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.

The objective of this study was to isolate, identify, and characterize some lactic acid bacterial strains from human milk, infant feces, and fermented grapes and dates, as potential probiotics with antimicrobial activity against some human pathogenic strains. One hundred and forty bacterial strains were isolated and, after initial identification and a preliminary screening for acid and bile tolerance, nine of the best isolates were selected and further identified using 16 S rRNA gene sequences. The nine selected isolates were then characterized in vitro for their probiotic characteristics and their antimicrobial activities against some human pathogens. Results showed that all nine isolates belonged to the genus Lactobacillus. They were able to tolerate pH 3 for 3 h, 0.3% bile salts for 4 h, and 1.9 mg/mL pancreatic enzymes for 3 h. They exhibited good ability to attach to intestinal epithelial cells and were not resistant to the tested antibiotics. They also showed good antimicrobial activities against the tested pathogenic strains of humans, and most of them exhibited stronger antimicrobial activity than the reference strain L. casei Shirota. Thus, the nine Lactobacillus strains could be considered as potential antimicrobial probiotic strains against human pathogens and should be further studied for their human health benefits.

MicroRNAs (miRNAs) are small non-coding RNA molecules which function as critical post-transcriptional gene regulators of various biological functions. Generally, miRNAs negatively regulate gene expression by binding to their selective messenger RNAs (mRNAs), thereby leading to either mRNA degradation or translational repression, depending on the degree of complementarity with target mRNA sequences. Aberrant expression of these miRNAs has been linked etiologically with various human diseases including breast cancer. Different cellular pathways of breast cancer development such as cell proliferation, apoptotic response, metastasis, cancer recurrence and chemoresistance are regulated by either the oncogenic miRNA (oncomiR) or tumor suppressor miRNA (tsmiR). In this review, we highlight the current state of research into miRNA involved in breast cancer, with particular attention to articles published between the years 2000 to 2019, using detailed searches of the databases PubMed, Google Scholar, and Scopus. The post-transcriptional gene regulatory roles of various dysregulated miRNAs in breast cancer and their potential as therapeutic targets are also discussed.

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.

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Several techniques have been devised for the dissociation of tissues for primary culture. These techniques can affect the quantity and quality of the isolated cells. The aim of our study was to develop the most appropriate method for the isolation of human umbilical cord-derived mesenchymal (hUCM) cells. In the present study, we compared four methods for the isolation of hUCM cells: three enzymatic methods; collagenase/hyaluronidase/trypsin (CHT), collagenase/trypsin (CT) and trypsin (Trp), and an explant culture (Exp) method. The trypan blue dye exclusion test, the water-soluble tetrazolium salt-1 (WST-1) assay, flow cytometry, alkaline phosphatase activity and histochemical staining were used to evaluate the results of the different methods. The hUCM cells were successfully isolated by all methods but the isolation method used profoundly altered the cell number and proliferation capacity of the isolated cells. The cells were successfully differentiated into adipogenic and osteogenic lineages and alkaline phosphatase activity was detected in the hUCM cell colonies of all groups. Flow cytometry analysis revealed that CD44, CD73, CD90 and CD105 were expressed in all groups, while CD34 and CD45 were not expressed. The expression of C-kit in the enzymatic groups was higher than in the explant group, while the expression of Oct-4 was higher in the CT group compared to the other groups. We concluded that the collagenase/trypsin method of cell isolation yields a higher cell density than the others. These cells expressed a higher rate of pluripotent cell markers such as C-kit and Oct-4, while the explant method of cell isolation resulted in a higher cell proliferation rate and activity compared to the other methods.

Liver cancer has become one of the major types of cancer with high mortality and liver cancer is not responsive to the current cytotoxic agents used in chemotherapy. The purpose of this study was to examine the in vitro cytotoxicity of goniothalamin on human hepatoblastoma HepG2 cells and normal liver Chang cells. The cytotoxicity of goniothalamin against HepG2 and liver Chang cell was tested using MTT cell viability assay, LDH leakage assay, cell cycle flow cytometry PI analysis, BrdU proliferation ELISA assay and trypan blue dye exclusion assay. Goniothalamin selectively inhibited HepG2 cells [IC50 = 4.6 (±0.23) µM in the MTT assay; IC50 = 5.20 (±0.01) µM for LDH assay at 72 hours], with less sensitivity in Chang cells [IC50 = 35.0 (±0.09) µM for MTT assay; IC50 = 32.5 (±0.04) µM for LDH assay at 72 hours]. In the trypan blue dye exclusion assay, the Viability Indexes were 52 ± 1.73% for HepG2 cells and 62 ± 4.36% for Chang cells at IC50 after 72 hours. Cytotoxicity of goniothalamin was related to inhibition of DNA synthesis, as revealed by the reduction of BrdU incorporation. At 72 hours, the lowest concentration of goniothalamin (2.3 µL) retained 97.6% of normal liver Chang cells proliferation while it reduced HepG2 cell proliferation to 19.8% as compared to control. Besides, goniothalamin caused accumulation of hypodiploid apoptosis and different degree of G2/M arrested as shown in cell cycle analysis by flow cytometry. Goniothalamin selectively killed liver cancer cell through suppression of proliferation and induction of apoptosis. These results suggest that goniothalamin shows potential cytotoxicity against hepatoblastoma HepG2 cells.

A series of choline carboxylate ionic liquids (ILs) have been synthesized by neutralization of choline hydroxide solution with acetic, propanoic, butanoic, pivalic, and hexanoic acids. The salts so obtained were characterized by NMR spectroscopy, thermal methods, and elemental analysis. Key physical properties (density, viscosity, and refractive index) were measured for the propanoate, butanoate, and hexanoate salts at temperatures from (293.15 to 353.15) K. The densities were used to estimate the molecular volumes, standard entropies, crystal lattice energies, and thermal expansion coefficients. All five choline carboxylates were found to have cytotoxicities (IC 50 values) above 10 mM toward the human breast cancer cell line, MCF-7, indicating they are much less toxic than common imidazolium-based ILs.