Conventional electroporation approaches show limitations in the delivery of macromolecules in vitro and in vivo. These limitations include low efficiency, noticeable cell damage and nonuniform delivery of cells. Here, we present a simple 3D electroporation platform that enables massively parallel single-cell manipulation and the intracellular delivery of macromolecules and small molecules. A pyramid pit micropore array chip was fabricated based on a silicon wet-etching method. A controllable vacuum system was adopted to trap a single cell on each micropore. Using this chip, safe single-cell electroporation was performed at low voltage. Cargoes of various sizes ranging from oligonucleotides (molecular beacons, 22 bp) to plasmid DNA (CRISPR-Cas9 expression vectors, >9 kb) were delivered into targeted cells with a significantly higher transfection efficiency than that of multiple benchmark methods (e.g., commercial electroporation devices and Lipofectamine). The delivered dose of the chemotherapeutic drug could be controlled by adjusting the applied voltage. By using CRISPR-Cas9 transfection with this system, the p62 gene and CXCR7 gene were knocked out in tumor cells, which effectively inhibited their cellular activity. Overall, this vacuumassisted micropore array platform provides a simple, efficient, high-throughput intracellular delivery method that may facilitate on-chip cell manipulation, intracellular investigation and cancer therapy.

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CD97 is a member of the epidermal growth factor-seven transmembrane family. It affects tumor aggressiveness by binding its cellular ligand CD55 and exhibits adhesive properties. Previous studies have shown that CD97 and CD55 are involved in the dedifferentiation, migration, invasiveness and metastasis of tumors. However, little is known regarding the roles of CD97 and CD55 in pancreatic cancer. In this study, immunohistochemistry was used to analyze CD97 and CD55 protein expression in samples obtained from 37 pancreatic cancer patients. CD97 and CD55 were absent or only weakly expressed in the normal pancreatic tissues but strongly expressed in pancreatic cancer tissues (P<0.05), particularly in tissues with lymph node involvement, metastasis or vascular invasion (P<0.05). Notably, CD97 and CD55 were expressed consistently in pancreatic cancer tissues (r2=0.5422; P<0.05). In addition, CD97 and CD55 expression levels were found to significantly correlate with tumor aggressiveness (P<0.01). Multivariate analyses revealed that CD97 and CD55 expression levels were closely associated with prognosis (P<0.05). Taken together, these results indicated that CD97 and its ligand CD55 are upregulated in pancreatic cancers and are closely associated with lymph node involvement, metastasis and vascular invasion. Thus, analysis of both CD97 and CD55 expression may present potential prognostic value for pancreatic cancer.

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Osteoclast inhibitory lectin (OCIL) is a novel regulator of bone remodeling, however, little is known concerning how OCIL is regulated to date. In this study, approximately 4.4 kb of the 5'-flanking sequence of rat OCIL gene was cloned into the promoter-less reporter vector pGL3-basic and transiently transfected into three different cell lines. The differences in the levels of luciferase activity paralleled well with the levels of OCIL mRNA expression in these cells, suggesting that the regulation of rat OCIL gene expression occurs mainly at the transcriptional level. Additional luciferase assays using a series of constructs containing unidirectionally deleted fragments showed that the construct-1819/pGL3 (-1819 to +118) exhibited the highest luciferase activity, suggesting the presence of functional promoter in this region. The region from -4370 to -2805 might contain negative regulatory elements, while the region from -1819 to -1336 might have important positive regulatory elements that enhance OCIL transcription. Sequence analysis of the promoter revealed the absence of both TATA and CAAT boxes. However, in the proximal promoter region (-81 to +118), several potential transcription factor binding sites that may be responsible for the basal transcriptional activity of rat OCIL promoter were observed. The promoter contains several potential Sp1 binding sites, and cotransfection of a shRNA expression plasmid that knockdowns Sp1 significantly reduced OCIL promoter activity and endogenous gene expression and moreover, overexpressing Sp7, a Sp1 family member that also binds to Sp1 binding sequence, increased OCIL promoter activity and gene expression, suggesting a role of Sp1 family proteins in regulation of OCIL transcription.

Sweet taste, one of the five basic taste qualities, is not only important for evaluation of food quality, but also guides the dietary food choices of animals. Sweet taste involves a variety of chemical compounds and structures, including natural sugars, sugar alcohols, natural and artificial sweeteners, and sweet-tasting proteins. The preference for sweetness has induced the over-consumption of sugar, contributing to certain prevailing health problems, such as obesity, diabetes and cardiovascular disease. Non-nutritive sweeteners, including natural and synthetic sweeteners, and sweet-tasting proteins have been added to foods to reduce the caloric intake from sugar, but many of these sugar substitutes induce an off-taste or after taste that negatively impacts any pleasure derived from the sweet taste. Sweet taste is detected by sweet taste receptor, that also play an important role in the metabolic regulation of the body, such as glucose homeostasis and incretin hormone secretion. In this review, the role of sweet tastants and the sweet taste receptors involved in sweetness perception, and their effect on obesity and diabetes are summarized. Sweet taste enhancement, as a new way to solve the over-consumption of sugar, is discussed in this contribution. Sweet taste enhancers can bind with sweet tastans to potentiate the sweetness of food without producing any taste by itself. Various type of sweet taste enhancers, including synthetic compounds, food-processed substances and aroma compounds, are summarized. Notably, few natural, non-volatile compounds have been identified as sweetness enhancers.

cDNAs of certain target genes are difficult to obtain by traditional reverse transcription. Herein we describe a novel method to synthesize cDNA based upon the use of the class IIS restriction enzymes. Briefly, the exons of a certain gene are separately PCR-amplified, each using the primers containing a recognition sequence of a certain class IIS restriction enzyme. All the fragments are restricted using the enzyme(s), resulting in the cohesive end of each exon that is complementary to the one in its adjacent exon. Then the fragments can be assembled together in their naturally occurring order. We successfully applied this method to acquire the coding sequence of Hoxa7 gene. This approach is simple, highly efficient, less error prone and cost-effective, and can also be used to fuse different PCR-fragments from distinct genes to create a chimeric gene or to perform site-directed mutagenesis.

We have developed a novel protocol for directional cloning of PCR products into any vector. The target sequence is amplified in two parallel asymmetric PCRs using specially but simply designed primers. Two single-stranded products are produced and they are annealed to form a double-stranded DNA fragment bearing overhangs at both ends that correspond to the restriction overhangs of certain restriction enzymes. The fragment can then be cloned into a certain vector previously treated with the corresponding enzymes without restriction of the inserted fragment. Compared with previously published protocols, the procedure described in this paper is highly efficient and it is independent of the restriction sites of the insert and is therefore applicable to molecular biology and biotechnology studies.