Intracellular trafficking mechanism of cationic phospholipids including cationic liposomes in HeLa cells.

The development of gene delivery methods is essential for the achievement of effective gene therapy. Elucidation of the intracellular transfer mechanism for cationic carriers is in progress, but there are few reports regarding the intracellular trafficking processes of the cationic phospholipids taken up into cells. In the present work, the trafficking processes of a cationic phospholipid (1,2-dioleoyl-3-trimethylammonium-propane, DOTAP) were investigated from intracellular uptake to extracellular efflux using cationic liposomes in vitro. Following intracellular transport of liposomes via endocytosis, DOTAP was localized in the endoplasmic reticulum, Golgi apparatus, and mitochondria. Moreover, the proteins involved in DOTAP intracellular trafficking and extracellular efflux were identified. In addition, helper lipids of cationic liposomes were found to partially affect this intracellulartrafficking. These findings might provide valuable information for designing cationic carriers and avoiding unexpected toxic side effects derived from cationic liposomal components.

Enhancement of gene expression by transcriptional activation using doxorubicin-loaded liposome/pDNA complexes.

Gene therapy is a promising treatment option for cancers generated by mutation of oncogenes or tumor suppressor genes. The transcriptional process is activated by doxorubicin (DXR), and gene expression efficiency followed by gene transfection can be enhanced by the combination-use of DXR. Therefore, co-encapsulation of plasmid DNA (pDNA) and DXR into non-viral gene carriers can enhance gene expression. Here, we prepared DXR-loaded liposome/pDNA complexes (DXR-loaded PEGylated lipoplexes) by co-encapsulating pDNA and DXR into liposomes. Gene expression was enhanced by DXR encapsulation into lipoplexes in colon-26 cells and cultured mouse macrophages, and this gene expression level was significantly higher than that obtained by the combination of PEGylated lipoplexes and free DXR. Moreover, the activation profiles of transcriptional factors induced by DXR-loaded lipoplexes were different from those induced by free DXR; therefore, co-encapsulation of pDNA and DXR into gene carriers might be contributed to effective enhancement of gene expression. These findings provide a new approach for achieving effective gene transfection using PEGylated lipoplexes.

Preference of Food Saltiness and Willingness to Consume Low-Sodium Content Food in a Chinese Population.

OBJECTIVE: To compare the preference of food saltiness and the willingness to consume low-sodium food among hypertensive older people, non-hypertensive older people and non-hypertensive young people in a Chinese population. DESIGN: A cross-sectional study based on a quota sample. Three saltiness options (low-sodium, medium-sodium and high-sodium) of soup and bread were offered to each participant who rated the taste of each food on a 5-point Likert scale. Then, the participants rated their willingness to consume the low-sodium content foods on a 5-point Likert scale, given they were informed of the benefit of the low-sodium option. Generalised linear mixed model and multiple linear regression were used to analyse the data. SETTING: Elderly centres and community centres in Hong Kong. PARTICIPANTS: Sixty hypertensive older people, 49 non-hypertensive older people and 60 non-hypertensive young people were recruited from June to August 2014. MEASUREMENTS: The tastiness score and the willingness score were the primary outcome measures. The Chinese Health Literacy Scale for Low Salt Consumption - Hong Kong population (CHLSalt-HK) was also assessed. RESULTS: The tastiness rating of the high-sodium option of soup was significantly lower than the medium-sodium option (p<0.001), but there was no significant difference between the low-sodium and the medium-sodium options (p=0.204). For bread, tastiness rating of the low-sodium option and the high-sodium option were significantly lower than the medium-sodium option (p<0.001 for both options). The tastiness score of soup did not have significant difference across the groups (p=0.181), but that of bread from the hypertensive older adults (p=0.012) and the non-hypertensive older adults (p=0.006) was significantly higher than the non-hypertensive young adults. Higher willingness rating to consume the low-sodium option was significantly (p<0.001) associated with higher tastiness rating of the low-sodium option of soup and bread, and weakly associated with higher health literacy of low salt intake (soup: p=0.041; bread: p=0.024). Hypertensive older adults tended to be more willing to consume the low-sodium option than non-hypertensive older adults for soup (p=0.009), there was insignificant difference between non-hypertensive older adults and non-hypertensive young adults (p=0.156). For bread, there was insignificant difference in willingness rating to consume low-sodium option (p=0.375). CONCLUSION: Older people are at a higher risk of hypertension, reduction of salt intake is important for them to reduce their risk of cardiovascular diseases. There is room for reducing the sodium content of soup, while the sodium in bread should be reduced progressively. Improving the taste of low-sodium food may help to promote reduction in dietary sodium intake.

Retinoic acid-induced human secretin gene expression in neuronal cells is mediated by cyclin-dependent kinase 1.

Previously, we found that secretin transcript levels were induced by all-trans retinoic acid (RA) in a neuroblastoma cell model, SH-SY5Y. In this article, this RA-dependent upregulation process was further investigated. In the cyclin-dependent kinase 1 (Cdk1) inhibitor-treated cells, the RA-dependent induction of secretin gene expression was inhibited. Together with our previous works, we propose here that the RA responsiveness of the secretin promotor is mediated by two different pathways. The first pathway is by changing the expression levels of NFI-C and Sp proteins while the second pathway is by modifying the phosphorylation status of both NFI-C and Sp proteins via Cdk1.

The study of sequence configuration and functional impact of the (AC)n(AT)xTy motif in human beta-globin gene promoter.

In this report we examine the (AC)n(AT)xTy motif residing -530 bp 5' upstream of the beta-globin gene in Chinese thalassaemic patients. This motif is a putative binding site for a repressor protein, termed beta protein 1 (BP1) (Berg et al., Nucleic Acids Res 1989;17:8833-8852). Variations in the (AC)n(AT)xTy repeats affect the binding affinity of BP1, thereby altering the expression of the beta-globin gene. Eight different configurations of this repeat motif are identified in our population of Chinese beta-thalassaemia patients. A (AC)3(AT)7T5 motif was identified among these thalassaemia patients and its influence in beta-globin gene expression was studied using stable transfection assay in murine erythroleukemia (MEL) cells. Our data demonstrated that the (AC)3(AT)7T5 motif has a moderately strong repressor effect on the expression of the cis-linked beta-globin gene. The high affinity of BP1 for this motif may result in the suppression of the transcription of the beta-globin gene (Berg et al., Am J Hematol 1991;36:42-47). We postulate that silencer elements in the beta-globin promoter play an important role in modifying the clinical presentation of the disease.

Differential lineage-specific regulation of murine CD45 transcription by Oct-1 and PU.1.

Although it has been established that CD45 expression is regulated at the transcriptional level, neither the regulatory elements that are responsible for its unique expression pattern nor the relevance of its three distinct transcriptional start sites (P1a, P1b, and P2) has been fully characterized. We studied the contribution of the three start sites to CD45 mRNA production in haematopoietic cell lines and primary haematopoietic cells. In myeloid and lymphoid cells and cell lines most CD45 transcripts originate from P1b with the exception of the thymoma-derived T cell line EL4, in which approximately 90% of CD45 transcripts originate from P1a. The degree of contribution of P1a is highest in lymphoid cells and increases in T cells following mitogen stimulation. In vitro evaluation of sequence upstream of the start sites shows that the P2 start site is sufficient for CD45 expression in lymphoid but not in myeloid cells, confirms the presence of a PU.1-binding site essential for myeloid expression of CD45, and reveals an Octamer-binding site that interacts with both Oct-1 and Oct-2 and activates CD45 transcription in lymphoid and myeloid cells. These findings are the first evidence that Octamer-binding factors are involved in the control of CD45 expression.

Real-time size distribution, concentration, and biomass measurement of marine phytoplankton with a novel dual-beam laser fluorescence Doppler cytometer.

A novel cytometer is reported for measuring particle-size distribution, concentration, and biomass of marine phytoplankton containing chlorophyll a. The system utilizes optical fibers to carry light to and from a flow tube for measuring phytoplankton taken directly from the ocean. A unique feature of this system is in the simple optical detection scheme for which sample handling and preparation are not required. This simplicity makes the system especially suitable for field measurements. The system utilizes sophisticated digital signal processors to handle and reduce the large amount of data gathered. The signal-processing algorithms are vigorously streamlined to process the signals in real time, thus computing size and flow velocity information instead of logging the raw data. The high efficiency of the signal processors gives the system a performance throughput of ~250 particles/s. The system was tested both in the laboratory and in the field, yielding good discrimination of size distribution and sensitivity of concentration.

An evaluation of three-dimensional diarthrodial joint contact using penetration data and the finite element method.

We have developed an approximate method for simulating the three-dimensional contact of soft biphasic tissues in diarthrodial joints under physiological loading. Input to the method includes: (i) kinematic information describing an in vitro joint articulation, measured while the cartilage is deformed under physiological loads, (ii) geometric properties for the relaxed (undeformed) cartilage layers, obtained for the analyses in this study via stereophotogrammetry, and (iii) material parameters for the biphasic constitutive relations used to represent cartilage. Solid models of the relaxed tissue layers are assembled in physiological positions, resulting in a mathematical overlap of the cartilage layers. The overlap distribution is quantified and converted via the biphasic governing equations into applied traction boundary conditions for both the solid and fluid phases for each of the contacting layers. Linear, biphasic, three-dimensional, finite element analysis is performed using the contact boundary conditions derived for each of the contacting layers. The method is found to produce results consistent with the continuity requirements of biphasic contact. Comparison with results from independent, biphasic contact analyses of axisymmetric problems shows that the method slightly underestimates the contact area, leading to an overestimation of the total traction, but yields a good approximation to elastic stress and solid phase displacement.

Evaluation of beta-globin gene therapy constructs in single copy transgenic mice.

Effective gene therapy constructs based on retrovirus or adeno-associated virus vectors will require regulatory elements that direct expression of genes transduced at single copy. Most beta-globin constructs designed for therapy of beta-thalassemias are regulated by the 5'HS2 component of the locus control region (LCR). Here we show that a human beta-globin gene flanked by two small 5'HS2 core elements or flanked by a 5'HS3 (footprints 1-3) core and a 5'HS2 core are not reproducibly expressed in single copy transgenic mice. In addition, low copy transgene concatamers that contain only dimer 5'HS2 cores fail to express, whereas those that contain monomer 5'HS2 cores express at 14% per copy. These data suggest that spacing between HS cores is crucial for LCR activity. We therefore constructed a novel 3.0 kb LCR cassette in which the 5'HS2, 5'HS3 and 5'HS4 cores are each separated by approximately 700 bp. When linked to the 815 bp beta-globin promoter this LCR directs 45% levels of expression from four independent single copy transgenes. However, the 3.0 kb LCR linked to the 265 bp promoter expresses variable levels, averaging 18%, from three single copy transgenes. Our findings suggest that sequences in the distal promoter play a role in single copy transgene activation and that larger LCR and promoter elements are most suitable for gene therapy applications.

A dominant chromatin-opening activity in 5' hypersensitive site 3 of the human beta-globin locus control region.

Single-copy human beta-globin transgenes are very susceptible to suppression by position effects of surrounding closed chromatin. However, these position effects are overcome by a 20 kbp DNA fragment containing the locus control region (LCR). Here we show that the 6.5 kbp microlocus LCR cassette reproducibly directs full expression from independent single-copy beta-globin transgenes. By testing individual DNase I-hypersensitive sites (HS) present in the microlocus cassette, we demonstrate that the 1.5 kbp 5'HS2 enhancer fragment does not direct beta-globin expression from single-copy transgenes. In contrast, the 1.9 kbp 5'HS3 fragment directs beta-globin expression in five independent single-copy transgenic mouse lines. Moreover, the 5'HS3 core element and beta-globin proximal promoter sequences are DNase I hypersensitive in fetal liver nuclei of these expressing transgenic lines. Taken together, these results demonstrate that LCR activity is the culmination of at least two separable functions including: (i) a novel activity located in 5'HS3 that dominantly opens and remodels chromatin structure; and (ii) a recessive enhancer activity residing in 5'HS2. We postulate that the different elements of the LCR form a 'holocomplex' that interacts with the individual globin genes.

Heterochromatin effects on the frequency and duration of LCR-mediated gene transcription.

Locus control regions (LCRs) are responsible for initiating and maintaining a stable tissue-specific open chromatin structure of a locus. In transgenic mice, LCRs confer high level expression on linked genes independent of position in the mouse genome. Here we show that an incomplete LCR loses this property when integrated into heterochromatic regions. Two disruption mechanisms were observed. One is classical position-effect variegation, resulting in continuous transcription in a clonal subpopulation of cells. The other is a novel mechanism resulting in intermittent gene transcription in all cells. We conclude that only a complete LCR fully overcomes heterochromatin silencing and that it controls the level of transcription by ensuring activity in all cells at all times rather than directly controlling the rate of transcription.

Comparative genome analysis delimits a chromosomal domain and identifies key regulatory elements in the alpha globin cluster.

We have cloned, sequenced and annotated segments of DNA spanning the mouse, chicken and pufferfish alpha globin gene clusters and compared them with the corresponding region in man. This has defined a small segment ( approximately 135-155 kb) of synteny and conserved gene order, which may contain all of the elements required to fully regulate alpha globin gene expression from its natural chromosomal environment. Comparing human and mouse sequences using previously described methods failed to identify the known regulatory elements. However, refining these methods by ranking identity scores of non-coding sequences, we found conserved sequences including the previously characterized alpha globin major regulatory element. In chicken and pufferfish, regions that may correspond to this element were found by analysing the distribution of transcription factor binding sites. Regions identified in this way act as strong enhancer elements in expression assays. In addition to delimiting the alpha globin chromosomal domain, this study has enabled us to develop a more sensitive and accurate routine for identifying regulatory elements in the human genome.

The molecular genetic analysis of hemophilia A: a directed search strategy for the detection of point mutations in the human factor VIII gene.

A directed-search strategy for point mutations in the factor VIII gene causing hemophilia A was used to screen eight potentially hypermutable CpG dinucleotides occurring at sites deemed to be of functional importance. Polymerase chain reaction-amplified DNA samples from 793 unrelated individuals with hemophilia A were screened by discriminant oligonucleotide hybridization. Point mutations were identified in 16 patients that were consistent with a model of 5-methylcytosine (5mC) deamination. Four new examples of recurrent mutation were demonstrated at the following codons: 336 (CGA----TGA), 372 (CGC----TGC), 372 (CGC----CAC), and 1689 (CGC----TGC). These are functionally important cleavage sites for either activated protein C or thrombin. Further novel C----T transitions were identified in the remaining arginine codons screened (-5, 427, 583, 795, and 1696), resulting in the creation of TGA termination codons. Differences in mutation frequency were found both within and between the CpG sites and between ethnic groups. These differences are assumed to be due to differences in the level of cytosine methylation at these sites, although direct evidence for this inference is lacking.