Luteinizing hormone receptor (LHR) is a member of the seven-transmembrane (TM) receptor family. Several mutations in LHR have been identified in many mammalian species, leading to either constitutive activation or inactivation of the receptor. Mutations in highly conserved regions of the TM domain have been reported. In this study, we analyzed signal transduction by three constitutively active mutants (designated M410T, L469R, and D590Y) and two inactivating mutants (D383N and Y546F) of eelLHR known as naturally occurring in human LHR . To directly assess the functional effects of these mutations, site-directed mutant receptors were transiently expressed in CHO-K1 cells and cAMP accumulation stimulated by recombinant eelLH (rec-eelLH) was measured by homogeneous time-resolved fluorescence (HTRF) assays. The cAMP response in cells expressing eelLHR wild-type (eLHR-WT) increased in a dose-dependent manner with rec-eelLH ligand stimulation. Cells expressing the activating eelLHR mutants, M410T, L469R, and D590Y, exhibited a 4.0-, 19.1-, and 7.8-fold increase in basal cAMP response, respectively. However, their maximal responses were approximately 73, 53, and 92%, respectively, of the maximal response of LHR-WT. The L469R mutant exhibited a particularly marked increase in cAMP production in the absence of agonist. The maximal responses of the inactivating mutants, D383N and Y546F, were 32 and 24% of LHR-WT, respectively. However, the inactivating mutations did not completely impair signal transduction. Thus, we report here the first characterization of activating and inactivating mutations in eelLHR and we show that these mutations have similar effects as those reported for mammalian LHRs. Moreover, eelLHR with activating mutations showed constitutive cAMP responses. These results provide important data on the signal transduction of constitutively active and inactive LHR mutants. Further studies should aim to identify the mechanism responsible for the significant increase in basal cAMP response in the constitutively activated eelLHR mutants.

Fusion protein therapeutics engineering is advancing to meet the need for novel medicine. Herein, we further characterize the development of novel RTA & PAP-S1 antiviral fusion proteins. In brief, RTA/PAP-S1 and PAP-S1/RTA fusion proteins were produced in both cell free and E. coli in vivo expression systems, purified by His-tag affinity chromatography, and protein synthesis inhibitory activity assayed by comparison to the production of a control protein, CalmL3. Results showed that the RTA/PAP-S1 fusion protein is amenable to standardized production and purification and has both increased potency and less toxicity compared to either RTA or PAP-S1 alone. Thus, this research highlights the developmental potential of novel fusion proteins with reduced cytotoxic risk and increased potency.

We analyzed signal transduction of three constitutively activating mutants (M410T, L469R, and D590Y) and two inactivating mutants (D417N and Y558F) of the eel luteinizing hormone receptor (eel LHR), known to occur in human LHR. The objective of this study was to assess the functional effects of these mutations in signal transduction and cell surface loss of receptor. Mutant receptors were transiently expressed in Chinese hamster ovary (CHO-K1) cells. Eel LH-stimulated accumulation of cyclic adenosine monophosphate (cAMP) was measured by homogeneous time-resolved fluorescence (HTRF) assays. The loss of receptors from the cells surface was measured using an enzyme-linked immunosorbent assay (ELISA) in human embryonic kidney (HEK) 293 cells. The cAMP response in cells expressing the wild type eel LHR was increased in a dose-dependent manner using eel LH ligand stimulation. Compared with the wild type, cells expressing the activating mutants (M410T, L469R, and D590Y), exhibited a 4.0-, 19.1-, and 7.8-fold increase in basal cAMP response without agonist stimulation, respectively. Their maximal responses to agonist stimulation were approximately 65%, 52%, and 98%, respectively, of those of the wild type. The inactivating mutants (D417N and Y558F) did not completely impair signal transduction, and their maximal responses were only 33% and25 % of those of wild type. These data clearly showed that the eel LHR-L469R and D590Y, activating mutants enhanced the rate of the loss of cell surface receptors following treatment with eel LH. Thus, the loss of cell surface receptors in cells expressing mutant eel LHRs was consistent with the eel LH agonist-induced production of cAMP. Our results suggested that the activation of the eel LHR requires appropriate loss of LHR-ligand complexes from the cell surface.

This study comprehensively evaluates the DNA/RNA Defend Pro (DRDP) sample collection buffer designed to inactivate and stabilize patient samples. The primary objectives encompassed assessing DRDP's efficacy in ensuring sample stability, facilitating extraction-free polymerase chain reaction (PCR), and ensuring compatibility with rapid antigen testing.Ninety-five diagnostic nasopharyngeal swab samples tested for influenza A/B, RSV A/B, and/or SARS-CoV-2 were diluted 1/10 with DRDP and anonymized. Stability testing involved incubating 31 positive samples at 4 °C, 20 °C, and 37 °C for 7 days, with regular extraction-free nucleic acid amplification testing (NAAT).Results demonstrated that DRDP guaranteed viral RNA stability at all temperatures for influenza, SARS-CoV-2 and RSV showing stability up to 7 days at 4 °C. DRDP-diluted samples exhibited robust compatibility with direct PCR, as 72 out of 88 samples positive for cobas Liat also tested positive for direct PCR. Non-concordant results could be explained by the 200-fold lower input in the extraction-free NAAT.Compared to NAAT, rapid antigen testing demonstrated lower sensitivity, as expected, while maintaining perfect specificity. Clear cutoffs between positive and negative rapid antigen testing results were observed in both cobas Liat and direct PCR analyses, validating DRDP's compatibility with these techniques. In conclusion, DRDP is an effective stabilizing medium compatible with direct PCR and rapid antigen testing and shows great potential for optimizing diagnostic processes, particularly in resource-limited or time-sensitive scenarios.

The castor plant (Ricinus communis L.) has been known since time immemorial in traditional medicine in the pharmacopeia of Mediterranean and eastern ancient cultures. Moreover, it is still used in folk medicine worldwide. Castor bean has been mainly recommended as anti-inflammatory, anthelmintic, anti-bacterial, laxative, abortifacient, for wounds, ulcers, and many other indications. Many cases of human intoxication occurred accidentally or voluntarily with the ingestion of castor seeds or derivatives. Ricinus toxicity depends on several molecules, among them the most important is ricin, a protein belonging to the family of ribosome-inactivating proteins. Ricin is the most studied of this category of proteins and it is also known to the general public, having been used for biocrimes in several cases. Here, the main steps of ricin research are reported with particular regards to its enzymatic activity, structure and cytotoxicity. Moreover, we discuss ricin toxicity for animals and humans, as well as the relation amongst bioterrorism and ricin and its impact on environmental toxicity. Ricin has also been of great utility to develop a number of immunotoxins specific for the elimination of unwanted cells, mainly cancer cells; some of these immunotoxins gave promising results also in clinical trials.