PFN(K124N), and ACT1(M356L) SNPs into 3D7 parasites. Homology regions (HRs) were designed to the 5′ flank (HR1) and 3′ flank (HR2) whereby HR1 was made up of the endogenous genes’ sequence (HR1A) and recodonised fragments (HR1B), encompassing the resistant mutation alleles. A synthetic guide RNA (gRNA) was designed for either profilin or actin-1 to direct Cas9 to the cleavage site and induce double crossover homologous recombination. WR99210 was used to select for integrated parasites via the human hydrofolate reductase (hDHFR). ii Integration into the profilin or actin-1 locus was validated whereby a 5′ UTR primer (i/v) was used in combination with a primer located in the glmS region (k). B) i Integrated parasites were tested in a 72-hour LDH growth assay, which revealed the resistant mutations conferred resistance against MMV291 and confirmed the profilin and actin-1 proteins as involved in the MoA of the compound. Growth has been normalised to that of parasites grown in 0.1% DMSO, and error bars indicate the standard deviation of 3 biological replicates. Source data can be found in S1 Data. ii EC50 values derived from nonlinear regression curves in GraphPad Prism with 95% confidence intervals shown in brackets. und = undefined.</p

MMV291 series show limited activity against <i>T</i>. <i>gondii</i> invasion.

Nanoluciferase expressing parasites were liberated from their host cell and incubated with the MMV291 analogues before being added back to fibroblasts and allowed to invade for 1 hour before compounds were washed out. After a 24-hour incubation, cells were then lysed and the relative light units was quantified to correlate with T. gondii invasion rate. This showed MMV291 analogues S-W936 (A), R-W936 (B), S-MMV291 (C), and R-MMV291 (D) had some inhibitory activity against invasion at high concentrations. DMSO was included to the same amount as the highest concentration of analogue to account for DMSO-related effects (30% reduction in invasion). Error bars indicate the standard deviation from 2 biological repeats. Source data can be found in S1 Data. (TIF)</p

MMV291 does not affect actin filaments in HeLa cells.

HeLa cells labelled with SiR-Actin imaged by lattice light-sheet microscopy upon stimulation with DMSO Control, 5 μM Latrunculin B, 200 nM CytD, 2.5 μM MMV291, 10 μM MMV291, and 20 μM MMV291 over a time course of 3 hours. Movies represent maximum intensity projections with the contrast scaled between 100–400 counts. Time is presented as HH:MM, and the scale bar represents 20 μm. (MP4)</p