Functional Characterization Research Articles

Cloning and Functional Characterization of Novel Human Neutralizing Anti-IFN-α and Anti-IFN-β Antibodies.

Type I IFNs play a pivotal role in immune response modulation, yet dysregulation is implicated in various disorders. Therefore, it is crucial to develop tools that facilitate the understanding of their mechanism of action and enable the development of more effective anti-IFN therapeutic strategies. In this study, we isolated, cloned, and characterized anti-IFN-α and anti-IFN-β Abs from PBMCs of individuals treated with IFN-α or IFN-β, harboring confirmed neutralizing Abs. Clones AH07856 and AH07857 were identified as neutralizing anti-IFN-α-specific with inhibition against IFN-α2a, -α2b, and -αK subtypes. Clones AH07859 and AH07866 were identified as neutralizing anti-IFN-β1a-specific signaling and able to block lipopolysaccharide or S100 calcium-binding protein A14-induced IFN-β signaling effects. Cloned Abs bind rhesus but not murine IFNs. The specificity of inhibition between IFN-α and IFN-β suggests potential for diverse research and clinical applications.

Genome-wide identification of TCP transcription factors and their potential roles in hydrolyzable tannin production in Quercus variabilis cupule

Hydrolyzable tannins (HTs) have garnered significant attention due to their proven beneficial effects in the clinical treatment of various diseases. The cupule of Chinese cork oak (Quercus variabilis Blume) has been used as raw material of traditional medicine for centuries for its high content of HTs. Previous studies have identified UGT84A13 as a key enzyme in the HT biosynthesis pathway in Q. variabilis, but the transcriptional regulation network of UGT84A13 remains obscure. Here, we performed a comprehensive genome-wide identification of the TCP transcription factors in Q. variabilis, elucidating their molecular evolution and gene structure. Gene expression analysis showed that TCP3 from the CIN subfamily and TCP6 from the PCF subfamily were co-expressed with UGT84A13 in cupule. Further functional characterization using dual-luciferase assays confirmed that TCP3, rather than TCP6, played a role in the transcriptional regulation of UGT84A13, thus promoting HT biosynthesis in the cupule of Q. variabilis. Our work identified TCP family members in Q. variabilis for the first time, and provided novel insights into the transcriptional regulatory network of UGT84A13 and HT biosynthesis in Q. variabilis, explaining the reason why the cupule enriches HTs that could be used for traditional medicine.

Acinetobacter baumannii OmpA-like porins: functional characterization of bacterial physiology, antibiotic-resistance, and virulence

Acinetobacter baumannii is a critical opportunistic pathogen associated with nosocomial infections. The high rates of antibiotic-resistance acquisition make most antibiotics ineffective. Thus, new medical countermeasures are urgently needed. Outer membrane proteins (OMPs) are prime candidates for developing novel drug targets and antibacterial strategies. However, there are substantial gaps in our knowledge of A. baumannii OMPs. This study reports the impact of OmpA-like protein on bacterial physiology and virulence in A. baumannii strain AB5075. We found that PsaB (ABUW_0505) negatively correlates to stress tolerance, while ArfA (ABUW_2730) significantly affects bacterial stiffness, cell shape, and cell envelope thickness. Furthermore, we expand our knowledge on YiaD (ABUW_3045), demonstrating structural and virulence roles of this porin, in addition to meropenem resistance. This study provides solid foundations for understanding how uncharacterized OMPs contribute to A. baumannii's physiological and pathological processes, aiding the development of innovative therapeutic strategies against A. baumannii infections.

Process optimization in the pyroconversion of native sweet potato starch: structural and functional characterization of pyrodextrin

This work aims to produce dextrin from sweet potato (Ipomoea batatas) starch by pyroconversion. The effect of dextrinization process (roasting time, temperature, and hydrochloric acid concentration) on properties (structural and functional) of starch have been studied using central composite design of experiment. Reaction conditions included: roasting time (55 and 94 min), temperature (127 and 152 °C), and hydrochloric acid concentration (0.17 and 0.43 M). The temperature (quadratic effect X22) is highly significant on the three measured responses (p < 0.05) (Color Index (CI), Turbidity (Turb), and Saccharification Percentage (SP)). Also, the findings indicated that the two most important parameters influencing the pyroconversion of sweet potato starch in the current study were temperature (X2) and hydrochloric acid concentration (X3). The optimal conditions for dextrin production obtained are 94 min, 127 °C and 0.43 M for roasting time, temperature, and hydrochloric acid concentration respectively having 97% of solubility. The FTIR analysis revealed that OH, C-H, C-O, C–C and C-O–H were the main functional groups. These functional groups remained unaffected by the pyroconversion, since the bands extend and the spectrums of all native and pyrodextrin were similar. X-Ray Diffractometry analysis results of dextrin showed that the thermal treatment reduces the crystallinity of starch. Also, this analysis shows starch has CA-type XRD patterns. Scanning Electron Microscopy (SEM) analysis results showed that the defined optimal production maintains the morphology of starch. Differential scanning calorimetry (DSC) used for thermal analysis helps to obtain the glass transition temperature of modified starch equals to 139 °C. Optimal conditions of production of pyrodextrins from sweet potato granule starches could be easily produced with improved physicochemical properties to be used as encapsulating agent or additives in food and chemical industries.

47 Generation and Characterization of Ex Vivo Expanded Tumor-Infiltrating Lymphocytes from Renal Cell Carcinoma Tumors for Adoptive Cell Therapy

Abstract Background Autologous therapeutic tumor-infiltrating lymphocyte (TIL) therapy is a promising strategy to enhance anti-tumor immunity. Optimization of ex vivo TIL expansion could expand current immunotherapy options. Previous attempts to generate TIL in renal cell carcinoma (RCC) have been technically challenging. We applied a second-generation manufacturing process, currently used to generate the melanoma TIL product lifileucel, in RCC. Methods Resected primary and metastatic RCC samples were processed using the Gen 2 manufacturing process comprising of pre-Rapid Expansion Protocol (pre-REP) and REP steps. We assessed REP TILs for viability and performed phenotypic and functional characterization. We correlated the tumor immune microenvironment (TIME) with successful TIL expansion. Results Eight of 11 RCC samples underwent successful REP. Three failed cases demonstrated low CD8/FoxP3 ratio and high expression of PD-1 within FoxP3 cells. Expression of exhaustion markers differed between the TIME and expanded TILs; the latter had a TIM3-high/PD-1-low phenotype but retained functional capacity comparable to lifileucel. Figure: Assessment of tumor immune microenvironment (TIME) via multiplex immunofluorescence (IF). Cases that failed REP are indicated in red. (A) Representative image at 60x magnification, fluorophore colors as indicated. (B) Density of tumor-infiltrating CD8+ and FoxP3+ cells. (C) Expression of co-inhibitory exhaustion markers as a percentage of total CD8+ cells. (D) Expression of PD-1 in FoxP3+ cells. (E) Comparison of tissue TIME (dots) with TIL product post-REP (open circles) exhaustion profiles as a percentage of total CD8+ cells. Conclusions The Gen 2 manufacturing process used for lifileucel successfully expanded functional TILs from RCC samples, enabling further study in a clinical trial. TIME features such as low CD8/FoxP3 ratio and high PD-1 expression within FoxP3 cells warrant study as potential biomarkers of successful TIL expansion.

Disruption of KLHL6 Fuels Oncogenic Antigen Receptor Signaling in B-cell Lymphoma.

Pathomechanisms that activate oncogenic B-cell receptor (BCR) signaling in diffuse large B-cell lymphoma (DLBCL), are largely unknown. Kelch-like family member 6 (KLHL6) encoding a substrate-adapter for Cullin-3-RING E3 ubiquitin-ligase (CRL) with poorly established targets is recurrently mutated in DLBCL. By applying high-throughput protein interactome screens and functional characterization, we discovered that KLHL6 regulates BCR by targeting its signaling subunits CD79A and CD79B. Loss of physiological KLHL6 expression pattern was frequent among the MCD/C5-like activated B-cell DLBCLs and was associated with higher CD79B levels and dismal outcome. Mutations in the BTB domain of KLHL6 disrupted its localization and heterodimerization, and increased surface BCR levels and signaling, whereas Kelch domain mutants had the opposite effect. Malfunctions of KLHL6 mutants extended beyond proximal BCR signaling with distinct phenotypes from KLHL6 silencing. Collectively, our findings uncover how recurrent mutations in KLHL6 alter BCR signaling and induce actionable phenotypic characteristics in DLBCL.

Discovery and characterization of a novel LysinB from F2 sub-cluster mycobacteriophage RitSun

The escalating antibiotic resistance in mycobacterial species poses a significant threat globally, necessitating an urgent need to find alternative solutions. Bacteriophage-derived endolysins, which facilitate phage progeny release by attacking bacterial cell walls, present promising antibacterial candidates due to their rapid lytic action, high specificity and low risk of resistance development. In mycobacteria, owing to the complex, hydrophobic cell wall, mycobacteriophages usually synthesize two endolysins: LysinA, which hydrolyzes peptidoglycan; LysinB, which delinks mycolic acid-containing outer membrane and arabinogalactan, releasing free mycolic acid. In this study, we conducted domain analysis and functional characterization of a novel LysinB from RitSun, an F2 sub-cluster mycobacteriophage from our phage collection. Several key properties of RitSun LysinB make it an important antimycobacterial agent: its ability to lyse Mycobacterium smegmatis from without, a higher than previously reported specific activity of 1.36 U/mg and its inhibitory effect on biofilm formation. Given the impermeable nature of the mycobacterial cell envelope, dissecting RitSun LysinB at the molecular level to identify its cell wall-destabilizing sequence could be utilized to engineer other native lysins as fusion proteins, broadening their activity spectrum.

Gene Expression Analysis of the Early Flowering 6 Homologues in Apricot Reveals Their Potential Role in Developmental Stages

In higher plants, regulation of gene expression and chromatin formation occurs by histone methylation and demethylation. Genes encoding JmjC-JmjN domains belong to the histone demethylase family and have an important role in the regulation of plant growth and development. Early Flowering 6 (AtELF6), which encodes the JmjC-JmjN domain in Arabidopsis thaliana, is a demethylase that regulates growth and development as well as the transition to flowering, but it has not been identified in apricot so far. In this study, two genes homologous to AtELF6 were identified for the first time in apricot. Gene expression analysis by RT-qPCR revealed that both ELF6 homologs were expressed in 12 different developmental stages of three different tissues. The fact that both homologues were expressed, especially in the flower bud, suggested that they play a role in the transition to flowering, similar to Arabidopsis thaliana. In summary, the information obtained from this study will provide a unique resource for understanding the role of ELF 6 in apricot growth and development, as well as for future functional characterization studies for the manipulation of the flowering transition.

Insight into the role of TXNRD2 in steroidogenesis through a novel homozygous TXNRD2 splice variant.

Adrenal cortisol production occurs through a biosynthetic pathway which depend on NADH and NADPH for energy supply. The mitochondrial respiratory chain and the reactive oxygen species (ROS) detoxification system are therefore important for steroidogenesis. Mitochondrial dysfunction leading to oxidative stress has been implicated in the pathogenesis of several adrenal conditions. Nonetheless, only very few patients with variants in one gene of the ROS detoxification system, Thioredoxin Reductase 2 (TXNRD2), have been described with variable phenotypes. Clinical, genetic, structural, and functional characterization of a novel, biallelic TXNRD2 splice variant. On human biomaterial, we performed whole exome sequencing to identify and RNA analysis to characterize the specific TXNRD2 splice variant. Amino acid conservation analysis and protein structure modeling were performed in silico. Using patient's fibroblast-derived human induced pluripotent stem cells, we generated adrenal-like cells (iALC) to study the impact of wild-type (WT) and mutant TXNRD2 on adrenal steroidogenesis and ROS production. The patient had a complex phenotype of primary adrenal insufficiency (PAI), combined with genital, ophthalmological, and neurological features. He carried a homozygous splice variant c.1348-1G > T in TXNRD2 which leads to a shorter protein lacking the C-terminus and thereby affecting homodimerization and flavin adenine dinucleotide binding. Patient-derived iALC showed a loss of cortisol production with overall diminished adrenal steroidogenesis, while ROS production was significantly increased. Lack of TXNRD2 activity for mitochondrial ROS detoxification affects adrenal steroidogenesis and predominantly cortisol production.

Cell wall-associated receptor kinase GhWAKL26 positively regulates salt tolerance by maintaining Na+ and K+ homeostasis in cotton

Cell wall-associated receptor kinases (WAKs/WAKLs), are a specialized class of plant receptor kinases essential for signaling during stress conditions. However, there has been no report on the involvement of WAKs/WAKLs in salt tolerance in cotton. In this study, we report the functional characterization of GhWAKL26, whose expression is induced by salt, with its levels increasing over time and with higher salt concentrations. In addition, the fusion protein of GhWAKL26 and GFP was localized to the plasma membrane. In transgenic Arabidopsis, the dry weight, fresh weight, and root length were significantly higher than those of wild-type plants, indicating enhanced salt tolerance. While in GhWAKL26-silenced cotton seedlings, H2O2, O2−, and MDA content were increased, and chlorophyll content was reduced under salt stress, displaying compromised salt tolerance. RNA-seq analysis revealed that the silencing of GhWAKL26 resulted in the down-regulation of expression levels of certain ion transport-related genes under salt stress, concurrently leading to an increased Na+/K+ ratio in cotton seedlings. Overall, our findings indicate that GhWAKL26 enhanced plant resistance to salt stress in cotton by regulating the balance of Na+ and K+ ions.

Extracellular vesicles from the microalga Tetraselmis chuii are biocompatible and exhibit unique bone tropism along with antioxidant and anti-inflammatory properties

Extracellular vesicles (EVs) are membrane-enclosed bio-nanoparticles secreted by cells and naturally evolved to transport various bioactive molecules between cells and even organisms. These cellular objects are considered one of the most promising bio-nanovehicles for the delivery of native and exogenous molecular cargo. However, many challenges with state-of-the-art EV-based candidates as drug carriers still exist, including issues with scalability, batch-to-batch reproducibility, and cost-sustainability of the final therapeutic formulation. Microalgal extracellular vesicles, which we named nanoalgosomes, are naturally released by various microalgal species. Here, we evaluate the innate biological properties of nanoalgosomes derived from cultures of the marine microalgae Tetraselmis chuii, using an optimized manufacturing protocol. Our investigation of nanoalgosome biocompatibility in preclinical models includes toxicological analyses, using the invertebrate model organism Caenorhabditis elegans, hematological and immunological evaluations ex vivo and in mice. We evaluate nanoalgosome cellular uptake mechanisms in C. elegans at cellular and subcellular levels, and study their biodistribution in mice with accurate space-time resolution. Further examination highlights the antioxidant and anti-inflammatory bioactivities of nanoalgosomes. This holistic approach to nanoalgosome functional characterization demonstrates that they are biocompatible and innate bioactive effectors with unique bone tropism. These findings suggest that nanoalgosomes have significant potential for future therapeutic applications.

Protein-extending ACTN2 frameshift variants cause variable myopathy phenotypes by protein aggregation.

The objective of the study is to characterize the pathomechanisms underlying actininopathies. Distal myopathies are a group of rare, inherited muscular disorders characterized by progressive loss of muscle fibers that begin in the distal parts of arms and legs. Recently, variants in a new disease gene, ACTN2, have been shown to cause distal myopathy. ACTN2, a gene previously only associated with cardiomyopathies, encodes alpha-actinin-2, a protein expressed in both cardiac and skeletal sarcomeres. The primary function of alpha-actinin-2 is to link actin and titin to the sarcomere Z-disk. New ACTN2 variants are continuously discovered; however, the clinical significance of many variants remains unknown. Thus, lack of clear genotype-phenotype correlations in ACTN2-related diseases, actininopathies, persists. Functional characterization in C2C12 cell model of several ACTN2 variants is conducted, including frameshift and missense variants associated with dominant and recessive actininopathies. We assess the genotype-phenotype correlations of actininopathies using clinical data from several patients carrying these variants. The results show that the missense variants associated with a recessive form of actininopathy do not cause detectable alpha-actinin-2 aggregates in the cell model. Conversely, dominant frameshift variants causing a protein extension do form alpha-actinin-2 aggregates. The results suggest that alpha-actinin-2 aggregation is the disease mechanism underlying some dominant actininopathies, and thus, we recommend that protein-extending frameshift variants in ACTN2 should be classified as pathogenic. However, this mechanism is likely elicited by only a limited number of variants. Alternative functional characterization methods should be explored to further investigate other molecular mechanisms underlying actininopathies.

Characterization of Weyl Functions in the Class of Operator-Valued Generalized Nevanlinna Functions

Characterization of Weyl Functions in the Class of Operator-Valued Generalized Nevanlinna Functions

Functional characterization of BbEaf6 in Beauveria bassiana: Implications for fungal virulence and stress response

ABSTRACT The Eaf6 protein, a conserved component of the NuA4 and NuA3 complexes in yeast and MOZ/MORF complexes in humans, plays crucial roles in transcriptional activation, gene regulation, and cell cycle control. Despite its significance in other organisms, the functional role of Eaf6 in entomopathogenic fungi (EPF) remained unexplored. Here, we investigate the function of BbEaf6, the Eaf6 homolog in the entomopathogenic fungus Beauveria bassiana. We demonstrate that BbEaf6 is predominantly localized in nuclei, similar to its counterpart in other fungi. Deletion of BbEaf6 resulted in delayed conidiation, reduced conidial yield, and altered conidial properties. Transcriptomic analysis revealed dysregulation of the genes involved in asexual development and cell cycle progression in the ΔBbEaf6 mutant. Furthermore, the ΔBbEaf6 mutant exhibited decreased tolerance to various stresses, including ionic stress, cell wall perturbation, and DNA damage stress. Notably, the ΔBbEaf6 mutant displayed attenuated virulence in insect bioassays, accompanied by dysregulation of genes associated with cuticle penetration and haemocoel infection. Overall, our study elucidates the multifaceted role of BbEaf6 in stress response, development, and virulence in B. bassiana, providing valuable insights into the molecular mechanisms governing fungal pathogenesis and potential targets for pest management strategies.

Genome-Wide Identification of Glutathione S-Transferase Family from Dendrobium officinale and the Functional Characterization of DoGST5 in Cadmium Tolerance

Genome-Wide Identification of Glutathione S-Transferase Family from Dendrobium officinale and the Functional Characterization of DoGST5 in Cadmium Tolerance

H3K36 methyltransferase GhKMT3;1a and GhKMT3;2a promote flowering in upland cotton

BackgroundThe SET domain group (SDG) genes encode histone lysine methyltransferases, which regulate gene transcription by altering chromatin structure and play pivotal roles in plant flowering determination. However, few studies have investigated their role in the regulation of flowering in upland cotton.ResultsA total of 86 SDG genes were identified through genome-wide analysis in upland cotton (Gossypium hirsutum). These genes were unevenly distributed across 25 chromosomes. Cluster analysis revealed that the 86 GhSDGs were divided into seven main branches. RNA-seq data and qRT‒PCR analysis revealed that lysine methyltransferase 3 (KMT3) genes were expressed at high levels in stamens, pistils and other floral organs. Using virus-induced gene silencing (VIGS), functional characterization of GhKMT3;1a and GhKMT3;2a revealed that, compared with those of the controls, the GhKMT3;1a- and GhKMT3;2a-silenced plants exhibited later budding and flowering and lower plant heightwere shorter. In addition, the expression of flowering-related genes (GhAP1, GhSOC1 and GhFT) significantly decreased and the expression level of GhSVP significantly increased in the GhKMT3;1a- and GhKMT3;2a-silenced plants compared with the control plants.ConclusionA total of 86 SDG genes were identified in upland cotton, among which GhKMT3;1a and GhKMT3;2a might regulate flowering by affecting the expression of GhAP1, GhSOC1, GhFT and GhSVP. These findings will provide genetic resources for advanced molecular breeding in the future.

Multiplexed mapping of the interactome of GPCRs with receptor activity-modifying proteins.

Receptor activity-modifying proteins (RAMPs) form complexes with G protein-coupled receptors (GPCRs) and may regulate their cellular trafficking and pharmacology. RAMP interactions have been identified for about 50 GPCRs, but only a few GPCR-RAMP complexes have been studied in detail. To elucidate a comprehensive GPCR-RAMP interactome, we created a library of 215 dual epitope-tagged (DuET) GPCRs representing all GPCR subfamilies and coexpressed each GPCR with each of the three RAMPs. Screening the GPCR-RAMP pairs with customized multiplexed suspension bead array (SBA) immunoassays, we identified 122 GPCRs that showed strong evidence for interaction with at least one RAMP. We screened for interactions in three cell lines and found 23 endogenously expressed GPCRs that formed complexes with RAMPs. Mapping the GPCR-RAMP interactome expands the current system-wide functional characterization of RAMP-interacting GPCRs to inform the design of selective therapeutics targeting GPCR-RAMP complexes.

Dissection of the synthesis of polyunsaturated fatty acids in nematodes and Collembola of the soil fauna

It is becoming increasingly clear that not only unicellular, photoautotrophic eukaryotes, plants, and fungi, but also invertebrates are capable of synthesizing ω3 long-chain polyunsaturated fatty acids (LC-PUFA) de novo. However, the distribution of this anabolic capacity among different invertebrate groups and its implementation at the gene and protein level are often still unknown. This study investigated the PUFA pathways in common soil fauna, i.e. two nematode and two Collembola species. Of these, one species each (Panagrellus redivivus, Folsomia candida) was assumed to produce ω3 LC-PUFA de novo, while the others (Acrobeloides bodenheimeri, Isotoma caerulea) were supposed to be unable to do so. A highly labeled oleic acid (99 % 13C) was supplemented and the isotopic signal was used to trace its metabolic path. All species followed the main pathway of lipid biosynthesis. However, in A. bodenheimeri this terminated at arachidonic acid (ω6 PUFA), whereas the other three species continued the pathway to eicosapentaenoic acid (ω3 PUFA), including I. caerulea. For the nematode P. redivivus the identification and functional characterization of four new fatty acid desaturase (FAD) genes was performed. These genes encode the FAD activities Δ9, Δ6, and Δ5, respectively. Additionally, the Δ12 desaturase was analyzed, yet the observed activity of an ω3 FAD could not be attributed to a coding gene. In the Collembola F. candida, 11 potential first desaturases (Δ9) and 13 front-end desaturases (Δ6 or Δ5 FADs) have been found. Further sequence analysis indicates the presence of omega FADs, specifically Δ12, which are likely derived from Δ9 FADs.

Functional Characterization of the Paeonia ostii P5CS Gene under Drought Stress

Functional Characterization of the Paeonia ostii P5CS Gene under Drought Stress

Functional Characterization of ABCA4 Missense Variants Aids Variant Interpretation and Phenotype Prediction in Patients With ABCA4-Retinal Dystrophies.

Biallelic pathogenic variants in the gene encoding the ATP-binding cassette transporter ABCA4 are the leading cause of irreversible vision loss in inherited retinal dystrophies (IRDs). Interpretation of ABCA4 variants is challenging, due to cis-modifying and hypomorphic variants. We have previously detected 10 missense variants of unknown significance (VUS) in patients with suspected ABCA4-retinal dystrophies (ABCA4-RDs) in Norway. In this study, we functionally characterized the VUS to aid interpretation of the variants and to determine if they are associated with the disease. The ABCA4 VUS were expressed in HEK293T cells and the ABCA4 expression level and ATPase activity were determined and correlated with the patients' phenotype. The functional data further used for reclassification of the VUS following the American College of Medical Genetics and Genomics (ACMG) guidelines. Of the 10 VUSs, 2 variants, Cys205Phe and Asn415Thr, were categorized as functionally severe. The age at presentation in the 2 patients carrying these variants was divergent and seemed to be driven by the patients' second pathogenic variants Gly1961Glu and c.5461-10T>C, respectively. Three variants, Val643Gly, Pro799Leu, and Val1433Ile were categorized as functionally moderate, and were found in patients with intermediate/late age at presentation. The remaining five variants were categorized as functionally normal/mild. Based on our data, c.614G>T p.(Cys205Phe), c.1244A>C p.(Asn415Thr), and c.2396C>T p.(Pro799Leu) were reclassified to (likely) pathogenic, while 4 of the functionally normal/mild variants could be reclassified to likely benign. Functional analyses of ABCA4 variants are a helpful tool in variant classification and enable us to better predict the disease severity in patients with ABCA4-RDs.