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We examined a 26-month-old steer with neoplastic lesions in the spleen, lymph nodes, heart and kidneys, characterized by pleomorphic lymphoid cells that were immunohistochemically positive for CD20. The presence of bovine leukemia virus (BLV) at >200,000 copies per 100,000 cells by quantitative RT-PCR was considered to be due to random integration of the provirus into the neoplastic cells' genomes. Inverse PCR identified the presence of one, two, two and three different malignant clones in the heart, spleen, mesenteric node and blood, respectively. Because BLV can rapidly induce lymphoma and a high proviral load facilitates B-cell carcinogenesis, multiclonal tumor development was suspected in the present case. [ABSTRACT FROM AUTHOR]

Enzootic bovine leukosis (EBL) is characterized by systemic B-cell lymphoma associated with bovine leukemia virus (BLV) infection in cattle over 3 years old. This study evaluated the use of inverse polymerase chain reaction (PCR) as a diagnostic method for EBL. Inverse PCR revealed monoclonal or biclonal integration of BLV proviral DNA in the genomes of 71 of 82 cattle with EBL, and polyclonal integration of BLV proviral DNA in the genomes of 16 cattle with B-cell type sporadic bovine leukosis (SBL), 5 cattle with T-cell type SBL, 4 cattle with tumors other than lymphoma, 20 cattle with inflammatory diseases, 5 cattle with diseases other than tumors and inflammatory diseases, and 20 healthy cattle. The sensitivity, specificity, positive predictive value, and negative predictive value of inverse PCR for diagnosis of EBL were 86.6%, 100%, 100%, and 86.4%, respectively, suggesting that the risk of false positives is low. Inverse PCR can be a useful tool for definitive diagnosis of EBL in cattle with difficult diagnosis, such as young cattle. [ABSTRACT FROM AUTHOR]

We examined a 26-month-old steer with neoplastic lesions in the spleen, lymph nodes, heart and kidneys, characterized by pleomorphic lymphoid cells that were immunohistochemically positive for CD20. The presence of bovine leukemia virus (BLV) at >200,000 copies per 100,000 cells by quantitative RT-PCR was considered to be due to random integration of the provirus into the neoplastic cells´ genomes. Inverse PCR identified the presence of one, two, two and three different malignant clones in the heart, spleen, mesenteric node and blood, respectively. Because BLV can rapidly induce lymphoma and a high proviral load facilitates B-cell carcinogenesis, multiclonal tumor development was suspected in the present case.

The quail:chick chimera system is a classical research model in developmental biology. An improvement over the quail:chick chimera system would be a line of transgenic chickens expressing a reporter gene. Transgenic chickens carrying lacZ and expressing bacterial beta-galactosidase have been generated, but complete characterization of the insertion event and characterization of beta-galactosidase expression have not previously been available. The genomic sequences flanking the retroviral insertion site have now been identified by using inverse polymerase chain reaction (PCR), homozygous individuals have been identified by using PCR-based genotyping, and beta-galactosidase expression has been evaluated by using Western analysis and histochemistry. Based upon the current draft of the chicken genome, the viral insertion carrying the lacZ gene has been located on chromosome 11 within the predicted gene for neurotactin/fractalkine (CX3CL1); neurotactin mRNA expression appears to be missing from the brain of homozygous individuals. When Generation 2 (G2) lacZ-positive individuals were inter-mated, they generated 361 G3 progeny; 82 were homozyous for lacZ (22.7%), 97 were wild-type non-transgenic (26.9%), and 182 (50.4%) were hemizygous for lacZ. Western analysis revealed the highest expression in the muscle and liver. With the identification of homozygous birds, the line of chickens is now designated NCSU-Blue1. [ABSTRACT FROM AUTHOR]

Integration of hepatitis B virus-DNA (HBV-DNA) into the host genome, a phenomenon found frequently in hepatocellular carcinomas (HCC) and causally linked to oncogenesis, has not been well characterized in children. The aim of the present study was to determine the prevalence of HBV integration more accurately and to decide whether the integration rate varies at different stages of chronic HBV infection in children. Of 13 children with chronic hepatitis, 14 liver biopsy tissues were analyzed. One liver tissue with pure liver cirrhosis, nine non-tumor, and nine tumor liver tissues from children with HCC were analyzed by a very sensitive method, inverse polymerase chain reaction (IPCR). Thirteen genuine viral-host junctional sequences from 23 patients were successfully isolated and proved that IPCR is a useful method in this context. The results also indicated that the detection rate of HBV-DNA integration increased in parallel with the progress of liver histology towards the neoplastic transformation, with 0% in the liver of chronic hepatitis, 22.2% in non-tumor livers of HCC patients, and 66.7% in tumor liver tissues of HCC patients. The present results indicate that integration of HBV-DNA into the host genome was rarely confirmed at the early stage of chronic hepatitis in children until the stage of HCC formation. [ABSTRACT FROM AUTHOR]

Objectives: T cell receptor (TCR) variable regions and complementarity determining regions 3 (CDR3) of infiltrating T cells in the livers of patients with chronic hepatitis B were examined in detail. Methods: TCR usage was examined by the inverse polymerase chain reaction in 2 hepatitis B e antigen-positive patients, one at an early stage and the other at an advanced stage. Results: In 61 productively rearranged clones bearing TCRα genes in the liver of the patient with early stage chronic hepatitis B, the Vα 7.2 gene segment was most frequently used (16.4%), while the other Vα gene segments were used less frequently. All but one of the clones with the Vα7.2 gene were joined with the Jα33 gene, and the CDR3 regions of these clones were the same length and similar in amino acid sequence. In contrast, in 68 productively rearranged clones from the liver of the patient with advanced stage disease, all Vα gene segments were used less frequently, and combinations of the Vα and Jα gene segments varied. Conclusions: Our data suggest a relatively restricted usage of TCR in the liver in early stage chronic hepatitis B. [ABSTRACT FROM AUTHOR]

Nonmagnetic mutants of Magnetospirillum magneticum AMB-1 were recovered following mini-Tn5 transposon mutagenesis. Transconjugants with kanamycin resistance were obtained at a frequency of 2.7 × 10−7 per recipient. Of 3327 transconjugants, 62 were defective for bacterial magnetic particle (BMP) synthesis. The frequency of independent transposition events for nonmagnetic mutants was about 1.4% in transconju gants. Further analysis of DNA sequences flanking transposon by inverted polymerase chain reaction allowed isolation of at least 10 genes or DNA sequences involved in BMP synthesis in M. magneticum AMB-1. [ABSTRACT FROM AUTHOR]

We describe a method for the isolation of transcriptional control regions of developmentally regulated plant genes. This method is based on T-DNA mediated random insertion into the plant genome of a promoterless luciferase ( lux) marker gene fused to the right border of the T-DNA. Insertion of the coding region of the luciferase gene downstream of a transcriptional regulatory element of a plant gene allows detection of luciferase gene expression by low-light image analysis. Using these screening methods, transgenic plants with constitutive or regulated lux gene expression were identified in vivo at different developmental stages. Genomic DNa is isolated from transgenic plants with luciferase expression in one organ only. Amplification of the tagged plant DNA sequences upstream of the lux insertion site by the inverse polymerase chain reaction method facilitates cloning of the plant promoter region. The promoterless luciferase marker gene system as part of a functional insertional element can be used for easy screening of insertional mutants, followed by isolation of developmentally regulated genes and their control elements from plants and other multicellular organisms. [ABSTRACT FROM AUTHOR]

Objective: The COVID-19 pandemic caused by the SARS-CoV-2 virus has generated a great burden on health systems, and healthcare workers have been the most affected. In Spain, the Health Ministry issued common protocols regarding the return-to-work of healthcare workers presenting a negative PCR (Polymerase Chain Reaction) test result and complete resolution of symptoms. Some autonomous communities decided to include a CT (Cycle Threshold) value criterion as well. The aim of this paper was to compare the time intervals required to obtain a negative PCR result by means of both criteria., Methods: A multicenter, cross-sectional study was performed from March to September of 2020, in the health areas of Navarra, La Rioja and Vigo (Galicia), managed by their own Occupational Risk Prevention Units. The PCR negativization time intervals were analyzed according to gender, age, professional category and the PCR repetition criteria used (clinical or defined by CT). Mean difference calculation by Student's t-test and Kaplan-Meier survival analyzes were conducted., Results: The sample amounted to a total of 1,052 workers: 673 from Navarra, 335 from La Rioja and 44 from Vigo, with an average age of 46 years, and of which 81% were women, 35.48% nursing personnel and 23.65% physicians. No statistically significant differences were found between the PCR negativization time periods with any of the variables, except with the CT criterion, which presented a mean of 6.49 days (95% CI, 4.96-8.02), longer and displaying positively displaced survival curves., Conclusions: The CT criterion for carrying out the control PCR test lengthened the reincorporation of essential personnel in the fight against the pandemic., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

We developed a simple and easy method to isolate microsatellites without screening genomic libraries by hybridization. The method requires only three basic techniques: polymerase chain reaction, DNA cloning and sequencing. We applied this method to develop microsatellite markers for the Japanese squirrel and isolated 45 clones that contained repetitive sequences. Among the 22 clones that we tested further, we found 11 diagnostic microsatellite loci that are applicable to the molecular ecological study of Japanese squirrels. [ABSTRACT FROM AUTHOR]

Genes encoding cuticle proteins are helpful subjects to study the molecular mechanisms of insect molting and metamorphosis. In this study, we isolated and characterized a novel cuticle protein R&R gene, referred to as AccCPR1, from Apis cerana cerana. The open reading frame of AccCPR1 has a length of 573 nt and encodes a protein of 190 amino acids that contains a chitin binding region and is a typical cuticle R&R-2 protein. Five putative E74 binding sites and four BR-C binding sites were predicted in the 5'-flanking region, which suggests a potential function in molting and metamorphosis. RT-qPCR showed that AccCPR1 transcript occurred as the ecdysteroid titer decreased after reaching a peak, which suggests AccCPR1 expression requires a "pulse" regimen of ecdysteroids. This hypothesis was tested using different experimental strategies. When larvae were reared with different concentrations of 20E in their diet, the ecdysteroid peak repressed AccCPR1 expression. Exposure of the thoracic integument of the pupae in vitro to different concentration of 20E repressed AccCPR1 expression, which recovered after the removal of 20E. These results suggest that AccCPR1 is a typical cuticle R&R-2 protein that plays an important role in development, and an ecdysteroid pulse is critical for high AccCPR1 gene expression., (© 2013.)