Transient, Implantable, Ultrathin Biofuel Cells Enabled by Laser-Induced Graphene and Gold Nanoparticles Composite.

Transient power sources with excellent biocompatibility and bioresorablility have attracted significant attention. Here, we report high-performance, transient glucose enzymatic biofuel cells (TEBFCs) based on the laser-induced graphene (LIG)/gold nanoparticles (Au NPs) composite electrodes. Such LIG electrodes can be easily fabricated from polyimide (PI) with an infrared CO2 laser and exhibit a low impedance (16 Ω). The resulted TEBFC yields a high open circuit potential (OCP) of 0.77 V and a maximum power density of 483.1 µW/cm2. The TEBFC not only exhibits a quick response time that enables reaching the maximum OCP within 1 min but also owns a long lifetime over 28 days in vitro. The excellent biocompatibility and transient performance from in vitro and in vivo tests allow long-term implantation of TEBFCs in rats for energy harvesting. The TEBFCs with advanced processing methods provide a promising power solution for transient electronics.

Skin-Integrated Wireless Odor Message Delivery Electronics for the Deaf-blind.

Deaf-blindness limits daily human activities, especially interactive modes of audio and visual perception. Although the developed standards have been verified as alternative communication methods, they are uncommon to the nondisabled due to the complicated learning process and inefficiency in terms of communicating distance and throughput. Therefore, the development of communication techniques employing innate sensory abilities including olfaction related to the cerebral limbic system processing emotions, memories, and recognition has been suggested for reducing the training level and increasing communication efficiency. Here, a skin-integrated and wireless olfactory interface system exploiting arrays of miniaturized odor generators (OGs) based on melting/solidifying odorous wax to release smell is introduced for establishing an advanced communication system between deaf-blind and non-deaf-blind. By optimizing the structure design of the OGs, each OG device is as small as 0.24 cm3 (length × width × height of 11 mm × 10 mm × 2.2 mm), enabling integration of up to 8 OGs on the epidermis between nose and lip for direct and rapid olfactory drive with a weight of only 24.56 g. By generating single or mixed odors, different linked messages could be delivered to a user within a short period in a wireless and programmable way. By adopting the olfactory interface message delivery system, the recognition rates for the messages have been improved 1.5 times that of the touch-based method, while the response times were immensely decreased 4 times. Thus, the presented wearable olfactory interface system exhibits great potential as an alternative message delivery method for the deaf-blind.

Thin, soft, wearable system for continuous wireless monitoring of artery blood pressure.

Continuous monitoring of arterial blood pressure (BP) outside of a clinical setting is crucial for preventing and diagnosing hypertension related diseases. However, current continuous BP monitoring instruments suffer from either bulky systems or poor user-device interfacial performance, hampering their applications in continuous BP monitoring. Here, we report a thin, soft, miniaturized system (TSMS) that combines a conformal piezoelectric sensor array, an active pressure adaptation unit, a signal processing module, and an advanced machine learning method, to allow real wearable, continuous wireless monitoring of ambulatory artery BP. By optimizing the materials selection, control/sampling strategy, and system integration, the TSMS exhibits improved interfacial performance while maintaining Grade A level measurement accuracy. Initial trials on 87 volunteers and clinical tracking of two hypertension individuals prove the capability of the TSMS as a reliable BP measurement product, and its feasibility and practical usability in precise BP control and personalized diagnosis schemes development.

High Channel Temperature Mapping Electronics in a Thin, Soft, Wireless Format for Non-Invasive Body Thermal Analysis.

Hemodynamic status has been perceived as an important diagnostic value as fundamental physiological health conditions, including decisive signs of fatal diseases like arteriosclerosis, can be diagnosed by monitoring it. Currently, the conventional hemodynamic monitoring methods highly rely on imaging techniques requiring inconveniently large numbers of operation procedures and equipment for mapping and with a high risk of radiation exposure. Herein, an ultra-thin, noninvasive, and flexible electronic skin (e-skin) hemodynamic monitoring system based on the thermal properties of blood vessels underneath the epidermis that can be portably attached to the skin for operation is introduced. Through a series of thermal sensors, the temperatures of each subsection of the arrayed sensors are observed in real-time, and the measurements are transmitted and displayed on the screen of an external device wirelessly through a Bluetooth module using a graphical user interface (GUI). The degrees of the thermal property of subsections are indicated with a spectrum of colors that specify the hemodynamic status of the target vessel. In addition, as the sensors are installed on a soft substrate, they can operate under twisting and bending without any malfunction. These characteristics of e-skin sensors exhibit great potential in wearable and portable diagnostics including point-of-care (POC) devices.

On-chip multiplexed single-cell patterning and controllable intracellular delivery.

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 vacuum-assisted micropore array platform provides a simple, efficient, high-throughput intracellular delivery method that may facilitate on-chip cell manipulation, intracellular investigation and cancer therapy.

Cloning and analysis of rat osteoclast inhibitory lectin gene promoter.

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.

A universal method for directional cloning of PCR products based on asymmetric PCR.

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.

An efficient approach for constructing shRNA expression vectors based on short oligonucleotide synthesis.

Traditional strategies for establishing shRNA expression constructs are inefficient, error-prone, or costly. We describe a simple approach that overcomes these drawbacks. Briefly, the sense and antisense strands of the short hairpin RNA coding sequence are segmented into two parts, respectively, at asymmetric sites. The four resulting short oligonucleotides are synthesized. Each oligonucleotide is annealed with its opposite, resulting in a double-stranded fragment with sticky termini at both ends. The two fragments so generated can be easily spliced by simple ligation to reconstitute the full-length short hairpin RNA coding sequence which can then be cloned into an appropriately restricted vector.

An alternative approach to synthesize cDNA bypassing traditional reverse transcription.

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.

SORS: a universal one-round PCR-based method for site-directed mutagenesis.

We have developed a novel protocol for site-directed mutagenesis of double-stranded DNA. The procedure, termed SORS (named because it undergoes the sequential procedure of segmentation-overhang creating PCR-reannealing-splicing) mutagenesis, is exemplified by a substitution, a deletion, and an insertion of nucleotide(s) in target genes. The template DNA is PCR-amplified into two separate segments divided at the prospective mutation site, and each segment is amplified in two parallel PCRs using primers introducing the mutation. The primers are designed to be able to create protruding bases upon pooling, denaturing, and reannealing the two parallel reactions. The protruding bases at the prospective junction of the two segments are mutually complementary; therefore, the two segments can be re-spliced together to generate the mutated gene. Compared to previously published protocols, this procedure is rapid, restriction-independent and ensures higher success rate and lower potential to produce second-site mutations.

Expression and prognostic value of CD97 and its ligand CD55 in pancreatic cancer.

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.

[Expression and analysis of the extracellular domain of human glucocorticoid-induced tumor necrosis factor receptor ligand in Escherichia coli].

GITRL (Glucocorticoid-induced tumor necrosis factor receptor ligand) has been recently identified as a novel inhibitor of osteoclastogenesis and hence called Osteostat. In this study, we expressed recombinant extracellular domain of GITRL protein in Escherichia coli and analyzed its bioactivity. Using an Eco31I enzyme-based restriction and ligation method, we obtained an E. coli-preferred DNA sequence coding for the extracellular domain of human GITRL. The DNA was cloned into expression vector pQE-30Xa that encodes a fusion tag of 6xHis before the insert. The resultant recombinant expression vector pQE/GITRL was subsequently transformed into E. coli strain M15[pREP4]. After induction with Isopropyl beta-D-Thiogalactoside (IPTG), the cells produced the fusion protein mainly in the form of inclusion bodies as identified by SDS-PAGE. The recombinant protein was purified by affinity chromatography through Ni-NTA column and recognized by anti-His polyclonal antibody using Western blotting analysis. Moreover, we established a simple, efficient and sensitive reporter gene-based method to detect the activity of the recombinant protein. The results showed that the target protein was biologically active.