Efficient non-viral gene delivery often involves the conjugation of a cell-specific ligand to the vector, which directs the vector to its intended target through binding to a cellular receptor ...followed by internalization via endocytosis. However, little is known in terms of how the various endocytosis pathways affect the performance of the delivery vehicle. Previously, the Pack lab has demonstrated that caveolin-mediated endocytosis is important to in vitro polyethylenimine (PEI)- and polyamidoamine (PAMAM)-mediated gene delivery in HeLa cells through the use of small molecule drugs and small-interfering RNA 1-2. The goal of this thesis is to further elucidate the effects of cellular uptake mechanism on non-viral gene delivery in vitro and in vivo utilizing a small hairpin RNA (shRNA) Tet-on system. Specifically, we have investigated the effects of clathrin-dependent endocytosis and caveolin-dependent endocytosis on the efficacy of PEI and PEI-derivative gene delivery in HeLa and MDA-MB-231 cell cultures and xenograft tumors. First, we attached transferrin and folate ligands to PEI to direct internalization by clathrin- and caveolin-mediated mechanisms, respectively, and transfected HeLa and MDA-MB-231 cells with targeted and untargeted PEI in the presence of small molecule drugs that inhibit clathrin- or caveolin-mediated endocytosis. We demonstrated through these studies that caveolin-dependent endocytosis is important to successful PEI-mediated gene delivery in both HeLa and, for the first time, MDA-MB-231 cells. In addition, a similar endocytic pathway study was performed using biodegradable PEI and acetylated PEI, which have demonstrated superior gene delivery capability compared to unmodified PEI in previous work 3-4. The data show that regardless of structure, size, molecular weight, or zeta potential of the PEI-derivative polyplexes, caveolin-dependent endocytosis was critical to effective gene delivery. In order to further understand the impact of endocytic pathways on gene delivery efficiency in vitro, we modified HeLa and MDA-MB-231 cells to express small hairpin RNA (shRNA) that inhibit expression of clathrin, caveolin, or lamin A/C (negative control) protein expression (HeLa-CLTC, HeLa-CAV, HeLa-LAM, 231-CLTC, 231-CAV, and 231-LAM) in the presence of tetracycline. Using the shRNA Tet-on system, we demonstrated ~90% clathrin knockdown and ~60% caveolin knockdown in both modified HeLa and MDA-MB-231 cells. Subsequently, utilizing this inducible shRNA protein knockdown system, we transfected modified shRNA cells using both targeted and untargeted PEIs and confirmed that caveolin-dependent endocytosis is more important than clathrin-dependent endocytosis to PEI-mediated gene delivery, which corroborates the results observed in unmodified cell lines using small-molecule endocytosis inhibitors. Lastly, we investigated the effect of clathrin-dependent and caveolin-dependent endocytosis on in vivo PEI-mediated gene delivery in a xenograft murine tumor model. By inoculating modified shRNA cells into NIH-III nude mice, we created a xenograft tumor model that allows us to induce CLTC and CAV protein knockdown inside the tumor. Using this Tet inducible system, we demonstrated that the mice fed with Tet-containing water, compared to those fed with water only, show over 50% target protein knockdown in modified MDA-MB-231 cells and 90% CLTC knockdown and 35% CAV knockdown for modified HeLa cells. By transfecting shRNA modified tumors intratumorally with PEI polyplexes, PEI gene delivery efficacy was 15-fold and 2.5-fold higher for modified HeLa and MDA-MB-231 tumors, respectively, when comparing clathrin- versus caveolin-dependent endocytosis inhibition, although the improvement for unmodified MDA-MB-231 tumors was statistically insignificant. The results confirmed that caveolin-dependent endocytosis was more important to in vivo PEI-mediated gene delivery than clathrin-dependent endocytosis, which is in good agreement with in vitro data using both small molecule drugs and Tet inducible protein knockdown. In conclusion, these studies of intracellular trafficking and its effect on PEI gene delivery efficacy in cancer cell lines and tumors will aid researchers in designing more efficient non-viral gene delivery vectors for cancer gene therapy, by synergistically combining the design of cell targeting ligands and the understanding of cellular uptake mechanism for polymeric carriers.
The lin-12/Notch signaling pathway is conserved from worms to humans and is a master regulator of metazoan development. Here, we demonstrate that lin-12/Notch gain-of-function (gf) animals display ...precocious alae at the L4 larval stage with a significant increase in let-7 expression levels. Furthermore, lin-12(gf) animals display a precocious and higher level of let-7 gfp transgene expression in seam cells at L3 stage. Interestingly, lin-12(gf) mutant rescued the lethal phenotype of let-7 mutants similar to other known heterochronic mutants. We propose that lin-12/Notch signaling pathway functions in late developmental timing, upstream of or in parallel to the let-7 heterochronic pathway. Importantly, the human microRNA let-7a was also upregulated in various human cell lines in response to Notch1 activation, suggesting an evolutionarily conserved cross-talk between let-7 and the canonical lin-12/Notch signaling pathway.
Background: Vascular stiffening develops with both hypertension and aging, and is a strong predictor of end-organ damage. Excessive deposition of collagen by vascular smooth muscle cells (VSMCs) can ...lead to decreased compliance of vessels such as the aorta. The IRE1α arm of the unfolded protein response is activated in cells with a secretory phenotype due to its role in augmenting protein folding capacity. We hypothesize that by a similar mechanism, VSMCs transitioning to a collagen-secreting phenotype in response to TGF-β1 require the activation of IRE1. Inhibition of this pathway is hypothesized to reduce collagen secretion and hence prevent the development of fibrosis in the aorta. ;
Methods: Collagen deposition by VSMCs in vitro was measured using immunoblotting and a Picrosirius Red-based colorimetric assay. Western blot and qRT-PCR were used to assess the expression of ER stress markers. Ex vivo culture of aortic rings was also performed to determine the effect of 4µ8c on TGF-β1-induced vascular stiffening. 12-14 week old male spontaneously hypertensive rats were divided into three treatment groups: 1) No treatment, 2) L-NAME (50 mg/L), and 3) L-NAME and the IRE1α inhibitor 4µ8c (2.5 mg/kg/day i.p.). Aortic compliance after 18 days of treatment was measured ex vivo using a wire myograph to construct tension-diameter curves. ;
Results: Inhibition of IRE1α endonuclease activity by 4µ8c reduced collagen production in VSMCs stimulated with TGF-β1 or Ang II. A decrease in the expression of the collagen-associated chaperones PDI, GRP78 and GRP94 was observed. Aortic rings treated with TGF-β1 developed vascular stiffening, which was improved by co-treatment with 4µ8c. SHRs treated with L-NAME for 18 days developed aortic stiffening, which was prevented by daily injections of 4µ8c. ;
Conclusions: Our data suggest that inhibition of the IRE1α pathway can reduce vascular stiffening and fibrosis by disrupting the collagen biosynthesis pathway in VSMCs.
Thesis
Master of Science (MSc)
This article describes a formal proof of the Kepler conjecture on dense sphere packings in a combination of the HOL Light and Isabelle proof assistants. This paper constitutes the official published ...account of the now completed Flyspeck project.
This study aims to establish a highly adaptable workflow downstream of microfluidic enrichment for facilitating systematic CTC enumeration and genetic discovery.
To facilitate CTC enumeration, we ...established a CK/EPCAM-combined immunostaining strategy and an automated CTC analytical pipeline using an open-source image analyzer. By virtue of this workflow, we conducted a pilot study of 56 cancer patients and 21 healthy individuals using a high-throughput spiral microfluidic chip system. To facilitate genetic discovery of somatic mutations in CTCs, we integrated the CTC enumeration into next-generation sequencing and established a straightforward amplicon library comprising diversifier random sequences to sequence CTC samples.
The CTC staining and enumeration workflow achieved 80.4% sensitivity and 85.7% specificity (AUC = 0.87, p = 0.004, power = 0.985), as evaluated by ROC analysis. Univariate and multivariate analysis verified that the CTC (CK/EpCAM+CD45-), but not other cell populations, is a significant and independent biomarker for cancer patients (p < 0.01). Serial CTC monitoring of the patients revealed reduction in CTC numbers after treatments, suggesting its clinical utility in pharmacodynamic studies. Deep sequencing of CTC samples revealed somatic mutations in TP53 and ESR1.
The significance of this report is to demonstrate a systematic and adaptable workflow to bridge the gap between the microfluidic enrichment and CTC analyses, which fosters broader applications of CTCs in both clinical settings and academic studies.
Improved control of Plasmodium vivax malaria can be achieved with the discovery of new antimalarials with radical cure efficacy, including prevention of relapse caused by hypnozoites residing in the ...liver of patients. We screened several compound libraries against P. vivax liver stages, including 1565 compounds against mature hypnozoites, resulting in one drug-like and several probe-like hits useful for investigating hypnozoite biology. Primaquine and tafenoquine, administered in combination with chloroquine, are currently the only FDA-approved antimalarials for radical cure, yet their activity against mature P. vivax hypnozoites has not yet been demonstrated in vitro. By developing an extended assay, we show both drugs are individually hypnozonticidal and made more potent when partnered with chloroquine, similar to clinically relevant combinations. Post-hoc analyses of screening data revealed excellent performance of ionophore controls and the high quality of single point assays, demonstrating a platform able to support screening of greater compound numbers. A comparison of P. vivax liver stage activity data with that of the P. cynomolgi blood, P. falciparum blood, and P. berghei liver stages reveals overlap in schizonticidal but not hypnozonticidal activity, indicating that the delivery of new radical curative agents killing P. vivax hypnozoites requires an independent and focused drug development test cascade.
With an emphasis placed on supply‐side interventions such as skills training and incentives enhancement, active labor market polices (ALMPs) are strongly promoted by international organizations and ...widely adopted across different welfare regimes to boost employment rates. This article first presents the under‐examined relationship between ALMPs and employment precariousness, which has posed a challenge to the neoliberal notion of employability and activation. Youth‐focused employment policies tend to speed up employment entry whilst downplaying the risk of precariousness and the importance of job quality, and thus further reinforcing the belief that engaging in precarious employment is tolerable if not inevitable. The article then examines the case of Hong Kong, which illustrates that its relatively low rate of youth unemployment may conceal the unfavorable employment conditions confronted by youth. It is argued that the service‐led employment policies and short‐term vocational training define the employability of young workers in terms of labor flexibilities. The coined phase of “flexi‐employability” is characterized by promoting youth's readiness for, and adaptability to, the volatilities and changing demands of the labor market. Arguably, the disciplinary approach to youth activation would only strengthen the work‐first principle by enforcing young people to take up jobs available and leave welfare as soon as possible, but without thoroughly addressing the risks and insecurities generated by the labor market in undermining their well‐being.
Charcot-Marie-Tooth disease (CMT) is a common inherited peripheral neuropathy affecting up to 1 in 1214 of the general population with more than 60 nuclear genes implicated in its pathogenesis. ...Traditional molecular diagnostic pathways based on relative prevalence and clinical phenotyping are limited by long turnaround time, population-specific prevalence of causative variants and inability to assess multiple co-existing variants. In this study, a CMT gene panel comprising 27 genes was used to uncover the pathogenic mutations in two index patients. The first patient is a 15-year-old boy, born of consanguineous parents, who has had frequent trips and falls since infancy, and was later found to have inverted champagne bottle appearance of bilateral legs and foot drop. His elder sister is similarly affected. The second patient is a 37-year-old woman referred for pre-pregnancy genetic diagnosis. During early adulthood, she developed progressive lower limb weakness, difficulties in tip-toe walking and thinning of calf muscles. Both patients are clinically compatible with CMT, have undergone multiple genetic testings and have not previously received a definitive genetic diagnosis. Patients 1 and 2 were found to have pathogenic homozygous
:NM_001540:c.250G>A (p.G84R) variant and heterozygous
:NM_018972:c.358C>T (p.R120W) variant, respectively. Advantages and limitations of the current approach are discussed.
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