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PubMed2026

Molecular diagnostics and integrated management challenges of tobacco streak virus: Current status and future perspectives.

Tobacco streak virus (TSV) is an economically important viral pathogen causing severe yield and quality losses in several agricultural, horticultural and medicinal crops worldwide. Its complex epidemiology involving sap transmission, infected pollen and pollen-feeding thrips, together with symptom similarity to other necrosis-inducing pathogens, frequently results in misdiagnosis and delayed disease management. This review critically evaluates recent advances in TSV diagnostics and integrated disease management strategies. Particular emphasis is placed on the transition from conventional biological and serological assays to advanced molecular diagnostics including reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR, multiplex PCR and emerging isothermal amplification technologies such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP). The review also highlights emerging innovations including CRISPR/Cas-based diagnostics in addition, integrated management approaches involving phytosanitation, weed reservoir management, vector ecology-based, host resistance breeding, RNA interference (RNAi) and genome editing technologies are critically analysed. Major challenges including inadequate field validation, limited multiplex capability, poor assay standardization and scarcity of resistant cultivars are discussed. Future objectives to develop quick, field-adaptable and durable TSV detection and management methods are additionally discussed.

CRISPR/Cas diagnostics↗
PubMed2005

Platelet-activating factor enhances the expression of nerve growth factor in normal human astrocytes under hypoxia.

Nerve growth factor (NGF) is required for the survival of neurons. We have addressed the effect of platelet-activating factor (PAF), one of the mediators of ischemic injury of the brain, on NGF expression in astrocytes. Normal human astrocytes in culture were stimulated with PAF, and levels of NGF mRNA and protein were analyzed by reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative PCR and enzyme-linked immunosorbent assay (ELISA). PAF increased the expressions of NGF mRNA and protein in astrocytes in time- and concentration-dependent manners. After 48-h stimulation, 10 nmol/L PAF increased the levels of NGF protein in astrocyte-conditioned medium by 1.4-fold. The PAF-induced stimulation of NGF expression was further enhanced (2.1-fold of the control) in the cells under hypoxic culture condition. BN52021 (Ginkgolide B), an antagonist for PAF binding sites, suppressed the effect of PAF. We conclude that PAF enhances NGF gene expression in human astrocytes, and the PAF-induced increase in the expression of NGF under hypoxia may benefit the protection of the nervous tissue by promoting neuronal survival.

Astrocytes↗
PubMed2026

HPV circulating tumor DNA as a potential prognostic and predictive biomarker in head and neck squamous cell carcinoma: a systematic review.

PURPOSE: Human papillomavirus circulating tumor DNA (HPVctDNA) has emerged as a promising prognostic biomarker in HPV-related head and neck squamous cell carcinoma (HNSCC). This systematic review aimed to synthesize current evidence on the diagnostic accuracy and prognostic value of HPVctDNA in HNSCC management. MATERIAL/METHODS: We systematically reviewed a PubMed-indexed database of studies published between January 2012 and September 2025. Eligible studies were assessed for design, primary tumor site and stage, treatment modality, HPVctDNA detection method, diagnostic accuracy (sensitivity and specificity), and reported clinical endpoints. Descriptive syntheses were performed; sensitivity and specificity were standardized to proportions and summarized as median values per group. RESULTS: A total of 60 studies, including 8,234 patients were analyzed, of which 41 (68.3%) focused exclusively on oropharyngeal squamous cell carcinoma (OPSCC) and 17 (28.3%) included mixed HPV-related HNSCC subsites and HPV-positive cancers of unknown primary. The median follow-up across the included studies was 23 months. Among the included studies, 19 were retrospective (31.7%) and 33 were prospective (55.0%), with a small proportion of cross-sectional and randomized clinical trials. Overall, 40 (66.7%) evaluated the role of HPVctDNA in a curative setting. Plasma was the most common sample type, analyzed in 55 studies (91.7%), while 5 studies also included saliva. Detection methods varied: 40 employed droplet digital PCR (ddPCR), 16 used quantitative PCR (qPCR) and 4 applied NGS-based assays. Most of these studies (38, 63.3%) evaluated the prognostic utility of HPVctDNA, while only 4 (6.7%) assessed HPVctDNA in a screening or diagnostic setting. Regarding diagnostic accuracy, the median sensitivity across evaluable studies was 91.1%, while the median specificity was 99.4%. In OPSCC-only cohorts, the median sensitivity and specificity were 89.4% and 99.4%, respectively. Dynamic changes in HPVctDNA levels during or after treatment were consistently associated with outcomes: clearance or sustained negativity correlated with higher response rates, improved progression-free survival and overall survival, while persistent positivity or increasing levels predicted disease progression and recurrence. CONCLUSIONS: HPVctDNA demonstrates high diagnostic and prognostic accuracy in HPV-related HNSCC, especially OPSCC, supporting its use for prognosis, treatment monitoring and early detection of recurrence. However, prospective interventional studies are still required to demonstrate that HPVctDNA-guided treatment decisions improve clinical outcomes before routine implementation.

Humans↗
PubMed2026

Multiplexed CRISPR/Cas9 mediated knockdown of BCH gene in potato enhances beta-carotene to combat vitamin A deficiency.

The inadequate amounts of provitamin A carotenoids in crops contribute to the widespread vitamin A deficiency, leading to malnutrition and blindness in humans. Suppression of the β-carotene hydroxylase (BCH) increases β-carotene levels. In the current study, we utilized the multiplexed CRISPR/Cas9 approach by designing three targets against the BCH gene in a local potato cultivar. Transformation efficiency was recorded as 15%, the successful integration of the CRISPR/Cas9-BCH multiplex construct in potatoes was confirmed through PCR. When analysed using TIDE software, Sanger sequencing revealed the highest indel efficacy of 92.1% in plant 7 and 26.6% in plant 1. qRT-PCR (quantitative real-time PCR) analysis indicated a significant 89-fold reduction in BCH transcript levels in genome-edited potato lines compared to control plants. Spectrophotometry demonstrated a notable increase in beta-carotene levels in genome-edited potato plants, ranging from 0.831 µg/mL FW to 4.236 µg/mL FW, compared to the control plant with the lowest beta-carotene concentration (0.344 µg/mL FW). HPLC analysis further confirmed increased beta-carotene levels in genome-edited potato plants, ranging from 0.11 mg/mL FW to 0.36 mg/mL FW, compared to the unmodified control plant with a minimum beta-carotene value of 0.09 mg/mL. Our results revealed that the multiplexed CRISPR-Cas9 approach targeting the BCH gene results in enhanced beta-carotene contents in potato tubers.

Solanum tuberosum↗
PubMed2000

Ultrasensitive in-house reverse transcription-competitive PCR for quantitation of HIV-1 RNA in plasma.

An ultrasensitive version of an 'in-house' reverse transcription-competitive polymerase chain reaction assay described previously for quantitation of human immunodeficiency virus type 1 (HIV-1) RNA in plasma was developed. The increase in sensitivity from 400 to 50 HIV-1 RNA copies/ml was achieved by pelleting virus particles from 1.8 ml plasma by centrifugation prior to RNA extraction, modifying competitor DNA structure and amounts, and redesigning primers. Quantitation of HIV-1 RNA in 130 samples tested previously by the standard assay showed that the two procedures yield comparable results (mean absolute difference, 0.26+/-0.20 log) and that the ultrasensitive version detects HIV-1 RNA below the threshold of sensitivity of the standard method. The ultrasensitive 'in-house assay' and the reference QUANTIPLEX HIV-1 RNA 3.0 had the same sensitivity and gave equivalent results (mean absolute difference, 0.19+/-0.11 log), as shown by parallel blinded testing of 47 plasma samples. Titration experiments with reconstructed plasma samples allowed the determination of a dynamic range of 50-500000 HIV-1 RNA copies/ml for the 'in-house' system. The interassay coefficient of variation for samples nominally containing 200, 4000 and 80000 HIV-1 RNA copies/ml were 33.4, 22.9 and 38.2%, respectively. The performance, turnaround time, and cost-effectiveness of this system make it suitable for medium-scale clinical application.

Centrifugation↗
PubMed1999

A quantitative duplex PCR technique for measuring amounts of cell-associated Marek's disease virus: differences in two populations of lymphoma cells.

A duplex polymerase chain reaction (PCR) was developed to measure Marek's disease virus (MDV) load in two subpopulations of Marek's disease (MD) lymphoma cells from chickens. PCR primers were designed using the sequence of the MDV-ICP4 gene and the chicken IFNgamma gene. Each set of primers was present in the same reaction tube so that the 327 bp ICP4 product and the 420 bp IFNgamma product were co-amplified. Two different fluorescent dyes were used to 5'-end label one PCR primer of each pair to distinguish the IFNgamma and ICP4 products by colour. The IFNgamma PCR product was used as an internal standard enabling comparisons of MDV-ICP4 products between different samples. Neither duplex PCR product was preferentially amplified and both reactions were in their exponential phases when stopped. The products could be distinguished by both size and colour. MD lymphoma cells were taken ex vivo and separated on the basis of expressing a novel host surface antigen recognised by the monoclonal antibody AV37. AV37 + lymphoma cells had greater MDV-loads than AV37 lymphoma cells. The principles used here should be applicable to any cell phenotype and/or cell-associated DNA virus.

Animals↗
PubMed2001

Development of two real-time quantitative TaqMan PCR assays to detect circulating Aspergillus fumigatus DNA in serum.

Several PCR assays have been developed for detecting Aspergillus fumigatus DNA in blood of patients with invasive aspergillosis. However, the best blood fraction to be assayed has not been defined and the multicopy genes used as the DNA targets for amplification not characterized. Firstly, we developed a real-time PCR assays based on the TaqMan technology targeted to a single copy gene. To compare serum, white cell pellet, and plasma for effectiveness as blood assay fractions, we spiked whole blood with A. fumigatus DNA and processed these fractions similarly. The difference between white cell pellet and serum was not significant. In contrast, the yield from plasma was 10 times lower than from serum. Then, we compared serum processed immediately or after 24 h at room temperature and observed a lower yield after 24 h. Secondly, a real-time PCR assay targeted to a mitochondrial gene was also developed. The copy number was estimated between 9 and 10 mitochondrial genes per single copy gene. Therefore, we recommend serum, stored and frozen as soon as possible, to be used for detecting circulating A. fumigatus DNA for diagnosis. Moreover, the mitochondrial multicopy gene was characterized in order to compare results from different patients.

Aspergillosis↗
PubMed2002

Rapid type-specific diagnosis of adenovirus type 4 infection using a hexon-based quantitative fluorogenic PCR.

A hexon-based fluorogenic polymerase chain reaction (PCR) assay utilizing the 5'-nuclease activity of DNA Taqpolymerase was developed as a rapid and type-specific diagnostic system for adenovirus type 4 (Ad4) detection and quantification. The assay consists of a pair of flanking primers and an internal fluorescence labeled probe that allows real time amplification to quantify the Ad4 virus. One out of 12 flanking primer pairs evaluated (combinations of three forward primers and four reverse primers) was found to be optimal for Ad4 virus detection that yielded background-free operation, i.e., no fluorescent signal generated by non-template controls. The assay was employed to detect Ad4 reference virus strain RI-67, Wyeth Ad4 vaccine strain and 71 different clinical Ad4 isolates from US military recruits used in this study with consistent sensitivity (lower detection limit) of 2-4 pfu per PCR reaction. The assay showed linear Ad4 detection with a dynamic range of greater than five logs (from 2-4 pfu/assay to greater than 10(5) pfu/assay). This Ad4-specific assay did not crossreact with representative members of Ad subgroups A, B, C, D and F at viral concentrations greater than 10(8) pfu/ml. It was also demonstrated that Ad4 viruses could be efficiently detected from throat swabs (71/72 specimens or 98.6% detection sensitivity) of infected patients by the Ad4-specific PCR. In general, there was a good correlation between PCR determined viral titers in throat swabs and time required to detect viral cytopathic effects (CPE) in cell culture. Evaluation of the simple Ad4 specific assay developed in this study could be used to provide a rapid clinically relevant diagnosis of Ad4 infections in patients with acute respiratory disease (ARD).

Adenovirus Infections, Human↗
PubMed2000

Predictive value of quantitative PCR-based viral burden analysis for eight human herpesviruses in pediatric solid organ transplant patients.

Human herpesviruses can cause significant morbidity and mortality in pediatric solid organ transplant recipients. It was hypothesized that viral burden quantification by polymerase chain reaction using an internal calibration standard could aid in distinguishing between viral disease and latency. Here we report the results of a 2-year prospective study of 27 pediatric solid organ (liver, kidney, or heart) transplant recipients in which multiple samples were analyzed for levels of all eight human herpesviruses by internal calibration standard-polymerase chain reaction. Herpes simplex viruses 1 and 2, varicella-zoster virus, and Kaposi's sarcoma-associated herpesvirus were not detected in any of these samples. Human herpesvirus types 6 and 7 were detected in half of the patients, but were present at low levels, similar to those found in reference populations. Epstein-Barr virus (EBV) and cytomegalovirus (CMV) were detected in 89% and 56% of the patients, respectively. Viral burden analysis suggested distinct patient populations for CMV, with a natural cutoff of 10,000 viral targets/ml blood strongly associated with disease. In some cases, a dramatic increase in CMV levels preceded clinical evidence of disease by several weeks. EBV viral burden was relatively high in the only patient presenting with an EBV syndrome. However, two other patients without evidence of EBV disease had single samples with high EBV burden. Rapid reduction in both EBV and CMV burden occurred with antiviral treatment. These data suggest that viral burden analysis using internal calibration standard-polymerase chain reaction for CMV, and possibly other herpesviruses, is an effective method for monitoring pediatric transplant patients for significant herpesvirus infection and response to therapy.

Adolescent↗
PubMed2004

Differences between scirrhous and non-scirrhous human gastric carcinomas from the aspect of proMMP-2 activation regulated by TIMP-3.

Gastric carcinomas can be classified into scirrhous carcinomas (SC), i.e. 'linitis plastica' or Borrmann 4 gastric cancer, and non-scirrhous carcinomas (NSC). SC are characterized by diffuse invasive growth patterns with marked fibrosis, frequent peritoneal dissemination and lymph-node metastases and poor prognosis, while NSC show medullary growth patterns and common hematogenous metastases. To study the differences in local expression levels of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) between SC and NSC, we examined the expression of MMPs and TIMPs in human gastric carcinoma tissues by several methods including sandwich-enzyme immunoassay systems, gelatin zymography, reverse transcriptase-polymerase chain reaction (RT-PCR), real-time quantitative PCR, immunoblotting, immunohistochemistry and in situ zymography. Of the seven MMPs and two TIMPs tested, only proMMP-2 levels were remarkably higher in SC than in NSC (P < 0.01), and proMMP-2 activation ratio was significantly lower in SC than in NSC (P < 0.05). TIMP-3 mRNA levels were remarkably about 2-fold higher in SC than in NSC tissues (P < 0.01). TIMP-3 production in SC was confirmed by immunoblotting and TIMP-3 was immunolocalized to stromal fibroblasts in SC. TIMP-3 mRNA levels inversely correlated with proMMP-2 activation ratios, although the expression levels of MT1-MMP and MT2-MMP were not different in SC and NSC. By in situ zymography, gelatinolytic activity appeared to be weaker in SC than in NSC. All these data suggest that proMMP-2 activation is down-regulated by TIMP-3 expressed in scirrhous gastric carcinomas. Our findings may explain the differences in clinical behaviors of SC and NSC.

Adenocarcinoma, Scirrhous↗
PubMed2004

Interactions of EGFR and caveolin-1 in human glioblastoma cells: evidence that tyrosine phosphorylation regulates EGFR association with caveolae.

Epidermal growth factor receptor (EGFR) amplification and type III mutation (EGFRvIII), associated with constitutive tyrosine kinase activation and high malignancy, are commonly observed in glioblastoma tumors. The association of EGFR and EGFRvIII with caveolins was investigated in human glioblastoma cell lines, U87MG and U87MG-EGFRvIII. Caveolin-1 expression, determined by RT-PCR, real-time quantitative PCR and Western blot, was upregulated in glioblastoma cell lines (two-fold) and tumors (20-300-fold) compared to primary human astrocytes and nonmalignant brain tissue, respectively. U87MG-EGFRvIII expressed higher levels of caveolin-1 than U87MG. In contrast, the expression of caveolin-2 and -3 were downregulated in glioblastoma cells compared to astrocytes. A colocalization of EGFR, but not of EGFRvIII, with lipid rafts and caveolin-1 was observed by immunocytochemistry. Association of EGFR and EGFRvIII with caveolae, assessed in vitro by binding to caveolin scaffolding domain peptides and in vivo by immunocolocalization studies in cells and caveolae-enriched cellular fraction, was phosphorylation-dependent: ligand-induced phosphorylation of EGFR resulted in dissociation of EGFR from caveolae. In contrast, inhibition of the EGFRvIII constitutive tyrosine phosphorylation by AG1478 increased association of EGFRvIII with caveolin-1. AG1478 also increased caveolin-1 expression and reduced glioblastoma cell growth in a semi-solid agar. The evidence suggests that the phosphorylation-regulated sequestration of EGFR in caveolae may be involved in arresting constitutive or ligand-induced signaling through EGFR responsible for glial cell transformation.

Caveolin 1↗
PubMed2006

Gene expression patterns and catalytic properties of UDP-D-glucose 4-epimerases from barley (Hordeum vulgare L.).

UGE (UDP-Glc 4-epimerase or UDP-Gal 4-epimerase; EC 5.1.3.2) catalyses the interconversion of UDP-Gal and UDP-Glc. Both nucleotide sugars act as activated sugar donors for the biosynthesis of cell wall polysaccharides such as cellulose, xyloglucans, (1,3;1,4)-beta-D-glucan and pectins, together with other biologically significant compounds including glycoproteins and glycolipids. Three members of the HvUGE (barley UGE) gene family, designated HvUGE1, HvUGE2 and HvUGE3, have been characterized. Q-PCR (quantitative real-time PCR) showed that HvUGE1 mRNA was most abundant in leaf tips and mature roots, but its expression levels were relatively low in basal leaves and root tips. The HvUGE2 gene was transcribed at significant levels in all organs examined, while HvUGE3 mRNA levels were very low in all the organs. Heterologous expression of a near full-length cDNA confirmed that HvUGE1 encodes a functional UGE. A non-covalently bound NAD+ was released from the enzyme after denaturing with aqueous ethanol and was identified by its spectrophotometric properties and by electrospray ionization MS. The K(m) values were 40 microM for UDP-Gal and 55 muM for UDP-Glc. HvUGE also catalyses the interconversion of UDP-GalNAc and UDP-GlcNAc, although it is not known if this has any biological significance. A three-dimensional model of the HvUGE revealed that its overall structural fold is highly conserved compared with the human UGE and provides a structural rationale for its ability to bind UDP-GlcNAc.

Amino Acid Sequence↗
PubMed1999

Rapid enumeration of Escherichia coli in oysters by a quantitative PCR-ELISA.

Direct enumeration of Escherichia coli from oysters was achieved using a polymerase chain reaction (PCR) amplification of the lamB gene coupled with an enzyme-linked immunosorbent assay (ELISA). Amplified PCR products generated using a digoxigenin-labelled primer were heat denatured before being quantified by an ELISA. A biotinylated probe immobilized onto streptavidin-coated microplates was used to capture the digoxigenin-labelled fragments that were detected with a peroxidase antidigoxigenin conjugate. Subsequent enzymic conversion of substrate gave distinct absorbance differences when assaying oyster samples containing E. coli in the range 10-10(5) cfu g-1.

Animals↗
PubMed2002

Expression of T helper 1 and T helper 2 cytokine mRNAs in freshly isolated peripheral blood mononuclear cells from dogs with atopic dermatitis.

Using reverse transcription-polymerase chain reaction (RT-PCR) and semi-quantitative PCR techniques, mRNA expression for canine interferon (IFN)-gamma, interleukin (IL)-4, IL-5 and IL-10 in freshly isolated peripheral blood mononuclear cells (PBMCs) was examined in dogs with or without atopic dermatitis (AD). The expression of IFN-gamma mRNA in dogs with AD was lower than that in dogs without AD (healthy control). The expression of IL-5 mRNA was higher in dogs with AD than in control dogs, but there were no significant differences in IL-4 mRNA and IL-10 mRNA expression between the groups. The number of circulating eosinophils was higher in dogs with AD than in control dogs, although eosinophilia was found in only one dog with AD. These results suggest that there is a tendency for the PBMCs of atopic dogs to express a type 2 cytokine pattern that is similar to the pattern observed in human AD patients.

Animals↗
PubMed2006

Evaluation of a non-targeted "omic" approach in the safety assessment of genetically modified plants.

Genetically modified plants must be approved before release in the European Union, and the approval is generally based upon a comparison of various characteristics between the transgenic plant and a conventional counterpart. As a case study, focusing on safety assessment of genetically modified plants, we here report the development and characterisation of six independently transformed ARABIDOPSIS THALIANA lines modified in the flavonoid biosynthesis. Analyses of integration events and comparative analysis for characterisation of the intended effects were performed by PCR, quantitative Real-time PCR, and High Performance Liquid Chromatography. Analysis by cDNA microarray was used as a non-targeted approach for the identification of potential unintended effects caused by the transformation. The results revealed that, although the transgenic lines possessed different types of integration events, no unintended effects were identified. However, we found that the majority of genes showing differential expression were identified as stress-related genes and that environmental conditions had a large impact on the expression of several genes, proteins, and metabolites. We suggest that the microarray approach has the potential to become a useful tool for screening of unintended effects, but state that it is crucial to have substantial information on the natural variation in traditional crops in order to be able to interpret "omics" data correctly within the framework of food safety assessment strategies of novel plant varieties, including genetically modified plant varieties.

Arabidopsis↗
PubMedDate not supplied

Characterization of the human SLC2A11 (GLUT11) gene: alternative promoter usage, function, expression, and subcellular distribution of three isoforms, and lack of mouse orthologue.

GLUT11 (SLC2A11) is a class II sugar transport facilitator which exhibits highest similarity with the fructose transporter GLUT5 (about 42%). Here we demonstrate that separate exons 1 (exon 1A, exon 1B, and exon 1C) of the SLC2A11 gene generate mRNAs of three GLUT11 variants (GLUT11-A, GLUT11-B, and GLUT11-C) that differ in the amino acid sequence of their N-termini. All three 5'-flanking regions of exon 1A, exon 1B and exon 1C exhibited promoter activity when expressed as luciferase fusion constructs in COS-7 cells. 5'-RACE-PCR, quantitative real-time PCR, and Northern blot analysis performed with specific probes for exon 1A, 1B and 1C demonstrated that GLUT11-A is expressed in heart, skeletal muscle, and kidney, GLUT11-B in kidney, adipose tissue, and placenta, and GLUT11-C in adipose tissue, heart, skeletal muscle, and pancreas. Surprisingly, mice and rats lack the SLC2A11 gene. When expressed in Xenopus oocytes, all three GLUT11 isoforms transport glucose and fructose but not galactose. There was no apparent difference in the subcellular distribution of the three isoforms expressed in COS-7 cells. Our data indicate that different promoters and splicing of the human SLC2A11 gene generate three GLUT11 isoforms which are expressed in a tissue specific manner but do not appear to differ in their functional characteristics.

Alternative Splicing↗