KLK10/LIPH/PARD6B/SLC52A3 are promising molecular biomarkers for the prognosis of pancreatic cancer through a ceRNA network.

Pancreatic adenocarcinoma (PAAD) remains challenging to diagnose and treat clinically due to its difficult early diagnosis, low surgical resection rate, and high risk of postoperative recurrence and metastasis. SMAD4 is a classical mutated gene in pancreatic cancer and is lost in up to 60%-90 % of PAAD patients, and its mutation often predicts a poor prognosis and treatment resistance. In this study, based on the expression profile data in The Cancer Genome Atlas database, we identified a ceRNA network composed of 2 lncRNAs, 1 miRNA, and 4 mRNAs through differential expression analysis and survival prognosis analysis. Among them, high expression of KLK10/LIPH/PARD6B/SLC52A3 influenced the prognosis and overall survival of PAAD patients. We confirmed the high expression of these target genes in pancreatic tissue of pancreatic-specific SMAD4-deficient mice. In addition, immune infiltration analysis showed that the high expression of these target genes affects the tumor immune environment and contributes to the progression of PAAD. Abnormal overexpression of these target genes may be caused by hypermethylation. In conclusion, we found that KLK10/LIPH/PARD6B/SLC52A3 is a potential prognostic marker for PAAD based on a competing endogenous RNA-mediated mechanism and revealed the potential pathogenic mechanism by which deficient expression of SMAD4 promotes pancreatic cancer progression, which provides a new pathway and theoretical basis for targeted therapy or improved prognosis of pancreatic cancer. These data will help reveal potential therapeutic targets for pancreatic cancer and improve the prognosis of pancreatic cancer patients.

The power and the promise of CAR-mediated cell immunotherapy for clinical application in pancreatic cancer.

BACKGROUND: Pancreatic cancer, referred to as the "monarch of malignancies," is a neoplastic growth mostly arising from the epithelial cells of the pancreatic duct and acinar cells. This particular neoplasm has a highly unfavorable prognosis due to its marked malignancy, inconspicuous initial manifestation, challenging early detection, rapid advancement, and limited survival duration. Cellular immunotherapy is the ex vivo culture and expansion of immune effector cells, granting them the capacity to selectively target malignant cells using specialized techniques. Subsequently, these modified cells are reintroduced into the patient's organism with the purpose of eradicating tumor cells and providing therapeutic intervention for cancer. PRESENT SITUATION: Presently, the primary cellular therapeutic modalities employed in the treatment of pancreatic cancer encompass CAR T-cell therapy, TCR T-cell therapy, NK-cell therapy, and CAR NK-cell therapy. AIM OF REVIEW: This review provides a concise overview of the mechanisms and primary targets associated with various cell therapies. Additionally, we will explore the prospective outlook of cell therapy in the context of treating pancreatic cancer.

Fecal calprotectin: A novel predictor of ulcerated esophageal injury after atrial fibrillation catheter ablation.

BACKGROUND: Atrial esophageal fistula (AEF) is a lethal complication that can occur post atrial fibrillation (AF) ablation. Esophageal injury (EI) is likely to be the initial lesion leading to AEF. Endoscopic examination is the gold standard for a diagnosis of EI but extensive endoscopic screening is invasive and costly. This study was conducted to determine whether fecal calprotectin (Fcal), a marker of inflammation throughout the intestinal tract, may be associated with the existence of esophageal injury. METHODS: This diagnostic study was conducted in a cohort of 166 patients with symptomatic AF undergoing radiofrequency catheter ablation from May 2020 to June 2021. Fcal tests were performed 1-7 days after ablation. All patients underwent endoscopic ultrasonography 1 or 2 days after ablation. RESULTS: The levels of Fcal were significantly different between the EI and non-EI groups (404.9 µg/g (IQR 129.6-723.6) vs. 40.4 µg/g (IQR 15.0-246.2), p < .001). Analysis of ROC curves revealed that a Fcal level of 125 µg/g might be the optimal cut-off value for a diagnosis of EI, giving a 78.8% sensitivity and a 65.4% specificity. The negative predictive value of Fcal was 100% for ulcerated EI. CONCLUSIONS: The level of Fcal is associated with EI post AF catheter ablation. 125 µg/g might be the optimal cut-off value for a diagnosis of EI. Negative Fcal could predict the absence of ulcerated EI, which could be considered a precursor to AEF.

Solvent effects on the photoinduced charge separation dynamics of directly linked zinc phthalocyanine-perylenediimide dyads: a nonadiabatic dynamics simulation with an optimally tuned screened range-separated hybrid functional.

Herein, we have employed a combination of the optimally tuned screened range-separated hybrid (OT-SRSH) functional, the polarizable continuum model (PCM), and nonadiabatic dynamics (NAMD) simulations to investigate the photoinduced dynamics of directly linked donor-acceptor dyads formed using zinc phthalocyanine (ZnPc) and perylenediimide (PDI), in which ZnPc is the donor while PDI is the acceptor. Our simulations aim to analyze the behavior of these dyads upon local excitation of the ZnPc moiety in the gas phase and in benzonitrile. Our findings indicate that the presence of a solvent can significantly influence the excited state dynamics of ZnPc-PDI dyads. Specifically, the polar solvent benzonitrile effectively lowers the vertical excitation energies of the charge transfer (CT) state from ZnPc to PDI. As a result, the energetic order of the locally excited (LE) states of ZnPc and the CT states is reversed compared to the gas phase. Consequently, the photoinduced electron transfer (PET) dynamics from ZnPc to PDI, which is absent in the gas phase, takes place in benzonitrile with a time constant of 10.4 ps. Importantly, our present work not only qualitatively agrees with experimental results but also provides in-depth insights into the underlying mechanisms responsible for the photoinduced dynamics of ZnPc-PDI. Moreover, this study emphasizes the importance of appropriately considering solvent effects in NAMD simulation of organic donor-acceptor systems, taking into account the distinct excited state dynamics observed in the gas phase and benzonitrile. Furthermore, the combination of the OT-SRSH functional, the PCM solvent model, and nonadiabatic dynamics simulations shows promise as a strategy for investigating the complex excited state dynamics of organic donor-acceptor systems in solvents. These findings will be valuable for the future design of novel organic donor-acceptor structures with improved performance.

Identification and validation of functional roles for three MYC-associated genes in hepatocellular carcinoma.

BACKGROUND: Aberrations in MYC underlie a large proportion of liver hepatocellular carcinoma (LIHC) cases; however, MYC is difficult to target because of its undruggable structure. We aimed to uncover MYC-associated molecular targets to provide new strategies for LIHC treatment. METHODS: LIHC transcriptome datasets and clinical information were obtained from The Cancer Genome Atlas. A series of bioinformatics analyses were performed for 370 patients who were stratified based on the median MYC expression level (high-MYC group and low-MYC group). Correlation analysis was performed to determine relationships between the expression of key MYC-associated genes and prognosis, DNA promotor methylation, and immune cell infiltration. Gene ontology and Kyoto Encyclopedia of Genes and Genomes Pathway enrichment analyses were performed to elucidate the functions of these genes in LIHC. Their expression and functions in LIHC were further verified using transgenic mice overexpressing c-Myc under control of the hepatocyte-specific promoter (Alb-Cre). RESULTS: AURKB, CCNB2, and CDKN3 were overexpressed in LIHC patients with high MYC expression and were associated with poor prognosis. Upregulation of these 3 genes was significantly correlated with hypomethylated promoter status, advanced T stage, metastasis, and immune cell infiltration in LIHC patients. Functional enrichment analyses indicated that these genes participate in the "p53 signaling pathway" and "cell cycle". Furthermore, RT-PCR and IHC analysis revealed that their mRNA and protein expression levels were upregulated in an Alb-Cre;cMYClsl/- mouse model. Drugs that target these 3 MYC-related genes were identified. CONCLUSION: Taken together, our results identify biomarkers of potential utility for managing liver cancer therapy owing to their significance in tumorigenesis, proliferation, and tumor immunity.

Recent trends in the incidence and survival of stage I liver cancer: a surveillance, epidemiology, and end results analysis.

BACKGROUND: Improvements in screening and imaging technologies and treatment of liver disease have influenced the trend in diagnosis for stage I liver cancer. In this article, recent trends in age, incidence, tumour size, and survival of different stages of liver cancer are analysed. METHODS: Surveillance, Epidemiology, and end results data from the National Cancer Institute were used to analyse trends in age-adjusted incidence rate, mean tumour size at diagnosis, age at diagnosis, and 5-year survival probability for stage I liver cancer. RESULTS: Stage I cases of liver cancer increased most tremendously over the study period, with a greater increase from 2004 to 2012 following a smaller increase from 2012 to 2015. Moreover, the mean age of stage I liver cancer increased by 1.72 years from 2004 to 2015. The 5-year-overall survival for stage I liver cases worsened from 97.9% to 83.7% from 2004 to 2011, whereas the 10-year survival probability for stage I cases worsened from 97.3% in 2004 to 79.6% in 2006. Comparing with higher stage cases, stage I liver cancer were more likely to be females, be married, live in metro areas, receive chemotherapy, and carry medical insurance. CONCLUSIONS: The incidence of stage I liver cancer has increased over the study period, with an increase in age of diagnosis, decrease in tumour size, and generally stable overall survival rate with slight decrease. These trends emphasized the importance of early detection of liver cancer and regular screening and better treatment for high-risk populations.RESEARCH HIGHLIGHTSImprovements in screening and imaging technologies and treatment of liver disease have influenced the trend in diagnosis for liver cancer.Stage I cases of liver cancer increased most tremendously over the study period, with a greater increase from 2004 to 2012 following a smaller increase from 2012 to 2015.These trends emphasized the importance of early detection of liver cancer and regular screening and better treatment for high-risk populations.

Construction of a Novel MYC-Associated ceRNA Regulatory Network to Identify Prognostic Biomarkers in Colon Adenocarcinoma.

Colorectal cancer (CRC) includes colon adenocarcinoma (COAD) and rectal adenocarcinoma (READ). Competitive endogenous RNA (ceRNA) is crucial for cancer pathogenesis. Abnormal expression of MYC is generally associated with a poor colon adenocarcinoma prognosis. The present study aimed to identify a novel MYC-associated ceRNA regulatory network and identify potential prognostic markers associated with COAD. We obtained the transcriptome sequencing profiles of 462 COAD cases from the TCGA database and analyzed differentially expressed genes (DEGs) in MYC high expression (MYChigh) and MYC low expression (Myclow) tumors. We identified an important lncRNA, LINC00114, which effectively predicts overall survival and plays a protective role in COAD. Moreover, the LINC00114/miR-216a-5p axis was identified as a clinical prognostic model. The predicted target genes of the LINC00114/miR-216a-5p axis include uromodulin Like 1 (UMODL1) and oncoprotein induced transcript 3 (OIT3), which are closely related to the survival and prognosis of COAD patients. In summary, we constructed a novel ceRNA regulatory network and identified potential biomarkers for the targeted therapy and prognosis of COAD.

Liquid biopsy in lung cancer: significance in diagnostics, prediction, and treatment monitoring.

Primary lung cancer is one of the most common malignant tumors in China. Approximately 60% of lung cancer patients have distant metastasis at the initial diagnosis, so it is necessary to find new tumor markers for early diagnosis and individualized treatment. Tumor markers contribute to the early diagnosis of lung cancer and play important roles in early detection and treatment, as well as in precision medicine, efficacy monitoring, and prognosis prediction. The pathological diagnosis of lung cancer in small biopsy specimens determines whether there are tumor cells in the biopsy and tumor type. Because biopsy is traumatic and the compliance of patients with multiple biopsies is poor, liquid biopsy has become a hot research direction. Liquid biopsies are advantageous because they are nontraumatic, easy to obtain, reflect the overall state of the tumor, and allow for real-time monitoring. At present, liquid biopsies mainly include circulating tumor cells, circulating tumor DNA, exosomes, microRNA, circulating RNA, tumor platelets, and tumor endothelial cells. This review introduces the research progress and clinical application prospect of liquid biopsy technology for lung cancer.

Deciphering cell-cell interactions and communication in the tumor microenvironment and unraveling intratumoral genetic heterogeneity via single-cell genomic sequencing.

A tumor's heterogeneity has important implications in terms of its clonal origin, progression, stemness, and drug resistance. Therefore, because of its significance in treatment, it is important to understand the gene expression pattern of a single cell, track gene expression or mutation in heterogeneous cells, evaluate the clonal origin of cancer cells, and determine the selective evolution of different subpopulations of cancer cells. Researchers are able to trace a cell's mutation and identify different types of tumor cells by measuring the whole transcriptome with single-cell sequencing (scRNA-seq). This technology provides a better understanding of the molecular mechanisms driving tumor growth than that offered by traditional RNA sequencing methods. In addition, it has revealed changes in the mutations and functions of somatic cells as a tumor evolves; it has also clarified immune cell infiltration and activation. Research on scRNA-seq technology has recently advanced significantly, suggesting new strategies for the treatment of cancer. In short, cancer researchers have become increasingly dependent on scRNA-seq. This paper reviews the development, detection principles, and processes of scRNA-seq technology and their application in tumor research. It also considers potential clinical applications.

Heterogeneity helps us determine the clonal origin, progression, and drug resistance of cancer cells.The gene expression pattern of a single cell has important biological significance.scRNA-seq enables a better understanding of cancer cells' molecular mechanisms.scRNA-seq provides information about the entirety of a tumor from what we can learn about a single cell.

Tetrahydropalmatine has a therapeutic effect in a lipopolysaccharide-induced disseminated intravascular coagulation model.

OBJECTIVES: The aim of this study was to determine the therapeutic effects of tetrahydropalmatine (Tet) on disseminated intravascular coagulation (DIC) by exploring the role of Tet using a lipopolysaccharide (LPS)-induced DIC model. Methods/Materials: We established a mouse DIC model by injecting LPS. Hematoxylin-eosin (HE) staining was performed to detect liver and kidney damage. Blood samples were obtained to determine liver and kidney injury indexes, coagulation indexes, and inflammatory cytokines. An in vitro cell inflammation model was also established. Tumor necrosis factor-α (TNF-α) levels and nuclear factor kappa B (NF-κB) signaling pathway activation were determined by western blot. RESULT: Tet ameliorated the damage to organ tissues, improved coagulation indexes, and reduced the inflammatory cytokine production in LPS-induced mouse DIC. Tet also inhibited TNF-α expression by suppressing NF-κB signaling pathway activation in an in vitro LPS model using RAW 264.7 macrophages. CONCLUSIONS: Tet has a mitigating and therapeutic effect on the LPS-induced DIC model via anticoagulant and anti-inflammatory effects, showing its potential as an adjunct to DIC treatment.

An allosteric PGAM1 inhibitor effectively suppresses pancreatic ductal adenocarcinoma.

Glycolytic enzyme phosphoglycerate mutase 1 (PGAM1) plays a critical role in cancer metabolism by coordinating glycolysis and biosynthesis. A well-validated PGAM1 inhibitor, however, has not been reported for treating pancreatic ductal adenocarcinoma (PDAC), which is one of the deadliest malignancies worldwide. By uncovering the elevated PGAM1 expressions were statistically related to worse prognosis of PDAC in a cohort of 50 patients, we developed a series of allosteric PGAM1 inhibitors by structure-guided optimization. The compound KH3 significantly suppressed proliferation of various PDAC cells by down-regulating the levels of glycolysis and mitochondrial respiration in correlation with PGAM1 expression. Similar to PGAM1 depletion, KH3 dramatically hampered the canonic pathways highly involved in cancer metabolism and development. Additionally, we observed the shared expression profiles of several signature pathways at 12 h after treatment in multiple PDAC primary cells of which the matched patient-derived xenograft (PDX) models responded similarly to KH3 in the 2 wk treatment. The better responses to KH3 in PDXs were associated with higher expression of PGAM1 and longer/stronger suppressions of cancer metabolic pathways. Taken together, our findings demonstrate a strategy of targeting cancer metabolism by PGAM1 inhibition in PDAC. Also, this work provided "proof of concept" for the potential application of metabolic treatment in clinical practice.

Establishment of a human primary pancreatic cancer mouse model to examine and investigate gemcitabine resistance.

Pancreatic cancer is one of the most fatal types of cancer and is associated with a dismal prognosis. Gemcitabine-based chemotherapy is clinically used for the treatment of advanced pancreatic cancer. However, many forms of pancreatic cancer have acquired resistance to gemcitabine. In order to prevent patients from suffering from the side effects of chemotherapy and to have the chance to receive more effective intervention, assessment of whether the patient pancreatic cancer cells are resistant to gemcitabine before clinical practice is crucial. Recently, patient-derived xenograft (PDX) models have been regarded as a practical approach for preclinical drug resistance test. In the present study, we harvested tumor specimens from 28 pancreatic cancer patients to establish PDX models. The tumor formation rate of the xenografts was 100%, several of which could be re-implanted in nude mice for more than 10 passages. Primary cells were further obtained from the PDX xenografts to determine their morphological features and evaluate their proliferation rate, migration capacity and angiopoietic ability. In addition, the sensitivities of the primary cells and PDX xenografts to gemcitabine were correlated with each other. When compared to the gemcitabine-sensitive cells, the gemcitabine-resistant cells had a higher level of MCF2L expression, suggesting that MCF2L plays an important role in gemcitabine resistance.

Atractylenolide â protects against lipopolysaccharide-induced disseminated intravascular coagulation by anti-inflammatory and anticoagulation effect.

OBJECTIVE: To investigate whether atractylenolide Ⅰ (ATL-Ⅰ) has protective effect on lipopolysaccharide (LPS)-induced disseminated intravascular coagulation (DIC) in vivo and in vitro, and explore whether NF-κB signaling pathway is involved in ATL-Ⅰ treatment. METHODS: New Zealand white rabbits were injected with LPS through marginal ear vein over a period of 6 h at a rate of 600 µg/kg (10 mL/h). Similarly, in the treatment groups, 1.0, 2.0, or 5.0 mg/kg ATL-Ⅰ were given. Both survival rate and organ function were tested, including the level of alanine aminotransferase (ALT), blood urine nitrogen (BUN), and TNF-α were examined by ELISA. Also hemostatic and fibrinolytic parameters in serum were measured. RAW 264.7 macrophage cells were administered with control, LPS, LPS + ATL-Ⅰ and ATL-Ⅰ alone, and TNF-α, phosphorylation (P)-IκBα, phosphorylation (P)-NF-κB (P65) and NF-κB (P65) were determined by Western blot. RESULTS: The administration of LPS resulted in 73.3% mortality rate, and the increase of serum TNF-α, BUN and ALT levels. When ATL-Ⅰ treatment significantly increased the survival rate of LPS-induced DIC model, also improved the function of blood coagulation. And protein analysis indicated that ATL-I remarkably protected liver and renal as decreasing TNF-α expression. In vitro, ATL-I obviously decreased LPS-induced TNF-α production and the expression of P-NF-κB (P65), with the decrease of P-IκBα. CONCLUSIONS: ATL-Ⅰ has protective effect on LPS-induced DIC, which can elevate the survival rate, reduce organ damage, improve the function of blood coagulation and suppress TNF-α expression by inhibiting the activation of NF-κB signaling pathway.

Length to width ratio of the ductus venosus in simple screening for fetal congenital heart diseases in the second trimester.

Antenatal diagnosis of congenital heart disease (CHD) is still low even though screening was first introduced over 25 years ago. The purpose of our study was to determine the efficacy of a second-trimester prenatal ultrasonographic method of screening for CHD.From September 2012 to September 2013, the length and width of the fetal ductus venosus were measured sonographically in 1006 singleton fetuses, and the ratio of length to width was calculated. The accuracy of each fetal measurement and Doppler ultrasonography were determined. The standard fetal echocardiographic evaluations including 2-dimensional gray-scale imaging, color, and Doppler color flow mapping were performed. The transducer was aligned to the long axis of the fetal trunk to view the ductus venosus in its full length, including the inlet (isthmus) and outlet portions of the vessel. The diameters of the vessel inner wall and mid-point of the ductus venosus were measured using calipers. All scans and fetal measurements were conducted by a registered sonographer with more than 20 years of perinatal ultrasound screening experience.Of the 1006 singleton fetuses between 19 and 28 weeks' gestation, 36 had CHD. The ductus venosus length/width ratio (DVR) for the first CHD screening was extremely sensitive at 88.90%, with a specificity of 99.10% for the cardiac abnormalities included in this study. Chromosomal anomalies accompanied CHD in 0.4% (4/1006) of all cases and 11.11% (4/36) of the CHD cases.The DVR differed significantly between fetuses with CHD and normal fetuses during the second trimester. Careful assessment of the ratio should be a part of the sonographic examination of every fetus. In the case of a small DVR, advanced echocardiography and karyotype analysis should be performed. The ratio is a helpful tool for screening CHD abnormalities prenatally in the Chinese population.

Thioredoxin is a novel diagnostic and prognostic marker in patients with ischemic stroke.

Serum thioredoxin (TRX), a redox-regulating protein with antioxidant activity, was recognized as an oxidative-stress marker. The purpose of this study was to investigate the potential diagnostic and prognostic role of TRX in Chinese patients with acute ischemic stroke (AIS). From January 1, 2012, to December 31, 2013, all patients with first-ever acute ischemic stroke were recruited to participate in the study. Serum levels of TRX were assayed with solid-phase sandwich ELISA, and severity of stroke was evaluated with the National Institutes of Health Stroke Scale (NIHSS) score on admission. Short-term functional outcome was measured by a modified Rankin scale (mRS) 3 months after admission. Multivariate analyses were performed using logistic regression models. We found the serum TRX reflected the disease severity of AIS. There was a significant positive association between serum TRX levels and NIHSS scores (r= 0.476, P<0.0001). Based on the ROC curve, the optimal cutoff value of serum TRX levels as an indicator for auxiliary diagnosis of AIS was projected to be 11.0 ng/ml, which yielded a sensitivity of 80.3% and a specificity of 73.7%, with the area under the curve at 0.807 (95% CI, 0.766-0.847). Elevated TRX (≥ 20.0 ng/ml) was an independent prognostic marker of short-term functional outcome [odds ratio (OR) 9.482 (95% CI, 3.11-8.15) P<0.0001; adjusted for NIHSS, other predictors and vascular risk factors] in patients with AIS. TRX improved the area under the receiver operating characteristic curve of the NHISS score for functional outcome from 0.722 (95% CI, 0.662-0.782) to 0.905 (95% CI, 0.828-0.962; P<0.0001). Our study demonstrated that elevated serum TRX level at admission was a novel diagnostic and prognostic marker in patients with acute ischemic stroke.

Analysis of DNA methylation of perennial ryegrass under drought using the methylation-sensitive amplification polymorphism (MSAP) technique.

Perennial ryegrass (Lolium perenne), an excellent grass for forage and turf, is widespread in temperate regions. Drought is an important factor that limits its growth, distribution, and yield. DNA methylation affects gene expression and plays an important role in adaptation to adverse environments. In this study, the DNA methylation changes in perennial ryegrass under drought stress were assessed using methylation-sensitive amplified polymorphism (MSAP). After 15 days of drought stress treatment, the plant height was less than half of the control, and the leaves were smaller and darker. Genome-wide, a total of 652 CCGG sites were detected by MSAP. The total methylation level was 57.67 and 47.39 % in the control and drought treatment, respectively, indicating a decrease of 10.28 % due to drought exposure. Fifteen differentially displayed DNA fragments in MSAP profiles were cloned for sequencing analysis. The results showed that most of the genes involved in stress responses. The relative expression levels revealed that three demethylated fragments were up-regulated. The expression of a predicted retrotransposon increased significantly, changing from hypermethylation to non-methylation. Although the extent of methylation in two other genes decreased, the sites of methylation remained, and the expression increased only slightly. All of these results suggested that drought stress decreased the total DNA methylation level in perennial ryegrass and demethylation up-regulated related gene expressions and that the extent of methylation was negatively correlated with expression. Overall, the induced epigenetic changes in genome probably are an important regulatory mechanism for acclimating perennial ryegrass to drought and possibly other environmental stresses.

Comparative transcriptome analysis of tomato (Solanum lycopersicum) in response to exogenous abscisic acid.

BACKGROUND: Abscisic acid (ABA) can regulate the expressions of many stress-responsive genes in plants. However, in defense responses to pathogens, mounting evidence suggests that ABA plays variable roles. Little information exists about genome-wide gene expression in ABA responses in tomato (Solanum lycopersicum L.), a model fruit crop plant. RESULTS: Global transcriptome profiles of tomato leaf responses to exogenous ABA were generated using Illumina RNA-sequencing. More than 173 million base pair reads were mapped onto the tomato reference genome and the expression pattern differences between treated and control leaves were assessed. In total, 50,616 transcripts were generated. Among them, 42,583 were functionally annotated in the NCBI non-redundant database and 47,877 in the tomato genome reference. Additionally, 31,107 transcripts were categorized into 57 functional groups based on Gene Ontology terms, and 14,371 were assigned to 310 Kyoto Encyclopedia of Genes and Genomes pathways. In both the ABA treatment and control samples, 39,671 transcripts were available to analyze their expressions, of which 21,712 (54.73%) responded to exogenous ABA. Of these transcripts, 2,787 were significantly differently expressed genes (DEGs). Many known and novel ABA-induced and -repressed genes were found. Exogenous ABA can influence the ABA signaling pathway with PYR/PYL/RCARs-PP2Cs-SnRK2s as the center. Eighteen PYL genes were detected. A large number of genes related to various transcription factors, heat shock proteins, pathogen resistance, and the salicylic acid, jasmonic acid, and ethylene signaling pathways were up-regulated by exogenous ABA. CONCLUSIONS: The results indicated that ABA has the potential to improve pathogen-resistance and abiotic stress tolerance in tomato. This study presents the global expression analysis of ABA-regulated transcripts in tomato and provides a robust database for investigating the functions of genes induced by ABA.

[Cloning and sequence analysis of SLC25A13 transcripts in human amniocytes].

OBJECTIVE: This research intends to amplify the entire coding region sequences of SLC25A13 mRNA which encodes citrin, and to investigate sequence features of the transcripts for this gene in cultured human amniocytes. This study will provide laboratory evidence for prenatal diagnosis of neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) at mRNA level. METHODS: One amniocyte sample was collected from a pregnant woman who underwent prenatal diagnosis of citrin deficiency and whose fetus has proven a carrier of 851del4 mutation by genomic DNA analysis. Another amniocyte sample, as a control, was from a fetus without family history of citrin deficiency. Total RNA was extracted from cultured amniocytes, cDNA was synthesized, and then nested-PCR was performed to amplify the entire coding region sequences of SLC25A13. The PCR products were cloned and analyzed by sequencing. RESULTS: The entire coding region of SLC25A13 gene was successful amplified from two cultured human amniocytes. The splice variant of SLC25A13, SLCA (normal mRNA), was identified in the two samples. SLCB (CAG insertion between exon 9-10) was identified in the control. SLCC (exon 5-11 skipping), but not transcriptional product from the allele with 851del4 mutation, was identified in the 851del4 mutation carrier. CONCLUSIONS: This study demonstrated that the entire coding region of SLC25A13 cDNA can be successfully amplified from two cultured human amniocytes, and revealed exon 5-11 skipping as a novel SLC25A13 transcript. Normal mRNA predominated in the transcripts in normal control and 851del4 mutation carrier, suggesting that the two fetuses were not at risk for NICCD. These SLC25A13 transcription features provided laboratory evidence for prenatal diagnosis of NICCD.

Cloning and sequence analysis of SLC25A13 transcripts in human amniocytes.

OBJECTIVE: To amplify the entire ORF of SLC25A13 cDNA which encodes citrin, a liver-type mitochondrial aspartate-glutamate carrier, and to investigate sequence feature of the transcripts for this gene in cultured human amniocytes. This study will provide laboratory evidences for prenatal diagnosis of NICCD at mRNA level. METHODS: Total RNA was extracted from cultured amniocytes, and cDNA was synthesized, and then nest PCR was performed to amplify the entire ORF sequences of SLC25A13. The PCR products were purified, cloned, sequenced, and aligned with the genomic DNA of SLC25A13 to analyze the alternative splicing pattern. RESULTS: The entire ORF of SLC25A13 gene was successfully amplified. Three splice variants of SLC25A13, i.e., SLCA (normal mRNA), SLCB (CAG insertion between exon 9-10) and SLCC (exon 5-11 skipping), were identified in the subjects. However, no abnormal mRNA from the allele with mutation 851del4 was detected in the amniocytes cultured from a carrier fetus of this mutation. CONCLUSIONS: This study demonstrated that the entire ORF of SLC25A13 cDNA can be successfully amplified from cultured human amniocytes, and revealed exon 5-11 skipping as a novel SLC25A13 transcript. Normal mRNA occupied majority of the transcripts in normal control and heterozygous amniocytes which contained normal allele and 851del4 mutation, indicating that the two fetuses wouldn't suffer from NICCD. These SLC25A13 transcription features provided laboratory evidences for prenatal diagnosis of NICCD.

Prenatal diagnosis of citrin deficiency in a Chinese family with a fatal proband.

Citrin deficiency (CD) is an autosomal recessive disorder with SLC25A13 as causative gene that encodes citrin, the liver-type aspartate/glutamate carrier isoform 2 (AGC2). Neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD), the major CD phenotype at pediatric age, has been previously reported as a self-limiting condition with clinical presentations resolving between 6 months and 1 year of life. We report the prenatal diagnosis of CD in a family with a fatal NICCD proband. The proband was a 10-month-old male presenting cough for 8 days and jaundiced skin 1 day. Physical examination revealed fever, dark jaundiced sclera and skin, hoarse breathing sounds, and hepatosplenomegaly. Laboratory tests uncovered elevated cholestatic indices, increased ammonia, and prolonged activated partial thromboplastin time and prothrombin time, and reduced fibrinogen. Sonography showed the features of liver cirrhosis. Metabolome analysis uncovered large quantity of 4-hydroxyphenyllactate and dicarboxylates in urine and increased citrulline and methionine in blood. The patient passed away due to liver failure at his age of 13.5 months. Mutation analysis revealed him a homozygote of 851del4, a four-base deletion in exon 9 of SLC25A13 gene. On request of the parents who had a second fetus, prenatal diagnosis of CD was performed by PCR-electrophoresis following amniocentesis and amniocyte culture, and demonstrated the fetus a carrier of the same mutation. The fatal proband in the present report has provided clinical evidence challenging the traditional concept on NICCD prognosis. Moreover, as the first trial on CD prenatal diagnosis, this study might open a novel area for clinical management of CD.