To date, generation of single-chain fragment variable (scFv) has become an established technique used to produce a completely functional antigen-binding fragment in bacterial systems. The advances in antibody engineering have now facilitated a more efficient and generally applicable method to produce Fv fragments. Basically, scFv antibodies produced from phage display can be genetically fused to the marker proteins, such as fluorescent proteins or alkaline phosphatase. These bifunctional proteins having both antigen-binding capacity and marker activity can be obtained from transformed bacteria and used for one-step immunodetection of biological agents. Alternatively, antibody fragments could also be applied in the construction of immunotoxins, therapeutic gene delivery, and anticancer intrabodies for therapeutic purposes. This paper provides an overview of the current studies on the principle, generation, and application of scFv. The potential of scFv in breast cancer research is also discussed in this paper.

In comparison to monolayer cells, MCTS has been claimed as more suitable candidate for studying drug penetration due to the high resemblance to solid tumors. However, the cultivation of MCTS is cumbersome, time consuming, and most technique fail to generate spheroids with uniform sizes. Therefore, the application of spheroid cultures in high throughput screening has been rather limiting. Besides, the lack of a well established screening protocol method that is applicable to spheroid could also be attributed to this limitation. Here we report a simple way of cultivating homogenous MCTS cultures with compact and rigid structure from the MCF-7 cells. Besides, we had also made some modifications to the standard MTT assay to realize high throughput screening of these spheroids. Using the modified protocol, tamoxifen showed cytotoxicity effect towards MCTS cultures from MCF-7 with high consistency. The results correlated well with the cultures’ response assessed by LDH release assay but the latter assay was not ideal for detecting a wide range of cytotoxicity due to high basal background reading. The MTT assay emerged as a better indicator to apoptosis event in comparison to the LDH release assay. Therefore, the method for spheroid generation and the modified MTT assay we reported here could be potentially applied to high throughput screening for response of spheroid cultures generated from MCF-7 as well as other cancer cell lines towards cytotoxic stimuli.

With increasing public health awareness worldwide, demand for functional food with multiple health benefits has also increased. The use of medicinal food from folk medicine to prevent diseases such as diabetes, obesity, and cardiovascular problems is now gaining momentum among the public. Seed from Salvia hispanica L. or more commonly known as chia is a traditional food in central and southern America. Currently, it is widely consumed for various health benefits especially in maintaining healthy serum lipid level. This effect is contributed by the presence of phenolic acid and omega 3/6 oil in the chia seed. Although the presence of active ingredients in chia seed warrants its health benefits, however, the safety and efficacy of this medicinal food or natural product need to be validated by scientific research. In vivo and clinical studies on the safety and efficacy of chia seed are still limited. This paper covers the up-to-date research on the identified active ingredients, methods for oil extraction, and in vivo and human trials on the health benefit of chia seed, and its current market potential.

Eupatorin has been reported with in vitro cytotoxic effect on several human cancer cells. However, reports on the mode of action and detail mechanism of eupatorin in vitro in breast cancer disease are limited. Hence, eupatorin’s effect on the human breast carcinoma cell line MCF-7 and MDA-MB-231 was investigated. MTT assay showed that eupatorin had cytotoxic effects on MCF-7 and MDA-MB-231 cells but was non-toxic to the normal cells of MCF-10a in a time-dose dependent manner. At 24 h, the eupatorin showed mild cytotoxicity on both MCF-7 and MDA-MB-231 cells with IC50 values higher than 20 μg/mL. After 48 h, eupatorin at 5 μg/mL inhibited the proliferation of MCF-7 and MDA-MB-231 cells by 50% while the IC50 of MCF-10a was significantly (p < 0.05) high with 30 μg/mL. The concentration of eupatorin at 5 μg/mL induced apoptosis mainly through intrinsic pathway by facilitating higher fold of caspase 9 compared to caspase 8 at 48 h. The cell cycle profile also showed that eupatorin (5 μg/mL) exerted anti-proliferation activity with the cell cycle arrest of MCF-7 and MDA-MB-231 cells at sub Gθ/G1 in a time-dependent manner. In addition, wound healing assay showed an incomplete wound closure of scratched MDA-MB-231 cells, and more than 60% of the MDA-MB-231 cells were prevented to migrate and invade the membrane in the Boyden chamber after 24 h. Eupatorin also inhibited angiogenic sprouting of new blood vessels in ex vivo mouse aorta ring assay. In gene expression assay, eupatorin up-regulated pro-apoptotic genes such as Bak1, HIF1A, Bax, Bad, cytochrome c and SMAC/Diablo and blocked the Phospho-Akt pathway. In conclusion, eupatorin is a potent candidate to induce apoptosis and concurrently inhibit the invasion, migration and angiogenesis of MDA-MB-231 and MCF-7 cells through inhibition of Phospho-Akt pathway and cell cycle blockade.

Transfection is a modern and powerful method used to insert foreign nucleic acids into eukaryotic cells. The ability to modify host cells’ genetic content enables the broad application of this process in studying normal cellular processes, disease molecular mechanism and gene therapeutic effect. In this review, we summarized and compared the findings from various reported literature on the characteristics, strengths, and limitations of various transfection methods, type of transfected nucleic acids, transfection controls and approaches to assess transfection efficiency. With the vast choices of approaches available, we hope that this review will help researchers, especially those new to the field, in their decision making over the transfection protocol or strategy appropriate for their experimental aims.