RyR1 is an intracellular Ca²⁺ channel important in excitable cells such as neurons and muscle fibers. Ca²⁺ activates it at low concentrations and inhibits it at high concentrations. Mg²⁺ is the main physiological RyR1 inhibitor, an effect that is overridden upon activation. Despite the significance of Mg²⁺-mediated inhibition, the molecular-level mechanisms remain unclear. In this work we determined two cryo-EM structures of RyR1 with Mg²⁺ up to 2.8 Å resolution, identifying multiple Mg²⁺ binding sites. Mg²⁺ inhibits at the known Ca²⁺ activating site and we propose that the EF hand domain is an inhibitory divalent cation sensor. Both divalent cations bind to ATP within a crevice, contributing to the precise transmission of allosteric changes within the enormous channel protein. Notably, Mg²⁺ inhibits RyR1 by interacting with the gating helices as validated by molecular dynamics. This structural insight enhances our understanding of how Mg²⁺ inhibition is overcome during excitation.

Peptide-induced disruption of lipid membranes is central to both amyloid diseases and the activity of antimicrobial peptides. Here, we combine all-atom molecular dynamics simulations with biophysical experiments to investigate how four amphipathic peptides interact with lipid bilayers. All peptides adsorb on the membrane surface. Peptide M01 [Ac-(FKFE)₂-NH₂] self-assembles into $\beta$-sheet nanofibrils that span both leaflets of the membrane, creating water-permeable channels. The other three peptides adopt $\alpha$-helical structures at the water–lipid interface. Peptide M02 [Ac-FFKKFFEE-NH₂], a sequence isomer of M01, does not form $\beta$-sheet aggregates and is too short to span the bilayer, resulting in no observable water permeation across the membrane. Peptides M03 and M04 are $\alpha$-helical isomers long enough to span the bilayer, with a polar face that allows the penetration of water deep inside the membrane. For the M03 peptide [Ac-(FFKKFFEE)₂-NH₂], insertion into the bilayer starts with the nonpolar N-terminal amino acids penetrating the hydrophobic core of the bilayer, while electrostatic interactions hold negative residues at the C-terminus on the membrane surface. The M04 peptide, [Ac-FFKKFFEEFKKFFEEF-NH₂], is made by relocating a single nonpolar residue from the central region of M03 to the C-terminus. This nonpolar residue becomes unfavorably exposed to the solvent upon insertion of the N-terminal region of the peptide into the membrane. Consequently, higher concentrations of M04 peptides are required to induce water permeation compared to M03. Overall, our comparative analysis reveals how subtle rearrangements of polar and nonpolar residues modulate peptide-induced water permeation. This provides mechanistic insights relevant to amyloid pathology and antimicrobial peptide design.

Rhipicephalus (Boophilus) microplus is one of the most widespread ticks causing a massive loss to livestock production. The long-term use of acaracides rapidly develops acaracide resistance. In R. microplus, enhancing the metabolic activity of glutathione S-transferase (RmGST) is one of the mechanisms underlying acaracide resistance. RmGST catalyzes the conjugation of glutathione (GSH) to insecticides causing an easy-to-excrete conjugate. The active RmGST dimer contains two active sites (hydrophobic co-substrate binding site (H-site) and GSH binding site (G-site)) in each monomer. To preserve the insecticide efficacy, s-hexyl glutathione (GTX), a GST inhibitor, has been used as a synergist. To date, no molecular information on the RmGST-GSH/GTX complex is available. The insight is important for developing a novel RmGST inhibitor. Therefore, in this work, molecular dynamics simulations (MD) were performed to explore the binding of GTX and GSH to RmGST. GSH binds tighter and sits rigidly inside the G-site, while flexible GTX occupies both active sites. In GSH, the backbone mainly interacts with W8, R43, W46, K50, N59, L60, Q72, and S73, while its thiol group directs to Y7. In contrast, the aliphatic hexyl of GTX protrudes into the H-site and allows a flexible peptide core to form various interactions. Such high GTX flexibility and the protrusion of its hexyl moiety to the H-site suggest the dual role of GTX in preventing the conjugation reaction and the binding of acaracide. This insight can provide a better understanding of an important insecticide-resistance mechanism, which may in turn facilitate the development of novel approaches to tick control.

Phylogenetic and population genetic analyses were conducted on tick specimens collected from cattle in northern, northeastern, central, and southern regions of Thailand. Morphological identification indicated these ticks consisted of three species, Rhipicephalus microplus from all four regions, R. sanguineus from the northern and northeastern regions, and a Haemaphysalis species only collected from the northeastern region. Analysis of cytochrome c oxidase subunit I gene ( COI ) sequences identified R. microplus clades A and C, while clade B was not detected in this study. The same analysis indicated specimens morphologically identified as Haemaphysalis were H. bispinosa, confirming previous reports of their prevalence in northeastern Thailand. H. bispinosa showed low haplotype and nucleotide diversity, suggesting either a bottleneck or founder effect. Both R. microplus clades displayed high haplotype diversity and low nucleotide diversity, a pattern associated with population expansion. Genetic structural analysis revealed significant genetic differences in R. microplus clade A, especially between mainland (northern, northeastern, and central regions) and peninsular (southern region) populations, which indicated limited gene flow between these areas while suggesting movement of these ticks across the mainland. The sequence analyses described in this report enhance understanding of the natural history of ticks in Thailand and are expected to guide and strengthen tick control strategies across Southeast Asia.

Unfolding of a coarse-grained COVN protein from its native configuration shows a linear response with increasing temperature followed by non-monotonic double peaks in its radius of gyration. The protein conforms to a random coil of folded segments in native state with increasingly tenuous and globular structures in specific temperature regimes where the effective dimensions of corresponding structures D ≈ 1.6–2.4. Thermal agitation alone is not sufficient to fully eradicate its segmental folding as few local folds are found to persist around such residues as ⁶⁵L, ¹¹⁰Y, ²²⁴L, ³⁷⁴P even at high temperatures.

Toxoplasma gondii, a pathogen of significant concern in animal production, companion animal health, and public health, particularly affects immunocompromised individuals and pregnant women. Current diagnostic techniques employ both direct and indirect methods, with serological assays widely used for detecting T. gondii infections in humans and animals. In this study, the TIM-barrel structure of Br2 β-glucosidase was engineered to create 10 chimeric multi-epitope proteins for T. gondii serological detection. Indirect ELISA screening identified three promising candidate proteins, V4Z, SFF, and S7V-V4Z-SFF, with sensitivities ranging from 71–86% and specificities ranging from 68–76%. Among these, ELISA-V4Z achieved the highest concordance with the reference IFAT method (Kappa = 0.58, 95% CI = 0.32–0.84) and demonstrated a moderate positive predictive value (PPV, 67%) and strong negative predictive value (NPV, 90%). These results suggest that the V4Z chimeric protein demonstrated the strongest performance among the tested candidates for T. gondii detection, exhibiting the highest sensitivity and specificity along with moderate agreement with the reference IFAT. However, its overall diagnostic performance remains limited. These findings highlight the need for further refinement and validation to enhance its diagnostic potential and assess its applicability for broader serological testing.