High-throughput FastCloning technology: A low-cost method for parallel cloning.

FastCloning, a reliable cloning technique for plasmid construction, is a widely used protocol in biomedical research laboratories. Only two-step molecular manipulations are required to add a gene (cDNA) of interest into the desired vector. However, parallel cloning of the gene into multiple vectors is still a labor-intensive operation, which requires a range of primers for different vectors in high-throughput cloning projects. The situation could even be worse if multiple fragments of DNA are required to be added into one plasmid. Here, we describe a high-throughput FastCloning (HTFC) method, a protocol for parallel cloning by adding an adaptor sequence into all vectors. The target gene and vectors were PCR amplified separately to obtain the insert product and linear vectors with 18-base overlapping at each end of the DNAs required for FastCloning. Furthermore, a method for generating polycistronic bacterial constructs based on the same strategy as that used for HTFC was developed. Thus, the HTFC technique is a simple, effective, reliable, and low-cost tool for parallel cloning.

T-cell immunoglobulin mucin-3 expression in invasive ductal breast carcinoma: Clinicopathological correlations and association with tumor infiltration by cytotoxic lymphocytes.

As a negative regulatory molecule, T-cell immunoglobulin and mucin domain-3 (Tim-3) is closely associated with tumor immunological tolerance. The aim of this study was to investigate Tim-3 expression in invasive ductal breast cancer (IDC), its effect on clinicopathological parameters and its association with cytotoxic lymphocyte infiltration. Tim-3 protein expression was measured in 150 paraffin-embedded IDC specimens and 100 paired normal breast tissue specimens by immunohistochemistry. It was demonstrated that the infiltration of the tumor by CD8+ T cells was significantly higher compared with that of normal tissue, and the Tim-3 expression on CD8+ T cells was higher in IDC tissue compared with that in normal tissue; the differences were statistically significant (both P-values=0.000). The median expression level of Tim-3 on tumor cells was significantly associated with clinicopathological parameters such as age, axillary lymph node metastasis and TNM stage (P=0.015, 0.001 and 0.027, respectively). The expression of Tim-3 on CD8+ T cells was correlated with lymph node metastasis, World Health Organization (WHO) grade and molecular classification (P=0.000, 0.004 and 0.000, respectively). Additionally, the number of tumor-infiltrating CD8+ T cells was associated with primary tumor size, lymph node metastasis, WHO grade, Ki-67 and molecular classification (P=0.017, 0.002, 0.007, 0.003 and 0.000, respectively). Thus, Tim-3 may promote the development and progression of breast cancer and affect the tumor microenvironment; thus, it may be used as an independent prognostic factor for IDC patients.

Hybrid use of combined and sequential delivery of growth factors and ultrasound stimulation in porous multilayer composite scaffolds to promote both vascularization and bone formation in bone tissue engineering.

In this study, a multilayer coating technology would be adopted to prepare a porous composite scaffold and the growth factor release and ultrasound techniques were introduced into bone tissue engineering to finally solve the problems of vascularization and bone formation in the scaffold whilst the designed multilayer composite with gradient degradation characteristics in the space was used to match the new bone growth process better. The results of animal experiments showed that the use of low intensity pulsed ultrasound (LIPUS) combined with growth factors demonstrated excellent capabilities and advantages in both vascularization and new bone formation in bone tissue engineering. The degradation of the used scaffold materials could match new bone formation very well. The results also showed that only RGD-promoted cell adhesion was insufficient to satisfy the needs of new bone formation while growth factors and LIPUS stimulation were the key factors in new bone formation.

Synthesis and characterisation of multifunctional alginate microspheres via the in situ formation of ZnO quantum dots and the graft of 4-(1-pyrenyl) butyric acid to sodium alginate.

Growth factor-loaded fluorescent alginate microspheres, which can realise sustained growth factor release and fluorescence imaging, were synthesised by in situ formation of ZnO quantum dots (QDs) and covalent graft of 4-(1-pyrenyl) butyric acid (PBA). BSA was chosen as a growth factor model protein to study the release kinetic of growth factors from alginate microspheres. The microsphere size and fluorescent properties were also investigated. Investigations of cell culture were used for evaluating biocompatibility of BSA-loaded fluorescent microspheres and fluorescence imaging property of ZnO QDs and PBA-grafted sodium alginate from the microspheres. The results show that they have good fluorescent property either to microspheres or to cells and fluorescent microspheres have good biocompatibility and property in sustained release of growth factors. The obtained microspheres will be expected to realise the imaging of cells and materials and also the release of growth factor in tissue engineering or in cell culture.