Thioredoxin-like Protein Research Articles

A symbiont fungal effector relocalizes a plastidic oxidoreductase to nuclei to induce resistance to pathogens and salt stress

The root endophytic fungus Serendipita indica establishes beneficial symbioses with a broad spectrum of plants and enhances host resilience against biotic and abiotic stresses. However, little is known about the mechanisms underlying S.indica-mediated plant protection. Here, we report S.indica effector (SIE) 141 and its host target CDSP32, a conserved thioredoxin-like protein, and underlying mechanisms for enhancing pathogen resistance and abiotic salt tolerance in Arabidopsis thaliana. SIE141 binding interfered with canonical targeting of CDSP32 to chloroplasts, leading to its re-location into the plant nucleus. This nuclear translocation is essential for both their interaction and resistance function. Furthermore, SIE141 enhanced oxidoreductase activity of CDSP32, leading to CDSP32-mediated monomerization and activation of NON-EXPRESSOR OF PATHOGENESIS-RELATED 1 (NPR1), a key regulator of systemic resistance. Our findings provide functional insights on how S.indica transfers well-known beneficial effects to host plants and indicate CDSP32 as a genetic resource to improve plant resilience to abiotic and biotic stresses.

Overexpression of thioredoxin-like protein ACHT2 leads to negative feedback control of photosynthesis in Arabidopsis thaliana

Thioredoxin (Trx) is a small redox mediator protein involved in the regulation of various chloroplast functions by modulating the redox state of Trx target proteins in ever-changing light environments. Using reducing equivalents produced by the photosynthetic electron transport chain, Trx reduces the disulfide bonds on target proteins and generally turns on their activities. While the details of the protein-reduction mechanism by Trx have been well investigated, the oxidation mechanism that counteracts it has long been unclear. We have recently demonstrated that Trx-like proteins such as Trx-like2 and atypical Cys His-rich Trx (ACHT) can function as protein oxidation factors in chloroplasts. Our latest study on transgenic Arabidopsis plants indicated that the ACHT isoform ACHT2 is involved in regulating the thermal dissipation of light energy. To understand the role of ACHT2 in vivo, we characterized phenotypic changes specifically caused by ACHT2 overexpression in Arabidopsis. ACHT2-overexpressing plants showed growth defects, especially under high light conditions. This growth phenotype was accompanied with the impaired reductive activation of Calvin–Benson cycle enzymes, enhanced thermal dissipation of light energy, and decreased photosystem II activity. Overall, ACHT2 overexpression promoted protein oxidation that led to the inadequate activation of Calvin–Benson cycle enzymes in light and consequently induced negative feedback control of the photosynthetic electron transport chain. This study highlights the importance of the balance between protein reduction and oxidation in chloroplasts for optimal photosynthetic performance and plant growth.

Selection and Verification of Standardized Reference Genes of Angelica dahurica under Various Abiotic Stresses by Real-Time Quantitative PCR.

In traditional Chinese medicine, Angelica dahurica is a valuable herb with numerous therapeutic applications for a range of ailments. There have not yet been any articles on the methodical assessment and choice of the best reference genes for A. dahurica gene expression studies. Real-time quantitative PCR (RT-qPCR) is widely employed as the predominant method for investigating gene expression. In order to ensure the precise determination of target gene expression outcomes in RT-qPCR analysis, it is imperative to employ stable reference genes. In this study, a total of 11 candidate reference genes including SAND family protein (SAND), polypyrimidine tract-binding protein (PTBP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), TIP41-like protein (TIP41), cyclophilin 2 (CYP2), elongation factor 1 α (EF1α), ubiquitin-protein ligase 9 (UBC9), tubulin β-6 (TUB6), thioredoxin-like protein YLS8 (YLS8), and tubulin-α (TUBA) were selected from the transcriptome of A. dahurica. Subsequently, three statistical algorithms (geNorm, NormFinder, and BestKeeper) were employed to assess the stability of their expression patterns across seven distinct stimulus treatments. The outcomes obtained from these analyses were subsequently amalgamated into a comprehensive ranking using RefFinder. Additionally, one target gene, phenylalanine ammonia-lyase (PAL), was used to confirm the effectiveness of the selected reference genes. According to the findings of this study, the two most stable reference genes for normalizing the expression of genes in A. dahurica are TIP41 and UBC9. Overall, our research has determined the appropriate reference genes for RT-qPCR in A. dahurica and provides a crucial foundation for gene screening and identifying genes associated with the biosynthesis of active ingredients in A. dahurica.

TXNL1 has dual functions as a redox active thioredoxin-like protein as well as an ATP- and redox-independent chaperone

TXNL1 (also named TRP32, for thioredoxin related protein of 32 kDa) is a cytosolic thioredoxin-fold protein expressed in all cell types and conserved from yeast to mammals, but with yet poorly known function. Here, we expressed and purified human TXNL1 together with several Cys-to-Ser variants, characterizing their enzymatic properties. TXNL1 could reduce disulfides in insulin, cystine and glutathione disulfide (GSSG) in reactions coupled to thioredoxin reductase (TXNRD1, TrxR1) using NADPH, similarly to thioredoxin (TXN, Trx1), but with lower catalytic efficacy due to at least one order of magnitude higher Km of TrxR1 for TXNL1 compared to Trx1. However, in sharp contrast to Trx1, we found that TXNL1 also had efficient chaperone activity that did not require ATP. TXNL1 made non-covalent complexes with reduced insulin, thereby keeping it in solution, and TXNL1 provided chaperone function towards whole cell lysate proteins by preventing their aggregation during heating. The chaperone activities of TXNL1 did not require its redox activity or any dithiol-disulfide exchange reactions, as revealed using Cys-to-Ser substituted variants, as well as a maintained chaperone activity of TXNL1 also in the absence of TrxR1 and NADPH. These results reveal that TXNL1 has dual functions, supporting TrxR1-driven redox activities in disulfide reduction reactions, as well as being an ATP-independent chaperone that does not require involvement of its redox activity.

The chloroplast protein HCF164 is predicted to be associated with Coffea SH9 resistance factor against Hemileia vastatrix

To explore the connection between chloroplast and coffee resistance factors, designated as SH1 to SH9, whole genomic DNA of 42 coffee genotypes was sequenced, and entire chloroplast genomes were de novo assembled. The chloroplast phylogenetic haplotype network clustered individuals per species instead of SH factors. However, for the first time, it allowed the molecular validation of Coffea arabica as the maternal parent of the spontaneous hybrid “Híbrido de Timor”. Individual reads were also aligned on the C. arabica reference genome to relate SH factors with chloroplast metabolism, and an in-silico analysis of selected nuclear-encoded chloroplast proteins (132 proteins) was performed. The nuclear-encoded thioredoxin-like membrane protein HCF164 enabled the discrimination of individuals with and without the SH9 factor, due to specific DNA variants linked to chromosome 7c (from C. canephora-derived sub-genome). The absence of both the thioredoxin domain and redox-active disulphide center in the HCF164 protein, observed in SH9 individuals, raises the possibility of potential implications on redox regulation. For the first time, the identification of specific DNA variants of chloroplast proteins allows discriminating individuals according to the SH profile. This study introduces an unexplored strategy for identifying protein/genes associated with SH factors and candidate targets of H. vastatrix effectors, thereby creating new perspectives for coffee breeding programs.

Molecular features, antioxidant potential, and immunological expression assessment of thioredoxin-like protein 1 (TXNL1) in yellowtail clownfish (Amphiprion clarkii)

Thioredoxin-like protein 1 (TXNL1) is a redox-active protein belonging to the thioredoxin family, which mainly controls the redox status of cells. The TXNL1 gene from Amphiprion clarkii (AcTXNL1) was obtained from a pre-established transcriptome database. The AcTXNL1 is encoded with 289 amino acids and is predominantly localized in the cytoplasm and nucleus. The TXN domain of AcTXNL1 comprises a34CGPC37 motif with redox-reactive thiol (SH-) groups. The spatial distribution pattern of AcTXNL1 mRNA was examined in different tissues, and the muscle was identified as the highest expressed tissue. AcTXNL1 mRNA levels in the blood and gills were significantly increased in response to different immunostimulants. In vitro antioxidant capacity of the recombinant AcTXNL1 protein (rACTXNL1) was evaluated using the ABTS free radical-scavenging activity assay, cupric ion reducing antioxidant capacity assay, turbidimetric disulfide reduction assay, and DNA nicking protection assay. The potent antioxidant activity of rAcTXNL1 exhibited a concentration-dependent manner in all assays. Furthermore, in the cellular environment, overexpression of AcTXNL1 increased cell viability under H2O2 stress and reduced nitric oxide (NO) production induced by lipopolysaccharides (LPS). Collectively, the experimental results revealed that AcTXNL1 is an antioxidant and immunologically important gene in A. clarkii.

The role of thioredoxin proteins in Mycobacterium tuberculosis probed by proteome-wide target profiling

Mycobacterium tuberculosis encounters diverse microenvironments, including oxidative assault (ROS and RNS), when it attempts to establish itself within its human host. Therefore, redox sensory and regulation processes are assumed significant importance, as these are essential processes for M. tuberculosis to survive under these hostile conditions. M. tuberculosis contains thioredoxin system to maintain redox homeostasis, which establish a balance between the thiol/dithiol couple.Still very less is known about it. In the present study, we attempted to capture the targets of all the M. tuberculosis thioredoxin proteins (viz., TrxB and TrxC) and a thioredoxin-like protein, NrdH, under aerobic and hypoxic conditions by performing thioredoxin trapping chromatography followed by mass spectrometry. We found that TrxC captured the maximum number of targets in both the physiological conditions and most of the targets of TrxB and NrdH showing overlap with targets of TrxC, indicating that TrxC acts as main thioredoxin. Further the PANTHER classification system provides involvement of targets in various metabolic processes and Gene Ontology analysis suggests that glutamine biosynthetic process and Fe–S cluster biosynthesis are the most enriched processes in the target list of TrxC and TrxB respectively. Also, we suggest that the thioredoxin system might play an important role under hypoxia by targeting those proteins which are responsible to sense and maintain hypoxic conditions. Furthermore, our studies establish a link between TrxB and iron-sulfur cluster biogenesis in M. tuberculosis. Ultimately, these findings open a new direction to target the thioredoxin system for screening new anti-mycobacterial drug targets.

Bacillus subtilis YtpP and Thioredoxin A Are New Players in the Coenzyme-A-Mediated Defense Mechanism against Cellular Stress.

Coenzyme A (CoA) is an important cellular metabolite that is critical for metabolic processes and the regulation of gene expression. Recent discovery of the antioxidant function of CoA has highlighted its protective role that leads to the formation of a mixed disulfide bond with protein cysteines, which is termed protein CoAlation. To date, more than 2000 CoAlated bacterial and mammalian proteins have been identified in cellular responses to oxidative stress, with the majority being involved in metabolic pathways (60%). Studies have shown that protein CoAlation is a widespread post-translational modification which modulates the activity and conformation of the modified proteins. The induction of protein CoAlation by oxidative stress was found to be rapidly reversed after the removal of oxidizing agents from the medium of cultured cells. In this study, we developed an enzyme-linked immunosorbent assay (ELISA)-based deCoAlation assay to detect deCoAlation activity from Bacillus subtilis and Bacillus megaterium lysates. We then used a combination of ELISA-based assay and purification strategies to show that deCoAlation is an enzyme-driven mechanism. Using mass-spectrometry and deCoAlation assays, we identified B. subtilis YtpP (thioredoxin-like protein) and thioredoxin A (TrxA) as enzymes that can remove CoA from different substrates. With mutagenesis studies, we identified YtpP and TrxA catalytic cysteine residues and proposed a possible deCoAlation mechanism for CoAlated methionine sulfoxide reducatse A (MsrA) and peroxiredoxin 5 (PRDX5) proteins, which results in the release of both CoA and the reduced form of MsrA or PRDX5. Overall, this paper reveals the deCoAlation activity of YtpP and TrxA and opens doors to future studies on the CoA-mediated redox regulation of CoAlated proteins under various cellular stress conditions.

Distinct Roles of SELENOF in Different Human Cancers.

SELENOF, previously known as SEP15, is a selenoprotein that contains selenium in the form of the amino acid selenocysteine. Like other selenoproteins, the role for SELENOF in carcinogenesis has been investigated due to its altered expression compared to the corresponding normal tissue, its molecular function, and the association of genetic variations in the SELENOF gene to cancer risk or outcome. This review summarizes SELENOF's discovery, structure, cellular localization, and expression. SELENOF belongs to a new family of thioredoxin-like proteins. Published data summarized here indicate a likely role for SELENOF in redox protein quality control, and in the regulation of lipids, glucose, and energy metabolism. Current evidence indicates that loss of SELENOF contributes to the development of prostate and breast cancer, while its loss may be protective against colon cancer. Additional investigation into SELENOF's molecular mechanisms and its impact on cancer is warranted.

Tat-Thioredoxin-like protein 1 attenuates ischemic brain injury by regulation of MAPKs and apoptosis signaling

Thioredoxin-like protein 1 (TXNL1), one of the thioredoxin superfamily known as redox-regulator, plays an essential in maintaining cell survival via various antioxidant and anti-apoptotic mechanisms. It is well known that relationship between ischemia and oxidative stress, however, the role of TXNL1 protein in ischemic damage has not been fully investigated. In the present study, we aimed to determine the protective role of TXNL1 against on ischemic injury in vitro and in vivo using cell permeable Tat-TXNL1 fusion protein. Transduced Tat-TXNL1 inhibited ROS production and cell death in H2O2-exposed hippocampal neuronal (HT-22) cells and modulated MAPKs and Akt activation, and pro-apoptotic protein expression levels in the cells. In an ischemia animal model, Tat-TXNL1 markedly decreased hippocampal neuronal cell death and the activation of astrocytes and microglia. These findings indicate that cell permeable Tat-TXNL1 protects against oxidative stress in vitro and in vivo ischemic animal model. Therefore, we suggest Tat-TXNL1 can be a potential therapeutic protein for ischemic injury.

The atypical thioredoxin 'Alr2205', a newly identified partner of the typical 2-Cys-Peroxiredoxin, safeguards the cyanobacterium Anabaena from oxidative stress.

Thioredoxins (Trxs) are ubiquitous proteins that play vital roles in several physiological processes. Alr2205, a thioredoxin-like protein from Anabaena PCC 7120, was evolutionarily closer to the Trx-domain of the NADPH-Thioredoxin Reductase C than the other thioredoxins. The Alr2205 protein showed disulphide reductase activity despite the presence a non-canonical active site motif 'CPSC'. Alr2205 not only physically interacted with, but also acted as a physiological reductant of Alr4641 (2-Cys-Peroxiredoxin), supporting its peroxidase function. Structurally, Alr2205 was a monomeric protein that formed an intramolecular disulphide bond between the two active site cysteines. However, the Alr2205C41S protein, wherein the resolving cysteine was mutated to serine, was capable of forming intermolecular disulphide bond and exist as a dimer when treated with H2O2. Overproduction of Alr2205 in E. coli protected cells from heavy metals, but not oxidative stress. To delve into its physiological role, Alr2205/Alr2205C41S was overexpressed in Anabaena, and the ability of the corresponding strains (An2205+ or An2205C41S+) to withstand environmental stresses was assessed. An2205+ showed higher resistance to H2O2 than An2205C41S+, indicating that the disulphide reductase function of this protein was critical to protect cells from this peroxide. Although, An2205+ did not show increased capability to withstand cadmium stress, An2205C41S+ was more susceptible to this heavy metal. This is the first study that provides a vital understanding into the function of atypical thioredoxins in countering the toxic effects of heavy metals/H2O2 in prokaryotes.

Molecular cloning and characterization of a thioredoxin-like protein gene in rotifer Brachionus plicatilis

The thioredoxin-like protein exists widely, in various organisms, as a regulator of redox homeostasis. In this study, the full-length cDNA of a thioredoxin-like protein gene from rotifer Brachionus plicatilis (designated as BpTXNL) was obtained by 5′ rapid amplification of cDNA end (RACE) technology. The complete cDNA of BpTXNL was 1111 bp, and contained a 5′ untranslated region (UTR) of 69 bp, a 3’ UTR of 163 bp with a polyadenylate additional signal and a polyadenylation site (PAS), and an open reading frame (ORF) of 878 bp, encoding 292 amino acids. The calculated molecular weight and the theoretical isoelectric point (pI) of the deduced BpTXNL peptide were 32.7 kDa and 4.97, respectively. The deduced protein sequence of BpTXNL contained a thioredoxin domain with the conserved redox-active site at 33CGPC36 and a proteasome-interacting thioredoxin (PITH) domain. Phylogenetic analysis demonstrated that BpTXNL was clustered with TXNLs of Strongyloides ratti and Caenorhabditis elegans. The temporal mRNA expression level of BpTXNL significantly decreased at 6 h, then increased to the peak 24h after the 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) challenge, while the mRNA transcripts of BpTXNL significantly increased and reached the peaks twice, at 6 h and 24 h after the lipopolysaccharide (LPS) challenge. The recombinant BpTXNL protein quickly exhibited a concentration-dependent antioxidant capacity and the peak occurred at 55 min in the 20 μM group. All these results showed that BpTXNL possesses an antioxidant capacity, and that it may be involved in the regulation of excessive reactive oxygen species (ROS) during environmental stress or pathogen invading.

Trx CDSP32-overexpressing tobacco plants improves cadmium tolerance by modulating antioxidant mechanism

The effects of overexpression of the thioredoxin-like protein CDSP32 (Trx CDSP32) on reactive oxygen species (ROS) metabolism in tobacco leaves exposed to cadmium (Cd) were studied by combining physiological measures and proteomics technology. Thus, the number of differentially expressed proteins (DEPs) in plants overexpressing the Trx CDSP32 gene in tobacco (OE) was observed to be evidently lower than that in wild-type (WT) tobacco under Cd exposure, especially the number of down-regulated DEPs. Cd exposure induced disordered ROS metabolism in tobacco leaves. Although Cd exposure inhibited the activities of superoxide dismutase (SOD), catalase (CAT), and l-ascorbate peroxidase (APX) and the expression of proteins related to the thioredoxin-peroxiredoxin (Trx-Prx) pathway, the increase in the activities of peroxidase (POD), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione reductase (GR), glutathione peroxidase (GPX), and glutathione S-transferase (GST) and their protein expression levels played an important role in the physiological response to Cd exposure. Notably, Trx CDSP32 was observed to alleviate the decrease in the expression and activities of SOD and CAT caused by Cd exposure and enhance the function of POD. Trx CDSP32 was observed to increase the H2O2 scavenging capacity of the ascorbic acid-glutathione (AsA-GSH) cycle and Trx-Prx pathway under Cd exposure, and it can especially regulate 2-Cys peroxiredoxin (2-Cys Prx) protein expression and thioredoxin peroxidase (TPX) activity. Thus, overexpression of the Trx CDSP32 gene can alleviate the oxidative damage that occurs in tobacco leaves under Cd exposure by modulating antioxidant defense systems.

Peripheral membrane proteins modulate stress tolerance by safeguarding cellulose synthases.

Controlled primary cell wall remodeling allows plant growth under stressful conditions, but how these changes are conveyed to adjust cellulose synthesis is not understood. Here, we identify the TETRATRICOPEPTIDE THIOREDOXIN-LIKE (TTL) proteins as new members of the cellulose synthase complex (CSC) and describe their unique and hitherto unknown dynamic association with the CSC under cellulose-deficient conditions. We find that TTLs are essential for maintaining cellulose synthesis under high-salinity conditions, establishing a stress-resilient cortical microtubule array, and stabilizing CSCs at the plasma membrane. To fulfill these functions, TTLs interact with CELLULOSE SYNTHASE 1 (CESA1) and engage with cortical microtubules to promote their polymerization. We propose that TTLs function as bridges connecting stress perception with dynamic regulation of cellulose biosynthesis at the plasma membrane.

Dissipation of the proton electrochemical gradient in chloroplasts promotes the oxidation of ATP synthase by thioredoxin-like proteins

Chloroplast FoF1-ATP synthase (CFoCF1) uses an electrochemical gradient of protons across the thylakoid membrane (ΔμH+) as an energy source in the ATP synthesis reaction. CFoCF1 activity is regulated by the redox state of a Cys pair on its central axis, that is, the γ subunit (CF1-γ). When the ΔμH+ is formed by the photosynthetic electron transfer chain under light conditions, CF1-γ is reduced by thioredoxin (Trx), and the entire CFoCF1 enzyme is activated. The redox regulation of CFoCF1 is a key mechanism underlying the control of ATP synthesis under light conditions. In contrast, the oxidative deactivation process involving CFoCF1 has not been clarified. In the present study, we analyzed the oxidation of CF1-γ by two physiological oxidants in the chloroplast, namely the proteins Trx-like 2 and atypical Cys-His-rich Trx. Using the thylakoid membrane containing the reduced form of CFoCF1, we were able to assess the CF1-γ oxidation ability of these Trx-like proteins. Our kinetic analysis indicated that these proteins oxidized CF1-γ with a higher efficiency than that achieved by a chemical oxidant and typical chloroplast Trxs. Additionally, the CF1-γ oxidation rate due to Trx-like proteins and the affinity between them were changed markedly when ΔμH+ formation across the thylakoid membrane was manipulated artificially. Collectively, these results indicate that the formation status of the ΔμH+ controls the redox regulation of CFoCF1 to prevent energetic disadvantages in plants.

The Emergence of Rod-Cone Cellular Interaction

We studied the origin of rod-derived cone viability factor (RdCVF) during evolution. In mammals, the nucleoredoxin-like 1 gene (NXNL1) produces a truncated thioredoxin-like protein, RdCVF, by intron retention in rod photoreceptors of the retina. This protein prevents the secondary cone degeneration in animal models of rod-cone degeneration. Extracellular RdCVF binds to a complex at the surface of the cones, composed of the basigin-1, a photoreceptor specific alternative splicing product of the basigin gene, and GLUT1, the glucose transporter. RdCVF accelerates glucose uptake allosterically. Glucose is either metabolized by aerobic glycolysis to sustain cone outer segment renewal or by the pentose phosphate pathway to support redox power to the thioredoxin RdCVFL. RdCVF signaling predates the appearance of the eye and evolved through two alternative splicing events. RdCVF signaling is observed first in hydra where it regulates an unknown signaling. A scallop RdCVF protein is produced by ciliated photoreceptors of the retina and binds its receptor, BSG1, the first occurrence of RdCVF/BSG1 signaling. In the lamprey, RdCVF metabolic signaling between rod and cones is fully operational. In the mouse, the production of BSG1 is regulated through alternative splicing. This signaling was extended to other regions of the brain, via its paralogue NXNL2.

Analysis of phylogenetic markers for classification of a hydrogen peroxide producing Streptococcus oralis isolated from saliva by a newly devised differential medium

Hydrogen peroxide (H2O2) is produced by alpha-hemolytic streptococci in aerobic conditions. However, the suitable method for detection of H2O2-producing streptococci in oral microbiota has not been setup. Here we show that o-dianisidine dye and horseradish peroxidase were useful in tryptic soy agar medium to detect and isolate H2O2-producing bacteria with the detection limit of one target colony in > 106 colony-forming units. As a proof, we isolated the strain HP01 (KCTC 21190) from a saliva sample using the medium and analyzed its characteristics. Further tests showed that the strain HP01 belongs to Streptococcus oralis in the Mitis group and characteristically forms short-chain streptococcal cells with a high capacity of acid tolerance and biofilm formation. The genome analysis revealed divergence of the strain HP01 from the type strains of S. oralis. They showed distinctive phylogenetic distances in their ROS-scavenging proteins, including superoxide dismutase SodA, thioredoxin TrxA, thioredoxin reductase TrxB, thioredoxin-like protein YtpP, and glutaredoxin-like protein NrdH, as well as a large number of antimicrobial resistance genes and horizontally transferred genes. The concatenated ROS-scavenging protein sequence can be used to identify and evaluate Streptococcus species and subspecies based on phylogenetic analysis.

Selection and validation of reference genes for quantitative real-time PCR normalization in Psoralea corylifolia (Babchi) under various abiotic stress

Psoralea corylifolia L. is a popular herb and has long been used in traditional Ayurvedic and Chinese medicine owing to its extensive pharmacological activities, especially in the treatment of various shin diseases. To date, the systematic evaluation and selection of the optimum reference genes for gene expression analysis of P. corylifolia were not reported. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a method for gene expression quantification. Selecting appropriate reference genes is the prerequisite for accurate normalization of RT-qPCR results. This study assessed the expression stability of 10 candidate reference genes under different abiotic stresses. First, amplification primers for RT-qPCR were designed and received testing and optimization. Then, expression data from each candidate gene were evaluated based on three statistical algorithms, and their results were further integrated into a comprehensive ranking based on the geometric mean. Additionally, one target gene, i.e., 1-aminocyclopropane-1-carboxylate oxidase (ACO), was used to validate the effectiveness of the selected reference. Our analysis suggested that thioredoxin-like protein YLS8 (YLS8), TIP41-like family protein (TIP41), and cyclophilin 2 (CYP2) genes provided superior expression normalization under different abiotic stresses. Overall, this work constitutes the first effort to select optimal endogenous controls for RT-qPCR studies of gene expression in P. corylifolia. It also provides a reasonable normalization standard and basis for further analysis of the gene expression of bioactive components in P. corylifolia.

A Novel Thioredoxin-Like Protein of Babesia microti Involved in Parasite Pathogenicity.

Babesiosis poses a serious threat to immunocompromised individuals and the major etiological species of Babesia for human babesiosis is Babesia microti. Merozoites are a critical stage in the life cycle of Babesia microti. Several merozoite proteins have been demonstrated to play important roles in this process; however, most of the merozoite proteins of B. microti remain unknown. In the present study, we identified a novel merozoite protein of B. microti with similar structure to the thioredoxin (Trx)-like domain of the Trx family, which was named as B. microti Trx-like protein (BmTLP). Western blot assays demonstrated that this protein was expressed by B. microti during the erythrocytic infection process, and its expression peaked on day 7 post-infection in vivo. Immunofluorescence assay further showed that this protein is mainly expressed in B. microti merozoites. BmTLP hold both heparin- and erythrocyte-binding properties, which are critical functions of invasion-related proteins. Immunization with recombinant BmTLP imparted significant protection against B. microti infection in mice. Taken together, these results suggest that the novel merozoite protein, BmTLP, is an important pathogenic molecule of B. microti and may be a possible target for the design of babesiosis control strategy.

SELENOT Deficiency in the Mouse Brain Impacts Catecholaminergic Neuron Density: An Immunohistochemical, in situ Hybridization and 3D Light-Sheet Imaging Study

Background: Selenoprotein T (SELENOT), a PACAP-regulated thioredoxin-like protein, plays a role in catecholamine secretion and protects dopaminergic neurons. However, the role of SELENOT in the establishment of the catecholaminergic (CA) neuronal system is not known yet. Methods: We analyzed by immunohistochemistry and RNAscope in situ hybridization the distribution of SELENOT and the expression of its mRNA, respectively. In addition, 3D imaging involving immunostaining in toto, clearing through the iDISCO+ method, acquisitions by light-sheet microscopy, and processing of 3D images was performed to map the CA neuronal system. A semi-automatic quantification of 3D images was carried out. Results: SELENOT protein and mRNA are widely distributed in the mouse brain, with important local variations. Three-dimensional mapping, through tyrosine hydroxylase (TH) labeling, and semi-automated quantification of CA neurons in brain-specific SELENOT knockout mice showed a significant decrease in the number of TH-positive neurons in the area postrema (AP-A2), the A11 cell group (A11), and the zona incerta (ZI-A13) of SELENOT-deficient females, and in the hypothalamus (Hyp-A12-A14-A15) of SELENOT-deficient females and males. Conclusion: These results showed that SELENOT is diffusely expressed in the mouse brain and that its deficiency impacts CA neuron distribution in different brain areas including Hyp-A12-A14-A15, in both male and female mice.