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KRAS, the most frequently mutated oncogene in human cancer, produces two isoforms, KRAS4a and KRAS4b, through alternative splicing. These isoforms differ in exon 4, which encodes the final 15 residues of the G-domain and hypervariable regions (HVRs), vital for trafficking and membrane localization. While KRAS4b has been extensively studied, KRAS4a has been largely overlooked. Our multidisciplinary study compared the structural and functional characteristics of KRAS4a and KRAS4b, revealing distinct structural properties and thermal stability. Position 151 influences KRAS4as thermal stability, while position 153 affects binding to RAF1 CRD protein. Nuclear magnetic resonance analysis identified localized structural differences near sequence variations and provided a solution-state conformational ensemble. Notably, KRAS4a exhibits substantial transcript abundance in bile ducts, liver, and stomach, with transcript levels approaching KRAS4b in the colon and rectum. Functional disparities were observed in full-length KRAS variants, highlighting the impact of HVR variations on interaction with trafficking proteins and downstream effectors like RAF and PI3K within cells.

The RAS proteins are GTP-dependent switches that regulate signaling pathways and are frequently mutated in cancer. RAS proteins concentrate in the plasma membrane via lipid-tethers and hypervariable region side-chain interactions in distinct nano-domains. However, little is known about RAS membrane dynamics and the details of RAS activation of downstream signaling. Here, we characterize RAS in live human and mouse cells using single-molecule-tracking methods and estimate RAS mobility parameters. KRAS4b exhibits confined mobility with three diffusive states distinct from the other RAS isoforms (KRAS4a, NRAS, and HRAS); and although most of the amino acid differences between RAS isoforms lie within the hypervariable region, the additional confinement of KRAS4b is largely determined by the protein's globular domain. To understand the altered mobility of an oncogenic KRAS4b, we used complementary experimental and molecular dynamics simulation approaches to reveal a detailed mechanism.

This article discusses a study on the detection of Neoparamoeba perurans, a parasite that causes amoebic gill disease (AGD) in salmon. The study compares the use of DNA-based and RNA-based assays for detecting N. perurans in seawater samples. The researchers found that the RNA-based assay was more sensitive and reliable than the DNA-based assay, detecting N. perurans even at low concentrations. The study suggests that targeting intracellular RNA may provide a more accurate measure of amoeba abundance. Further research is needed to determine if RNA-based quantification of N. perurans can be used as an early indicator of AGD. [Extracted from the article]

The Tasmanian salmon industry had remained relatively free of major viral diseases until the emergence of pilchard orthomyxovirus (POMV). Originally isolated from wild pilchards, POMV is of concern to the industry as it can cause high mortality in farmed salmon (Salmo salar). Field observations suggest the virus can spread from pen to pen and between farms, but evidence of passive transmission in sea water was unclear. Our aim was to establish whether direct contact between infected and naïve fish was required for transmission, and to examine viral infection dynamics. Atlantic salmon post‐smolts were challenged with POMV by either direct exposure via cohabitation or indirect exposure via virus‐contaminated sea water. POMV was transmissible in sea water and direct contact between fish was not required for infection. Head kidney and heart presented the highest viral loads in early stages of infection. POMV survivors presented low viral loads in most tissues, but these remained relatively high in gills. A consistent feature was the infiltration of viral‐infected melanomacrophages in different tissues, suggesting an important role of these in the immune response to POMV. Understanding POMV transmission and host–pathogen interactions is key for the development of improved surveillance tools, transmission models and ultimately for disease prevention. [ABSTRACT FROM AUTHOR]

Hydrogen peroxide (H2O2) is a commonly used treatment for a range of parasitic diseases of marine finfish, including amoebic gill disease (AGD). While this treatment is partially effective at reducing parasite load, H2O2 can have detrimental effects on the host under certain conditions. Treatment temperature and dose concentration are two factors that are known to influence the toxicity of H2O2; however, their impact on the outcome of AGD treatment remains unclear. Here, we investigated the effects of treatment temperature (8, 12 or 16°C) and dose concentration (750, 1,000, 1,250 mg/L) on the efficacy of H2O2 to treat AGD. We demonstrated that a 20‐min bath treatment of H2O2 at all doses reduced both parasite load and gross gill score significantly. Parasite load and gross gill score were lowest in the 1,000 mg/L treatment performed at 12°C. At the high dose and temperature combinations, H2O2 caused moderate gill damage and a significant increase in the plasma concentration of electrolytes (sodium, chloride and potassium). Taken together, our study demonstrates that higher H2O2 treatment temperatures can adversely affect the host and do not improve the effectiveness of the treatment. [ABSTRACT FROM AUTHOR]

HER2 and αVβ6 integrin are independent predictors of breast cancer survival and metastasis. We identify an αVβ6/HER2 cross-talk mechanism driving invasion, which is dysregulated in drug-resistant HER2+ breast cancer cells. Proteomic analyses reveal ligand-bound αVβ6 recruits HER2 and a trafficking subnetwork, comprising guanosine triphosphatases RAB5 and RAB7A and the Rab regulator guanine nucleotide dissociation inhibitor 2 (GDI2). The RAB5/RAB7A/GDI2 functional module mediates direct cross-talk between αVβ6 and HER2, affecting receptor trafficking and signaling. Acute exposure to trastuzumab increases recruitment of the subnetwork to αVβ6, but trastuzumab resistance decouples GDI2 recruitment. GDI2, RAB5, and RAB7A cooperate to regulate migration and transforming growth factor-ß activation to promote invasion. However, these mechanisms are dysregulated in trastuzumab-resistant cells. In patients, RAB5A, RAB7A, and GDI2 expression correlates with patient survival and αVβ6 expression predicts relapse following trastuzumab treatment. Thus, the RAB5/RAB7A/GDI2 subnetwork regulates αVβ6-HER2 cross-talk to drive breast cancer invasion but is subverted in trastuzumab-resistant cells to drive αVβ6-independent and HER2-independent tumor progression. [ABSTRACT FROM AUTHOR]

ABSTRACT Nodular gill disease (NGD) is a serious proliferative gill condition that affects farmed salmonids, particularly in Europe. While the cause of NGD remains unknown (and maybe multifactorial), various amoebae are often isolated from the gills of affected fish and can in some cases be seen associated with lesions by histopathology. The present study aimed to quantify the abundance of different amoeba species directly from the gills of rainbow trout affected by NGD and healthy controls. An 18S rRNA amplicon metagenomic approach was employed to profile the diversity and abundance of micro‐eukaryotes (including amoebae) while suppressing the amplification of host DNA using a salmonid‐specific C3 spacer blocking primer. The 18S rRNA metagenomics approach identified a diversity of micro‐eukaryotes on the gills of rainbow trout, including the phylum's Amoebozoa, Diatomea, Platyhelminthes and Ciliophora. Rainbow trout clinically affected by NGD had a significantly higher abundance of a specific sequence (zOTU2) classified as Vannella sp. compared to healthy controls. A quantitative PCR assay was then developed and validated which accurately quantified the abundance of this Vannella sp. sequence from a NGD outbreak in a Swiss rainbow trout farm. Additional PCR and Sanger sequencing analysis of the zOTU2 sequence demonstrated that this sequence is most likely derived from Vannella mustalahtiana. Our study highlights the potential role of Vannella mustalahtiana in NGD in Switzerland and further describes a specific and validated diagnostic PCR assay for accurate detection of this Vannella species. [ABSTRACT FROM AUTHOR]

With recent advances proving that effective inhibition of KRAS is possible, there have been significant efforts made to develop inhibitors of specific mutant alleles. Here we describe a detailed protocol that employs homogeneous time-resolved fluorescence (HTRF) to identify compounds acting on KRAS signaling in malignant cell lines. This method allows for high-throughput, cell-based screens of large compound libraries for the development of RAS-targeted therapeutics., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)