Activation of the toll-like receptor 2 signaling pathway by GAPDH from bacterial strain RD055328.

We characterized the membrane vesicle fraction (RD-MV fraction) from bacterial strain RD055328, which is related to members of the genus Companilactobacillus and Lactiplantibacillus plantarum. RD-MVs and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were detected in the RD-MV fraction. Immunoglobulin A (IgA) was produced by Peyer's patch cells following the addition of the RD-MV fraction. In the presence of the RD-MV fraction, RAW264 cells produced the pro-inflammatory cytokine IL-6. Recombinant GAPDH probably induced the production of IL-6 by RAW264 cells via superficial toll-like receptor 2 (TLR2) recognition. A confocal laser scanning microscopy image analysis indicated that RD-MVs and GAPDH were taken up by RAW264 cells. GAPDH wrapped around RAW264 cells. We suggest that GAPDH from strain RD055328 enhanced the production of IgA by acquired immune cells via the production of IL-6 by innate immune cells through TLR2 signal transduction.

Pleiotropic effects of trans-regulatory mutations on fitness and gene expression.

Variation in gene expression arises from cis- and trans-regulatory mutations, which contribute differentially to expression divergence. We compare the impacts on gene expression and fitness resulting from cis- and trans-regulatory mutations in Saccharomyces cerevisiae, with a focus on the TDH3 gene. We use the effects of cis-regulatory mutations to infer effects of trans-regulatory mutations attributable to impacts beyond the focal gene, revealing a distribution of pleiotropic effects. Cis- and trans-regulatory mutations had different effects on gene expression with pleiotropic effects of trans-regulatory mutants affecting expression of genes both in parallel to and downstream of the focal gene. The more widespread and deleterious effects of trans-regulatory mutations we observed are consistent with their decreasing relative contribution to expression differences over evolutionary time.

Screening and stability analysis of reference genes in fasting caecotrophy model in rabbits.

BACKGROUND: The selection and validation of stably expressed reference genes is key for accurately quantifying the mRNA abundance of genes under different treatments. In the rabbit model of fasting caecotrophy, reports about the selection of stable reference genes are not available. METHODS AND RESULTS: This study aims to screen suitable reference genes in different tissues (including uterus, cecum, and liver) of rabbits between control and fasting caecotrophy groups. RT-qPCR was used to analyze the expression levels of eight commonly used reference genes (including GAPDH, 18S rRNA, B2M, CYP, HPRT1, ß-actin, H2afz, Ywhaz), and RefFinder (including geNorm, NormFinder, and BestKeeper) was used to analyze the expression stability of these reference genes. Our results showed that the most stable reference genes were different in different tissues and treatments. In the control and fasting caecotrophy groups, CYP, GAPDH and HPRT1 were proven to be the top stable reference genes in the uterus, cecum, and liver tissues, respectively. GAPDH and Ywhaz were proven to be the top two stable reference genes among uterus, cecum, and liver in both control and fasting caecotrophy groups. CONCLUSIONS: Our results indicated that the combined analysis of three or more reference genes (GAPDH, HPRT1, and Ywhaz) are recommended to be used for RT-qPCR normalization in the rabbit model of fasting caecotrophy, and that GAPDH is a better choice than the other reference genes for normalizing the relative expression of target genes in different tissues of fasting caecotrophy rabbits.

Identification and evaluation of an appropriate housekeeping gene for real time gene profiling of hepatocellular carcinoma cells cultured in three dimensional scaffold.

BACKGROUND: Assessing an optimal reference gene as an internal control for target gene normalization is important during quantitative real time polymerase chain reaction (RT-qPCR) of three dimensional (3D) cell culture. Especially, gene profiling of cancer cells under a complex 3D microenvironment in a polymer scaffold provides a deeper understanding of tumor functioning in vivo. METHODS AND RESULTS: Expression of six housekeeping genes (HKG's): Glyceraldehyde-3-phosphodehydrogenase (GAPDH), ß-actin (ACTB), beta-2-microglobulin (B2M), 18S ribosomal RNA (18S rRNA), peptidyl-propyl-isomerase A (PPIA), and ribosomal protein L13 (RPL-13) during two dimensional (2D) culture, and alginate-carboxymethylcellulose scaffold based 3D culture conditioned up to 21 days was analysed for hepatocellular carcinoma (Huh-7) cells. The gene expression studies were performed by determining primer efficiency, melting curve and threshold cycle analysis. Further, RT-qPCR data was validated statistically using geNorm and NormFinder softwares. The study indicated RPL-13, 18S rRNA and B2M to be stable among selected referral HKG candidates. CONCLUSION: An exploration of a reliable HKG is necessary for normalization of gene expression in RT-qPCR during varying cell culture conditions.

Establishment and validation of five autophagy-related signatures for predicting survival and immune microenvironment in glioma.

BACKGROUND: Gliomas, especially Glioblastoma multiforme, are the most frequent type of primary tumors in central nervous system. Increasing researches have revealed the relationship between autophagy and tumor, while the molecular mechanism of autophagy in glioma is still rarely reported. OBJECTIVE: Our research aims to conform the autophagy-related genes (ARGs) implicated in the development and progression of glioma and improve our understanding of autophagy in glioma. METHODS: 20 candidate ARGs were screened through the protein-protein interaction network. We also downloaded the publicly accessible glioma data for 665 individuals from TCGA and 970 individuals from CGGA with RNA sequences and clinicopathological information. Subsequently, univariate and multivariate Cox regression analysis identified 5 key ARGs among the 20 candidate genes as key prognostic genes for survival, GSEA and immune response analysis. RESULTS: ATG5, BCL2L1, CASP3, CASP8, GAPDH were identified as key ARGs in our research. Further studies showed that the high-risk population was linked to a dismal prognosis and suggested an immune-inhibitory microenvironment. GSEA results demonstrated that high risk population was closely related to DNA repair, hypoxia pathways, implicated in immunosuppression and carcinogenesis. Through CMap, we finally identified 14 candidate drugs for the ARG high risk population. CONCLUSIONS: This study established and verified an ARG risk model, which can serve as an independent predictor for prognosis, reflect on the strength of the immune response and predict the potential drugs in glioma. Our findings offer new understandings of ARG molecular mechanism and promising therapeutic targets for glioma treatment.

Development and Validation of an Autophagy-Related Gene Signature for Predicting the Prognosis of Hepatocellular Carcinoma.

PURPOSE: Autophagy is a lysosomal degradation pathway that is essential for maintaining the homeostasis of the intracellular environment. Mounting evidence indicates that autophagy plays an essential role in the occurrence and development of hepatocellular cancer (HCC). This research is aimed at exploring the prognostic value of autophagy-related genes (ARGs) in HCC patients. METHODS: The Wilcoxon test was used to identify differentially expressed ARGs in The Cancer Genome Atlas (TCGA) HCC cohort. Then, the TCGA cohort was randomly divided into training and testing groups. Cox and LASSO regression models were used to screen for autophagy-related genes that affect overall survival (OS) in the TCGA training group. Based on the coefficient of risk genes, we constructed an autophagy-related gene signature for predicting the prognosis of HCC patients. Finally, we validated the prognostic significance of autophagy-related gene signature using the TCGA testing group and three external datasets. RESULTS: ATG10, BIRC5, GAPDH, and TMEM74 are risk genes for OS. According to the optimal cutoff value of risk score in each HCC dataset, HCC patients can divide into high- and low-risk groups. ARG risk score can significantly distinguish HCC patients with different survival outcomes. Meanwhile, the ARG risk score is independently correlated with OS in multiple HCC cohorts. CONCLUSIONS: The autophagy-related risk score can effectively screen high-risk HCC patients and provide guidance for clinical prevention and treatment of HCC.

Characterization of endogenous promoters of GapC1 and GS for recombinant protein expression in Phaeodactylum tricornutum.

Although diatoms have been utilized as a cellular factory to produce biopharmaceuticals, recombinant proteins, and biofuels, only a few numbers of gene promoters are available. Therefore, the development of novel endogenous promoters is essential for the production of a range of bioactive substances. Here, we characterized the activities of endogenous promoters glyceraldehyde-3-phosphate dehydrogenase (GapC1) and glutamine synthetase (GS) of Phaeodactylum tricornutum using green fluorescent protein (GFP) under different culture conditions. Compared with the widely used fucoxanthin chlorophyll-binding protein A (fcpA) promoter, the GS promoter constitutively drove the expression of GFP throughout all growth phases of P. tricornutum, regardless of culture conditions. Additionally, the GFP level driven by the GapC1 promoter was the highest at the log phase, similar to the fcpA promoter, and increased light and nitrogen-starvation conditions reduced GFP levels by inhibiting promoter activity. These results suggested that the GS promoter could be utilized as a strong endogenous promoter for the genetic engineering of P. tricornutum.

Mutual stimulatory signaling between human myogenic cells and rat cerebellar neurons.

Insight into the bidirectional signaling between primary human myogenic cells and neurons is lacking. For this purpose, human myogenic cells were derived from the semitendinosus and gracilis muscles of five healthy individuals and co-cultured with cerebellar granule neurons from two litters of 7-day-old Wistar rat pups, in muscle medium or neural medium, alongside monocultures of myogenic cells or neurons. RT-PCR was performed to determine human mRNA levels of GAPDH, Ki67, myogenin, and MUSK, and the acetylcholine receptor subtypes CHRNA1, CHRNB1, CHRNG, CHRND, and CHRNE, and rat mRNA levels of GAPDH, Fth1, Rack1, vimentin, Cdh13, and Ppp1r1a. Immunocytochemistry was used to evaluate neurite outgrowth (GAP43) in the presence and absence of myogenic cells. Co-culture with primary neurons lead to higher myogenic cell gene expression levels of GAPDH, myogenin, MUSK, CHRNA1, CHRNG, and CHRND, compared to myogenic cells cultured alone. It appeared that neurons preferentially attached to myotubes and that neurite outgrowth was enhanced when neurons were cultured with myogenic cells compared to monoculture. In neural medium, rat mRNA levels of GAPDH, vimentin, Cdh13, and Ppp1r1a were greater in co-culture, versus monoculture, whereas in muscle medium co-culture lead to lower levels of Fth1, Rack1, vimentin, and Cdh13 than monoculture. These findings demonstrate mutually beneficial stimulatory signaling between rat cerebellar granule neurons and human myogenic cells, providing support for an active role for both the neuron and the muscle cell in stimulating neurite growth and myogenesis. Bidirectional muscle nerve signaling.

Molecular Determinants for RNA Release into Extracellular Vesicles.

Extracellular vesicles (EVs) are important for intercellular communication and act as vehicles for biological material, such as various classes of coding and non-coding RNAs, a few of which were shown to selectively target into vesicles. However, protein factors, mechanisms, and sequence elements contributing to this specificity remain largely elusive. Here, we use a reporter system that results in different types of modified transcripts to decipher the specificity determinants of RNAs released into EVs. First, we found that small RNAs are more efficiently packaged into EVs than large ones, and second, we determined absolute quantities for several endogenous RNA transcripts in EVs (U6 snRNA, U1 snRNA, Y1 RNA, and GAPDH mRNA). We show that RNA polymerase III (pol III) transcripts are more efficiently secreted into EVs compared to pol II-derived transcripts. Surprisingly, our quantitative analysis revealed no RNA accumulation in the vesicles relative to the total cellular levels, based on both overexpressed reporter transcripts and endogenous RNAs. RNA appears to be EV-associated only at low copy numbers, ranging between 0.02 and 1 molecule per EV. This RNA association may reflect internal EV encapsulation or a less tightly bound state at the vesicle surface.

Highly sensitive fusion detection using plasma cell-free RNA in non-small-cell lung cancers.

ALK, ROS1, and RET kinase fusions are important predictive biomarkers of tyrosine kinase inhibitors (TKIs) in non-small-cell lung cancer (NSCLC). Analysis of cell-free DNA (cfDNA) provides a noninvasive method to identify gene changes in tumor cells. The present study sought to use cfRNA and cfDNA for identifying fusion genes. A reliable protocol was established to detect fusion genes using cfRNA and assessed the analytical validity and clinical usefulness in 30 samples from 20 cases of fusion-positive NSCLC. The results of cfRNA-based assays were compared with tissue biopsy and cfDNA-based liquid biopsy (Guardant360 plasma next-generation sequencing [NGS] assay). The overall sensitivity of the cfRNA-based assay was 26.7% (8/30) and that of cfDNA-based assay was 16.7% (3/18). When analysis was limited to the samples collected at chemo-naïve or progressive disease status and available for both assays, the sensitivity of the cfRNA-based assay was 77.8% (7/9) and that of cfDNA-based assay was 33.3% (3/9). Fusion gene identification in cfRNA was correlated with treatment response. These results suggest that the proposed cfRNA assay is a useful diagnostic test for patients with insufficient tissues to facilitate effective administration of first-line treatment and is a useful tool to monitor the progression of NSCLC for consideration of second-line treatments.

Identification of the Hub Genes in Alzheimer's Disease.

PURPOSE: Alzheimer's disease (AD) is considered to be the most common neurodegenerative disease and also one of the major fatal diseases affecting the elderly, thus bringing a huge burden to society. Therefore, identifying AD-related hub genes is extremely important for developing novel strategies against AD. MATERIALS AND METHODS: Here, we extracted the gene expression profile GSE63061 from the National Center for Biotechnology Information (NCBI) GEO database. Once the unverified gene chip was removed, we standardized the microarray data after quality control. We utilized the Limma software package to screen the differentially expressed genes (DEGs). We conducted Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of DEGs. Subsequently, we constructed a protein-protein interaction (PPI) network using the STRING database. RESULT: We screened 2169 DEGs, comprising 1313 DEGs with upregulation and 856 DEGs with downregulation. Functional enrichment analysis showed that the response of immune, the degranulation of neutrophils, lysosome, and the differentiation of osteoclast were greatly enriched in DEGs with upregulation; peptide biosynthetic process, translation, ribosome, and oxidative phosphorylation were dramatically enriched in DEGs with downregulation. 379 nodes and 1149 PPI edges were demonstrated in the PPI network constructed by upregulated DEGs; 202 nodes and 1963 PPI edges were shown in the PPI network constructed by downregulated DEGs. Four hub genes, including GAPDH, RHOA, RPS29, and RPS27A, were identified to be the newly produced candidates involved in AD pathology. CONCLUSION: GAPDH, RHOA, RPS29, and RPS27A are expected to be key candidates for AD progression. The results of this study can provide comprehensive insight into understanding AD's pathogenesis and potential new therapeutic targets.

Single-Cell Transcriptome Analysis Uncovers Intratumoral Heterogeneity and Underlying Mechanisms for Drug Resistance in Hepatobiliary Tumor Organoids.

Molecular heterogeneity of hepatobiliary tumor including intertumoral and intratumoral disparity always leads to drug resistance. Here, seven hepatobiliary tumor organoids are generated to explore heterogeneity and evolution via single-cell RNA sequencing. HCC272 with high status of epithelia-mesenchymal transition proves broad-spectrum drug resistance. By examining the expression pattern of cancer stem cells markers (e.g., PROM1, CD44, and EPCAM), it is found that CD44 positive population may render drug resistance in HCC272. UMAP and pseudo-time analysis identify the intratumoral heterogeneity and distinct evolutionary trajectories, of which catenin beta-1 (CTNNB1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and nuclear paraspeckle assembly transcript 1 (NEAT1) advantage expression clusters are commonly shared across hepatobiliary organoids. CellphoneDB analysis further implies that metabolism advantage organoids with enrichment of hypoxia signal upregulate NEAT1 expression in CD44 subgroup and mediate drug resistance that relies on Jak-STAT pathway. Moreover, metabolism advantage clusters shared in several organoids have similar characteristic genes (GAPDH, NDRG1 (N-Myc downstream regulated 1), ALDOA, and CA9). The combination of GAPDH and NDRG1 is an independent risk factor and predictor for patient survival. This study delineates heterogeneity of hepatobiliary tumor organoids and proposes that the collaboration of intratumoral heterogenic subpopulations renders malignant phenotypes and drug resistance.

Transcriptomic Analysis of Rice Plants Overexpressing PsGAPDH in Response to Salinity Stress.

In plants, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a main enzyme in the glycolytic pathway. It plays an essential role in glycerolipid metabolism and response to various stresses. To examine the function of PsGAPDH (Pleurotus sajor-caju GAPDH) in response to abiotic stress, we generated transgenic rice plants with single-copy/intergenic/homozygous overexpression PsGAPDH (PsGAPDH-OX) and investigated their responses to salinity stress. Seedling growth and germination rates of PsGAPDH-OX were significantly increased under salt stress conditions compared to those of the wild type. To elucidate the role of PsGAPDH-OX in salt stress tolerance of rice, an Illumina HiSeq 2000 platform was used to analyze transcriptome profiles of leaves under salt stress. Analysis results of sequencing data showed that 1124 transcripts were differentially expressed. Using the list of differentially expressed genes (DEGs), functional enrichment analyses of DEGs such as Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were performed. KEGG pathway enrichment analysis revealed that unigenes exhibiting differential expression were involved in starch and sucrose metabolism. Interestingly, trehalose-6-phosphate synthase (TPS) genes, of which expression was enhanced by abiotic stress, showed a significant difference in PsGAPDH-OX. Findings of this study suggest that PsGAPDH plays a role in the adaptation of rice plants to salt stress.

Molecular phylogenetic and in silico analysis of glyceraldeyde-3-phosphate dehydrogenase (GAPDH) gene from northern bobwhite quail (Colinus virginianus).

Many recent studies have been focused on prevalence and impact of two helminth parasites, eyeworm Oxyspirura petrowi and caecal worm Aulonocephalus pennula, in the northern bobwhite quail (Colinus virginianus). However, few studies have attempted to examine the effect of these parasites on the bobwhite immune system. This is likely due to the lack of proper reference genes for relative gene expression studies. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that is often utilized as a reference gene, and in this preliminary study, we evaluated the similarity of bobwhite GAPDH to GAPDH in other avian species to evaluate its potential as a reference gene in bobwhite. GAPDH was identified in the bobwhite full genome sequence and multiple sets of PCR primers were designed to generate overlapping PCR products. These products were then sequenced and then aligned to generate the sequence for the full-length open reading frame (ORF) of bobwhite GAPDH. Utilizing this sequence, phylogenetic analyses and comparative analysis of the exon-intron pattern were conducted that revealed high similarity of GAPDH encoding sequences among bobwhite and other Galliformes. Additionally, This ORF sequence was also used to predict the encoded protein and its three-dimensional structure which like the phylogenetic analyses reveal that bobwhite GAPDH is similar to GAPDH in other Galliformes. Finally, GAPDH qPCR primers were designed, standardized, and tested with bobwhite both uninfected and infected with O. petrowi, and this preliminary test showed no statistical difference in expression of GAPDH between the two groups. These analyses are the first to investigate GAPDH in bobwhite. These efforts in phylogeny, sequence analysis, and protein structure suggest that there is > 97% conservation of GADPH among Galliformes. Furthermore, the results of these in silico tests and the preliminary qPCR indicate that GAPDH is a prospective candidate for use in gene expression analyses in bobwhite.

Determining the quantitative relationship between glycolysis and GAPDH in cancer cells exhibiting the Warburg effect.

Previous studies have identified GAPDH as a promising target for treating cancer and modulating immunity because its inhibition reduces glycolysis in cells (cancer cells and immune cells) with the Warburg effect, a modified form of cellular metabolism found in cancer cells. However, the quantitative relationship between GAPDH and the aerobic glycolysis remains unknown. Here, using siRNA-mediated knockdown of GAPDH expression and iodoacetate-dependent inhibition of enzyme activity, we examined the quantitative relationship between GAPDH activity and glycolysis rate. We found that glycolytic rates were unaffected by the reduction of GAPDH activity down to 19% ± 4.8% relative to untreated controls. However, further reduction of GAPDH activity below this level caused proportional reductions in the glycolysis rate. GAPDH knockdown or inhibition also simultaneously increased the concentration of glyceraldehyde 3-phosphate (GA3P, the substrate of GAPDH). This increased GA3P concentration countered the effect of GAPDH knockdown or inhibition and stabilized the glycolysis rate by promoting GAPDH activity. Mechanistically, the intracellular GA3P concentration is controlled by the Gibbs free energy of the reactions upstream of GAPDH. The thermodynamic state of the reactions along the glycolysis pathway was only affected when GAPDH activity was reduced below 19% ± 4.8%. Doing so moved the reactions catalyzed by GAPDH + PGK1 (phosphoglycerate kinase 1, the enzyme immediate downstream of GAPDH) away from the near-equilibrium state, revealing an important biochemical basis to interpret the rate control of glycolysis by GAPDH. Collectively, we resolved the numerical relationship between GAPDH and glycolysis in cancer cells with the Warburg effect and interpreted the underlying mechanism.

Validation of common reference genes stability in exosomal mRNA-isolated from liver and breast cancer cell lines.

Accurate relative gene expression analysis by reverse transcription-quantitative polymerase chain reaction relies on the usage of suitable reference genes for data normalization. The RNA content of small extracellular vesicles including exosomes is growingly considered as cancer biomarkers. So, reliable relative quantification of exosomal messenger RNA (mRNA) is essential for cancer diagnosis and prognosis applications. However, suitable reference genes for accurate normalization of a target gene in exosomes derived from cancer cells are not depicted yet. Here, we analyzed the expression and stability of eight well-known reference genes namely GAPDH, B2M, HPRT1, ACTB, YWHAZ, UBC, RNA18S, and TBP in exosomes-isolated from the liver (Huh7, HepG2, PLC/PRF/5) and breast (SK-BR-3) cancer cell lines using five different algorithms including geNorm, BestKeeper, Delta Ct, NormFinder, and RefFinder. Our results showed that ACTB, TBP, and HPRT1 were not expressed in exosomes-isolated from studied liver and breast cancer cell lines. The geNorm and BestKeeper algorithms indicated GAPDH and UBC as the most stable candidates. Moreover, Delta Ct and NormFinder algorithms showed YWHAZ as the most stable reference genes. Comprehensive ranking calculated by the RefFinder algorithm also pointed out GAPDH, YWHAZ, and UBC as the first three stable reference genes. Taken together, this study validated the common reference genes stability in exosomal mRNA derived from liver and breast cancer cell lines for the first time. We believe that this study would be the first step in finding more stable reference genes in exosomes that triggers more accurate detection of exosomal biomarkers.

Hydrogen sulfide-induced GAPDH sulfhydration disrupts the CCAR2-SIRT1 interaction to initiate autophagy.

The deacetylase SIRT1 (sirtuin 1) has emerged as a major regulator of nucleocytoplasmic distribution of macroautophagy/autophagy marker MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3). Activation of SIRT1 leads to the deacetylation of LC3 and its translocation from the nucleus into the cytoplasm leading to an increase in the autophagy flux. Notably, hydrogen sulfide (H2S) is a cytoprotective gasotransmitter known to activate SIRT1 and autophagy; however, the underlying mechanism for both remains unknown. Herein, we demonstrate that H2S sulfhydrates the active site cysteine of the glycolytic enzyme GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Sulfhydration of GAPDH leads to its redistribution into the nucleus. Importantly, nuclear localization of GAPDH is critical for H2S-mediated activation of autophagy as H2S does not induce autophagy in cells with GAPDH ablation or cells overexpressing a GAPDH mutant lacking the active site cysteine. Importantly, we observed that nuclear GAPDH interacts with CCAR2/DBC1 (cell cycle activator a nd apoptosis regulator 2) inside the nucleus. CCAR2 interacts with the deacetylase SIRT1 to inhibit its activity. Interaction of GAPDH with CCAR2 disrupts the inhibitory effect of CCAR2 on SIRT1. Activated SIRT1 then deacetylates MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 beta) to induce its translocation into the cytoplasm and activate autophagy. Additionally, we demonstrate this pathway's physiological role in autophagy-mediated trafficking of Mycobacterium tuberculosis into lysosomes to restrict intracellular mycobacteria growth. We think that the pathway described here could be involved in H2S-mediated clearance of intracellular pathogens and other health benefits.Abbreviations: ATG5: autophagy related 5; ATG7: autophagy related 7; BECN1: beclin 1, autophagy related; CCAR2/DBC1: cell cycle activator and apoptosis regulator 2; CFU: colony-forming units; DLG4/PSD95: discs large MAGUK scaffold protein 4; EX-527: 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; H2S: hydrogen sulfide; HEK: human embryonic kidney cells; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; Mtb: Mycobacterium tuberculosis; MTOR: mechanistic target of rapamycin kinase; MOI: multiplicity of infection; NO: nitric oxide; PI3K: phosphatidylinositol-4,5-bisphosphate 3-kinase; PLA: proximity ligation assay; PRKAA: protein kinase, AMP-activated, alpha catalytic subunit; SIAH1: siah E3 ubiquitin protein ligase 1A; SIRT1: sirtuin 1; TB: tuberculosis; TP53INP2/DOR: transformation related protein 53 inducible nuclear protein 2; TRP53/TP53: transformation related protein 53.

Assessment of the potential allergenicity and toxicity of Pichia proteins in a novel leghemoglobin preparation.

The production of soy leghemoglobin C2 (LegH) by Pichia pastoris (syn. K. phaffii) was developed by Impossible Foods to serve as a sustainable source of flavor and aroma in plant-based meats. The potential allergenicity and toxicity of a LegH from a new production process was analyzed using bioinformatics, proteomics and a pepsin digestion assay on leghemoglobin, and residual host proteins. LegH in the new preparation had the same proteoform as in the previous preparations as well as in soy root nodule extracts. Results of seven Pichia proteins, each representing ≥1% of the total protein content, showed no significant sequence matches to any known allergens with the exception of one, which matched the highly conserved wheat GAPDH, whose protein homolog is found in fungi and humans. Based on the data, it is unlikely that there is any risk of cross reactivity between LegH Prep and GAPDH. Pichia protein sequences showed very good alignment to homologous proteins from many common yeasts including Saccharomyces sp. In addition, LegH and Pichia proteins were all rapidly digested in a pepsin digest assay. In conclusion, LegH Prep from this P. pastoris production process is unlikely to pose a risk of food allergenicity.

Reference gene selection for transcriptional profiling in Cryptocercus punctulatus, an evolutionary link between Isoptera and Blattodea.

The subsocial life style and wood-feeding capability of Cryptocercus gives us an evolutionary key to unlock some outstanding questions in biology. With the advent of the Genomics Era, there is an unprecedented opportunity to address the evolution of eusociality and the acquisition of lignocellulases at the genetic level. However, to quantify gene expression, an appropriate normalization strategy is warranted to control for the non-specific variations among samples across different experimental conditions. To search for the internal references, 10 housekeeping genes from a gut transcriptome of a wood-feeding cockroach, Cryptocercus punctulatus, were selected as the candidates for the RT-qPCR analysis. The expression profiles of these candidates, including ACT, EF1α, GAPDH, HSP60, HSP70, αTUB, UBC, RPS18, ATPase and GST, were analyzed using a panel of analytical tools, including geNorm, NormFinder, BestKeeper, and comparative ΔCT method. RefFinder, a comprehensive ranking system integrating all four above-mentioned algorithms, rated ACT as the most stable reference gene for different developmental stages and tissues. Expression analysis of the target genes, Hex-1 and Cell-1, using the most or the least appropriate reference genes and a single or multiple normalizers signified this research. Our finding is the first step toward establishing a standardized RT-qPCR analysis in Cryptocercus.

BDNF Val66Met Polymorphism, the Allele-Specific Analysis by qRT-PCR - a Novel Protocol.

Background: Alteration in brain-derived neurotrophic factor (BDNF) production is a marker of neuropathological conditions, which has led to the investigation of Val66Met polymorphism occurring in the human BDNF gene (BDNF). Presently, there are no reported methods available for the analysis of Val66Met impact on human BDNF functioning. Purpose: To develop a qRT-PCR protocol for the allele-specific expression evaluation of the Val66Met polymorphism in BDNF. Methods: Using RNA extracted from muscle samples of 9 healthy volunteers (32.9 ± 10.3 y) at rest and following a maximal effort aerobic capacity exercise test, a protocol was developed for the detection of Val66/Met66 allele-specific BDNF expression in Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) - relative to housekeeping genes - and validated by absolute quantification in Droplet Digital Polymerase Chain Reaction (ddPCR). Results: Differences in the relative values of BDNF mRNA were confirmed by ddPCR analysis. HPRT1 and B2M were the most stable genes expressed in muscle tissue among different metabolic conditions, while GAPDH revealed to be metabolic responsive. Conclusion: Our qRT-PCR protocol successfully determines the allele-specific detection and changes in BDNF expression regarding the Val66Met polymorphism.