A comparative study and evaluation of anti-EGFR nanobodies expressed in Pichia pastoris and Escherichia coli as antitumor moieties.

Anti-EGFR nanobodies have been successfully applied as antitumor moieties in the photodynamic therapy and drug delivery systems. But the yields of nanobodies were still limited due to the volumetric capacity of the periplasmic compartments and inclusion bodies of Escherichia coli. A comparative study of Pichia pastoris and Escherichia coli was done through characterizing their products. Nanobody 7D12 and 7D12-9G8 were successfully expressed in Pichia pastoris with 6-13.6-fold higher yield. Both two types of nanobodies had internalization ability to be developed as antitumor moieties.

Ultrasensitive detection of trypsin in serum via nanochannel device.

A highly sensitive trypsin sensing system in serum was developed by using an anodic alumina oxide (AAO)-based, trypsin substrate-decorated hybrid ion permeation membrane. Owing to the trypsin-triggered peptide hydrolyzation reaction, the surface electrical feature of the peptide-decorated hybrid ion membrane changed. The electric double layer effect reduces the effective ion current diameter in the AAO nano unit, so that the ion current rectification ratio will be enhanced, realizing the quantitative detection of trypsin. The lowest detection concentration can be achieved as low as 0.1 pM. This method is no need for sample pre-preparation, easy to operate, highly sensitive, and also applicable to other enzyme evaluation systems by changing corresponding substrates. This study provides a new idea for selective measurements of proteases in complex biological samples.

Circular RNA-circLRP6 protects cardiomyocyte from hypoxia-induced apoptosis by facilitating hnRNPM-mediated expression of FGF-9.

Coronary atherosclerosis-induced myocardial ischemia leads to cardiomyocyte apoptosis. The regulatory mechanisms for cardiomyocyte apoptosis have not been fully understood. Circular RNAs are non-coding RNAs which play important roles in heart function maintenance and progression of heart diseases by regulating gene transcription and protein translation. Here, we reported a conserved cardiac circular RNA, which is generated from the second exon of LRP6 and named circLRP62-2 . CircLRP62-2 can protect cardiomyocyte from hypoxia-induced apoptosis. The expression of circLRP62-2 in cardiomyocytes was down-regulated under hypoxia, while forced expression of circLRP62-2 inhibited cell apoptosis. Normally, circLRP62-2 was mainly localized in the nucleus. Under hypoxia, circLRP62-2 is associated with heterogeneous nuclear ribonucleoprotein M (hnRNPM) to be translocated into the cytoplasm. It recruited hnRNPM to fibroblast growth factor 9 (FGF9) mRNA to enhance the expression of FGF9 protein, promoting hypoxia-adaption and viability of cardiomyocytes. In summary, this study uncovers a new inhibitor of apoptosis and reveals a novel anti-apoptotic pathway composed of circLRP62-2 , hnRNPM, and FGF9, which may provide therapeutic targets for coronary heart disease and ischemic myocardial injury.

Mitochondrial Non-Coding RNAs Are Potential Mediators of Mitochondrial Homeostasis.

Mitochondria are the energy production center in cells, which regulate aerobic metabolism, calcium balance, gene expression and cell death. Their homeostasis is crucial for cell viability. Although mitochondria own a nucleus-independent and self-replicating genome, most of the proteins, which fulfill mitochondrial functions and mitochondrial quality control, are encoded by the nuclear genome and are imported into mitochondria. Hence, the regulation of mitochondrial protein expression and translocation is considered essential for mitochondrial homeostasis. By means of high-throughput RNA sequencing and bioinformatic analysis, non-coding RNAs localized in mitochondria have been generally identified. They are either generated from the mitochondrial genome or the nuclear genome. The mitochondrial non-coding RNAs can directly interact with mitochondrial DNAs or transcripts to affect gene expression. They can also bind nuclear genome-encoded mitochondrial proteins to regulate their mitochondrial import, protein level and combination. Generally, mitochondrial non-coding RNAs act as regulators for mitochondrial processes including oxidative phosphorylation and metabolism. In this review, we would like to introduce the latest research progressions regarding mitochondrial non-coding RNAs and summarize their identification, biogenesis, translocation, molecular mechanism and function.

Direct MYD88L265P gene detection for diffuse large B-cell lymphoma (DLBCL) via a miniaturised CRISPR/dCas9-based sensing chip.

Traditional methods for single-nucleotide variants based on amplification and fluorescence signals require expensive reagents and cumbersome instruments, and they are time-consuming for each trial. Here, a porous anodised aluminium (PAA)-based sensing chip modified with deactivated Cas9 (dCas9) proteins and synthetic guide RNA (sgRNA) as the biorecognition receptor is developed, which can be used for the label-free sensing of the diffuse large B-cell lymphoma (DLBCL) MYD88L265P gene by integrating with electrochemical ionic current rectification (ICR) measurement. The sgRNA that can specifically identify and capture the MYD88L265P gene was screened, which has been proved to be workable to activate dCas9 for the target MYD88L265P. In the sensing process, the dCas9 proteins can capture the genome sequence, thus bringing negative charges over the PAA chip and correspondingly resulting in a variation in the ICR value due to the uneven transport of potassium anions through the ion channels of the PAA chip. The whole sensing can be finished within 40 min, and there is no need for gene amplification. The CRISPR/dCas9-based sensor demonstrates ultrasensitive detection performance in the concentration range of 50 to 200 ng µL-1 and it has been proved to be feasible for the genome sequence of patient tissues. This sensor shows the potential of targeting other mutations by designing the corresponding sgRNAs and expands the applications of CRISPR/dCas9 technology to the on-chip electrical detection of nucleic acids, which will be very valuable for rapid diagnosis of clinically mutated genes. This makes the hybrid CRISPR-PAA chip an ideal candidate for next-generation nucleic acid biosensors.

Identification of a novel anti-EGFR nanobody by phage display and its distinct paratope and epitope via homology modeling and molecular docking.

Since EGFR is an important and effective target for tumor therapy in the clinic. Several monoclonal antibodies and nanobodies were proved to target domain III of EGFR. Regarding the increased attention on nanobodies, the present study aimed to generate nanobodies specifically against domain III. After camel immunization, a gene repertoire of sdAb fragments with a diversity of 3×109 clones was produced. Following the construction of two sdAb phage display libraries, the successful epitope binning was carried out to identify the nanobody with the designated epitope. Modelling of the identified nanobody and molecular docking studies illustrated the paratope and epitope. Docking analysis revealed that the paratope focused on CDR2 loop of the identified nanobody. The identified nanobody potently cover part of the epitope of Matuzumab and Nb 9G8, which indicated that it blocked EGFR by preventing dimerization of the receptors.