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PubMed1996

Hepatic glutathione deficiency in chronic hepatitis C: quantitative evaluation in patients who are HIV positive and HIV negative and correlations with plasmatic and lymphocytic concentrations and with the activity of the liver disease.

OBJECTIVES: Reduced glutathione (GSH) is decreased in patients affected by chronic hepatitis C (CHC) as well as in patients who are HIV positive. Because the liver is the most important source of plasmatic GSH, we measured the concentrations of GSH in the liver (H-GSH) of patients with CHC who were either HIV positive or negative, correlating it to the concentrations of GSH in plasma (P-GSH) and in peripheral blood mononuclear cells (PBMCs) (L-GSH), to the replication activity of hepatitis C virus (HCV) in PBMCs, to the activity of the liver disease, and to the state of immunodeficiency in patients who were HIV positive. METHODS: One hundred, five patients with serologically and histologically demonstrated CHC (55 HIV positive and 50 HIV negative) entered the trial. Fifty-one healthy individuals made up a control group for P-GSH and L-GSH concentrations. H-GSH concentration was determined by high performance liquid chromatography on liver specimens obtained by ultrasound-guided biopsy according to the method described by Reed et al. The concentrations of P-GSH and L-GSH were determined according to the method described by Suarez et al. The detection of HCV RNA strands in PBMCs was performed according to the method described by Qian et al. Histological findings and degree of fibrosis were scored according to the numerical scoring system proposed by Scheuer and by Knodell et al. RESULTS: H-GSH, P-GSH, and L-GSH were significantly reduced in patients affected by CHC compared with healthy controls (p < 0.001). H-GSH and particularly L-GSH were more significantly reduced in patients who were HIV positive compared with those who were HIV negative (p < 0.001), without significant correlation with the values of the T cell subset CD4+. The reductions in H-GSH, P-GSH, and L-GSH were significantly correlated to the replication activity of HCV in PBMCs (p < 0.001) and to the grade of activity of the liver disease assessed by the values of ALT (p < 0.001) and by histological and fibrosis scores of CHC (p < 0.001). In both groups of patients with CHC, H-GSH, P-GSH, and L-GSH were more reduced in patients addicted to drugs than in patients who were not addicted. CONCLUSIONS: In patients with CHC, particularly those who are HIV positive, a systemic depletion of GSH is present. This depletion may be a factor underlying the resistance to interferon therapy and, in patients who are HIV positive, to antiretroviral drugs, fostering HCV and/or HIV replication. This may represent the biological basis for GSH replacement therapy.

Adult↗

PubMed1994

The apolipoprotein B messenger RNA editing enzyme.

The editing of apolipoprotein (apo)B messenger RNA (mRNA) involves a novel C to U modification, which creates an in-frame stop-translation codon, thereby generating the carboxyl-terminal of apoB48. The 27 kDa catalytic subunit of the editing enzyme has been cloned and established to be a zinc-containing cytidine deaminase. The catalytic subunit is guided to the editing site by a second targeting subunit or subunits. A candidate for the targeting subunit is a 60 kDa protein that can be UV crosslinked to the sequence UGAU, which is part of a motif downstream of the editing site that is essential for editing.

Amino Acid Sequence↗

PubMed2001

In situ hybridization: use of 35S-labeled probes on paraffin tissue sections.

The following protocol is for radioactive in situ hybridization detection of RNA using paraffin-embedded tissue sections on glass microscope slides. Steps taken to inhibit RNase activity such as diethyl pyrocarbonate (DEPC) treatment of solutions and baked glassware are unnecessary. The tissue is fixed using 4% paraformaldehyde, hybridized with (35)S-labeled RNA probes, and exposed to nuclear-track emulsion. The entire procedure takes 2-3 days prior to autoradiography. The time required for autoradiography is variable with an average time of 10 days. Parameters that affect the length of the autoradiography include: (1) number of copies of mRNA in the tissue, (2) incorporation of label into the probe, and (3) amount of background signal. Additional steps involved in the autoradiography process, including development of the emulsion, cleaning of the microscope slides, counterstaining of the tissue, and mounting coverslips on the microscope slides, are discussed. In addition, a general guide to the interpretation of the in situ results is provided.

Acetic Anhydrides↗

PubMed2003

Low-dose indinavir in combination with low-dose ritonavir: steady-state pharmacokinetics and long-term clinical outcome follow-up.

OBJECTIVES: To evaluate the long-term efficacy and pharmacokinetics of indinavir (IDV)/ritonavir (RTV) 400/100 mg twice a day in combination with two nucleoside reverse transcriptase inhibitors. METHODS: The study was retrospective with a prospective pharmacokinetic study at a single centre. All HIV-1-infected patients who started the regimen in the period from January 1999 to February 2001 were included in the study. Plasma HIV RNA and CD4 cell counts were recorded from baseline to week 120. Results were evaluated as intention-to-treat and on-treatment analyses with separate analyses for protease inhibitor naive and experienced patients. Patients who were still on the regimen by August 2001 were asked to participate in a pharmacokinetic evaluation. RESULTS: Twenty-one patients started treatment with the regimen (median follow-up: 116 weeks). The percentage of patients with below 20 HIV-1 RNA copies/mL was 70.0% at week 120 and the median CD4 cell count increased from 320 to 607 cells/microL (P=0.062). The median IDV morning and evening Cmin were 434 ng/mL and 220 ng/mL, respectively. CONCLUSIONS: Treatment with the IDV/RTV 400/100 mg regimen appears to be efficacious for up to 2 years. However, rather low IDV Cmin suggests that the regimen should be evaluated further before its widespread use and that the regimen probably should be guided by pharmacokinetic evaluation.

Adolescent↗

PubMed1997

Inter-domain cross-linking and molecular modelling of the hairpin ribozyme.

The hairpin ribozyme is a small catalytic RNA composed of two helical domains containing a small and a large internal loop and, thus, constitutes a valuable paradigm for the study of RNA structure and catalysis. We have carried out molecular modelling of the hairpin ribozyme to learn how the two domains (A and B) might fold and approach each other. To help distinguish alternative inter-domain orientations, we have chemically synthesized hairpin ribozymes containing 2'-2' disulphide linkages of known spacing (12 or 16 A) between defined ribose residues in the internal loop regions of each domain. The abilities of cross-linked ribozymes to carry out RNA cleavage under single turnover conditions were compared to the corresponding disulphide-reduced, untethered ribozymes. Ribozymes were classed in three categories according to whether their cleavage rates were marginally, moderately, or strongly affected by cross-linking. This rank order of activity guided the docking of the two domains in the molecular modelling process. The proposed three-dimensional model of the hairpin ribozyme incorporates three different crystallographically determined structural motifs: in domain A, the 5'-GAR-3'-motif of the hammerhead ribozyme, in domain B, the J4/5 motif of group I ribozymes, and connecting the two domains, a "ribose zipper", another group I ribozyme feature, formed between the hydroxyl groups of residues A10, G11 of domain A and C25, A24 of domain B. This latter feature might be key to the selection and precise orientation of the inter-domain docking necessary for the specific phosphodiester cleavage. The model provides an important basis for further studies of hairpin ribozyme structure and function.

Computer Simulation↗

PubMed2025

Viral viability markers of SARS-CoV-2: a comparison of cell culture, genomic RNA RT-PCR, and subgenomic RNA RT-PCR.

UNLABELLED: Accurate methods to assess viral viability are crucial for determining isolation duration and antiviral therapy in immunocompromised patients. Although cell culture (CC) is the gold standard, it has limitations. Cycle threshold (Ct) values from genomic RNA (gRNA) RT-PCR and subgenomic RNA (sgRNA) RT-PCR have been proposed as markers of active viral replication. This study evaluated the correlation between CC, gRNA Ct values, and sgRNA to identify the best viral viability marker. This study aimed to evaluate the correlation between CC, gRNA Ct values, and sgRNA to identify the best viral viability marker. We conducted a prospective study on immunocompromised patients with suspected SARS-CoV-2 infection at a tertiary hospital (May 2021 to May 2023). Nasopharyngeal swabs were inoculated into Vero E6 cells and tested for gRNA using RT-PCR (Cobas 6800, Roche) and for sgRNA (non-commercial RT-PCR). The sensitivity (S), specificity (SP), positive (PPV) and negative predictive value (NPV), and accuracy were calculated using CC as the gold standard. Among 285 samples from 108 patients, gRNA RT-PCR had high S and NPV (1.0) but low SP (0.24) and moderate PPV (0.63). Ct analysis improved performance in extreme but not intermediate values. A Ct ≤ 30 maximized S but had low SP; Ct ≤ 25 yielded S (0.88), SP (0.89), PPV (0.92), NPV (0.84), and accuracy (0.88); sgRNA showed the highest S (0.99), SP (0.96), PPV (0.97), NPV (0.99), and accuracy (0.98). sgRNA detection is the best marker for identifying viable SARS-CoV-2, aiding decisions on isolation, antiviral treatment, or delaying chemotherapy in immunocompromised patients. IMPORTANCE: Identifying whether a patient still has contagious SARS-CoV-2 is essential for managing isolation, antiviral treatment, and other clinical decisions-especially in immunocompromised individuals. While viral culture is the gold standard for confirming viral viability, it is slow, expensive, and not widely available. Many hospitals rely on RT-PCR tests, but these detect viral genetic material whether or not the virus is still active. This study shows that detecting subgenomic RNA (sgRNA), a molecule only present when the virus is actively replicating, is a highly accurate way, a molecule only present when the virus is actively replicating, is a highly accurate way to determine whether the virus is still viable. Compared to standard PCR or viral culture, sgRNA testing better predicts who is truly infectious. These findings support sgRNA as a useful tool to guide clinical management and infection control in vulnerable patients.

Humans↗

PubMed2026

Enhancing pan-cancer spatial transcriptomics at single-cell resolution with stPainter.

Subcellular spatial transcriptomics can resolve tissue architecture at cellular scale, but sparse gene panels and limited detection sensitivity constrain downstream analysis. Existing enhancement methods often require tissue-matched single-cell RNA sequencing (scRNA-seq) references and dataset-specific retraining. Here we show that stPainter, a conditional generative model pretrained on a pan-cancer scRNA-seq atlas, can enhance spatial transcriptomics data without matched references or retraining. Using a latent diffusion architecture guided by Stochastic Differential Equations (SDE), stPainter reconstructs expanded expression profiles from sparse measurements and produces latent representations for clustering and cell-state analysis. When we apply stPainter upon 6 spatial transcriptomics datasets of different cancer types, we demonstrate that our model empowers downstream biological analyses, including fine-grained subpopulation clustering and pathway enrichment. Comparison with spatially resolved proteomics (CODEX) provided independent support for regional agreement between imputed cellular compositions and protein-level tissue organization. These results establish stPainter as a scalable approach for analyzing tumor microenvironments without auxiliary sequencing data.

Spatial Transcriptomics↗

PubMed2006

Hepatitis C virus serologic and virologic tests and clinical diagnosis of HCV-related liver disease.

The use of serological and virological tests has become essential in the management of hepatitis C virus (HCV) infection in order to diagnose infection, guide treatment decisions and assess the virological response to antiviral therapy. Virological tools include serological assays for anti-HCV antibody detection and serological determination of the HCV genotype, and molecular assays that detect and quantify HCV RNA and determine the HCV genotype. Anti-HCV antibody testing and HCV RNA testing are used to diagnose acute and chronic hepatitis C. Only patients with detectable HCV RNA should be considered for pegylated interferon alfa and ribavirin therapy and the HCV genotype should be systematically determined before treatment, as it determines the indication, the duration of treatment, the dose of ribavirin and the virological monitoring procedure. HCV RNA monitoring during therapy is used to tailor treatment duration in HCV genotype 1 infection, and molecular assays are used to assess the end-of-treatment and, most importantly the sustained virological response, i.e. the endpoint of therapy.

Journal Article↗

PubMed1997

Control of gene expression in viruses and protozoan parasites by antisense oligonucleotides.

Chemically-modified oligonucleotides are now routinely used to prevent gene expression in cell-free media and in cultured cells. The binding of an antisense sequence to a complementary RNA target may lead to the selective inhibition of the encoded information. This may occur at different levels: splicing; transport of the mature RNA from the nucleus to the cytoplasm; translation. Antisense oligonucleotides constitute an interesting tool to shed some light on gene function. They are also potential new therapeutic agents against pathogenic organisms. This review discusses the rules that guide the design of an antisense oligomer and the choice of a target sequence. Examples of the potential use of antisense oligonucleotides in the fields of virology and parasitology, in particular in relation to trypanosomatids, are described.

Animals↗

PubMed1999

Intrapericardial treatment of inflammatory and neoplastic pericarditis guided by pericardioscopy and epicardial biopsy--results from a pilot study.

From a registry of 136 patients undergoing pericardiocentesis, 14 patients with autoimmune and 15 patients with neoplastic effusions were selected. All underwent pericardioscopy, epicardial and pericardial biopsy with histologic, immunohistologic, and polymerase chain reaction/or in situ hybridization analysis for microbial DNAs and RNA. Pericardioscopy identified neoplastic effusions by the high occurrence of protrusions. Fibrin threads and layers and neovascularization were found in both groups. For identification of the inflammatory and neoplastic process, the combined analysis of the cytology of the effusion and epicardial biopsy evaluation proved to be most important. Epicardial biopsy demonstrated a slightly higher sensitivity for identifying neoplastic disorders in the pericardium than cytology alone. Pericardial biopsy was inconclusive. Intrapericardial administration of 1 g of crystalloid triamcinolone in autoreactive pericarditis prevented recurrence in 13 of the 14 cases after 3 months and in 12 of the 14 cases after 1 year. In neoplastic effusion, intrapericardial administration of 50 mg cis-platin for 24 h prevented recurrence of a hemodynamically relevant effusion after 3 months in all, and after 6-12 months in 14 of 15 patients. Mortality in neoplastic effusion due to noncardiac tumor progression was 47 and 80%, respectively, after 3 and 6 months, as can be expected in endstage neoplastic disease. This pilot study demonstrates that local drug application is feasible, life-saving, and well tolerated by the patients. It opens perspectives for local drug application in other cardiac disorders as well.

Adolescent↗

PubMed2006

Development of a quantitative-competitive PCR for quantification of human cytomegalovirus load and comparison with antigenaemia, viraemia and pp67 RNA detection by nucleic acid sequence-based amplification.

AIM: The human cytomegalovirus (HCMV) is an important pathogen in immunocompromised patients, such as transplant recipients. The use of sensitive and rapid diagnostic assays can have a great impact on antiviral prophylaxis and therapy monitoring and diagnosing active disease. Quantification of HCMV DNA may additionally have prognostic value and guide routine management. The aim of this study was to develop a reliable internally-controlled quantitative-competitive PCR (QC-PCR) for the detection and quantification of HCMV DNA viral load in peripheral blood and compare it with other methods: the HCMV pp65 antigenaemia assay in leukocyte fraction, the HCMV viraemia, both routinely employed in our laboratory, and the nucleic acid sequence-based amplification (NASBA) for detection of HCMV pp67-mRNA. METHODS: Quantitative-competitive PCR is a procedure for nucleic acid quantification based on co-amplification of competitive templates, the target DNA and a competitor functioning as internal standard. In particular, a standard curve is generated by amplifying 10(2) to 10(5) copies of target pCMV-435 plasmid with 10(4) copies of competitor pCMV-C plasmid. Clinical samples derived from 40 kidney transplant patients were tested by spiking 10(4) copies of pCMV-C into the PCR mix as internal control, and comparing results with the standard curve. RESULTS: Of the 40 patients studied, 39 (97.5%) were positive for HCMV DNA by QC-PCR. While the correlation between the number of pp65-positive cells and the number of HCMV DNA genome copies/mL and the former and the pp67mRNA-positivity were statistically significant, there was no significant correlation between HCMV DNA viral load assayed by QC-PCR and HCMV viraemia. CONCLUSIONS: The QC-PCR assay could detect from 10(2) to over 10(7) copies of HCMV DNA with a range of linearity between 10(2) and 10(5) genomes.

Adult↗

PubMed2001

Monitoring the response to antiretroviral therapy in HIV-1 group O infected patients using two new RT-PCR assays.

Failure to recognise infection caused by human immunodeficiency virus type 1 (HIV-1) group O variants has been described using both serological and genetic procedures. Moreover, monitoring the response to antiretroviral therapy is a difficult task in patients infected with HIV-1 group O since commercial tests are not available so far for the quantitation of this virus. In this study, the virological response to antiretroviral therapy were assessed in five HIV-1 group O-infected patients living in Spain by using two new and different RT-PCR methods (MUPROVAMA and LCx). Twenty-four plasma samples belonging to these five patients were selected. As reference, p24 antigenaemia levels and CD4+ cell counts were used. All samples yielded positive viral load values using MUPROVAMA (range: 138 to 595,500 HIV-RNA copies/ml) and 23 of 24 using LCx (range: < 178 to 98,356 HIV-RNA copies/ml). Overall, the results obtained using both assays showed a good correlation among themselves, and in respect to p24 antigenaemia and CD4+ cell counts. However, the values provided by LCx were significantly lower (0.33 logs on average) than those provided by MUPROVAMA. In conclusion, both the highly sensitive MUPROVAMA and LCx Quantitative assays might represent an useful tool for guiding the decision on when start treatment and for monitoring the response to antiretroviral therapy in HIV-1 group O-infected patients.

Adolescent↗

PubMed1998

The pathophysiological role of estrogen receptor variants in human breast cancer.

The accumulated evidence supports the expression of estrogen receptor variants at both the mRNA and protein levels. The relative level of expression of some estrogen receptor variant mRNAs and possibly progesterone receptor variant mRNAs is altered during breast tumorigenesis and breast cancer progression. The altered expression of estrogen receptor variants may effect estrogen signal transduction as well as the interpretation of assays where the estimation of estrogen receptor levels is used as a guide to treatment strategies and prognosis.

Breast Neoplasms↗

PubMed2000

The '30K' superfamily of viral movement proteins.

Relationships among the amino acid sequences of viral movement proteins related to the 30 kDa ('30K') movement protein of tobacco mosaic virus - the 30K superfamily - were explored. Sequences were grouped into 18 families. A comparison of secondary structure predictions for each family revealed a common predicted core structure flanked by variable N- and C-terminal domains. The core consisted of a series of beta-elements flanked by an alpha-helix on each end. Consensus sequences for each of the families were generated and aligned with one another. From this alignment an overall secondary structure prediction was generated and a consensus sequence that can recognize each family in database searches was obtained. The analysis led to criteria that were used to evaluate other virus-encoded proteins for possible membership of the 30K superfamily. A rhabdoviral and a tenuiviral protein were identified as 30K superfamily members, as were plant-encoded phloem proteins. Parsimony analysis grouped tubule-forming movement proteins separate from others. Establishment of the alignment of residues of diverse families facilitates comparison of mutagenesis experiments done on different movement proteins and should serve as a guide for further such experiments.

Amino Acid Sequence↗

PubMed2012

MRI roadmap-guided transendocardial delivery of exon-skipping recombinant adeno-associated virus restores dystrophin expression in a canine model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) cardiomyopathy patients currently have no therapeutic options. We evaluated catheter-based transendocardial delivery of a recombinant adeno-associated virus (rAAV) expressing a small nuclear U7 RNA (U7smOPT) complementary to specific cis-acting splicing signals. Eliminating specific exons restores the open reading frame resulting in translation of truncated dystrophin protein. To test this approach in a clinically relevant DMD model, golden retriever muscular dystrophy (GRMD) dogs received serotype 6 rAAV-U7smOPT via the intracoronary or transendocardial route. Transendocardial injections were administered with an injection-tipped catheter and fluoroscopic guidance using X-ray fused with magnetic resonance imaging (XFM) roadmaps. Three months after treatment, tissues were analyzed for DNA, RNA, dystrophin protein, and histology. Whereas intracoronary delivery did not result in effective transduction, transendocardial injections, XFM guidance, enabled 30±10 non-overlapping injections per animal. Vector DNA was detectable in all samples tested and ranged from <1 to >3000 vector genome copies per cell. RNA analysis, western blot analysis, and immunohistology demonstrated extensive expression of skipped RNA and dystrophin protein in the treated myocardium. Left ventricular function remained unchanged over a 3-month follow-up. These results demonstrated that effective transendocardial delivery of rAAV-U7smOPT was achieved using XFM. This approach restores an open reading frame for dystrophin in affected dogs and has potential clinical utility.

Animals↗

PubMed1999

The tmRNA website.

tmRNA (10Sa RNA) has a central role in trans -translation, in which a peptide tag encoded in tmRNA is added to the abnormally short protein product of a broken mRNA, as a signal for proteolysis of the entire tagged protein. The tmRNA website was established in 1997 as a resource for phylogenetic considerations of tmRNA structure and function. Since then, three partial tmRNA sequences have been completed, and sequences from 13 more species have been identified. Forty-six species from 10 bacterial phyla and chloroplasts are now represented in the database. Provisional sequence alignments and predicted proteolysis tag sequences are provided, as well as a literature review and a guide to searching for new tmRNA sequences. The tmRNA website is accessible via WWW at a new URL: http://sunflower.bio.indiana.edu/kwilliam/tmRNA /home.html

Bacteria↗

PubMed2002

The Schizosaccharomyces pombe mgU6-47 gene is required for 2'-O-methylation of U6 snRNA at A41.

Through a computer search of DNA databases, we have identified the homologs of the mgU6-47 snoRNA gene from the yeast Schizosaccharomyces pombe, the fly Drosophila melanogaster and human. The three box C/D-containing snoRNA genes showed no significant similarity in their sequences except for an 11 nt long complementarity to U6 snRNA, suggesting that the mechanism of snoRNA guided snRNA methylation is conserved from mammals to yeast. The corresponding snoRNAs have been positively detected by reverse transcription and northern blotting. Taking advantage of the fission yeast system, we have disrupted the yeast mgU6-47 gene and demonstrated that it is absolutely required for site-specific 2'-O-methylation of U6 at position A41. No growth differences between mgU6-47 gene-disrupted and wild-type cells were observed, suggesting that the mgU6-47 gene, as for most rRNA methylation guides, is dispensable in yeast. Nevertheless, it was revealed by temperature shift assay that abolition of A41 methylation in yeast U6 snRNA might cause a small decrease in mRNA splicing efficiency. The timing of S.pombe U6 pre-RNA transport in the nucleus for splicing and methylation was also analyzed and is described.

Active Transport, Cell Nucleus↗

PubMed1995

[The diagnostic significance of anti-HCV IgM in chronic hepatitis C viral infection].

The significance of anti-HCV IgM testing in the diagnosis and evaluation of treatment of chronic hepatitis C was studied. The serum samples from 82 patients with chronic hepatitis C were tested for ALT, anti-HCV IgG and IgM and HCV RNA. All the 82 cases were positive for anti-HCV IgG and 51 (62.19%) positive for anti-HCV IgM. The emergence of anti-HCV IgM might be of four types: (1) persistent positive type, 24 (29.26%) cases; (2) intermittent positive type, 8 (9.75%) cases; (3) transient positive type, 19 (23.17%) cases; (4) persistent negative type, 31 (37.80%) cases. Usually there was progressive liver damage in type 1 and 2. If the patients were of transient positive type, they would usually recover. There was a significant correlation between the levels of serum anti-HCV IgM and ALT, while no correlation existed between the levels of serum ALT anti-HCV IgG. It is suggested that in patients with chronic hepatitis C, detection of anti-HCV IgM may be useful for estimating the extent of progressive liver damage and guiding the antiviral therapy.

Adult↗