How can we use the endocytosis pathways to design nanoparticle drug-delivery vehicles to target cancer cells over healthy cells?

Targeted drug delivery in cancer typically focuses on maximising the endocytosis of drugs into the diseased cells. However, there has been less focus on exploiting the differences in the endocytosis pathways of cancer cells versus non-cancer cells. An understanding of the endocytosis pathways in both cancer and non-cancer cells allows for the design of nanoparticles to deliver drugs to cancer cells whilst restricting healthy cells from taking up anticancer drugs, thus efficiently killing the cancer cells. Herein we compare the differences in the endocytosis pathways of cancer and healthy cells. Second, we highlight the importance of the physicochemical properties of nanoparticles (size, shape, stiffness, and surface chemistry) on cellular uptake and how they can be adjusted to selectively target the dominated endocytosis pathway of cancer cells over healthy cells and to deliver anticancer drug to the target cells. The review generates new thought in the design of cancer-selective nanoparticles based on the endocytosis pathways

Improved genome editing in human cell lines using the CRISPR method

The Cas9/CRISPR system has become a popular choice for genome editing. In this system, binding of a single guide (sg) RNA to a cognate genomic sequence enables the Cas9 nuclease to induce a double-strand break at that locus. This break is next repaired by an error-prone mechanism, leading to mutation and gene disruption. In this study we describe a range of refinements of the method, including stable cell lines expressing Cas9, and a PCR based protocol for the generation of the sgRNA. We also describe a simple methodology that allows both elimination of Cas9 from cells after gene disruption and re-introduction of the disrupted gene. This advance enables easy assessment of the off target effects associated with gene disruption, as well as phenotype-based structure-function analysis. In our study, we used the Fan1 DNA repair gene as control in these experiments. Cas9/CRISPR-mediated Fan1 disruption occurred at frequencies of around 29%, and resulted in the anticipated spectrum of genotoxin hypersensitivity, which was rescued by re-introduction of Fan1

Vaccines against toxoplasma gondii : challenges and opportunities

Development of vaccines against Toxoplasma gondii infection in humans is of high priority, given the high burden of disease in some areas of the world like South America, and the lack of effective drugs with few adverse effects. Rodent models have been used in research on vaccines against T. gondii over the past decades. However, regardless of the vaccine construct, the vaccines have not been able to induce protective immunity when the organism is challenged with T. gondii, either directly or via a vector. Only a few live, attenuated T. gondii strains used for immunization have been able to confer protective immunity, which is measured by a lack of tissue cysts after challenge. Furthermore, challenge with low virulence strains, especially strains with genotype II, will probably be insufficient to provide protection against the more virulent T. gondii strains, such as those with genotypes I or II, or those genotypes from South America not belonging to genotype I, II or III. Future studies should use animal models besides rodents, and challenges should be performed with at least one genotype II T. gondii and one of the more virulent genotypes. Endpoints like maternal-foetal transmission and prevention of eye disease are important in addition to the traditional endpoint of survival or reduction in numbers of brain cysts after challenge

Acquisition of pneumococci specific effector and regulatory Cd4+ T cells localising within human upper respiratory-tract mucosal lymphoid tissue

The upper respiratory tract mucosa is the location for commensal Streptococcus (S.) pneumoniae colonization and therefore represents a major site of contact between host and bacteria. The CD4(+) T cell response to pneumococcus is increasingly recognised as an important mediator of immunity that protects against invasive disease, with data suggesting a critical role for Th17 cells in mucosal clearance. By assessing CD4 T cell proliferative responses we demonstrate age-related sequestration of Th1 and Th17 CD4(+) T cells reactive to pneumococcal protein antigens within mucosal lymphoid tissue. CD25(hi) T cell depletion and utilisation of pneumococcal specific MHCII tetramers revealed the presence of antigen specific Tregs that utilised CTLA-4 and PDL-1 surface molecules to suppress these responses. The balance between mucosal effector and regulatory CD4(+) T cell immunity is likely to be critical to pneumococcal commensalism and the prevention of unwanted pathology associated with carriage. However, if dysregulated, such responses may render the host more susceptible to invasive pneumococcal infection and adversely affect the successful implementation of both polysaccharide-conjugate and novel protein-based pneumococcal vaccines

To respond or not to respond - a personal perspective of intestinal tolerance

For many years, the intestine was one of the poor relations of the immunology world, being a realm inhabited mostly by specialists and those interested in unusual phenomena. However, this has changed dramatically in recent years with the realization of how important the microbiota is in shaping immune function throughout the body, and almost every major immunology institution now includes the intestine as an area of interest. One of the most important aspects of the intestinal immune system is how it discriminates carefully between harmless and harmful antigens, in particular, its ability to generate active tolerance to materials such as commensal bacteria and food proteins. This phenomenon has been recognized for more than 100 years, and it is essential for preventing inflammatory disease in the intestine, but its basis remains enigmatic. Here, I discuss the progress that has been made in understanding oral tolerance during my 40 years in the field and highlight the topics that will be the focus of future research

The interaction of HAb18G/CD147 with integrin α6β1 and its implications for the invasion potential of human hepatoma cells

<p>Abstract</p> <p>Background</p> <p>HAb18G/CD147 plays pivotal roles in invasion by hepatoma cells, but the underlying mechanism remains unclear. Our previous study demonstrated that overexpression of HAb18G/CD147 promotes invasion by interacting with integrin α3β1. However, it has never been investigated whether α3β1 is solely responsible for this process or if other integrin family members also interact with HAb18G/CD147 in human hepatoma cells.</p> <p>Methods</p> <p>Human SMMC-7721 and FHCC98 cells were cultured and transfected with siRNA fragments against HAb18G/CD147. The expression levels of HAb18G/CD147 and integrin α6β1 were determined by immunofluorescent double-staining and confocal imaging analysis. Co-immunoprecipitation and Western blot analyses were performed to examine the native conformations of HAb18G/CD147 and integrin α6β1. Invasion potential was evaluated with an invasion assay and gelatin zymography.</p> <p>Results</p> <p>We found that integrin α6β1 co-localizes and interacts with HAb18G/CD147 in human hepatoma cells. The enhancing effects of HAb18G/CD147 on invasion capacity and secretion of matrix metalloproteinases (MMPs) were partially blocked by integrin α6β1 antibodies (<it>P </it>< 0.01). Wortmannin, a specific phosphatidylinositol kinase (PI3K) inhibitor that reverses the effect of HAb18G/CD147 on the regulation of intracellular Ca²⁺mobilization, significantly reduced cell invasion potential and secretion of MMPs in human hepatoma cells (<it>P </it>< 0.05). Importantly, no additive effect between Wortmannin and α6β1 antibodies was observed, indicating that α6β1 and PI3K transmit the signal in an upstream-downstream relationship.</p> <p>Conclusion</p> <p>These results suggest that α6β1 interacts with HAb18G/CD147 to mediate tumor invasion and metastatic processes through the PI3K pathway.</p

Rapid assembly of customized TALENs into multiple

Transcriptional activator-like effector nucleases (TALENs) have become a powerful tool for genome editing. Here we present an efficient TALEN assembly approach in which TALENs are assembled by direct Golden Gate ligation into Gateway® Entry vectors from a repeat variable di-residue (RVD) plasmid array. We constructed TALEN pairs targeted to mouse Ddx3 subfamily genes, and demonstrated that our modified TALEN assembly approach efficiently generates accurate TALEN moieties that effectively introduce mutations into target genes. We generated "user friendly" TALEN Entry vectors containing TALEN expression cassettes with fluorescent reporter genes that can be efficiently transferred via Gateway (LR) recombination into different delivery systems. We demonstrated that the TALEN Entry vectors can be easily transferred to an adenoviral delivery system to expand application to cells that are difficult to transfect. Since TALENs work in pairs, we also generated a TALEN Entry vector set that combines a TALEN pair into one PiggyBac transposon-based destination vector. The approach described here can also be modified for construction of TALE transcriptional activators, repressors or other functional domains. © 2013 Zhang et al

LSK Derived LSK– Cells Have a High Apoptotic Rate Related to Survival Regulation of Hematopoietic and Leukemic Stem Cells

A balanced pool of hematopoietic stem cells (HSCs) in bone marrow is tightly regulated, and this regulation is disturbed in hematopoietic malignancies such as chronic myeloid leukemia (CML). The underlying mechanisms are largely unknown. Here we show that the Lin−Sca-1+c-Kit- (LSK−) cell population derived from HSC-containing Lin−Sca-1+c-Kit+ (LSK) cells has significantly higher numbers of apoptotic cells. Depletion of LSK cells by radiation or the cytotoxic chemical 5-fluorouracil results in an expansion of the LSK− population. In contrast, the LSK− population is reduced in CML mice, and depletion of leukemia stem cells (LSCs; BCR-ABL-expressing HSCs) by deleting Alox5 or by inhibiting heat shock protein 90 causes an increase in this LSK− population. The transition of LSK to LSK− cells is controlled by the Icsbp gene and its downstream gene Lyn, and regulation of this cellular transition is critical for the survival of normal LSK cells and LSCs. These results indicate a potential function of the LSK− cells in the regulation of LSK cells and LSCs

Attitudes on the donation of human embryos for stem cell research among Chinese IVF patients and students

Bioethical debates on the use of human embryos and oocytes for stem cell research have often been criticized for the lack of empirical insights into the perceptions and experiences of the women and couples who are asked to donate these tissues in the IVF clinic. Empirical studies that have investigated the attitudes of IVF patients and citizens on the (potential) donation of their embryos and oocytes have been scarce and have focused predominantly on the situation in Europe and Australia. This article examines the viewpoints on the donation of embryos for stem cell research among IVF patients and students in China. Research into the perceptions of patients is based on in-depth interviews with IVF patients and IVF clinicians. Research into the attitudes of students is based on a quantitative survey study (n=427). The empirical findings in this paper indicate that perceptions of the donation of human embryos for stem cell research in China are far more diverse and complex than has commonly been suggested. Claims that ethical concerns regarding the donation and use of embryos and oocytes for stem cell research are typical for Western societies but absent in China cannot be upheld. The article shows that research into the situated perceptions and cultural specificities of human tissue donation can play a crucial role in the deconstruction of politicized bioethical argumentation and the (often ill-informed) assumptions about “others” that underlie socio-ethical debates on the moral dilemmas of technology developments in the life sciences