Plasmid vectors have been widely used for DNA vaccines and gene therapy. Following intramuscular injection, the plasmid that persists is extrachromosomal and integration into host DNA, if it occurs at all, is negligible. However, new technologies for improving DNA delivery could increase the frequency of integration. In the present study, we tested the effect of electroporation on plasmid uptake and potential integration following intramuscular injection in mice, using a plasmid containing the mouse erythropoietin gene. Electroporation increased plasmid tissue levels by approximately six-to 34-fold. Using a quantitative gel-purification assay for integration, electroporation was found to markedly increase the level of plasmid associated with high-molecular-weight genomic DNA. To confirm integration and identify the insertion sites, we developed a new assay -referred to as repeat-anchored integration capture (RAIC) PCR -that is capable of detecting rare integration events in a complex mixture in vivo. Using this assay, we identified four independent integration events. Sequencing of the insertion sites suggested a random integration process, but with short segments of homology between the vector breakpoint and the insertion site in three of the four cases. This is the first definitive demonstration of integration of plasmid DNA into genomic DNA following injection in vivo.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. Although recent advances in therapeutic approaches for treating HCC have improved the prognoses of patients with HCC, this cancer is still associated with a poor survival rate mainly due to late diagnosis. Therefore, a diagnosis must be made sufficiently early to perform curative and effective treatments. There is a need for a deeper understanding of the molecular mechanisms underlying the initiation and progression of HCC because these mechanisms are critical for making early diagnoses and developing novel therapeutic strategies. Over the past decade, much progress has been made in elucidating the molecular mechanisms underlying hepatocarcinogenesis. In particular, recent advances in next-generation sequencing technologies have revealed numerous genetic alterations, including recurrently mutated genes and dysregulated signaling pathways in HCC. A better understanding of the genetic alterations in HCC could contribute to identifying potential driver mutations and discovering novel therapeutic targets in the future. In this article, we summarize the current advances in research on the genetic alterations, including genomic instability, single-nucleotide polymorphisms, somatic mutations and deregulated signaling pathways, implicated in the initiation and progression of HCC. We also attempt to elucidate some of the genetic mechanisms that contribute to making early diagnoses of and developing molecularly targeted therapies for HCC.

A variety of factors could affect the frequency of integration of plasmid DNA vaccines into host cellular DNA, including DNA sequences within the plasmid, the expressed gene product (antigen), the formulation, delivery method, route of administration, and the type of cells exposed to the plasmid. In this report, we examined the tissue distribution and potential integration of plasmid DNA vaccines following intramuscular administration in mice and guinea pigs. We compared needle versus Biojector (needleless jet) delivery, examined the effect of aluminum phosphate adjuvants, compared the results of different plasmid DNA vaccines, and tested a gene (the human papilloma virus E7 gene) whose protein product is known to increase integration frequency in vitro. Six weeks following intramuscular injection, the vast majority of the plasmid was detected in the muscle and skin near the injection site; lower levels of plasmid were also detected in the draining lymph nodes. At early time points (1–7 days) after injection, a low level of systemic exposure could be detected. Occasionally, plasmid was detected in gonads, but it dissipated rapidly and was extrachromosomal – indicating a low risk of germline transmission. Aluminum phosphate adjuvant had no effect on the tissue distribution and did not result in a detectable increase in integration frequency. Biojector delivery, compared with needle injection, greatly increased the uptake of plasmid (particularly in skin at the injection site), but did not result in a detectable increase in integration frequency. Finally, injection of a plasmid DNA vaccine containing the human papilloma virus type 16 E7 gene, known to increase integration in vitro, did not result in detectable integration in mice. These results suggest that the risk of integration following intramuscular injection of plasmid DNA is low under a variety of experimental conditions.

CGRP is a neuropeptide that has previously been described to possess immunosuppressive activities. CGRP is released from peripheral nerves that, in the skin, are in close physical association with dendritic APC. We sought to investigate the mechanisms by which CGRP can inhibit immune responses by studying its effects on human peripheral blood mononuclear cells (PBMC). Using allogeneic monocytes as stimulator cells, CGRP could inhibit the proliferation of PBMC by 47% when CGRP was present for the duration of culture. Interestingly, when the stimulator monocytes were incubated with CGRP for 2 h prior to irradiation then washed, the observed inhibition increased to 85%, suggesting that CGRP was exerting a direct effect on the monocyte stimulator population. Finally, the recall response to tetanus toxoid (TT) by PBMC from individuals vaccinated with TT 14 d prior was inhibited by 25-50% in the presence of CGRP. Also, CGRP decreased the levels of B7.2 but not B7.1 on treated monocytes, and this inhibition could be abrogated by the addition of anti-IL-10 antibody, suggesting that the inhibition was mediated by an increase in IL-10 production. Moreover, increased IL-10 production was confirmed by ELISA. Both IL-12 p40 and IFN-gamma levels in CGRP-treated cultures were found to be decreased by approximately 30%. The decrease in IL-12 p40 levels could be reversed by addition of anti-IL-10. These data suggest that CGRP inhibits PBMC proliferation, in part, through the release of IL-10, which in turn can downregulate important co-stimulatory molecules and the cytokines IL-12 and IFN-gamma.

Long non-coding RNAs (lncRNAs) are a subgroup of non-coding RNA transcripts greater than 200 nucleotides in length with little or no protein-coding potential. Emerging evidence indicates that lncRNAs may play important regulatory roles in the pathogenesis and progression of human cancers, including hepatocellular carcinoma (HCC). Certain lncRNAs may be used as diagnostic or prognostic markers for HCC, a serious malignancy with increasing morbidity and high mortality rates worldwide. Therefore, elucidating the functional roles of lncRNAs in tumors can contribute to a better understanding of the molecular mechanisms of HCC and may help in developing novel therapeutic targets. In this review, we summarize the recent progress regarding the functional roles of lncRNAs in HCC and explore their clinical implications as diagnostic or prognostic biomarkers and molecular therapeutic targets for HCC.

Hypericin is a photodynamic compound activated by either visible (400-700 nm) or UVA (320-400 nm) light, and has been shown to inhibit the growth of a variety of neoplastic cell types. In this study, hypericin was found to inhibit proliferative responses of malignant T cells derived from the blood of patients with cutaneous T cell lymphoma. Control cells included peripheral blood mononuclear cells (PBMC) from normal volunteers or Epstein-Barr virus-transformed lymphocytes. Cells from each of these populations were incubated with serial dilutions of hypericin or 8-methoxypsoralen and then stimulated with the mitogen ConA (10 microg per ml). Cultures were prepared in the dark to minimize photoactivation of the hypericin. Proliferation was measured by [3H]thymidine labeling after 72 h. Hypericin, photoactivated with 1.1-3.3 J white light per cm2, inhibited cellular proliferation of malignant T cells with IC50 values from 0.34 to 0.53 microM, normal PBMC with IC50 values of 0.11-0.76 microM, and Epstein-Barr virus-transformed cells with IC50 values of 0.75-3.2 microM. UVA-photoactivated hypericin (0.5-2.0 J per cm2) could also inhibit proliferation with IC50 values of 0.57-1.8 microM, 0.7-4.6 microM, and 2.0-3.7 microM for malignant, normal, or Epstein-Barr virus-transformed cells, respectively. Hypericin, photoactivated with either UVA or white light, could induce near complete apoptosis (94%) in malignant cutaneous T cell lymphoma T cells, whereas lower levels of apoptosis (37-88%) were induced in normal PBMC. These data indicate that hypericin inhibits mitogen-induced proliferation of malignant T cells from patients with cutaneous T cell lymphoma, PBMC from normal individuals, as well as Epstein-Barr virus-transformed lymphocytes, and that inhibition of cell proliferation is dependent on the concentration of hypericin used and the dose of light required to photoactivate the compound. Induction of apoptosis is, in part, one mechanism by which photoactivated hypericin inhibits malignant T cell proliferation.

Apoptosis, or programmed cell death (PCD), recently has emerged as an important homeostatic mechanism within several hematopoietic lineages. This process is subject to both positive and negative modulation by cytokines and within the erythroid lineage is inhibited by interleukin-3, stem cell factor, and erythropoietin (Epo). Through the expression of carboxyl-truncated Epo receptor mutants in FDC-P1 cells, a receptor form possessing 80 membrane-proximal cytoplasmic residues is shown to efficiently mediate Epo-dependent inhibition of PCD. This is in contrast to previous studies that attributed this activity to a distal carboxyl-terminal receptor subdomain (and/or heterodimerization of wild type Epo receptors with a truncated non-functional receptor form). Epo-dependent inhibition of PCD also is shown to be blocked by ectopic expression of kinase-deficient dominant-negative forms of Jak2 (Jak2 delta VIII and Jak2-829), further underlining a role of this membrane-proximal subdomain of the Epo receptor in the inhibition of PCD. To our knowledge, this comprises the first direct evidence for an essential role for a Jak tyrosine kinase (Jak2) in this apoptotic response pathway.

Despite the extensive developments of flexible capacitive pressure sensors, it is still elusive to simultaneously achieve excellent linearity over a broad pressure range, high sensitivity, and ultrahigh pressure resolution under large pressure preloads. Here, we present a programmable fabrication method for microstructures to integrate an ultrathin ionic layer. The resulting optimized sensor exhibits a sensitivity of 33.7 kPa−1 over a linear range of 1700 kPa, a detection limit of 0.36 Pa, and a pressure resolution of 0.00725% under the pressure of 2000 kPa. Taken together with rapid response/recovery and excellent repeatability, the sensor is applied to subtle pulse detection, interactive robotic hand, and ultrahigh-resolution smart weight scale/chair. The proposed fabrication approaches and design toolkit from this work can also be leveraged to easily tune the pressure sensor performance for varying target applications and open up opportunities to create other iontronic sensors.