Validation of RNA Extraction Methods and Suitable Reference Genes for Gene Expression Studies in Developing Fetal Human Inner Ear Tissue.

A comprehensive gene expression investigation requires high-quality RNA extraction, in sufficient amounts for real-time quantitative polymerase chain reaction and next-generation sequencing. In this work, we compared different RNA extraction methods and evaluated different reference genes for gene expression studies in the fetal human inner ear. We compared the RNA extracted from formalin-fixed paraffin-embedded tissue with fresh tissue stored at -80 °C in RNAlater solution and validated the expression stability of 12 reference genes (from gestational week 11 to 19). The RNA from fresh tissue in RNAlater resulted in higher amounts and a better quality of RNA than that from the paraffin-embedded tissue. The reference gene evaluation exhibited four stably expressed reference genes (B2M, HPRT1, GAPDH and GUSB). The selected reference genes were then used to examine the effect on the expression outcome of target genes (OTOF and TECTA), which are known to be regulated during inner ear development. The selected reference genes displayed no differences in the expression profile of OTOF and TECTA, which was confirmed by immunostaining. The results underline the importance of the choice of the RNA extraction method and reference genes used in gene expression studies.

Determination of internal controls for quantitative gene expression of Spodoptera litura under microbial pesticide stress.

Quantitative real-time polymerase chain reaction (qRT-PCR) has become a commonly used method for the quantification of gene expression. However, accurate qRT-PCR analysis requires a valid internal reference for data normalization. To determine the valid reference characterized with low expression variability among Spodoptera litura samples after microbial pesticide treatments, nine housekeeping genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), arginine kinase, ubiquitin C, actin-5C (ACT5C), actin, ribosomal protein S13 (RPS13), tubulin, acidic ribosomal protein P0 (RPLP0) and ubiquinol-cytochrome c reductase, were evaluated for their suitability using geNorm, Normfinder, BestKeeper, RefFinder and the comparative delta CT methods in this study. S. litura larvae after direct treatment (larvae were immersed in biopesticides), indirect treatment (larvae were fed with biopesticide immersed artificial diets) and comprehensive treatment (larvae were treated with the first two treatments in sequence), respectively with Metarhizium anisopliae, Empedobacter brevis and Bacillus thuringiensis, were investigated. The results indicated that the best sets of internal references were as follows: RPLP0 and ACT5C for direct treatment conditions; RPLP0 and RPS13 for indirect treatment conditions; RPS13 and GAPDH for comprehensive treatment conditions; RPS13 and RPLP0 for all the samples. These results provide valuable bases for further genetic researches in S. litura.

Identification of suitable reference genes for RT-qPCR studies in human parathyroid tissue glandular cells.

Identifying a proper reference gene allows us to understand fundamental changes in many biological processes. Normalization during gene expression analyses is essential for every tissue/cell type, including parathyroid tissue glandular cells. Quantitative method of gene expression analyses via qRT-PCR method provides the accurate examination of every target gene. There are limited reports to present commonly used reference genes in human parathyroid tissues rather than for glandular cell types. This study aims to determine and compare the most stable to least stable genes for parathyroid tissue cells. 43 human parathyroid tissue obtained from primary and secondary hyperparathyroidism patients and glandular cells isolated enzymatically by the removal of extracellular matrix components. After extraction of the total RNA, cDNA synthesis was performed, then qRT-PCR evaluated 14 candidate reference genes. Stability was determined by RefFinder software (Delta ct, BestKeeper, Genorm, and NormFinder algorithms), and the outcome was evaluated for five groups. Even if assessed with different groups, the most stable genes were RPLP0 and GAPDH, while the CLTC and RNA 18S were the least stable. We have confirmed the comprehensive ranking of the most stable three genes alone with the NormFinder algorithm to understand intergroup variation and found out that RPLP0>GAPDH>PGK1. Lastly, comparisons of relative target gene (GCM2) expression revealed similar expression patterns for the most stable reference genes. The most stable reference gene is recommended for the stages where stability is evaluated using the results of four different approaches using RefFinder. We aspire for this study to assist future research to conduct thorough assessments of appropriate reference genes before engaging in gene expression analyses for parathyroid tissue.

Evaluation of reference genes for gene expression analysis by real-time quantitative PCR (qPCR) in different tissues from mice infected by Ascaris suum.

Human ascariasis is the most prevalent helminth infection, affecting 445 million people worldwide. To better understand the impact of the immune system on the pathophysiology of individuals infected with Ascaris suum, mice have been used as experimental models. The RT-qPCR technique is a critical auxiliary tool of investigation used to quantify mRNA levels. However, proper normalization using reference genes is essential to ensure reliable outcomes to avoid analytical errors and false results. Despite the importance of reference genes for experimental A. suum infection studies, no specific reference genes have been identified yet. Therefore, we conducted a study to assess five potential reference genes (GAPDH, 18s, ACTB, B2M, and HPRT1) in different tissues (liver, lungs, small and large intestines) affected by A. suum larval migration in C57BL/6j mice. Tissue collection was carried out to analyze parasite burden and confirm the presence of larvae during the peak of migration in each tissue. Upon confirmation, we analyzed different genes in the tissues and found no common gene with stable expression. Our results highlight the importance of analyzing different genes and using different software programs to ensure reliable relative expression results. Based on our findings, B2M was ranked as the ideal reference gene for the liver, while 18S was the most stable gene in the lung and small intestine. ACTB, or a combination of ACTB with GAPDH, was deemed suitable as reference genes for the large intestine due to their stable expression and less variation between the control and infected groups. To further demonstrate the impact of using different reference genes, we normalized the expression of a chemokine gene (CXCL9) in all tissues. Significant differences in CXCL9 expression levels were observed between different groups in all tissues except for the large intestine. This underscores the importance of selecting appropriate reference genes to avoid overestimating target gene expression levels and encountering normalization-related issues that can lead to false results. In conclusion, our study highlights the significance of using reliable reference genes for accurate RT-qPCR analysis, especially in the context of A. suum infection studies in different tissues. Proper normalization is crucial to ensure the validity of gene expression data and avoid potential pitfalls in interpreting results.

Validation of Endogenous Control Genes by Real-Time Quantitative Reverse Transcriptase Polymerase Chain Reaction for Acute Leukemia Gene Expression Studies.

Reference genes are used as internal reaction controls for gene expression analysis, and for this reason, they are considered reliable and must meet several important criteria. In view of the absence of studies regarding the best reference gene for the analysis of acute leukemia patients, a panel of genes commonly used as endogenous controls was selected from the literature for stability analysis: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Abelson murine leukemia viral oncogene human homolog 1 (ABL), Hypoxanthine phosphoribosyl-transferase 1 (HPRT1), Ribosomal protein lateral stalk subunit P0 (RPLP0), ß-actin (ACTB) and TATA box binding protein (TBP). The stability of candidate reference genes was analyzed according to three statistical methods of assessment, namely, NormFinder, GeNorm and R software (version 4.0.3). From this study's analysis, it was possible to identify that the endogenous set composed of ACTB, ABL, TBP and RPLP0 demonstrated good performances and stable expressions between the analyzed groups. In addition to that, the GAPDH and HPRT genes could not be classified as good reference genes, considering that they presented a high standard deviation and great variability between groups, indicating low stability. Given these findings, this study suggests the main endogenous gene set for use as a control/reference for the gene expression in peripheral blood and bone marrow samples from patients with acute leukemias is composed of the ACTB, ABL, TBP and RPLP0 genes. Researchers may choose two to three of these housekeeping genes to perform data normalization.

Exploration of reference genes for the development of a diagnostic kit on vascular aging in human saliva.

Identifying reliable biomarkers in saliva can be a promising approach to developing a rapid diagnostic kit for detecting vascular aging. This study investigated the most suitable reference gene for polymerase chain reaction (PCR) in saliva that is not affected by vascular aging variables. Whole saliva samples were collected to assess the expression of reference genes: actin beta (ACTB), 18S ribosomal RNA (18S rRNA), beta-2-microglobulin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The most abundantly expressed gene was 18S rRNA, and the least expressed gene was GAPDH. Four genes were ranked according to their relative stability, as determined by mathematical algorithms, indicating that ACTB and 18S rRNA were stably expressed as reference genes. 18S rRNA was identified as the most promising reference gene for detecting systemic diseases using saliva from patients with vascular aging in these limited experimental conditions.

Multi-omics analysis reveals GAPDH posttranscriptional regulation of IFN-γ and PHGDH as a metabolic checkpoint of microglia polarization.

A "switch" in the metabolic pattern of microglia is considered to be required to meet the metabolic demands of cell survival and functions. However, how metabolic switches regulate microglial function remains controversial. We found here that exposure to amyloid-ß triggers microglial inflammation accompanied by increasing GAPDH levels. The increase of GAPDH, a glycolysis enzyme, leads to the reduced release of interferon-γ (IFN-γ) from inflammatory microglia. Such alternation is translational and is regulated by the binding of glycolysis enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to IFN-γ mRNA. GAPDH, by engaging/disengaging glycolysis and through influencing IFN-γ expression, regulates microglia functions, including phagocytosis and cytokine production. Phosphoglycerate dehydrogenase (PHGDH), screened from different state microglia by metabolomics combined with METARECON analysis, is a metabolic enzyme adjacent downstream of GAPDH and synthesizes serine on the collateral pathway derived from glycolysis. Polarization of microglial with PHGDH as a metabolic checkpoint can be bidirectionally regulated by adding IL-4 or giving PHGDH inhibitors. Therefore, regulation of metabolic enzymes not only reprograms metabolic patterns, but also manipulates microglia functions. Further study should be performed to explore the mechanism of metabolic checkpoints in human microglia or more in vivo animal experiments, and may expand to the effects of various metabolic substrates or enzyme, such as lipids and amino acids, on the functions of microglia.

Structure-based functional analysis of a novel NADPH-producing glyceraldehyde-3-phosphate dehydrogenase from Corynebacterium glutamicum.

Corynebacterium glutamicum is an industrial workhorse applied in the production of valuable biochemicals. In the process of bio-based chemical production, improving cofactor recycling and mitigating cofactor imbalance are considered major solutions for enhancing the production yield and efficiency. Although, glyceraldehyde-3-phosphate dehydrogenase (GapDH), a glycolytic enzyme, can be a promising candidate for a sufficient NADPH cofactor supply, however, most microorganisms have only NAD-dependent GapDHs. In this study, we performed functional characterization and structure determination of novel NADPH-producing GapDH from C. glutamicum (CgGapX). Based on the crystal structure of CgGapX in complex with NADP cofactor, the unique structural features of CgGapX for NADP stabilization were elucidated. Also, N-terminal additional region (Auxiliary domain, AD) appears to have an effect on enzyme stabilization. In addition, through structure-guided enzyme engineering, we developed a CgGapX variant that exhibited 4.3-fold higher kcat, and 1.2-fold higher kcat/KM values when compared with wild-type. Furthermore, a bioinformatic analysis of 100 GapX-like enzymes from 97 microorganisms in the KEGG database revealed that the GapX-like enzymes possess a variety of AD, which seem to determine enzyme stability. Our findings are expected to provide valuable information for supplying NADPH cofactor pools in bio-based value-added chemical production.

Reprogramming the translatome during daily light transitions as affected by cytosolic glyceraldehyde-3-phosphate dehydrogenases GAPC1/C2.

Dark-light and light-dark transitions during the day are switching points of leaf metabolism that strongly affect the regulatory state of the cells, and this change is hypothesized to affect the translatome. The cytosolic glyceraldehyde-3-phosphate dehydrogenases GAPC1 and GAPC2 function in glycolysis, and carbohydrate and energy metabolism, but GAPC1/C2 also shows moonlighting functions in gene expression and post-transcriptional regulation. In this study we examined the rapid reprogramming of the translatome that occurs within 10 min at the end of the night and the end of the day in wild-type (WT) Arabidopsis and a gapc1/c2 double-knockdown mutant. Metabolite profiling compared to the WT showed that gapc1/c2 knockdown led to increases in a set of metabolites at the start of day, particularly intermediates of the citric acid cycle and linked pathways. Differences in metabolite changes were also detected at the end of the day. Only small sets of transcripts changed in the total RNA pool; however, RNA-sequencing revealed major alterations in polysome-associated transcripts at the light-transition points. The most pronounced difference between the WT and gapc1/c2 was seen in the reorganization of the translatome at the start of the night. Our results are in line with the proposed hypothesis that GAPC1/C2 play a role in the control of the translatome during light/dark transitions.

Role of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in autophagy activation following subarachnoid hemorrhage.

BACKGROUND: Early brain injury (EBI) refers to a severe brain injury that occurs within hours to days after subarachnoid hemorrhage (SAH). Neuronal damage in EBI is considered a key factor leading to poor prognosis. Currently, our understanding of the mechanisms of neuronal damage, such as neuronal autophagy, is still incomplete. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in metabolism and plays an important role in autophagy. Based on this, this study will further explore the regulation of autophagy by GAPDH after SAH, which may provide a new treatment strategy for improving the prognosis of SAH patients. METHODS: The rat SAH model was established by endovascular puncturing, and the trend of autophagy in hippocampal neurons at different time points was discussed. Additionally, an in vitro SAH model was created using the oxygenated hemoglobin and hippocampal neuronal HT22 cell line. Through siRNA and overexpression adenovirus techniques, we further investigated the relationship between the key enzyme GAPDH and autophagy in the in vitro SAH model. RESULTS: We observed significant neuronal damage in the hippocampus 24 h after SAH, and the proteomics showed significant enrichment of autophagy-related pathways at this time point. Further studies showed that the expression of LC3 and Beclin1 peaked at 24 h, and the nuclear translocation of GAPDH occurred simultaneously with SAH-induced neuronal autophagy. Our in vitro SAH model confirmed the role of GAPDH in regulating the level of autophagy in HT22 cells. Knockdown of GAPDH significantly reduced the level of autophagy, while overexpression of GAPDH increased the level of autophagy. CONCLUSION: This study shows the trend of autophagy in hippocampal neurons after SAH, and reveals the regulatory role of GAPDH in SAH-induced autophagy. However, further studies are needed to reveal the exact mechanism of GAPDH in the nuclear translocation regulation of autophagy and validate in animal models.

Oxidation of the active site cysteine residue of glyceraldehyde-3-phosphate dehydrogenase to the hyper-oxidized sulfonic acid form is favored under crowded conditions.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key cellular enzyme, with major roles in both glycolysis, and 'moonlighting' activities in the nucleus (uracil DNA glycosylase activity, nuclear protein nitrosylation), as a regulator of mRNA stability, a transferrin receptor, and as an antimicrobial agent. These activities are dependent, at least in part, on the integrity of an active site Cys residue, and a second neighboring Cys. These residues are differentially sensitive to oxidation, and determine both its catalytic activity and the redox signaling capacity of the protein. Such Cys modification is critical to cellular adaptation to oxidative environments by re-routing metabolic pathways to favor NADPH generation and antioxidant defenses. Despite the susceptibility of GAPDH to oxidation, it remains a puzzle as to how this enzyme acts as a redox signaling hub for oxidants such as hydrogen peroxide (H2O2) in the presence of high concentrations of specialized high-efficiency peroxide-removing enzymes. One possibility is that crowded environments, such as the cell cytosol, alter the oxidation pathways of GAPDH. In this study, we investigated the role of crowding (induced by dextran) on H2O2- and SIN-1-induced GAPDH oxidation, with data for crowded and dilute conditions compared. LC-MS/MS data revealed a lower extent of modification of the catalytic Cys under crowded conditions (i.e. less monomer units modified), but enhanced formation of the sulfonic acid resulting from hyper-oxidation. This effect was not observed with SIN-1. These data indicate that molecular crowding can modulate the oxidation pathways of GAPDH and its extent of oxidation and inactivation.

Trypanosoma sp. infection in Boa constrictor snakes: morphological, hematological, clinical biochemistry, molecular, and phylogenetic characteristics.

There are few reports of Trypanosoma in snakes, as well as little information about its pathogenicity in these animals. Thus, the present study aimed to characterize Trypanosoma found in Boa constrictor snakes, to verify the influence of the parasitism on hematological and clinical biochemistry parameters, and to perform a phylogenetic study of the isolates. Blood samples from sixty-one boas were analyzed for the presence of trypanosomatids and by hematological and clinical biochemistry assays. The flagellates that were found in this analysis were used for cell culture, morphometry, and molecular analysis. Later, molecular typing phylogenetic studies were performed. Nine positive animals (14.75%) were identified by microscopy analysis. The hematological results showed that parasitized animals presented significantly lower levels of packed cell volume, hemoglobin, mean corpuscular volume, and mean corpuscular hemoglobin. In the leukogram, eosinophils and heterophils counts were higher in parasitized animals. Considering the molecular analyses, the isolates presented a higher identity of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the 18S small subunit ribosomal RNA (SSU rRNA) gene fragments with Trypanosoma serpentis. The phylogenetic tree, using the GAPDH, clustered all isolates with T. serpentis and Trypanosoma cascavelli. This is the first description of T. serpentis parasitizing boas and of the clinical changes caused by trypanosomatid infection in snakes.

[Selection and validation of reference genes for quantitative real-time PCR analysis in Paeonia veitchii].

Paeonia veitchii and P. lactiflora are both original plants of the famous Chinese medicinal drug Paeoniae Radix Rubra in the Chinese Pharmacopoeia. They have important medicinal value and great potential in the flower market. The selection of stable and reliable reference genes is a necessary prerequisite for molecular research on P. veitchii. In this study, two reference genes, Actin and GAPDH, were selected as candidate genes from the transcriptome data of P. veitchii. The expression levels of the two candidate genes in different tissues(phloem, xylem, stem, leaf, petiole, and ovary) and different growth stages(bud stage, flowering stage, and dormant stage) of P. veitchii were detected using real-time fluorescence quantitative technology(qRT-PCR). Then, the stability of the expression of the two reference genes was comprehensively analyzed using geNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that the expression patterns of Actin and GAPDH were stable in different tissues and growth stages of P. veitchii. Furthermore, the expression levels of eight genes(Pv-TPS01, Pv-TPS02, Pv-CYP01, Pv-CYP02, Pv-CYP03, Pv-BAHD01, Pv-UGT01, and Pv-UGT02) in different tissues were further detected based on the transcriptome data of P. veitchii. The results showed that when Actin and GAPDH were used as reference genes, the expression trends of the eight genes in different tissues of P. veitchii were consistent, validating the reliability of Actin and GAPDH as reference genes for P. veitchii. In conclusion, this study finds that Actin and GAPDH can be used as reference genes for studying gene expression levels in different tissues and growth stages of P. veitchii.

Assessment of Candidate Reference Genes for Gene Expression Studies Using RT-qPCR in Colletotrichum fructicola from Litchi.

Litchi (Litchi chinensis Sonn.) is a tropical fruit originating from southern China that is currently cultivated in subtropical and tropical regions worldwide. Litchi anthracnose, caused by Colletotrichum fructicola, a dominant species of Colletotrichum spp., is an important disease of litchi that damages the fruits in fields and in post-harvest storage. Real-time quantitative PCR (RT-qPCR) is a common technique with which to detect the expression of and function of target genes quickly and precisely, and stable reference genes are crucial. However, there is no comprehensive information on suitable reference genes of C. fructicola present. Here, we designed eight candidate genes (GAPDH, α-tubulin, 18S, ß-tubulin, EF1a, TATA, RPS5, and EF3) using RefFinder software (programs: geNorm, ΔCt, BestKeeper, and NormFinder) to investigate their reliability in the detection of C. fructicola under five different treatments (fungal development stage, temperature, UV, culture medium, and fungicide). The results showed the optimal reference genes under different conditions: EF1a and α-tubulin for developmental stage; α-tubulin and ß-tubulin for temperature; α-tubulin and RPS5 for UV treatment; RPS5 and α-tubulin for culture medium; α-tubulin, GAPDH, and TATA for fungicide treatments. The corresponding expression patterns of HSP70 (Heat shock protein 70) were significantly different when the most and the least stable reference genes were selected when treated under different conditions. Our study provides the first detailed list of optimal reference genes for the analysis of gene expression in C. fructicola via RT-qPCR, which should be useful for future functional studies of target genes in C. fructicola.

The variations in gene expression of GAPDH in Ocimum basilicum cultivars under drought-induced stress conditions.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) holds a pivotal role within the glycolytic pathway of higher plants. It has garnered attention as a significant target protein in instances of oxidative stress, where it can engage in thiolation reactions within its active site. Numerous genes encoding cytosolic iterations of GAPDH have been identified and analyzed in specific plant species. This investigation was conducted to gain insights into GAPDH's function amidst drought-induced stress. Within this framework, the basil plant (Ocimum basilicum) was chosen for focused exploration, encompassing the cloning of the comprehensive cDNA of basil GAPDH (ObGAPDH) and scrutinizing its patterns of expression. The complete sequence of Ob-GAPDH spanned 1315 base pairs. The resultant protein derived from this sequence comprised 399 amino acids, projecting a molecular weight of approximately 42.54 kDa and an isoelectric point (pI) of 6.01. An examination of the evolutionary connections among various GAPDH proteins unveiled ObGAPDH's shared lineage with GAPDH proteins sourced from other plants, such as Salvia splendens and Sesamum indicum. Furthermore, computational methodologies were harnessed to predict the potential oxidative role of ObGAPDH in response to external signals. Molecular docking simulations illuminated the interaction between ObGAPDH and hydrogen peroxide (H2O2) as a ligand. Scrutinizing the expression patterns of the ObGAPDH gene under conditions of water scarcity stress brought to light diverse levels of transcriptional activity. Collectively, these findings underscore the notion that the regulation of ObGAPDH expression is contingent upon both the specific plant cultivar and the presence of stress stemming from drought conditions.

Structure-Guided Protein Engineering of Glyceraldehyde-3-phosphate Dehydrogenase from Corynebacterium glutamicum for Dual NAD/NADP Cofactor Specificity.

Since the discovery of l-glutamate-producing Corynebacterium glutamicum, it has evolved to be an industrial workhorse. For biobased chemical production, suppling sufficient amounts of the NADPH cofactor is crucial. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme that converts glyceraldehyde-3-phosphate (G3P) to 1,3-bisphosphoglycerate and produces NADH, is a major prospective solution for the cofactor imbalance issue. In this study, we determined the crystal structure of GAPDH from C. glutamicum ATCC13032 (CgGAPDH). Based on the structural information, we generated six CgGAPDH variants, CgGAPDHL36S, CgGAPDHL36S/T37K, CgGAPDHL36S/T37K/P192S, CgGAPDHL36S/T37K/F100V/P192S, CgGAPDHL36S/T37K/F100L/P192S, and CgGAPDHL36S/T37K/F100I/P192S, that can produce both NADH and NAPDH. The final CgGAPDHL36S/T37K/F100V/P192S variant showed a 212-fold increase in enzyme activity for NADP as well as 200% and 30% increased activity for the G3P substrate under NAD and NADP cofactor conditions, respectively. In addition, crystal structures of CgGAPDH variants in complex with NAD(P) permit the elucidation of differences between wild-type CgGAPDH and variants in relation to cofactor stabilization.

Glyceraldehyde-3-phosphate dehydrogenase Gh_GAPDH9 is associated with drought resistance in Gossypium hirsutum.

Background: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is the central enzyme of glycolysis and plays important regulatory roles in plant growth and development and responses to adverse stress conditions. However, studies on the characteristics and functions of cotton GAPDH family genes are still lacking. Methods: In this study, genome-wide identification of the cotton GAPDH gene family was performed, and the phylogeny, gene structures, promoter progenitors and expression profiles of upland cotton GAPDH gene family members were explored by bioinformatics analysis to highlight potential functions. The functions of GhGAPDH9 in response to drought stress were initially validated based on RNA-seq, qRT‒PCR, VIGS techniques and overexpression laying a foundation for further studies on the functions of GAPDH genes. Results: This study is the first systematic analysis of the cotton GAPDH gene family, which contains a total of 84 GAPDH genes, among which upland cotton contains 27 members. Quantitative, phylogenetic and covariance analyses of the genes revealed that the GAPDH gene family has been conserved during the evolution of cotton. Promoter analysis revealed that most cis-acting elements were related to MeJA and ABA. Based on the identified promoter cis-acting elements and RNA-seq data, it was hypothesized that Gh_GAPDH9, Gh_GAPDH11, Gh_GAPDH19 and Gh_GAPDH21 are involved in the response of cotton to abiotic stress. The expression levels of the Gh_GAPDH9 gene in two drought-resistant and two drought-sensitive materials were analyzed by qRT‒PCR and found to be high early in the treatment period in the drought-resistant material. The silencing of Gh_GAPDH9 based on virus-induced gene silencing (VIGS) technology resulted in significant leaf wilting or whole-plant dieback in silenced plants after drought stress compared to the control. The content of-malondialdehyde (MDA) in cotton leaves was significantly increased, and the content of proline (Pro) and chlorophyll (Chl) was reduced. In addition, the leaf wilting and dryness of transgenic lines under drought stress were lower than those of wild-type Arabidopsis, indicating that Gh_GAPDH9 is a positive regulator of drought resistance. In conclusion, our results demonstrate that GAPDH genes play an important role in the response of cotton to abiotic stresses and provide preliminary validation of the function of the Gh_GAPDH9 gene under drought stress. These findings provide an important theoretical basis for further studies on the function of the Gh_GAPDH9 gene and the molecular mechanism of the drought response in cotton.

Three consecutive cytosolic glycolysis enzymes modulate autophagic flux.

Autophagy serves as an important recycling route for the growth and survival of eukaryotic organisms in nutrient-deficient conditions. Since starvation induces massive changes in the metabolic flux that are coordinated by key metabolic enzymes, specific processing steps of autophagy may be linked with metabolic flux-monitoring enzymes. We attempted to identify carbon metabolic genes that modulate autophagy using VIGS screening of 45 glycolysis- and Calvin-Benson cycle-related genes in Arabidopsis (Arabidopsis thaliana). Here, we report that three consecutive triose-phosphate-processing enzymes involved in cytosolic glycolysis, triose-phosphate-isomerase (TPI), glyceraldehyde-3-phosphate dehydrogenase (GAPC), and phosphoglycerate kinase (PGK), designated TGP, negatively regulate autophagy. Depletion of TGP enzymes causes spontaneous autophagy induction and increases AUTOPHAGY-RELATED 1 (ATG1) kinase activity. TGP enzymes interact with ATG101, a regulatory component of the ATG1 kinase complex. Spontaneous autophagy induction and abnormal growth under insufficient sugar in TGP mutants are suppressed by crossing with the atg101 mutant. Considering that triose-phosphates are photosynthates transported to the cytosol from active chloroplasts, the TGP enzymes would be strategically positioned to monitor the flow of photosynthetic sugars and modulate autophagy accordingly. Collectively, these results suggest that TGP enzymes negatively control autophagy acting upstream of the ATG1 complex, which is critical for seedling development.

Selection and Validation of Reference Genes in Virus-Infected Sweet Potato Plants.

Quantitative real-time PCR (qRT-PCR) in sweet potatoes requires accurate data normalization; however, there are insufficient studies on appropriate reference genes for gene expression analysis. We examined variations in the expression of eight candidate reference genes in the leaf and root tissues of sweet potatoes (eight nonvirus-infected or eight virus-infected samples). Parallel analyses with geNorm, NormFinder, and Best-Keeper show that different viral infections and origin tissues affect the expression levels of these genes. Based on the results of the evaluation of the three software, the adenosine diphosphate-ribosylation factor is suitable for nonvirus or virus-infected sweet potato leaves. Cyclophilin and ubiquitin extension proteins are suitable for nonvirus-infected sweet potato leaves. Phospholipase D1 alpha is suitable for virus-infected sweet potato leaves. Actin is suitable for roots of nonvirus-infected sweet potatoes. Glyceraldehyde-3-phosphate dehydrogenase is suitable for virus-infected sweet potato roots. The research provides appropriate reference genes for further analysis in leaf and root samples of viruses in sweet potatoes.

Efficient tagging of endogenous proteins in human cell lines for structural studies by single-particle cryo-EM.

CRISPR/Cas9-based genome engineering has revolutionized our ability to manipulate biological systems, particularly in higher organisms. Here, we designed a set of homology-directed repair donor templates that enable efficient tagging of endogenous proteins with affinity tags by transient transfection and selection of genome-edited cells in various human cell lines. Combined with technological advancements in single-particle cryogenic electron microscopy, this strategy allows efficient structural studies of endogenous proteins captured in their native cellular environment and during different cellular processes. We demonstrated this strategy by tagging six different human proteins in both HEK293T and Jurkat cells. Moreover, analysis of endogenous glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in HEK293T cells allowed us to follow its behavior spatially and temporally in response to prolonged oxidative stress, correlating the increased number of oxidation-induced inactive catalytic sites in GAPDH with its translocation from cytosol to nucleus.