ABSTRACT
Cas9 can cleave DNA in both blunt and staggered configurations, resulting in distinct editing outcomes, but what dictates the type of Cas9 incisions is largely unknown. In this study, we developed BreakTag, a versatile method for profiling Cas9-induced DNA double-strand breaks (DSBs) and identifying the determinants of Cas9 incisions. Overall, we assessed cleavage by SpCas9 at more than 150,000 endogenous on-target and off-target sites targeted by approximately 3,500 single guide RNAs. We found that approximately 35% of SpCas9 DSBs are staggered, and the type of incision is influenced by DNA:gRNA complementarity and the use of engineered Cas9 variants. A machine learning model shows that Cas9 incision is dependent on the protospacer sequence and that human genetic variation impacts the configuration of Cas9 cuts and the DSB repair outcome. Matched datasets of Cas9 and engineered variant incisions with repair outcomes show that Cas9-mediated staggered breaks are linked with precise, templated and predictable single-nucleotide insertions, demonstrating that a scission-based gRNA design can be used to correct clinically relevant pathogenic single-nucleotide deletions.
ABSTRACT
Somatic copy number alterations (SCNAs) are pervasive in advanced human cancers, but their prevalence and spatial distribution in early-stage, localized tumors and their surrounding normal tissues are poorly characterized. Here, we perform multi-region, single-cell DNA sequencing to characterize the SCNA landscape across tumor-rich and normal tissue in two male patients with localized prostate cancer. We identify two distinct karyotypes: 'pseudo-diploid' cells harboring few SCNAs and highly aneuploid cells. Pseudo-diploid cells form numerous small-sized subclones ranging from highly spatially localized to broadly spread subclones. In contrast, aneuploid cells do not form subclones and are detected throughout the prostate, including normal tissue regions. Highly localized pseudo-diploid subclones are confined within tumor-rich regions and carry deletions in multiple tumor-suppressor genes. Our study reveals that SCNAs are widespread in normal and tumor regions across the prostate in localized prostate cancer patients and suggests that a subset of pseudo-diploid cells drive tumorigenesis in the aging prostate.
Subject(s)
DNA Copy Number Variations , Prostatic Neoplasms , Single-Cell Analysis , Humans , Male , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Aneuploidy , Prostate/pathology , Prostate/metabolism , Clone Cells , Diploidy , AgedABSTRACT
Glioblastoma (GB) is the most aggressive primary malignant brain tumor and is associated with short survival. O-GlcNAcylation is an intracellular glycosylation that regulates protein function, enzymatic activity, protein stability, and subcellular localization. Aberrant O-GlcNAcylation is related to the tumorigenesis of different tumors, and mounting evidence supports O-GlcNAc transferase (OGT) as a potential therapeutic target. Here, we used two human GB cell lines alongside primary human astrocytes as a non-tumoral control to investigate the role of O-GlcNAcylation in cell proliferation, cell cycle, autophagy, and cell death. We observed that hyper O-GlcNAcylation promoted increased cellular proliferation, independent of alterations in the cell cycle, through the activation of autophagy. On the other hand, hypo O-GlcNAcylation inhibited autophagy, promoted cell death by apoptosis, and reduced cell proliferation. In addition, the decrease in O-GlcNAcylation sensitized GB cells to the chemotherapeutic temozolomide (TMZ) without affecting human astrocytes. Combined, these results indicated a role for O-GlcNAcylation in governing cell proliferation, autophagy, cell death, and TMZ response, thereby indicating possible therapeutic implications for treating GB. These findings pave the way for further research and the development of novel treatment approaches which may contribute to improved outcomes and increased survival rates for patients facing this challenging disease.
ABSTRACT
The improvement of laboratory diagnosis is a critical step for the reduction of syphilis cases around the world. In this paper, we present the development of an impedance-based method for detecting T. pallidum antigens and antibodies as an auxiliary tool for syphilis laboratory diagnosis. We evaluate the voltammetric signal obtained after incubation in carbon or gold nanoparticle-modified carbon electrodes in the presence or absence of Poly-L-Lysine. Our results indicate that the signal obtained from the electrodes was sufficient to distinguish between infected and non-infected samples immediately (T0') or 15 min (T15') after incubation, indicating its potential use as a point-of-care method as a screening strategy.
Subject(s)
Metal Nanoparticles , Syphilis , Humans , Treponema pallidum , Gold , Antibodies, Bacterial , Syphilis/diagnosis , CarbonABSTRACT
BACKGROUND: Chikungunya virus (CHIKV) causes a febrile syndrome with intense and debilitating arthralgia that can persist for several months or years after complete virus clearance. As there is no specific antiviral treatment or vaccine against CHIKV, identification of serological markers that help clinical management of CHIKV patients is urgent. The High Mobility Group Box 1 (HMGB1) protein is secreted to extracellular milieu and triggers an intense inflammatory process by inducing the overexpression of pro-inflammatory cytokines. HMGB1 plays an important role in several virus diseases as well as in rheumatoid arthritis. OBJECTIVES: This study focus on the investigation of HMGB1 serum levels in a sera panel from CHIKV-infected patients in an attempt to assess its potential as a biomarker for chikungunya clinical management. STUDY DESIGN: Eighty CHIKV-positive samples and 32 samples from healthy donors were subjected to a quantitative HMGB1 ELISA assay to assess the HMGB1 circulating levels. RESULTS: HMGB1 levels were significantly higher in CHIKV-positive samples (516.12 ng/mL, SEM ± 48.83 ng/mL) compared to negative control (31.20 ng/mL, SEM ± 3.24 ng/mL, p < 0.0001). Circulating levels of HMGB1 persisted elevated during the whole acute-phase of disease and correlated with virus titer (p < 0.05). CONCLUSIONS: The present study is the first to describe increased serum levels of HMGB1 in CHIKV infection and its positive correlation with virus titer, suggesting its potential use as a biomarker for diagnosis and treatment of chikungunya fever.
Subject(s)
Chikungunya Fever , Chikungunya virus , HMGB1 Protein , Biomarkers , Case-Control Studies , Enzyme-Linked Immunosorbent Assay , HMGB1 Protein/blood , HumansABSTRACT
Transcription poses a threat to genomic stability through the formation of R-loops that can obstruct progression of replication forks. R-loops are three-stranded nucleic acid structures formed by an RNA-DNA hybrid with a displaced non-template DNA strand. We developed RNA-DNA Proximity Proteomics to map the R-loop proximal proteome of human cells using quantitative mass spectrometry. We implicate different cellular proteins in R-loop regulation and identify a role of the tumor suppressor DDX41 in opposing R-loop and double strand DNA break accumulation in promoters. DDX41 is enriched in promoter regions in vivo, and can unwind RNA-DNA hybrids in vitro. R-loop accumulation upon loss of DDX41 is accompanied with replication stress, an increase in the formation of double strand DNA breaks and transcriptome changes associated with the inflammatory response. Germline loss-of-function mutations in DDX41 lead to predisposition to acute myeloid leukemia in adulthood. We propose that R-loop accumulation and genomic instability-associated inflammatory response may contribute to the development of familial AML with mutated DDX41.
Subject(s)
DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Genomic Instability , Proteomics , R-Loop Structures , Transcription, Genetic , Adult , Cell Line, Tumor , DNA/metabolism , DNA Breaks, Double-Stranded , Gene Knockdown Techniques , Genes, Tumor Suppressor , HEK293 Cells , Humans , Leukemia, Myeloid, Acute , Nucleic Acid Conformation , Nucleic Acid Hybridization , Promoter Regions, Genetic , R-Loop Structures/genetics , RNA/metabolismABSTRACT
ABSTRACT Introduction: Studies suggest the association between antibody production and the severity of coronavirus disease 2019 (Covid-19). Objectives: To evaluate the concentrations of immunoglobulins class A (IgA) and class G (IgG) during the hospitalization period of Covid-19 patients according to the outcome (survival vs death). Materials and methods: Patients with severe acute respiratory syndrome of coronavirus 2 (Sars-CoV-2) infection confirmed by reverse transcriptase reaction followed by polymerase chain reaction (RT-PCR) were included in this prospective study. Samples were obtained weekly during the follow-up of individuals, considering symptom onset. Titers of anti-Sars-CoV-2 IgA and IgG were measured using a commercial immunoassay. Correlations between IgA/IgG and cycle threshold (Ct) values for N1 and N2 target genes were also assessed. Results: We studied 55 Covid-19 patients (59.7±16.2 years, 63.6% male), of which 28 (50.9%) died. We observed IgA and IgG positivity (IgA+ and IgG+) in 90.9% and 80% of patients, respectively. The highest IgA+ frequency was observed at weeks 2 and 3 and the highest IgG+ at weeks 3 and 4. It is important to note that patients who died presented lower IgA titers in the first two weeks (p < 0.05); however, a significant increase in IgA levels was observed in the subsequent weeks. Lastly, we identified that significant correlations between Ct values and immunoglobulins levels, both IgA and IgG were correlated with Ct N2 in patients who died. Conclusion: Our results suggest that lower IgA titers in early Covid-19, which is associated with lower Ct values, may indicate patients at higher risk for death.
RESUMEN Introducción: Los estudios sugieren una asociación entre la producción de anticuerpos y la gravedad de la enfermedad por coronavirus 2019 (Covid-19). Objetivos: Evaluar las concentraciones de inmunoglobulinas clase A (IgA) y clase G (IgG) durante la hospitalización de pacientes con Covid-19 según el desenlace (supervivencia vs muerte). Materiales y métodos: Se incluyeron en este estudio prospectivo pacientes con síndrome respiratorio agudo severo de infección por coronavirus 2 (Sars-CoV-2) confirmado por la reacción en cadena de la polimerasa con transcriptasa inversa (RT-PCR). Las muestras se obtuvieron semanalmente durante el seguimiento de los individuos, considerando la aparición de los síntomas. Los títulos de IgA e IgG anti-Sars-CoV-2 se midieron usando un inmunoensayo comercial. También se evaluaron las correlaciones entre IgA/IgG y los valores de los umbrales de ciclo [cycle threshold (Ct)] para los genes N1 y N2. Resultados: Se estudiaron 55 pacientes Covid-19 (59,7 ± 16,2 años, 63,6% varones), de los cuales 28 (50,9%) fallecieron. Observamos positividad de IgA e IgG (IgA+ e IgG+) en el 90,9% y el 80% de los pacientes, respectivamente. La frecuencia más alta de IgA+ se observó en las semanas dos y tres y la IgG + más alta en las semanas tres y cuatro. Es importante señalar que los pacientes que fallecieron presentaron títulos de IgA más bajos en las dos primeras semanas (p < 0,05); sin embargo, se observó un aumento significativo en los niveles de IgA en las semanas siguientes. Conclusión: Identificamos correlaciones significativas entre los valores de Ct y los niveles de Ig, tanto IgA como IgG se correlacionaron con Ct N2 en los pacientes que fallecieron. Nuestros resultados sugieren que los títulos de IgA más bajos en Covid-19 temprano, que se asocia con valores de Ct más bajos, pueden indicar que los pacientes tienen un mayor riesgo de muerte.
RESUMO Introdução: Estudos sugerem a associação entre a produção de anticorpos e a gravidade da coronavirus disease 2019 (Covid-19). Objetivos: Avaliar as concentrações de imunoglobulinas da classe A (IgA) e da classe G (IgG) durante a internação de pacientes com Covid-19 de acordo com o desfecho (sobrevida vs óbito). Materiais e métodos: Pacientes com infecção pela síndrome respiratória aguda grave do coronavírus 2 (Sars-CoV-2) confirmada por reação da transcriptase reversa seguida de reação em cadeia da polimerase (RT-PCR) foram incluídos neste estudo prospectivo. As amostras foram obtidas semanalmente durante o acompanhamento dos indivíduos, considerando o início dos sintomas. Os títulos de IgA e IgG anti-Sars-CoV-2 foram mensurados por meio de um imunoensaio comercial. Correlações entre IgA/IgG e valores de limiar de detecção [cycle thresholds (Ct)] para os genes alvos N1 e N2 também foram avaliadas. Resultados: Estudamos 55 pacientes com Covid-19 (59,7 ± 6,2 anos; 63,6% do sexo masculino); destes, 28 (50,9%) morreram. Observamos positividade para IgA e IgG (IgA+/IgG+) em 90,9% e 80% dos pacientes, respectivamente. A maior frequência de IgA+ foi verificada nas semanas 2 e 3, e a maior frequência de IgG+, nas semanas 3 e 4. É importante observar que os pacientes que morreram apresentaram títulos de IgA mais baixos nas primeiras duas semanas (p < 0,05); no entanto, um aumento significativo na concentração de IgA foi observado nas semanas subsequentes. Por fim, identificamos correlações significativas entre os valores de Ct e imunoglobulinas; tanto IgA quanto IgG foram correlacionadas com Ct N2 em pacientes que morreram. Conclusão: Nossos resultados sugerem que títulos mais baixos de IgA no início da Covid-19 - que estão associados a valores mais baixos de Ct - podem indicar pacientes com risco elevado de evoluir para óbito.
ABSTRACT
Structural variation (SV), involving deletions, duplications, inversions and translocations of DNA segments, is a major source of genetic variability in somatic cells and can dysregulate cancer-related pathways. However, discovering somatic SVs in single cells has been challenging, with copy-number-neutral and complex variants typically escaping detection. Here we describe single-cell tri-channel processing (scTRIP), a computational framework that integrates read depth, template strand and haplotype phase to comprehensively discover SVs in individual cells. We surveyed SV landscapes of 565 single cells, including transformed epithelial cells and patient-derived leukemic samples, to discover abundant SV classes, including inversions, translocations and complex DNA rearrangements. Analysis of the leukemic samples revealed four times more somatic SVs than cytogenetic karyotyping, submicroscopic copy-number alterations, oncogenic copy-neutral rearrangements and a subclonal chromothripsis event. Advancing current methods, single-cell tri-channel processing can directly measure SV mutational processes in individual cells, such as breakage-fusion-bridge cycles, facilitating studies of clonal evolution, genetic mosaicism and SV formation mechanisms, which could improve disease classification for precision medicine.
Subject(s)
Computational Biology/methods , Genomic Structural Variation , Leukemia/genetics , Single-Cell Analysis/methods , Cell Line , Chromothripsis , Clonal Evolution , Gene Rearrangement , Humans , INDEL Mutation , Sequence Inversion , Translocation, GeneticABSTRACT
Capacitation (CAP) and acrosome reaction (AR) are sequential processes of sperm activation. Beside the known ionic, membrane, and transduction events and final release of proteolytic enzymes that help sperm movement toward the egg, chromatin changes, such as a physiological remodeling, are also possible. Our aims were to ascertain that CAP and AR do not induce DNA damage and to evaluate changes occurring in the human sperm head during these physiological processes using cytochemical stains. Percollpurified spermatozoa from normal donors were incubated in Biggers, Whitten, and Whittingham medium ± fetal cord serum ultrafiltrate (CAP inducer) and then with lysophosphatidylcholine (AR inducer). CAP and AR were associated with increases in aniline blue (AB, for histones; â¼70%) and toluidine blue (TB, for chromatin compaction; â¼40%) staining but had no influence on that of chromomycin A3 (for protamines). The increase (â¼40%) in iodoacetamide-fluorescein (IAF, for sulfhydryl groups) staining observed during CAP was absent after AR. CAP and AR did not damage DNA (percentage of DNA fragmentation index remained low) nor affect histone content. CAP, and even more AR, primed sperm heads to decondense (â¼80% and â¼140% increases, respectively) when challenged with sodium dodecyl sulfate + dithiothreitol. Interestingly, induced decondensation correlated with all other tests (CAP, AB, TB, and IAF). Therefore, the data strongly support a physiological remodeling of nondamaged human sperm chromatin during CAP and AR, and modifications are probably interlinked and help prepare chromatin for postfertilization events.
Subject(s)
Acrosome Reaction , Chromatin Assembly and Disassembly , Sperm Capacitation , Sperm Head/metabolism , Acrosome Reaction/drug effects , Chromatin Assembly and Disassembly/drug effects , DNA Damage , Dithiothreitol/pharmacology , Histones/metabolism , Humans , Lysophosphatidylcholines/pharmacology , Male , Protamines/metabolism , Sodium Dodecyl Sulfate/pharmacology , Sperm Capacitation/drug effects , Sperm Head/drug effects , Sperm Head/pathology , Staining and Labeling/methodsABSTRACT
Recurrent haemarthroses often lead to chronic synovitis in patients with haemophilia and von Willebrand disease. Radioactive synovectomy with yttrium-90 (9°Y) citrate is frequently used to treat this complication, usually with good results. Since 2006, the Nuclear Energy Research Institute (IPEN, Sao Paulo, Brazil) has produced hydroxyapatite particles labelled with 9°Y for radioactive synovectomy. The aim of this study was to compare the results achieved by both forms of 9°Y in the treatment of haemophilic synovitis. We included 221 joints from 136 patients (age range: 6-20 years), treated by one of the two radiopharmaceuticals, at the Hemocenter of Mato Grosso, Brazil. The outcomes analysed were the annual frequency of haemarthrosis, articular pain and joint range of motion before and 1 year after RS. Similar results were achieved regardless of whether 9°Y hydroxyapatite or 9°Y citrate was used, and results were independent of the joint type, age, gender, radiologic stage and presence of inhibitors. 9°Y hydroxyapatite appears to be equivalent to the reference product 9°Y citrate in the treatment of chronic synovitis associated with bleeding disorders.
Subject(s)
Citrates/therapeutic use , Durapatite/therapeutic use , Hemophilia A/complications , Organometallic Compounds/therapeutic use , Synovitis/radiotherapy , Yttrium Radioisotopes/therapeutic use , Adolescent , Arthralgia/radiotherapy , Brazil , Child , Female , Hemarthrosis/complications , Humans , Male , Pain Measurement , Radiography , Range of Motion, Articular , Synovitis/diagnostic imaging , Synovitis/etiology , Young AdultABSTRACT
The reproductive effects of the coadministration of di-2-(ethylhexyl) phthalate (DEHP) and di-butyl phthalate (DBP) were studied in both foetal and adult male rat offspring exposed in utero. Pregnant Wistar rats were treated by oral gavage from gestation day 13 to 21 with vehicle control, 150 mg DEHP/kg body weight (bw)/day, 100 mg DBP/kg bw/ or a combination of the two compounds (DEHP 150 + DBP 100 mg/kg bw/day). An additional group of dams received 500 mg DBP/kg bw/day. A significant decrease in foetal testicular testosterone levels was observed in animals exposed to 500 mg DBP/kg/day or the phthalate mixture. Similarly, histological analysis of the foetal testis revealed that the coadministration of DEHP and DBP was able to increase the diameter of seminiferous cords and induce gonocyte multinucleation at doses that individually had no significant effects on these variables. However, in the phthalate mixture group, no significant changes were observed in anogenital distance and nipple retention, variables that are used to indicate possible anti-androgenic effects. Also, the adult endpoints investigated, that included reproductive organ weights and the number of spermatids per testis, were unaffected by any treatment regimen. Overall, coadministration of DEHP and DBP in utero significantly reduced testicular testosterone levels and resulted in misshapen seminiferous cords and gonocyte multinucleation in rat foetal testis. Our results also confirm that these foetal endpoints seem to be the most sensitive markers of prenatal phthalate exposure.
Subject(s)
Androgen Antagonists/pharmacology , Fetus/drug effects , Testis/drug effects , Animals , Dibutyl Phthalate/pharmacology , Diethylhexyl Phthalate/pharmacology , Female , Fertility , Male , Organ Size/drug effects , Phthalic Acids , Pregnancy , Rats , Rats, Wistar , Reproduction/drug effects , Testis/pathology , Testosterone/pharmacologySubject(s)
Actinomycosis/diagnosis , Brain Abscess/microbiology , Actinomyces/metabolism , Actinomycosis/etiology , Actinomycosis/pathology , Actinomycosis/therapy , Adult , Brain Abscess/etiology , Brain Abscess/pathology , Brain Abscess/therapy , Focal Infection, Dental/complications , Humans , MaleABSTRACT
A canine calicivirus (CaCV) isolated in Japan, designated as CaCV No. 48 strain, was propagated in MDCK cells and purified by CsCl equilibrium gradient centrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the purified samples revealed the presence of only one major species of viral protein of about 60 kilodaltons after Coomassie staining. The same band, presumably that of the capsid protein, was detected by western blotting using a mouse hyperimmune serum. This capsid protein was synthesized in MDCK cells as early as 2 hr post-inoculation. Experimental infection of dogs resulted in the production of anti-CaCV antibodies which were detected by microneutralization test and western blotting. Likewise, serosurvey revealed not only the presence of neutralizing antibodies but also reactivity of the field sera against the capsid protein of the purified virus. These results indicate that the capsid protein of CaCV No. 48 strain is immunogenic and could be detected by antibodies in western blotting.
Subject(s)
Caliciviridae Infections/veterinary , Caliciviridae/isolation & purification , Capsid/analysis , Capsid/immunology , Dog Diseases/diagnosis , Animals , Antibodies, Viral/blood , Blotting, Western/methods , Blotting, Western/veterinary , Caliciviridae/immunology , Caliciviridae/metabolism , Caliciviridae Infections/diagnosis , Caliciviridae Infections/immunology , Capsid/metabolism , Cell Line , Dog Diseases/blood , Dog Diseases/immunology , Dogs , Electrophoresis, Polyacrylamide Gel/methods , Electrophoresis, Polyacrylamide Gel/veterinary , Kidney/pathology , Kidney/virology , Mice , Specific Pathogen-Free Organisms , Time FactorsABSTRACT
Twenty-seven feline parvovirus (FPV) isolates were recovered from cats clinically diagnosed with feline panleukopenia (FPL) for assessing antigenic and genomic properties of FPL viruses (FPLV) recently prevalent among cats in Japan. All isolates, with the exception of one novel isolate, FPV-314, possessed homologous properties, and their subgroups in FPVs were identified as FPLV. The FPV-314 isolate, which was from a 1.5-year-old cat which manifested clinical signs of FPL and died on the 13th day after the first medical examination, was finally identified as canine parvovirus (CPV) because it lacked a specific antigenic epitope commonly detected in FPLV and mink enteritis virus and because the nucleotide sequence of the capsid protein gene was almost identical to those of CPV-2a and -2b antigenic type strains recently prevalent among dogs in Japan. The present result together with our previous findings (M. Mochizuki, R. Harasawa, and H. Nakatani. Vet. Microbiol. 38:1-10, 1993) indicates the possibility that CPV and FPLV undergo mutual interspecies transmission between dogs and cats, and it is postulated that they may cause disease in some adventitious hosts.
Subject(s)
DNA, Viral/analysis , Feline Panleukopenia/virology , Parvovirus, Canine/isolation & purification , Animals , Cats/virology , Dogs , Molecular Sequence DataABSTRACT
The genes for Flaviviridae structural proteins are located at the 5' terminus of the genome, while the 3' terminus contains the genes for the non-structural proteins. The first protein product of the large ORF of pestiviruses, the p20 protein, is however a non-structural protein which possess an autoproteolytic activity. Here we report the cloning of the p20/p14 genes behind the strong Trc promotor and expression of the p20 at high levels in E. coli. The autoprotease p20 was responsible for its own release from the nascent polyprotein in E. coli and was further purified by chromatography techniques.
Subject(s)
Classical Swine Fever Virus/enzymology , Escherichia coli/metabolism , Gene Expression , Viral Structural Proteins/genetics , Viral Structural Proteins/isolation & purification , Animals , Classical Swine Fever Virus/genetics , Cloning, Molecular , Genetic Vectors , Recombinant Fusion Proteins/geneticsABSTRACT
The metatarsophalangeal joint of the great toe often requires an arthroplasty to correct joint disease and pain. Today, joint replacement systems are combinations of components manufactured to optimize biological ingrowth, mechanical interlock, press fit, and cementing. Three different types of arthroplasties are available to foot surgeons: the double stem hinged silicone implant, the two-component joint mimicking implant, and a hemi-implant available for the phalanx. No comprehensive studies on very large populations have been conducted to accurately evaluate the beneficial long-term effects of these implants. This review article describes the development of the toe arthroplasty, details the commercially available implants, and addresses the advantages and adverse effects of each implant.
Subject(s)
Joint Prosthesis , Metatarsophalangeal Joint/surgery , Humans , Joint Diseases/surgery , Joint Prosthesis/instrumentation , Joint Prosthesis/methods , Prosthesis Design , SiliconesABSTRACT
A polymerase chain reaction (PCR) assay, which specifically amplifies the capsid gene of canine parvovirus (CPV), was compared as a diagnostic method for detecting CPV in faeces, with virus isolation (VI) on Crandell feline kidney (CRFK) or Madin-Darby canine kidney (MDCK) cells, and a faecal haemagglutination (HA) assay confirmed by inhibition with a CPV-specific antiserum. Although a false-negative result was obtained in one of 59 faecal samples (1.7 per cent) tested by the PCR assay, it was as sensitive as the VI assay using MDCK cells, and more sensitive than the VI assay using CRFK cells or the HA assay. These results indicate that the PCR assay may be useful as a routine diagnostic method for detecting CPV in faecal specimens.
Subject(s)
Dog Diseases , Feces/microbiology , Hemagglutination Tests/veterinary , Parvoviridae Infections/veterinary , Parvoviridae/isolation & purification , Polymerase Chain Reaction/veterinary , Animals , Base Sequence , Cell Line , DNA, Viral/analysis , Dogs , False Negative Reactions , Kidney , Molecular Sequence Data , Oligodeoxyribonucleotides , Parvoviridae Infections/diagnosis , Polymerase Chain Reaction/methodsABSTRACT
After amplification by PCR, the 5' region of the genome of hog cholera virus (HCV) strain Alfort 187 was cloned and sequenced. The nucleotide and deduced amino acid sequences were compared with the ones of other pestiviruses. By in vitro translation experiments we were able to demonstrate the protease activity of the p 20 protein of HCV.
Subject(s)
Classical Swine Fever Virus/genetics , Viral Structural Proteins/genetics , Base Sequence , Classical Swine Fever Virus/enzymology , Cloning, Molecular , Molecular Sequence Data , Pestivirus/genetics , Polymerase Chain Reaction , Protein Biosynthesis , Viral Structural Proteins/metabolismABSTRACT
A method for the measurement of fast, intensity-dependent refractive-index changes with the use of a modified Sagnac ring interferometer is presented. The measurement is not degraded by slowly responding background index changes. Nonlinear refractive-index changes in an undoped silicon wafer, and in poly-bis toluene sulfonate polydiacetylene and dye-doped polymethyl methacrylate waveguides, were measured with the use of a cw mode-locked Nd:YAG laser.