Improved method for calculating CO2 emission from industrial solid wastes combustion system based on fossil and biogenic carbon fraction.

Waste-to-Energy (WtE) technology is the most effective solution for managing non-recyclable wastes through mass burning and energy recovery. Owing to the significant volumes of plastics in China's industrial solid wastes (ISW), a large amount of greenhouse gases (GHG) is generated during the incineration process. Therefore, monitoring GHG emissions from WtE facilities is essential. Owing to the lack of suitable accounting models and characterized fossil carbon fraction (FCF) data, current studies use default values provided by the Intergovernmental Panel on Climate Change's (IPCC), which increases calculation inaccuracies. Therefore, this study established an improved method to accurately account for carbon emissions during solid waste incineration by firstly using radiocarbon dating by accelerator mass spectrometry (AMS) technique to determine the FCF of the solid waste components in China. Monte Carlo analysis was employed to perform the sensitivity analysis, and the results indicated that there was a significant deviation between the measured value and IPCC's default values of FCF, 3.2, 32.48, 93.39, 93.76, 90.49, and 93.8 % for paper, cotton, synthetic textiles, artificial rubber, artificial leather, and plastics, respectively. By replacing coal with ISW in a 2 × 110 t/h circulating fluidized bed boilers, 9.251 × 104 t CO2-eq emissions were reduced, and the carbon emission factor reached 0.56 t CO2-eq/t waste. This study complements the research gap fossil carbon data of wastes in the IPCC guidelines and provides a more accurate and effective way to calculate carbon emissions during ISW incineration treatment.

Partial substitution of wheat flour with soybean and gluten powder: impact on flavor characteristics of Pixian Douban-Meju and its quality.

BACKGROUND: In this study, different proportions of soybean flour and gluten flour were used as partial replacements for wheat flour for the fermentation of Pixian Douban-Meju (PXDB). The aim was to study the effects of soybean flour/gluten flour on the quality improvement of PXDB. RESULTS: In comparison with the control group (CT) (0% substitution of wheat flour), substitution of wheat flower with 12.5% soybean flour (the H2 group), 7.5% gluten flour (G2), and 10% gluten flour (G3) improved the amino acid nitrogen content by 3.8%, 5.6%, and 9.4% respectively. The mixtures of wheat flour and gluten flour (G2 or G3) increased the organic acid and free amino acid content. The results of two-dimensional gas chromatography mass spectrometry (GC × GC-MS) showed that the amount of key aroma substances increased about sixfold in comparison with the CT group (194.61 g.kg-1 ), achieving 1283.67, 1113.883, and 1160.19 g.kg-1 in the H2, G2, and G3 groups, respectively. There were also more aldehydes and pyrazines in all the substitution groups. Quantitative descriptive analysis indicated that the G3 sample presented the best organoleptic quality with a reddish-brown color and a more mellow aroma than the control sample. CONCLUSION: In conclusion, the fermentation of G3 resulted in higher quality PXDB-meju, showing that partial substitution of wheat flour with gluten improved the quality of PXDB. © 2023 Society of Chemical Industry.

Profiles of Small Regulatory RNAs at Different Growth Phases of Streptococcus thermophilus During pH-Controlled Batch Fermentation.

Small regulatory RNA (sRNA) has been shown to play an important role under various stress conditions in bacteria, and it plays a vital role in regulating growth, adaptation and survival through posttranscriptional control of gene expression in bacterial cells. Streptococcus thermophilus is widely used as a starter culture in the manufacture of fermented dairy products. However, the lack of reliable information on the expression profiles and potential physiological functions of sRNAs in this species hinders our understanding of the importance of sRNAs in S. thermophilus. The present study was conducted to assess the expression profiles of sRNAs in S. thermophilus and to identify sRNAs that exhibited significant changes. A total of 530 potential sRNAs were identified, including 198 asRNAs, 135 sRNAs from intergenic regions, and 197 sRNAs from untranslated regions (UTRs). Significant changes occurred in the expression of 238, 83, 194, and 139 sRNA genes during the lag, early exponential growth, late exponential growth, and stationary phases, respectively. The expression of 14 of the identified sRNAs was verified by qRT-PCR. Predictions of the target genes of these candidate sRNAs showed that the primary metabolic pathways targeted were involved in carbon metabolism, biosynthesis of amino acids, ABC transporters, the metabolism of amino and nucleotide sugars, purine metabolism, and the phosphotransferase system. The expression of the predicted target genes was further analyzed to better understand the roles of sRNAs during different growth stages. The results suggested that these sRNAs play crucial roles by regulating biological pathways during different growth phases of S. thermophilus. According to the results, sRNAs sts141, sts392, sts318, and sts014 are involved in the regulation of osmotic stress. sRNAs sts508, sts087, sts372, sts141, sts375, and sts119 are involved in the regulation of starvation stress. sRNAs sts129, sts226, sts166, sts231, sts204, sts145, and sts236 are involved in arginine synthesis. sRNAs sts033, sts341, sts492, sts140, sts230, sts172, and sts377 are involved in the ADI pathway. The present study provided valuable information for the functional study of sRNAs in S. thermophilus and indicated a future research direction for sRNA in S. thermophilus. Overall, our results provided new insights for understanding the complex regulatory network of sRNAs in S. thermophilus.

The nutrient requirements of Lactobacillus acidophilus LA-5 and their application to fermented milk.

Lactobacillus acidophilus LA-5 is a suitable probiotic for food application, but because of its slow growth in milk, an increase in its efficiency is desired. To shorten the time required for fermentation, the nutrient requirements of L. acidophilus LA-5 were analyzed, including the patterns of consumption of amino acids, purines, pyrimidines, vitamins, and metal ions. The nutrients required by L. acidophilus LA-5 were Asn, Asp, Cys, Leu, Met, riboflavin, guanine, uracil, and Mn2+, and when they were added to milk, the fermentation time of fermented milk prepared by L. acidophilus LA-5 alone was shortened by 9 h, with high viable cell counts that were maintained during storage of nutrient-supplemented fermented milk compared with the control. For fermented milk prepared by fermentation with Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, and L. acidophilus LA-5, viable cell counts of L. acidophilus LA-5 increased 1.3-fold and were maintained during storage of nutrient-supplemented fermented milk compared with the control. Adding nutrients had no negative effect on the quality of the fermented milk. The results indicated that suitable nutrients enhanced the growth of L. acidophilus LA-5 and increased its viable cell counts in fermented milk prepared by L. acidophilus LA-5 alone and mixed starter culture, respectively.

Metabolic Profiles of Carbohydrates in Streptococcus thermophilus During pH-Controlled Batch Fermentation.

Revealing the metabolic profiles of carbohydrates with their regulatory genes and metabolites is conducive to understanding their mechanism of utilization in Streptococcus thermophilus MN-ZLW-002 during pH-controlled batch fermentation. Transcriptomics and metabolomics were used to study carbohydrate metabolism. More than 200 unigenes were involved in carbohydrate transport. Of these unigenes, 55 were involved in the phosphotransferase system (PTS), which had higher expression levels than those involved in ABC protein-dependent systems, permeases, and symporters. The expression levels of the genes involved in the carbohydrate transport systems and phosphate transport system were high at the end-lag and end-exponential growth phases, respectively. In addition, 166 differentially expressed genes (DEGs) associated with carbohydrate metabolism were identified. Most genes had their highest expression levels at the end-lag phase. The pfk, ldh, zwf, and E3.2.1.21 genes involved in the glycolytic pathway had higher expression levels at the end-exponential growth phase than the mid-exponential growth phase. The results showed high expression levels of lacZ and galKTM genes and reabsorption of extracellular galactose. S. thermophilus MN-ZLW-002 can metabolize and utilize galactose. Overall, this comprehensive network of carbohydrate metabolism is useful for further studies of the control of glycolytic pathway during the high-density culture of S. thermophilus.

Characterization of Volatile Compounds by SPME-GC-MS During the Ripening of Kedong Sufu, a Typical Chinese Traditional Bacteria-Fermented Soybean Product.

The purpose of the present study was to evaluate the volatile profile of Kedong sufu, which is a typical bacteria-fermented soybean product in China, using solid phase microextraction coupled to gas chromatography and mass spectrometry and to reveal the evolution and diversity of flavor substances for this specialty. A total of 75 compounds were identified, including 35 esters, 4 alcohols, 4 phenols, 4 aldehydes, 7 acids, 10 ketones, and 11 other compounds from sufu samples during ripening. Some volatile compounds increased with ripening time, especially hexadecenoic acid ethyl ester, methoxy acetic acid pentyl ester, benzene propanoic acid ethyl ester, ethyl 9-hexadecenoate, ethyl oleate, ethanol, 3-methyl-1-butanol, 5-methoxy-1-pentanol, and eugenol; these compounds enriched the flavors and provided the typical savory taste of Kedong sufu. PRACTICAL APPLICATION: This research elucidated the formation of flavor substances in sufu. For traditional fermented foods, this study provides a scientific basis for promoting the generation of typical flavor substances and for the precise determination of maturity time.

Transcriptomic and proteomic profiling revealed global changes in Streptococcus thermophilus during pH-controlled batch fermentations.

Understanding global changes of physiological processes at the molecular level during the growth of Streptococcus thermophilus is essential for the rational design of cultivation media and the optimization of bioprocesses. Transcriptomics and proteomics were combined to investigate the global changes at the transcript and protein level during the growth of S. thermophilus. The expression of 1396 genes (FDR ≤ 0.001) and 876 proteins (P < 0.05) changed significantly over time. The most remarkable growth phase dependent changes occurred in the late-lag phase and were related to heterofermentation, glycolysis, peptidoglycan biosynthesis, conversion between amino acids and stress response. The present results could provide theoretical guidance for high-cell-density culture, help design cultivation media, and help attain a high biomass of S. thermophilus.

The nutrient requirements of Lactobacillus rhamnosus GG and their application to fermented milk.

Lactobacillus rhamnosus GG (LGG) performs many physiological functions, but the fermentation time is long when fermented milk is prepared using LGG alone. To shorten the fermentation time, we analyzed the nutrient requirement profiles of LGG. Based on nutrient requirement profiles, we evaluated the effects on the fermentation time, quality, and sensory properties of unmodified cow's milk fermented by LGG alone. According to the consumption and necessary patterns of amino acids and those of purine, pyrimidine, vitamins, metal ions, and nutrients essential to LGG, we selected Cys, Ser, Arg, Pro, Asp, Glu, guanine, uracil, and xanthine with which to supplement milk. Compared with fermented milk prepared using LGG alone in unmodified milk, the fermentation time of supplemented milk was shortened by 5 h. Viable cell counts, titratable acidity, and water-retaining capability of the fermented milk were improved by addition of nutrient supplements. Supplementation with nutrients did not obviously change the sensory and textural characteristics of fermented milk.

Profiles of Streptococcus thermophilus MN-ZLW-002 nutrient requirements in controlled pH batch fermentations.

This study aimed to evaluate the profiles of Streptococcus thermophilus nutrient requirements to guide the design of media for high cell density culturing. The growth kinetics, physiological state, and nutrient requirement profiles of S. thermophilus were analyzed in chemically defined media. The results showed that the intracellular ATP concentration, H+ -ATPase activity, NADH/NAD+ , and NH3 concentrations varied with intracellular pH. The nutrient components with the highest amounts required were Leu and Asp; ascorbic acid and p-amino benzoic acid; K+ and PO43- ; and guanine and uracil. The nutrient components with the largest required ratios were Arg, His, and Met; folic acid, cyanocobalamine, biotin, and nicotinic acid; Ca2+ and Mg2+ ; and guanine and uracil. In this study, different nutrient components were primarily used at different phase. Trp, Tyr, calcium pantothenate, thiamine, guanine, and Mg2+ were mainly used from late-lag to midexponential phase. Met, Pro, Phe, Ala, Gly, nicotinic acid, and riboflavin were mainly used from midexponential to late-exponential phase. The highest bioavailabilities of nutrient components were also found at diverse phase. Met, Leu, Ile, Asn, Glu, Lys, Pro, Gly, riboflavin, nicotinic acid, adenine, uracil, inosine, and Ca2+ had the highest bioavailability from late-lag to midexponential phase. Lactose, Glu, Asp, His, Trp, Cys, Val, Arg, Phe, Ala, Ser, Thr, Tyr, folate and cobalamin, calcium pantothenate, ascorbic acid, thiamine, biotin, p-amino benzoic acid, vitamin B6 , K+ , Mg2+ , guanine, xanthine, and PO43- had the highest bioavailability from midexponential to late-exponential phase. This study elucidated the nutrient requirement profiles with culture time and biomass at various average growth rates during the growth of S. thermophilus. The present results will help to formulate complex media for high cell density cultivation and provide the theoretical basis for S. thermophilus feeding strategies.

Metabolic Pathway Profiling in Intracellular and Extracellular Environments of Streptococcus thermophilus During pH-Controlled Batch Fermentations.

Elucidating the metabolite profiles during the growth of Streptococcus thermophilus is beneficial for understanding its growth characteristics. The changes in the intracellular and extracellular concentrations of carbohydrates, nucleotides, amino sugars, nucleoside sugars, fatty acids, and amino acids, as well as their metabolites over time, were investigated by metabolomics technology. Most metabolites of nucleotides were highly accumulated in the intracellular environment after the mid-exponential phase. Increases in the intracellular unsaturated fatty acids and N-acetyl-glucosamine and N-acetyl-muramoate recycling provided potential evidence that cell envelope remodeling occurred after the mid-exponential phase. At the later fermentation stages, potentially functional metabolite produced by glycine was highly accumulated in the intracellular environment. Additionally, potential toxic metabolites produced by phenylalanine and tyrosine could not be excreted into the extracellular environment in a timely basis. The accumulation of large amounts of these metabolites might be the primary cause of the overconsumption of amino acids and influence the growth of S. thermophilus.

Metabolic profiles of cysteine, methionine, glutamate, glutamine, arginine, aspartate, asparagine, alanine and glutathione in Streptococcus thermophilus during pH-controlled batch fermentations.

Elucidating the amino acid (AA) metabolism patterns of Streptococcus thermophilus has important effects on the precise design of nitrogen sources for high-cell-density culture. Transcriptomics and metabolomics were combined to reveal the cysteine, methionine, glutamate, glutamine, arginine, aspartate, asparagine and alanine metabolic pathways in S. thermophilus MN-ZLW-002, including glutathione. The changes in the synthesis, consumption and concentration of AAs and their metabolites, as well as regulatory genes with time were revealed. The metabolism of L-cysteine, L-glutamate, L-aspartate and L-alanine generated some potential functional metabolites. The metabolism of methionine and glutamate generated potential harmful metabolites. S. thermophilus MN-ZLW-002 can synthesize glutathione. Some potential functional metabolites have similar biological functions, indicating that S. thermophilus can resist environmental stresses through multiple mechanisms. The expression of some key genes in synthesis pathway of AA indicated that cysteine, methionine, asparagine, aspartate, arginine and lysine were insufficient or imbalance between nutrient components. The accumulation of large amounts of AA metabolites might be the primary cause of the overconsumption of AAs and influence the growth of S. thermophilus. The present study revealed the metabolic profiles of abovementioned AAs as well as those of regulatory genes and metabolites. These results were beneficial to the precise design of nitrogen sources and regulation of functional metabolites for the high-cell-density culture of S. thermophilus.

Short communication: Nutrient consumption patterns of Lactobacillus acidophilus KLDS 1.0738 in controlled pH batch fermentations.

This work focused on elucidating the nutrient consumption patterns of Lactobacillus acidophilus to guide the design of media for high-cell-density culture. We investigated the nutrient consumption patterns of L. acidophilus KLDS 1.0738 in chemically defined media in controlled pH batch fermentations. The most abundantly consumed amino acids, vitamins, ions, and purines and pyrimidines were Glu and Gly, pyridoxine and nicotinamide, K+ and PO43-, and guanine and uracil, respectively. The highest consumption rates for amino acids, vitamins, ions, and purines and pyrimidines were Asp and Arg, folic acid and pyridoxine, Fe2+ and Mn2+, and uracil and thymine, respectively. Furthermore, most of the amino acids, as well as guanine, thymine, pyridoxine, folic acid, nicotinamide, Mg2+, PO43-, and K+ had the highest bioavailability from the end of the lag growth phase to the mid-exponential growth phase. The overall consumption of glucose, adenine nucleotides, 2'-deoxyguanosine monohydrate, calcium pantothenate, Fe2+ and Mn2+ decreased with increasing average growth rate, indicating more effective use of these nutritional components at a higher average growth rate, as biomass yield based on nutritional component consumption increased. Our findings help to formulate complex media for high-cell-density cultivation and provide a theoretical basis for L. acidophilus feeding strategies.

Oxidized low-density lipoprotein suppresses expression of prostaglandin E receptor subtype EP3 in human THP-1 macrophages.

EP3, one of four prostaglandin E2 (PGE2) receptors, is significantly lower in atherosclerotic plaques than in normal arteries and is localized predominantly in macrophages of the plaque shoulder region. However, mechanisms behind this EP3 expression pattern are still unknown. We investigated the underlying mechanism of EP3 expression in phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophages with oxidized low-density lipoprotein (oxLDL) treatment. We found that oxLDL decreased EP3 expression, in a dose-dependent manner, at both the mRNA and protein levels. Moreover, oxLDL inhibited nuclear factor-κB (NF-κB)-dependent transcription of the EP3 gene by the activation of peroxisome proliferator-activated receptor-γ (PPAR-γ). Finally, chromatin immunoprecipitation revealed decreased binding of NF-κB to the EP3 promoter with oxLDL and PPAR-γ agonist treatment. Our results show that oxLDL suppresses EP3 expression by activation of PPAR-γ and subsequent inhibition of NF-κB in macrophages. These results suggest that down-regulation of EP3 expression by oxLDL is associated with impairment of EP3-mediated anti-inflammatory effects, and that EP3 receptor activity may exert a beneficial effect on atherosclerosis.

Overexpression of WRAP53 is associated with development and progression of esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is a highly aggressive cancer whose underlying molecular mechanisms are poorly understood. The natural antisense transcript (NAT) WRAP53 regulates p53 expression and WRAP53 protein is a component of telomerase. NATs play key roles in carcinogenesis, and although WRAP53 is known to increase cancer cell survival, its role in ESCC clinicopathology is unknown. The aim of this study was to investigate WRAP53 expression in ESCC and to correlate it with clinicopathological characteristics. METHODS: WRAP53 mRNA and protein expression was measured by quantitative PCR (qRT-PCR) and western blotting, respectively, in 4 ESSC cells lines and in 45 paired ESCC and non-neoplastic esophageal mucosa tissues. To correlate WRAP53 protein expression with clinicopathological characteristics, immunohistochemistry (IHC) was performed on 134 ESCC and 85 non-neoplastic esophageal mucosa tissues. RESULTS: Expression of WRAP53 was detected in all ESCC cell lines and was upregulated in the ESCC tissues compared with the corresponding non-neoplastic tissues (P<0.01). More cells expressed WRAP53 protein in the ESCC tissues than in the non-neoplastic tissues (P<0.01). Overexpression of WRAP53 was significantly correlated with tumor infiltration depth (P = 0.000), clinical stage (P = 0.001), and lymph node metastasis (P = 0.025). Wrap53 expression was not correlated with age, gender, or tumor differentiation. CONCLUSION: This report indicates increased expression of WRAP53 in ESCC and that WRAP53 overexpression is correlated with tumor progression. WRAP53 may play a significant role in ESCC; accordingly, WRAP53 could be a useful biomarker for ESCC.

Tumor suppression by miR-31 in esophageal carcinoma is p21-dependent.

microRNA regulation network is important for the cancer genetic heterogeneity. Relative to the increasing numbers of microRNA's targets identified, upstream regulatory mechanisms that control functional microRNAs are less well-documented. Here, we investigated the function of miR-31, a pleiotropically-acting microRNA, in esophageal squamous cell cancer (ESCC). We demonstrated that miR-31 only exerted tumor-suppressive effects in TE-7 ESCC cells, but not in TE-1 ESCC cells, although both of these cell lines harbor inactive p53. Interestingly, TE-1 cells highly expressed p21, while p21 levels were virtually undetectable in TE-7 cells, suggesting a p21-dependent mechanism of miR-31-mediated tumor suppression. Accordingly, knockdown of p21 in TE-1 cells reversed the tumor suppressive actions of miR-31. In patient ESCC specimens, real-time RT-PCR analysis revealed that expression of E2F2 and STK40, two known miR-31 target oncogenes, was negatively correlated with the expression of miR-31 in a p21-dependent manner, supporting the conclusion that miR-31 only downregulates its target oncogenes when p21 levels are low. Collectively, these data suggest a novel mechanism through which the tumor-suppressive effect of miR-31 is p21-dependent. In addition, we speculate that delivery of miR-31 could provide therapeutic benefit in the personalized management of a subgroup of ESCC patients with p21-deficient tumors.

Conversion of a heme-based oxygen sensor to a heme oxygenase by hydrogen sulfide: effects of mutations in the heme distal side of a heme-based oxygen sensor phosphodiesterase (Ec DOS).

The heme-based oxygen-sensor phosphodiesterase from Escherichia coli (Ec DOS), is composed of an N-terminal heme-bound oxygen sensing domain and a C-terminal catalytic domain. Oxygen (O2) binding to the heme Fe(II) complex in Ec DOS substantially enhances catalysis. Addition of hydrogen sulfide (H2S) to the heme Fe(III) complex in Ec DOS also remarkably stimulates catalysis in part due to the heme Fe(III)-SH and heme Fe(II)-O2 complexes formed by H2S. In this study, we examined the roles of the heme distal amino acids, M95 (the axial ligand of the heme Fe(II) complex) and R97 (the O2 binding site in the heme Fe(II)-O2 complex) of the isolated heme-binding domain of Ec DOS (Ec DOS-PAS) in the binding of H2S under aerobic conditions. Interestingly, R97A and R97I mutant proteins formed an oxygen-incorporated modified heme, verdoheme, following addition of H2S combined with H2O2 generated by the reactions. Time-dependent mass spectroscopic data corroborated the findings. In contrast, H2S did not interact with the heme Fe(III) complex of M95H and R97E mutants. Thus, M95 and/or R97 on the heme distal side in Ec DOS-PAS significantly contribute to the interaction of H2S with the Fe(III) heme complex and also to the modification of the heme Fe(III) complex with reactive oxygen species. Importantly, mutations of the O2 binding site of the heme protein converted its function from oxygen sensor to that of a heme oxygenase. This study establishes the novel role of H2S in modifying the heme iron complex to form verdoheme with the aid of reactive oxygen species.

Human NRDRB1, an alternatively spliced isoform of NADP(H)-dependent retinol dehydrogenase/reductase enhanced enzymatic activity of benzil.

AIMS: Human NRDRB1, a 226 amino acid alternatively spliced isoform of the NADP(H)- dependent retinol dehydrogenase/reductase (NRDR), lacks the complete coding region of exon 3, but preserves all the important functional motifs for NRDR catalytic activity. Nevertheless, its tissue distribution and physiological function remain to be elucidated. METHODS: Expression of NRDRB1 and NRDR in cells and tissues was analyzed by semi-quantitative polymerase chain reaction (PCR) and western blot. NRDRB1 was expressed as a His(6) fusion protein and subjected to kinetics assays. RESULTS: Recombinant NRDRB1 had 1.2 to 8.6 fold higher k(cat)/K(m) values than recombinant NRDR, depending on the substrate. NRDRB1 catalyzed the NADPH-dependent reduction of α-dicarbonyl compounds, such as isatin, 9,10-phenanthrenequinone, and especially benzil. The significantly high catalytic activity and the relatively high expression in human liver of NRDRB1 conferred cellular resistance to benzil-induced cell toxicity and over-expression of NRDRB1 in low expressing Ec109 cells significantly enhanced cell tolerance toward benzil. CONCLUSIONS: Based on its substrate specificity, catalytic activity and relatively high expression in human liver tissue, our results suggest that NRDRB1, an alternatively spliced isoform of NRDR in vivo functions better than NRDR as a dicarbonyl reductase for xenobiotics containing reactive carbonyls. Our study is the first reporting this phenomenon of the enzymes involved in biochemical reactions.

AS1DHRS4, a head-to-head natural antisense transcript, silences the DHRS4 gene cluster in cis and trans.

The human genome, like other mammalian genomes, encodes numerous natural antisense transcripts (NATs) that have been classified into head-to-head, tail-to-tail, or fully overlapped categories in reference to their sense transcripts. Evidence for NAT-mediated epigenetic silencing of sense transcription remains scanty. The DHRS4 gene encodes a metabolic enzyme and forms a gene cluster with its two immediately downstream homologous genes, DHRS4L2 and DHRS4L1, generated by gene duplication. We identified a head-to-head NAT of DHRS4, designated AS1DHRS4, which markedly regulates the expression of these three genes in the DHRS4 gene cluster. By pairing with ongoing sense transcripts, AS1DHRS4 not only mediates deacetylation of histone H3 and demethylation of H3K4 in cis for the DHRS4 gene, but also interacts physically in trans with the epigenetic modifiers H3K9- and H3K27-specific histone methyltransferases G9a and EZH2, targeting the promoters of the downstream DHRS4L2 and DHRS4L1 genes to induce local repressive H3K9me2 and H3K27me3 histone modifications. Furthermore, AS1DHRS4 induces DNA methylation in the promoter regions of DHRS4L2 by recruiting DNA methyltransferases. This study demonstrates that AS1DHRS4, as a long noncoding RNA, simultaneously controls the chromatin state of each gene within the DHRS4 gene cluster in a discriminative manner. This finding provides an example of transcriptional control over the multiple and highly homologous genes in a tight gene cluster, and may help explain the role of antisense RNAs in the regulation of duplicated genes as the result of genomic evolution.

Identification of a novel isoform of DHRS4 protein with a nuclear localization signal.

The DHRS4 gene encodes an NADP(H)-dependent retinol dehydrogenase/reductase (NRDR) and plays an important role in regulating the synthesis of retinoic acid. In the present study, we identified a novel splice RNA variant, designated NRDRA2, of the human DHRS4 gene by RT-PCR, 3' RACE, and 5' RACE. NRDRA2 mRNA lacked exons 4 and 6, and had a shift in the reading frame when compared to DHRS4 mRNA, resulting in loss of the peroxisomal targeting signal of NRDR and gain of a nuclear localization signal in the predicted NRDRA2 protein. Endogenous NRDRA2 protein was identified in the human cervical carcinoma cell line HeLa by immunoprecipitation and mass spectrometric assay. A green fluorescent protein reporter assay showed that NRDRA2 protein mainly localized to the nuclei, confirming the sequence at its C-terminus as a legitimate nuclear localization signal sequence. This study identifies the alternative transcript variant NRDRA2 encoding a subcellular nuclear localized NRDRA2 protein.

Bioinformatic analysis of the human DHRS4 gene cluster and a proposed mechanism for its transcriptional regulation.

BACKGROUND: The human DHRS4 gene cluster consists of three genes, DHRS4, DHRS4L2 and DHRS4L1. Among them, DHRS4 encodes NADP(H)-dependent retinol dehydrogenase/reductase. In a previous study, we investigated the alternative splicing of DHRS4 and DHRS4L2. DHRS4L1 was added to the gene cluster recently, but little is known about its structure and expression. To reveal the regulatory mechanism of the DHRS4 gene cluster expression, we studied the structure and transcription of DHRS4L1 in the context of the transcriptional behaviors of the human DHRS4 gene cluster. Based on the results of bioinformatics analysis, we propose a possible mechanism for the transcriptional regulation of the human DHRS4 gene cluster. RESULTS: The homologous comparison analysis suggests that DHRS4, DHRS4L2 and DHRS4L1 are three homologous genes in human. DHRS4L1 and DHRS4L2 are paralogues of DHRS4, and DHRS4L2 is the most recent member of the DHRS4 gene cluster. In the minus strand of the human DHRS4 gene cluster, a gene transcribed in an antisense direction was found containing a 5' sequence overlapping the region of exon 1 and promoter of DHRS4. By cloning the full length of RNA variants through 5'RACE and 3'RACE, we identified two transcription start sites, within exon a2 and exon 1, of this newly named gene DHRS4L1 using neuroblastoma cell line BE(2)-M17. Analysis of exon composition in the transcripts of DHRS4 gene cluster revealed that exon 1 was absent in all the transcripts initiated from exon a1 of DHRS4L2 and exon a2 of DHRS4L1. CONCLUSIONS: Alternatively spliced RNA variants are prevalent in the human DHRS4 gene cluster. Based on the analysis of gene transcripts and bioinformatic prediction, we propose here that antisense transcription may be involved in the transcriptional initiation regulation of DHRS4 and in the posttranscriptional splicing of DHRS4L2 and DRHS4L1 for the homologous identity of DHRS4 gene cluster. Beside the alternative transcriptional start sites, the antisense RNA is novel possible factor serving to remove exon 1 from the transcripts initiated from exon a1 and exon a2.