An Initial In Vitro Investigation into the Potential Therapeutic Use of SupT1 Cells to Prevent AIDS in HIV-Seropositive Individuals

HIV infection usually leads to a progressive decline in number and functionality of CD4+ T lymphocytes, resulting in AIDS development. In this study, I investigated the strategy of using inoculated SupT1 cells to move infection from HIV-1 X4 strains toward the inoculated cells, which should theoretically prevent infection and depletion of normal CD4+ T cells, preventing the development of AIDS-related pathologies. Interestingly, the persistent in vitro replication in SupT1 cells renders the virus less cytopathic and more sensitive to antibody-mediated neutralization, suggesting that replication of the virus in the inoculated SupT1 cells may have a vaccination effect in the long run. In order to mimic the scenario of a therapy in which SupT1 cells are inoculated in an HIV-seropositive patient, I used infected SupT1/PBMC cocultures and a series of control experiments. Infections were done with equal amounts of the wild type HIV-1 LAI virus. The SupT1 CD4+CD8+ T cell population was distinguished from the PBMC CD4+CD8− T cell population by FACS analysis. The results of this study show that the virus-mediated killing of primary CD4+ T cells in the SupT1/PBMC cocultures was significantly delayed, suggesting that the preferential infection of SupT1 cells can induce the virus to spare primary CD4+ T cells from infection and depletion. The preferential infection of SupT1 cells can be explained by the higher viral tropism for the SupT1 cell line. In conclusion, this study demonstrates that it's possible in an in vitro system to use SupT1 cells to prevent HIV infection of primary CD4+ T cells, suggesting that further exploration of the SupT1 cell line as a cell-based therapy against HIV-1 may prove worthwhile

Differences in Disease Severity but Similar Telomere Lengths in Genetic Subgroups of Patients with Telomerase and Shelterin Mutations

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Zebrafish as a Model System to Study the Physiological Function of Telomeric Protein TPP1

Telomeres are specialized chromatin structures at the end of chromosomes. Telomere dysfunction can lead to chromosomal abnormalities, DNA damage responses, and even cancer. In mammalian cells, a six-protein complex (telosome/shelterin) is assembled on the telomeres through the interactions between various domain structures of the six telomere proteins (POT1, TPP1, TIN2, TRF1, TRF2 and RAP1), and functions in telomere maintenance and protection. Within the telosome, TPP1 interacts directly with POT1 and TIN2 and help to mediate telosome assembly. Mechanisms of telomere regulation have been extensively studied in a variety of model organisms. For example, the physiological roles of telomere-targeted proteins have been assessed in mice through homozygous inactivation. In these cases, early embryonic lethality has prevented further studies of these proteins in embryogenesis and development. As a model system, zebrafish offers unique advantages such as genetic similarities with human, rapid developmental cycles, and ease of manipulation of its embryos. In this report, we detailed the identification of zebrafish homologues of TPP1, POT1, and TIN2, and showed that the domain structures and interactions of these telosome components appeared intact in zebrafish. Importantly, knocking down TPP1 led to multiple abnormalities in zebrafish embryogenesis, including neural death, heart malformation, and caudal defect. And these embryos displayed extensive apoptosis. These results underline the importance of TPP1 in zebrafish embryogenesis, and highlight the feasibility and advantages of investigating the signaling pathways and physiological function of telomere proteins in zebrafish

Transcriptome profile analysis of flowering molecular processes of early flowering trifoliate orange mutant and the wild-type [Poncirus trifoliata (L.) Raf.] by massively parallel signature sequencing

<p>Abstract</p> <p>Background</p> <p>After several years in the juvenile phase, trees undergo flowering transition to become mature (florally competent) trees. This transition depends on the balanced expression of a complex network of genes that is regulated by both endogenous and environmental factors. However, relatively little is known about the molecular processes regulating flowering transition in woody plants compared with herbaceous plants.</p> <p>Results</p> <p>Comparative transcript profiling of spring shoots after self-pruning was performed on a spontaneously early flowering trifoliate orange mutant (precocious trifoliate orange, <it>Poncirus trifoliata</it>) with a short juvenile phase and the wild-type (WT) tree by using massively parallel signature sequencing (MPSS). A total of 16,564,500 and 16,235,952 high quality reads were obtained for the WT and the mutant (MT), respectively. Interpretation of the MPSS signatures revealed that the total number of transcribed genes in the MT (31,468) was larger than in the WT (29,864), suggesting that newly initiated transcription occurs in the MT. Further comparison of the transcripts revealed that 2735 genes had more than twofold expression difference in the MT compared with the WT. In addition, we identified 110 citrus flowering-time genes homologous with known elements of flowering-time pathways through sequencing and bioinformatics analysis. These genes are highly conserved in citrus and other species, suggesting that the functions of the related proteins in controlling reproductive development may be conserved as well.</p> <p>Conclusion</p> <p>Our results provide a foundation for comparative gene expression studies between WT and precocious trifoliate orange. Additionally, a number of candidate genes required for the early flowering process of precocious trifoliate orange were identified. These results provide new insight into the molecular processes regulating flowering time in citrus.</p

A novel chemiluminescence assay of organophosphorous pesticide quinalphos residue in vegetable with luminol detection

<p>Abstract</p> <p>Background</p> <p>Organophosphorous pesticides are the most popular pesticides used in agriculture. As acetylcholinesterase inhibitors, organophosphorous pesticides are toxic organic chemicals. The control and detection of organophosphorous pesticide residue in food, water, and environment therefore plays a very important role in maintaining physical health. A sensitive, rapid, simple chemiluminescence(CL) method has been developed for the determination of quinalphos based on the reaction of quinalphos with luminol-H₂O₂in an alkaline medium. The method has been applied to detection of quinalphos in vegetable samples with satisfactory results.</p> <p>Results</p> <p>The CL method for the determination of organophosphorous pesticide quinalphos is based on the phenomenon that quinalphos can apparently enhance the CL intensity of the luminol-H₂O₂system. The optimal conditions were: luminol concentration 5.0 × 10^-4mol/L, H₂O₂concentration 0.05 mol/L.pH value 13. In order to restrain the interference from metal ions, 1.0 × 10^-3mol/L of EDTA was added to the luminol solution. The possible mechanism was proposed.</p> <p>Conclusion</p> <p>Under the optimum reaction conditions, CL was linear with the concentration of quinalphos in the range of 0.02 μg/mL -1.0 μg/mL and the detection limit was 0.0055 μg/mL (3σ). This method has been successfully applied to the detection of quinalphos in vegetable samples. According to the experimental data, the average recoveries for quinalphos in cherry tomato and green pepper 97.20% and 90.13%. Meanwhile, the possible mechanism was proposed.</p

Heterozygosity increases microsatellite mutation rate, linking it to demographic history

<p>Abstract</p> <p>Background</p> <p>Biochemical experiments in yeast suggest a possible mechanism that would cause heterozygous sites to mutate faster than equivalent homozygous sites. If such a process operates, it could undermine a key assumption at the core of population genetic theory, namely that mutation rate and population size are indpendent, because population expansion would increase heterozygosity that in turn would increase mutation rate. Here we test this hypothesis using both direct counting of microsatellite mutations in human pedigrees and an analysis of the relationship between microsatellite length and patterns of demographically-induced variation in heterozygosity.</p> <p>Results</p> <p>We find that microsatellite alleles of any given length are more likely to mutate when their homologue is unusually different in length. Furthermore, microsatellite lengths in human populations do not vary randomly, but instead exhibit highly predictable trends with both distance from Africa, a surrogate measure of genome-wide heterozygosity, and modern population size. This predictability remains even after statistically controlling for non-independence due to shared ancestry among populations.</p> <p>Conclusion</p> <p>Our results reveal patterns that are unexpected under classical population genetic theory, where no mechanism exists capable of linking allele length to extrinsic variables such as geography or population size. However, the predictability of microsatellite length is consistent with heterozygote instability and suggest that this has an important impact on microsatellite evolution. Whether similar processes impact on single nucleotide polymorphisms remains unclear.</p

Genome-Wide Divergence of DNA Methylation Marks in Cerebral and Cerebellar Cortices

Emerging evidence suggests that DNA methylation plays an expansive role in the central nervous system (CNS). Large-scale whole genome DNA methylation profiling of the normal human brain offers tremendous potential in understanding the role of DNA methylation in brain development and function.Using methylation-sensitive SNP chip analysis (MSNP), we performed whole genome DNA methylation profiling of the prefrontal, occipital, and temporal regions of cerebral cortex, as well as cerebellum. These data provide an unbiased representation of CpG sites comprising 377,509 CpG dinucleotides within both the genic and intergenic euchromatic region of the genome. Our large-scale genome DNA methylation profiling reveals that the prefrontal, occipital, and temporal regions of the cerebral cortex compared to cerebellum have markedly different DNA methylation signatures, with the cerebral cortex being hypermethylated and cerebellum being hypomethylated. Such differences were observed in distinct genomic regions, including genes involved in CNS function. The MSNP data were validated for a subset of these genes, by performing bisulfite cloning and sequencing and confirming that prefrontal, occipital, and temporal cortices are significantly more methylated as compared to the cerebellum.These findings are consistent with known developmental differences in nucleosome repeat lengths in cerebral and cerebellar cortices, with cerebrum exhibiting shorter repeat lengths than cerebellum. Our observed differences in DNA methylation profiles in these regions underscores the potential role of DNA methylation in chromatin structure and organization in CNS, reflecting functional specialization within cortical regions

sodC-Based Real-Time PCR for Detection of Neisseria meningitidis

Real-time PCR (rt-PCR) is a widely used molecular method for detection of Neisseria meningitidis (Nm). Several rt-PCR assays for Nm target the capsule transport gene, ctrA. However, over 16% of meningococcal carriage isolates lack ctrA, rendering this target gene ineffective at identification of this sub-population of meningococcal isolates. The Cu-Zn superoxide dismutase gene, sodC, is found in Nm but not in other Neisseria species. To better identify Nm, regardless of capsule genotype or expression status, a sodC-based TaqMan rt-PCR assay was developed and validated. Standard curves revealed an average lower limit of detection of 73 genomes per reaction at cycle threshold (Ct) value of 35, with 100% average reaction efficiency and an average R2 of 0.9925. 99.7% (624/626) of Nm isolates tested were sodC-positive, with a range of average Ct values from 13.0 to 29.5. The mean sodC Ct value of these Nm isolates was 17.6±2.2 (±SD). Of the 626 Nm tested, 178 were nongroupable (NG) ctrA-negative Nm isolates, and 98.9% (176/178) of these were detected by sodC rt-PCR. The assay was 100% specific, with all 244 non-Nm isolates testing negative. Of 157 clinical specimens tested, sodC detected 25/157 Nm or 4 additional specimens compared to ctrA and 24 more than culture. Among 582 carriage specimens, sodC detected Nm in 1 more than ctrA and in 4 more than culture. This sodC rt-PCR assay is a highly sensitive and specific method for detection of Nm, especially in carriage studies where many meningococcal isolates lack capsule genes

The Protein Network Surrounding the Human Telomere Repeat Binding Factors TRF1, TRF2, and POT1

Telomere integrity (including telomere length and capping) is critical in overall genomic stability. Telomere repeat binding factors and their associated proteins play vital roles in telomere length regulation and end protection. In this study, we explore the protein network surrounding telomere repeat binding factors, TRF1, TRF2, and POT1 using dual-tag affinity purification in combination with multidimensional protein identification technology liquid chromatography - tandem mass spectrometry (MudPIT LC-MS/MS). After control subtraction and data filtering, we found that TRF2 and POT1 co-purified all six members of the telomere protein complex, while TRF1 identified five of six components at frequencies that lend evidence towards the currently accepted telomere architecture. Many of the known TRF1 or TRF2 interacting proteins were also identified. Moreover, putative associating partners identified for each of the three core components fell into functional categories such as DNA damage repair, ubiquitination, chromosome cohesion, chromatin modification/remodeling, DNA replication, cell cycle and transcription regulation, nucleotide metabolism, RNA processing, and nuclear transport. These putative protein-protein associations may participate in different biological processes at telomeres or, intriguingly, outside telomeres