Protocol to reconstitute translationally arrested heat shock mRNPs and condensates in vitro.

Heat shock (HS) coincides with the assembly of translationally arrested heat shock messenger ribonucleoprotein particles (HS-mRNPs) and condensates. Here, we present a protocol to reconstitute HS-mRNPs and HS condensates with eIF4G, eIF4E, Pab1p, and mRNA in vitro. In addition, we describe the necessary steps to measure the effect of HS-mRNPs and HS condensates on translation in yeast extracts. The protocol can be modified to study mRNPs and condensates assembled with other proteins and to study translation in extracts prepared from different cells. For complete details on the use and execution of this protocol, please refer to Desroches Altamirano et al.1.

Structural and biochemical analysis of highly similar HLA-B allotypes differentially associated with type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease involving T cell-mediated destruction of the insulin-producing beta cells in the pancreatic islets of Langerhans. CD8+ T cells, responding to beta cell peptides presented by class I major histocompatibility complex (MHC) molecules, are important effectors leading to beta cell elimination. Human leukocyte antigen (HLA) B*39:06, B*39:01, and B*38:01 are closely related class I MHC allotypes that nonetheless show differential association with T1D. HLA-B*39:06 is the most predisposing of all HLA class I molecules and is associated with early age at disease onset. B*39:01 is also associated with susceptibility to T1D, but to a lesser extent, though differing from B*39:06 by only two amino acids. HLA-B*38:01, in contrast, is associated with protection from the disease. Upon identifying a peptide that binds to both HLA-B*39:06 and B*39:01, we determined the respective X-ray structures of the two allotypes presenting this peptide to 1.7 Å resolution. The peptide residues available for T cell receptor contact and those serving as anchors were identified. Analysis of the F pocket of HLA-B*39:06 and B*39:01 provided an explanation for the distinct peptide C-terminus preferences of the two allotypes. Structure-based modeling of the protective HLA-B*38:01 suggested a potential reason for its peptide preferences and its reduced propensity to present 8-mer peptides compared to B*39:06. Notably, the three allotypes showed differential binding to peptides derived from beta cell autoantigens. Taken together, our findings should facilitate identification of disease-relevant candidate T cell epitopes and structure-guided therapeutics to interfere with peptide binding.

CgIκB2 negatively regulates the expression of interferon-like protein by Rel/NF-κB signal in Crassostrea gigas.

Inhibitors of NF-κB (IκBs) have been implicated as major components of the Rel/NF-κB signaling pathway, playing an important negative regulatory role in host antiviral immunity such as in the activation of interferon (IFN) in vertebrates. In the present study, the immunomodulatory effect of IκB (CgIκB2) on the expression of interferon-like protein (CgIFNLP) was evaluated in Pacific oyster (Crassostrea gigas). After poly (I:C) stimulation, the mRNA expression level of CgIκB2 in haemocytes was significantly down-regulated at 3-12 h while up-regulated at 48-72 h. The mRNA expression of CgIκB2 in haemocytes was significantly up-regulated at 3 h after rCgIFNLP stimulation. In the CgIκB2-RNAi oysters, the mRNA expression of CgIFNLP, interferon regulatory factor-8 (CgIRF8) and NF-κB subunit (CgRel), the abundance of CgIFNLP and CgIRF8 protein in haemocytes, as well as the abundance of CgRel protein in nucleus were significantly increased after poly (I:C) stimulation. Immunofluorescence assay showed that nuclear translocation of CgIRF8 and CgRel protein was promoted in CgIκB2-RNAi oysters compared with that in EGFP-RNAi group. In the CgRel-RNAi oysters, the mRNA and protein expression level of CgIFNLP significantly down-regulated after poly (I:C) stimulation. The collective results indicated that CgIκB2 plays an important role in regulating CgIFNLP expression through its effects on Rel/NF-κB and IRF signaling pathways.

SCN1A intronic variants impact on Nav1.1 protein expression and sodium channel function, and associated with epilepsy phenotypic severity.

High-throughput sequencing has identified numerous intronic variants in the SCN1A gene in epilepsy patients. Abnormal mRNA splicing caused by these variants can lead to significant phenotypic differences, but the mechanisms of epileptogenicity and phenotypic differences remain unknown. Two variants, c.4853-1 G>C and c.4853-25 T>A, were identified in intron 25 of SCN1A, which were associated with severe Dravet syndrome (DS) and mild focal epilepsy with febrile seizures plus (FEFS+), respectively. The impact of these variants on protein expression, electrophysiological properties of sodium channels and their correlation with epilepsy severity was investigated through plasmid construction and transfection based on the aberrant spliced mRNA. We found that the expression of truncated mutant proteins was significantly reduced on the cell membrane, and retained in the cytoplasmic endoplasmic reticulum. The mutants caused a decrease in current density, voltage sensitivity, and an increased vulnerability of channel, leading to a partial impairment of sodium channel function. Notably, the expression of DS-related mutant protein on the cell membrane was higher compared to that of FEFS+-related mutant, whereas the sodium channel function impairment caused by DS-related mutant was comparatively milder than that caused by FEFS+-related mutant. Our study suggests that differences in protein expression levels and altered electrophysiological properties of sodium channels play important roles in the manifestation of diverse epileptic phenotypes. The presence of intronic splice site variants may result in severe phenotypes due to the dominant-negative effects, whereas deep intronic variants leading to haploinsufficiency could potentially cause milder phenotypes.

Molecular and serological investigations of Batai virus in cattle and goats in the border area of Yunnan, China (2021-2022).

Introduction: Batai virus (BATV), a zoonotic pathogen transmitted by mosquitoes, infects vertebrates, including livestock, birds, and humans. Although BATV has been detected and isolated in mosquitoes in Yunnan Province, China, there have been no reports of livestock infection. Thus, we conducted a molecular and serological investigation of BATV in cattle and goat sera collected in spring and autumn from 2021 to 2022 in Honghe Prefecture, Yunnan Province, on the China-Vietnam border. Methods: We used indirect enzyme-linked immunosorbent assays and reverse transcription real-time PCR (RT-qPCR) to test 929 cattle and 973 goat serum samples. Results: BATV antibodies were detected in 262/929 (28.2%) cattle and 263/973 (27.0%) goat serum samples. RT-qPCR did not detect BATV RNA. Discussion: The positive rate of BATV serum antibodies in cattle and goats in Luxi County was higher compared with other areas, and it was also higher in autumn compared with spring, which may be related to climate, temperature, and mosquito density. Although our findings indicated the presence of BATV infection in livestock in the region, RT-qPCR did not detect BATV RNA. Therefore, BATV monitoring in cattle and goats should be heightened in autumn, and the scope of host monitoring should be expanded to clarify the hosts and vectors of BATV infection.

Study of genotoxic and cytotoxic effects induced in human fibroblasts by exposure to pulsed and continuous 1.6 GHz radiofrequency.

Background: The widespread use of radiofrequency (RF) sources, ranging from household appliances to telecommunications devices and military equipment, raises concerns among people and regulatory agencies about the potential health risks of RF exposure. Consequently, several in vitro and in vivo studies have been done to investigate the biological effects, in particular non-thermal, of this non-ionizing radiation. To date, this issue is still being debated due to the controversial results that have been reported. Furthermore, the impact of different RF signal modulations on biological systems remains poorly investigated. The present in vitro study aims to evaluate the cytotoxicity and genotoxicity of continuous or pulsed 1.6 GHz RF in human dermal fibroblasts (HDF). Methods: HDF cultures were exposed to continuous and pulsed 1.6 GHz RF, for 2 h, with Specific Absorption Rate (SAR) of 0.4 W/kg. The potential biological effects of 1.6 GHz RF on HDF were assessed with a multi-methodological approach, analyzing the effects on cell cycle, ultrastructure, protein expression, mitotic spindle, CREST stained micronuclei, chromosome segregation and γ-H2AX/53BP1 foci. Results: 1.6 GHz RF exposure modified proteins expression and morphology of HDF. Specifically, the expression of different heat-shock proteins (HSP) (i.e., HSP-90, HSP-60, and HSP-25) and phospho-AKT were affected. In addition, both continuous and pulsed RF modified the cytoskeletal organization in HDF and increased the number of lysosomes, while the formation of autophagosomes was observed only after pulsed RF exposure. Mitotic spindle anomalies were also found after exposure. However, no significant effect was observed on cell cycle, chromosome segregation, CREST-stained micronuclei and γ-H2AX/53BP1 foci. Conclusion: The results of the present study show the absence of genotoxic damage in 1.6 GHz RF exposed HDF and, although mitotic spindle alterations were observed, they did not have an aneugenic effect. On the other hand, changes in some proteins expression and cell ultrastructure in exposed HDF suggest that RF can potentially induce cell alterations at the morphological and molecular levels.

IAPP - oligomerisation levels in plasma of people with type 2 diabetes.

Islet amyloid polypeptide (IAPP) is co-secreted with insulin from pancreatic ß-cells. Its oligomerisation is regarded as disease driving force in type 2 diabetes (T2D) pathology. Up to now, IAPP oligomers have been detected in affected tissues. IAPP oligomer concentrations in blood have not been analysed so far. Using the IAPP single-oligomer-sensitive and monomer-insensitive surface-based fluorescence intensity distribution analysis (sFIDA) technology, levels of IAPP oligomers in blood plasma from healthy controls and people with T2D in different disease stages where determined. Subsequently, the level of IAPP oligomerisation was introduced as the ratio between the IAPP oligomers determined with sFIDA and the total IAPP concentration determined with ELISA. Highest oligomerisation levels were detected in plasma of people with T2D without late complication and without insulin therapy. Their levels stand out significantly from the control group. Healthy controls presented with the lowest oligomerisation levels in plasma. In people with T2D without complications, IAPP oligomerisation levels correlated with disease duration. The results clearly demonstrate that IAPP oligomerisation in insulin-naïve patients correlates with duration of T2D. Although a correlation per se does not identify, which is cause and what is consequence, this result supports the hypothesis that IAPP aggregation is the driving factor of T2D development and progression. The alternative and conventional hypothesis explains development of T2D with increasing insulin resistance causing exhaustion of pancreatic ß-cells due to over-secretion of insulin, and thus IAPP, too, resulting in subsequent IAPP aggregation and fibril deposition in the pancreas. Further experiments and comparative analyses with primary tissues are warranted.

Development of a novel bacterial production system for recombinant bioactive proteins completely free from endotoxin contamination.

Endotoxins, or lipopolysaccharides (LPS), are potent immunostimulatory molecules of critical concern in bacterial recombinant protein expression systems. The gram-negative bacterium Acinetobacter baumannii exhibits an interesting and unique phenotype characterized by the complete loss of LPS. In this study, we developed a novel system for producing recombinant proteins completely devoid of endotoxin contamination using LPS-deficient A. baumannii. We purified endotoxin-free functional green fluorescent protein, which reduced endotoxin contamination by approximately three orders of magnitude, and also purified the functional cytokine tumor necrosis factor (TNF)-α. Additionally, utilization of the Omp38 signal peptide of A. baumannii enabled the extracellular production of variable domain of heavy chain of heavy chain (VHH) antibodies. With these advantages, mNb6-tri-20aa, a multivalent VHH that specifically binds to the spike protein of severe acute respiratory syndrome coronavirus 2, was purified from the culture supernatant, and endotoxin contamination was reduced by a factor of approximately 2 × 105 compared with that in conventional expression systems. A virus neutralization assay demonstrated the functionality of the purified antibody in suppressing viral infections. Moreover, we applied our system to produce ozoralizumab, a multispecific VHH that binds to human TNF-α and albumin and are marketed as a rheumatoid arthritis drug. We successfully purified a functional antibody from endotoxin contamination. This system establishes a new, completely endotoxin-free platform for the expression of recombinant proteins, which distinguishes it from other bacterial expression systems, and holds promise for future applications.

Molecular mechanism of regulating tat protein expression of pingganjiedu TCM in the treatment of AIDS based on network pharmacology.

AIDS is a serious disease with impaired immune function caused by human immunodeficiency virus (HIV) infection. The treatment of AIDS has always been the focus of global scientific research, and Tat protein is a key regulatory protein in the process of HIV infection. Its high expression is closely related to virus replication, disease progression, etc. The aim of this study is to explore the molecular mechanism of regulating Tat protein expression by using network pharmacology based traditional Chinese medicine for calming the liver and detoxifying. 129 AIDS patients were enrolled in the study and randomly divided into HAART combined with PGJDP treatment and HAART alone treatment groups. The virological response rate, immunological response status (CD4 + T cell level, CD4/CD8) and incidence of abnormal liver function were observed before and 48 weeks after treatment. Using the TCMSP database to obtain the chemical components and targets of the main traditional Chinese medicine components in PGJDP, clinical results indicate that the combination of HAART and PGJDP treatment can improve the virological response rate (P > 0.05); Increase the number of CD4 + T lymphocytes (P > 0.05); Significantly increased CD4/CD8 ratio (P < 0.01); Simultaneously, it significantly reduced the incidence of liver dysfunction (P < 0.01). After screening and analysis, the Chinese herbal medicine for calming liver and detoxifying has the potential to significantly regulate the expression of Tat protein. These Chinese herbal compounds can reduce the expression of Tat protein by affecting key pathways and regulating the expression of related genes, which has potential therapeutic effects on the treatment of AIDS.

Advances in silver nanoparticles: a comprehensive review on their potential as antimicrobial agents and their mechanisms of action elucidated by proteomics.

Nanoparticles play a crucial role in the field of nanotechnology, offering different properties due to their surface area attributed to their small size. Among them, silver nanoparticles (AgNPs) have attracted significant attention due to their antimicrobial properties, with applications that date back from ancient medicinal practices to contemporary commercial products containing ions or silver nanoparticles. AgNPs possess broad-spectrum biocidal potential against bacteria, fungi, viruses, and Mycobacterium, in addition to exhibiting synergistic effects when combined with certain antibiotics. The mechanisms underlying its antimicrobial action include the generation of oxygen-reactive species, damage to DNA, rupture of bacterial cell membranes and inhibition of protein synthesis. Recent studies have highlighted the effectiveness of AgNPs against various clinically relevant bacterial strains through their potential to combat antibiotic-resistant pathogens. This review investigates the proteomic mechanisms by which AgNPs exert their antimicrobial effects, with a special focus on their activity against planktonic bacteria and in biofilms. Furthermore, it discusses the biomedical applications of AgNPs and their potential non-preparation of antibiotic formulations, also addressing the issue of resistance to antibiotics.

Characterization of MAP c21873-1 as a new counter-selectable marker for unmarked genetic modification of Pichia pastoris.

BACKGROUND: Selection markers are useful in genetic modification of yeast Pichia pastoris. However, the leakage of the promoter caused undesired expression of selection markers especially those toxic proteins like MazF, halting the cell growth and hampering the genetic manipulation in procaryotic system. In this study, a new counter-selectable marker-based strategy has been established for seamless modification with high efficiency and low toxicity. RESULTS: At first, the leaky expression of the enhanced green fluorescent protein (EGFP) as a reporter gene under the control of six inducible promoters of P. pastoris was investigated in two hosts Escherichia coli and P. pastoris, respectively. The results demonstrated that the DAS1 and FDH1 promoters (PDAS1 and PFDH1) had the highest leakage expression activities in procaryotes and eukaryotes, and the DAS2 promoter (PDAS2) was inducible with medium strength but low leakage expression activity, all of which were selected for further investigation. Next, Mirabilis antiviral proteins (MAPs) c21873-1, c21873-1T (truncated form of c21873-1) and c23467 were mined as the new counter-selectable markers, and hygromycin B (Hyg B) resistance gene was used as the positive-selectable marker, respectively. Then, modular plasmids with MAP-target gene-Hyg B cassettes were constructed and used to transform into P. pastoris cells after linearization, and the target genes were integrated into its genome at the BmT1 locus through single-crossover homologous recombination (HR). After counter-selection induced by methanol medium, the markers c21873-1 and c21873-1T were recycled efficiently. But c23467 failed to be recycled due to its toxic effect on the P. pastoris cells. At last, the counter-selectable marker c21873-1 under the tightly regulated PDAS2 enabled the encoding genes of reporter EGFP and tested proteins to be integrated into the target locus and expressed successfully. CONCLUSIONS: We have developed MAP c21873-1 as a novel counter-selectable marker which could perform efficient gene knock-in by site-directed HR. Upon counter-selection, the marker could be recycled for repeated use, and no undesirable sequences were introduced except for the target gene. This unmarked genetic modification strategy may be extended to other genetic modification including but not limited to gene knock-out and site-directed mutagenesis in future.

Proteomic analysis of baicalin intervention on protein expression and modification in the hippocampus of Alzheimer's disease model rat.

OBJECTIVE: Aimed to explore the treatment effect and therapeutic mechanisms of baicalin in Alzheimer's disease (AD). METHODS: The AD rat model was established by intracerebroventricular injection of Aß1-40, with rats in the baicalin group receiving baicalin intraventricular injections. Morris Water Maze and hematoxylin-eosin (H&E) Staining were employed to detect the successful model construction and baicalin treatment effect. The proteins extracted from the hippocampus were subjected to proteomics analysis. Bioinformatics technology was employed for differential protein screening, functional classification, and enrichment. Western Blot was employed to validate the expressions of differentially expressed proteins (DEPs) and the protein modification alternations. RESULTS: Water maze test confirmed the successful AD model construction and baicalin can improve learning and memory abilities. 26 DEPs associated with 28 Gene Ontology (GO) functions were identified in the model and 32 DEPs were obtained between the baicalin group and the model. Bioinformatics analysis demonstrated that AD occurrence resulted in neuronal dysfunction and was associated with immune responses. The baicalin therapeutic effect on AD may be associated with metabolic processes, vitamin response, angiogenesis regulation, and fatty acid response. Immunoglobulin heavy constant mu (Ighm) and Immunoglobulin G2a (IgG2a) exhibited significant increases in AD and baicalin attenuated their expressions, while Fatty acid desaturase 1 (Fads1) exhibited a significantly diminished expression and baicalin could reverse the trend. Succinylation detection exhibited the differentially expressed at 35 kD between the model and baicalin group. CONCLUSION: Baicalin intervention may ameliorate cognitive impairment in AD rats by modulating the expressions of proteins and the succinylation modifications.

Expression of F1L, a vaccinia virus H3L transmembrane protein analogue of orf virus, and its successful purification as a diagnostic antigen.

Orf or contagious ecthyma is a highly contagious, zoonotic, and economically important global viral disease of small ruminants and is endemic in India. Vaccination of susceptible goats/sheep along with suitable recombinant protein-based serological assay will be useful in the control of the infection. In this study, the full-length and truncated versions of F1L encoding gene (ORF 059) of orf virus were cloned into pFasBac HT A vector, transformed in DH10Bac cells, and expressed in insect cells. The full-length and truncated recombinant F1L proteins were expressed as a 6 × histidine-tagged fusion protein for ease of purification by Ni-NTA affinity chromatography under denaturing conditions. A protein with ~ 40 kDa and ~ 35 kDa for full-length and truncated F1L protein, respectively, were expressed and confirmed by SDS-PAGE and western blot. The protein reactivity evaluated by western blot analysis and indirect ELISA using ORFV hyperimmune serum was also found to be reactive. The results of the present study showed that the purified recombinant F1L protein can be used as a diagnostic antigen in sero-surveillance of ORFV infection in small ruminants. To the best of authors' knowledge, this is the first report on the expression of ORFV F1L in insect cells using a baculovirus vector and its successful purification to use as the potential diagnostic antigen in ELISA.

Strategic approaches for designing yeast strains as protein secretion and display platforms.

Yeast has been established as a versatile platform for expressing functional molecules, owing to its well-characterized biology and extensive genetic modification tools. Compared to prokaryotic systems, yeast possesses advanced cellular mechanisms that ensure accurate protein folding and post-translational modifications. These capabilities are particularly advantageous for the expression of human-derived functional proteins. However, designing yeast strains as an expression platform for proteins requires the integration of molecular and cellular functions. By delving into the complexities of yeast-based expression systems, this review aims to empower researchers with the knowledge to fully exploit yeast as a functional platform to produce a diverse range of proteins. This review includes an exploration of the host strains, gene cassette structures, as well as considerations for maximizing the efficiency of the expression system. Through this in-depth analysis, the review anticipates stimulating further innovation in the field of yeast biotechnology and protein engineering.

Non-immune factors cause prolonged myofibroblast phenotype in implanted synthetic heart valve scaffolds.

The clinical application of heart valve scaffolds is hindered by complications associated with the activation of valvular interstitial cell-like (VIC-like) cells and their transdifferentiation into myofibroblasts. This study aimed to examine several molecular pathway(s) that may trigger the overactive myofibroblast phenotypes in the implanted scaffolds. So, we investigated the influence of three molecular pathways - macrophage-induced inflammation, the TGF-ß1-SMAD2, and WNT/ß-catenin ß on VIC-like cells during tissue engineering of heart valve scaffolds. We implanted electrospun heart valve scaffolds in adult sheep for up to 6 months in the right ventricular outflow tract (RVOT) and analyzed biomolecular (gene and protein) expression associated with the above three pathways by the scaffold infiltrating cells. The results showed a gradual increase in gene and protein expression of markers related to the activation of VIC-like cells and the myofibroblast phenotypes over 6 months of scaffold implantation. Conversely, there was a gradual increase in macrophage activity for the first three months after scaffold implantation. However, a decrease in macrophage activity from three to six months of scaffold tissue engineering suggested that immunological signal factors were not the primary cause of myofibroblast phenotype. Similarly, the gene and protein expression of factors associated with the TGF-ß1-SMAD2 pathway in the cells increased in the first three months but declined in the next three months. Contrastingly, the gene and protein expression of factors associated with the WNT/ß-catenin pathway increased significantly over the six-month study. Thus, the WNT/ß-catenin pathway could be the predominant mechanism in activating VIC-like cells and subsequent myofibroblast phenotype.

Protocol for the purification of replisomes from the Xenopus laevis egg extract system for single-particle cryo-EM analysis.

Here, we present a large-scale FLAG immunoprecipitation protocol to isolate large protein complexes driving DNA replication at replicating chromatin assembled in Xenopus laevis egg extract. We describe how to prepare demembranated sperm nuclei (DNA) and low-speed supernatant egg extract (LSS) and present detailed procedures for sample preparation and application onto grids for negative stain electron microscopy (NS-EM) and cryoelectron microscopy (cryo-EM). For complete details on the use and execution of this protocol, please refer to Cvetkovic et al.1.

Protocol for the production and reconstitution of VDAC1 for functional assays.

The voltage-dependent anion channel (VDAC) is an abundant and multifunctional outer mitochondrial membrane protein, playing key roles in neurodegeneration, apoptosis, and mitochondrial membrane biogenesis. Here, we present a protocol to produce and reconstitute high yields of detergent-solubilized VDAC, expressed as inclusion bodies in E. coli. We describe steps for purification by affinity chromatography and refolding in lauryldimethylamine-N-oxide (LDAO). We then detail procedures for reconstituting VDAC into membrane vesicles to assay its channel and phospholipid scramblase activity via fluorescence-based assays. For complete details on the use and execution of this protocol, please refer to Bergdoll et al.,1 Queralt-Martín et al., 2 and Jahn et al.3.

Strenuous expression of porcine epidemic diarrhea virus ORF3 protein suggests host resistance.

Porcine epidemic diarrhea virus is attenuated upon adaptation to cell culture. Exclusively genomic mutations have been traced to the ORF3 gene of the laboratory strains. Previous attempts to express the protein were unsuccessful. We sought to express the ORF3 protein in both mammalian and bacteria cells as a prerequisite for investigation of the protein's role. For prokaryotic expression, two vector systems, pET28-a(+) and pGEX-4T-1 were constructed and expressed in Escherichia coli cells. For eukaryotic analyses, ORF3/pEGFP-C1 vector constructs were expressed in human embryonic, green monkey kidney and mouse fibrous cells. Intriguingly, there was minimal expression of the ORF3 gene. Following a documented hint that truncated ORF3 revealed higher expression, ORF3 gene was truncated. The simple modular architecture research tool analysis predicted two transmembrane domains between amino acid (aa) 41-63 and aa 76-98. Consequently, we generated two fragments; ORF-N (aa 1-98) inclusive of transmembrane domains and ORF3-C (aa 99-224). These truncated sequences were constructed as the whole gene here referred to as ORF3 wild type (wt). Coomassie blue stained gels revealed bands of ORF3-C expressed as a fusion protein of 17.5 and 39 kDa in pET28-a(+) and pGEX-4T-1 vectors, respectively. In contrast, ORF3-N was not. Additionally, ORF3-N induction decreased total cellular proteins suggesting inhibition of protein synthesis or metabolism. Solubility tests carried out at 30 °C, 25 °C and 18 °C showed that ORF3 formed inclusion bodies. Similar findings were observed in mammalian cells. Noteworthy, morphological distortions appeared in mammalian cells expressing ORF3 protein or its truncated mutants suggesting significance in host viability.

RiboScreenTM Technology Delivers a Ribosomal Target and a Small-Molecule Ligand for Ribosome Editing to Boost the Production Levels of Tropoelastin, the Monomeric Unit of Elastin.

Elastin, a key structural protein essential for the elasticity of the skin and elastogenic tissues, degrades with age. Replenishing elastin holds promise for anti-aging cosmetics and the supplementation of elastic activities of the cardiovascular system. We employed RiboScreenTM, a technology for identifying molecules that enhance the production of specific proteins, to target the production of tropoelastin. We make use of RiboScreenTM in two crucial steps: first, to pinpoint a target ribosomal protein (TRP), which acts as a switch to increase the production of the protein of interest (POI), and second, to identify small molecules that activate this ribosomal protein switch. Using RiboScreenTM, we identified ribosomal protein L40, henceforth eL40, as a TRP switch to boost tropoelastin production. Drug discovery identified a small-molecule hit that binds to eL40. In-cell treatment demonstrated activity of the eL40 ligand and delivered increased tropoelastin production levels in a dose-dependent manner. Thus, we demonstrate that RiboScreenTM can successfully identify a small-molecule hit capable of selectively enhancing tropoelastin production. This compound has the potential to be developed for topical or systemic applications to promote skin rejuvenation and to supplement elastic functionality within the cardiovascular system.

Discordant ALK Status in Non-Small Cell Lung Carcinoma: A Detailed Reevaluation Comparing IHC, FISH, and NGS Analyses.

ALK detection was performed on 2813 EGFR-unmutated NSCLC cases by simultaneous use of immunohistochemistry (VENTANA® anti-ALK D5F3, Roche Molecular Systems, Inc., Rotkreuz, Switzerland) and fluorescence in situ hybridization with the ALK break apart and the ALK/EML4 fusion probe (ZytoVision, Bremerhaven, Germany). A total of 33 cases were positive discordant (FISH-positive, IHC-negative) and 17 cases were negative discordant (FISH-negative, IHC-positive). This study's aim was to reevaluate the methods used and compare discordant samples to positive concordant samples in order to ellucidate the differences. FISH signal variants were examined and compared. Positive discordant cases featured one pattern of ALK rearrangement in 41.4%, two patterns in 48.3%, and three patterns in 10.3% of analysed samples, with a higher variability of detected patterns and a higher number of ALK copy gains. Positive concordant cases displayed one pattern of rearrangement in 82%, two patterns in 17.8%, and three patterns in 0.6% of analysed samples. The association between number of patterns and concordance/discordance was statistically significant (p < 0.05). Eleven positive discordant and two negative concordant cases underwent NGS analysis, which resulted in identification of ALK fusion in one positive discordant and two negative discordant cases. Positive protein expression regardless of FISH result correlated more with a positive NGS result compared to samples with a positive FISH result with negative protein expression. FISH analysis was able to detect atypical or heterogenous patterns of rearrangement in a proportion of cases with negative protein expression, which may be associated with more extensive genetic alterations rather than true ALK rearrangement.