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Students always encounter difficulties in studying biochemical pathways. They are especially weak in understanding the relationships between metabolic pathways and their integration because these pathways are always taught one-by-one in class. In the past, various online resources have been developed to facilitate students' understanding toward energy metabolism. Although these learning materials enable students to understand individual metabolic pathway in a clearer manner, many of them fail to demonstrate the linkages between these pathways. The "AG City" is a self-learning tool which aims to arouse students' interest in exploring nucleotide metabolism. We have designed a metro map as a concept map to allow students to have an overview of different pathways as well as their integration. Major pathways are presented as railway lines in an easy-to-understand and interactive manner using navigation, animations, and narrations. Key molecules involved in the pathways are presented as "railway stations". Students can easily identify common "railway stations" presented in different pathways and link the concepts that they have learnt together. This interactive self-learning tool has been packaged as a courseware using the Articulate Storyline eLearning authoring software. © 2019 International Union of Biochemistry and Molecular Biology, 47(5):532-537, 2019.

To develop drugs to kill cancer cells, we chemically synthesized a number of anti-cancer agents by adding dif-ferent side chains to the core backbone of saponin. With the use of bioassay-guided methods, we found one agent that possessed a high cytotoxicity to a number of cancer cell lines. Interestingly, this compound was later found to be an active component of a tradition Chinese herb Paris polyphylla known as Polyphyllin D (PD) (diosgenyl -L-rhamnopyranosyl-(1 2)-( -L-ara-binofuranosyl-(1 4)- -D-glucopyranoside). In China, the rhizome of Paris polyphylla (Chong Lou) has been used as a traditional Chinese medicine to treat a number of cancers including pancreas and liver cancers for a long time. Results from our laboratory demonstrate that PD is a potent anti-cancer agent that bypasses multi-drug resistance (MDR) and induces programmed cell death in R-HepG2 cells over-expressing P-glycoprotein (P-gp). In this paper, we re-viewed the mechanisms how PD overcomes the MDR and exhibits a stronger cytotoxicity in the R-HepG2 than its parent line without P-gp through mitochondrial injury.

We previously showed that polyphyllin D (PD) produced a stronger apoptotic effect in R-HepG2 with multi-drug resistance (MDR) than that in its parent HepG2 cells without MDR. In this study, PD was found to elicit mitochondrial fragmentation in live cells by using total internal reflection fluorescence microscopy (TIRFM). When mitochondria were isolated and treated directly with PD, a stronger swelling, deeper transmembrane depolarization, and more apoptosis-inducing factor (AIF) release were observed from the mitochondria of R-HepG2 than that of HepG2. These observations suggest that PD is a potent anti-cancer agent that bypasses MDR and elicits apoptosis via mitochondrial injury.

Nuclear tubules (NTs) were found in the nucleus of HeLa cells. Although no function has been ascribed to these structures, our previous data has shown that they are the sites of Ca2+release with mitochondria shuttled around. In the present study, we further characterized these NTs through different fluorescent dye-labeling and red fluorescent protein transfection experiments. We found that doxorubicin (Dox) is a good indicator to demonstrate the NTs since Dox is fluorescent and DNA is able to quench its fluorescence. By using confocal and electron microscopy, we show that the number and nature of the NTs in HeLa vary from cell to cell, ranging from tubular to intricately branched structures. Additionally, these NTs are double-membrane invaginations of the nuclear envelope and usually lie close to nucleolus. At rest, NTs appeared to be stable and their mouths are always closed. Upon Ca2+ionomycin stimulation, various forms of dynamism, including membrane protrusion to the nucleus, enlargement and shrinkage of the NTs, and distortion of the nuclear envelope and NTs were observed over a time scale of minutes. These observations suggest that the NT represents a specialized and dynamic compartment inside the nucleus under the control of Ca2+.

We present a study on the surface-enhanced Raman scattering (SERS) properties of Ag nanoparticle island substrates (NIS) and their applications for target oligonucleotide (OND) detection. It has been found that the surface nanostructure of NIS samples can be controlled with a good degree of reproducibility, and a high SERS enhancement can be achieved when the peak extinction wavelength of NIS is tuned to a spectral window (approximately 60 nm) between the excitation wavelength and the scattered Raman wavelength. The highest SERS enhancement was obtained from the NIS substrates with a nominal thickness of 50 A. Detection of target OND was performed with a sandwich format in which the target OND was hybridized both to a capture OND immobilized on the NIS substrate, and a detection OND conjugated with a Raman-active dye for SERS signal generation. We compare the detection performance of two strategies based on the use of the detection OND with or without the gold nanoparticle (Au-NP). Our results confirm that, when the detection OND is coupled to the Au-NP, a better sensitivity for the target OND detection, in terms of a wider dynamic range and a lower detection limit (0.4 fM versus 1 nM without Au-NP), would be achieved.

Localized laser beams had been observed to generate reactive oxygen species (ROS) in human hepatocellular carcinoma cells (HepG2). The oxidative stress and thermal effect generated depended on the nature of the laser beam used. It was found that the ROS-induced fluorescence signal caused by femtosecond (fs) laser pulses were much smaller than that due to continuous-wave laser radiation around 1550nm. This implies fs laser pulses are relatively harmless to cells.

Summary form only given. This work presents a systematic study of the surface-enhanced Raman-scattering (SERS) properties of nanoparticle island substrates (NIS) and their application for oligonucleotide target detection. Using NIS that are structurally well characterized by atomic force microscopy (AFM) and UV-VIS extinction spectrum, it demonstrates the correlation of nanostructure of substrates and extinction maximum (?max) of substrates fabricated on different conditions, i.e. thickness of metal Ag. To effectively implement SERS on NIS and find out optimal condition for DNA detection, the relationship between extinction maximum (?max) and SERS enhancement factor (EF) was explored in detail. In fact, this work demonstrates high S/N ratio SERS spectra can be achieved for NIS with ?max within a spectrum window (~60 nm) consisting by the excitation wavelength (514 nm) and the scattered Raman wavelength, and highest EF measured is about 4 × 108 with thickness of Ag being 50 A. Detection of oligonucleotide target has been performed with a sandwich assay scheme. In this scheme a layer of thiol modified receptor oligonucleotide was assembled on NIS and 3' of oligonucleotide probe has been labeled with a Raman-active dye. Furthermore, We compared the detection performance of strategies using probe oligonucleotide with or without gold nanoparticles (Au-NPs, 20 nm) capped on 5'. The experimental results reveal that the DNA detection implemented with NIS can provide high sensitivity, and both dynamic range and diction limit can be amplified with the aid of Au-NPs on 5' of probes. The current detection limits of NIS with and without Au-NPs are 0.4 femtomolar and 1 nanomolar respectively.

Evanescent field based imaging and sensor technologies have been widely used for a long time, but only recently has TIRF been introduced for bio-imaging. This paper will discuss the reasons and give a brief summary of the recent development of TIRF which is also based on evanescent wave excitation, and which is well suited for ultra thin optical sectioning (hundreds of nanometres) at cell-substrate regions with an unusually high signal-to-noise ratio and image quality.

Nuclear tubules (NTs) were found in the nucleus of HeLa cells. Although no function has been ascribed to these structures, our previous data has shown that they are the sites of Ca(2+) release with mitochondria shuttled around. In the present study, we further characterized these NTs through different fluorescent dye-labeling and red fluorescent protein transfection experiments. We found that doxorubicin (Dox) is a good indicator to demonstrate the NTs since Dox is fluorescent and DNA is able to quench its fluorescence. By using confocal and electron microscopy, we show that the number and nature of the NTs in HeLa vary from cell to cell, ranging from tubular to intricately branched structures. Additionally, these NTs are double-membrane invaginations of the nuclear envelope and usually lie close to nucleolus. At rest, NTs appeared to be stable and their mouths are always closed. Upon Ca(2+) ionomycin stimulation, various forms of dynamism, including membrane protrusion to the nucleus, enlargement and shrinkage of the NTs, and distortion of the nuclear envelope and NTs were observed over a time scale of minutes. These observations suggest that the NT represents a specialized and dynamic compartment inside the nucleus under the control of Ca(2+).

We examined the effect of femtosecond (fs) and continuous wave (CW) lasers at near-infrared range on the creation of reactive oxygen species in a human liver cancer cell line. By controlling the mitochondria electron transport chain (ETC), it was found that a major part of the oxidative stress was generated by the laser induced thermal effect on the mitochondria while the remaining part was created by direct free electron liberation by the fs pulses, which could be observed after breaking the ETC. The study helps clarify the major effects produced on animal cells when excited by fs lasers.

We report the transfection of human hepatocarcinoma (HepG2) cells by femtosecond (fs) laser pulses at 1554 nm. It was found that HepG2 cells could be perforated transiently to admit propidium iodide by that laser. This photoporation was safe, as the membrane resealed itself within a short time and no mitochondrial depolarization was detected. The cells were next photoporated in the presence of DNA plasmids for the expression of green fluorescence protein, and about 80% of the exposed cells showed green fluorescence 24 h later. Thus it could be concluded that it is safe and efficient to use fs laser at 1554 nm to transfect foreign molecules into cells under a standard microscope.

Cell-cell fusion by physical methods has progressed slowly until the advent of femtosecond lasers in the near infrared range, which have an ultrahigh photon density and few side effects. Here we demonstrated using a fiber femtosecond laser at 1550 nm with an average power of 1.6×104 W per pulse to fuse human HepG2 and HeLa cells homotypically with an efficiency of 35%–37% in the absence of chemical fusogen. Heterohybrid HepG2-HeLa cells were also prepared. Our site-directed cell-cell fusion technique offers a precise tool for biotechnology and fundamental research in biomedical fields.