Small-Caliber Tissue-Engineered Vascular Grafts Based on Human-Induced Pluripotent Stem Cells: Progress and Challenges.

Small-caliber tissue-engineered vascular grafts (TEVGs, luminal diameter <6 mm) are promising therapies for coronary or peripheral artery bypassing surgeries or emergency treatments of vascular trauma, and a robust seed cell source is required for scalable manufacturing of small-caliber TEVGs with robust mechanical strength and bioactive endothelium in future. Human-induced pluripotent stem cells (hiPSCs) could serve as a robust cell source to derive functional vascular seed cells and potentially lead to generation of immunocompatible engineered vascular tissues. Up to date, this rising field of small-caliber hiPSC-derived TEVG (hiPSC-TEVG) research has received increasing attention and achieved significant progress. Implantable, small-caliber, hiPSC-TEVGs have been generated. These hiPSC-TEVGs displayed rupture pressure and suture retention strength approaching to those of human native saphenous veins, with vessel wall decellularized and luminal surface endothelialized with monolayer of hiPSC-endothelial cells. Meanwhile, a series of challenges remain in this field, including functional maturity of hiPSC-derived vascular cells, poor elastogenesis, suboptimal efficiency of obtaining hiPSC-derived seed cells, and relative low ready availability of hiPSC-TEVGs, which are waiting to be addressed. This review is conceived to introduce representative achievements and challenges in small-caliber TEVG generation using hiPSCs, and encapsulate the potential solution and future directions.

Mutations in the regulatory regions result in increased streptomycin resistance and keratinase synthesis in Bacillus thuringiensis.

Keratinases are a group of proteases of great industrial significance. To take full advantage of Bacillus species as an inherent superior microbial producer of proteases, we performed the ribosome engineering to improve the keratinase synthesis capacity of the wild-type Bacillus thuringiensis by inducing streptomycin resistance. Mutant Bt(Str-O) was identified as a stable keratinase overproducer. Comparative characterization of the two strains revealed that, although the resistance to Streptomycin increased by eight-fold in MIC, the mutant's resistance to other commonly used antibiotics was not affected. Furthermore, the mutant exhibited an enhanced keratinase synthesis (1.5-fold) when cultured in a liquid LB medium. In the whole feather degradation experiment, the mutant could secret twofold keratinase into the medium, reaching 640 U/mL per 107 CFU. By contrast, no significant differences were found in the scanning electron microscopic analysis and spore formation experiment. To understand the genetic factors causing these phenotypic changes, we cloned and analyzed the rpsL gene. No mutation was observed. We subsequently determined the genome sequences of the two strains. Comparing the rpsL gene revealed that the emergence of streptomycin resistance was not necessarily dependent on the mutation(s) in the generally recognized "hotspot." Genome-wide analysis showed that the phenotypic changes of the mutant were the collective consequence of the genetic variations occurring in the regulatory regions and the non-coding RNA genes. This study demonstrated the importance of genetic changes in regulatory regions and the effectiveness of irrational ribosome engineering in creating prokaryotic microbial mutants without sufficient genetic information.

A Thermostable Aluminum-Tolerant Protease Produced by Feather-Degrading Bacillus thuringiensis Isolated from Tea Plantation.

BACKGROUND: Proteases with keratinolytic activity are widely used in biotechnologies. The feather-degrading Bacillus thuringensis isolated from soil sample of a tea plantation produced high level of extracellular keratinase. OBJECTIVE: This study aimed to analyze the properties by biochemical and enzymological methods to gain information for better utilization of the enzyme. METHODS: The enzyme was purified with ion exchange and size exclusion chromatography. The substrate preference, optimal pH and temperature, and the effects of organic solvents and ions were checked. Circular dichroism was performed to compare the secondary structures of the native and apo-enzyme. RESULTS: The enzyme worked best at 50°C, and it was an acidic serine protease with an optimal pH of 6.2. Ions Ca2+ and Mg2+ were essential for its activity. Organic solvents and other metal ions generally deactivated the enzyme in a concentration-dependent manner. However, Mn2+ and DMSO, which were frequently reported as inhibitors of protease, could activate the enzyme at low concentration (0.01 to 2 mmol/L of Mn2+; DMSO <2%, v/v). The enzyme exhibited high resistance to Al3+, which might be explained by the soil properties of its host's residence. Circular dichroism confirmed the contribution of ions to the structure and activity. CONCLUSION: The enzyme was a thermostable aluminum-tolerant serine protease with unique biochemical properties.

Ribosome Hibernation as a Stress Response of Bacteria.

Ribosome is primarily regarded as the committing organelle for the translation process. Besides the expansion of its function from a translational machine for protein synthesis to a regulatory platform for protein quality control, the activity regulation and recycling of ribosome have been deepened significantly. Recent advances have confirmed a novel mechanism in the regulation of ribosome activity when a cell encounters adverse conditions. Due to the binding of certain protein factors onto a ribosome, the structural and functional change of the ribosome inside the cell will take place, thereby leading to the formation of inactive ribosomes (70S monomer or 100S dimer), or ribosome hibernation. By ribosome hibernation, the overall protein synthesis rate of a cell could be slowed down. The resistance to adverse conditions or chemicals of the host cell will be enhanced. In this paper, we discussed the phenomenon, molecular mechanism, and physiological effect of ribosome hibernation when cells are under stresses. And then, we discussed the resuscitation of a hibernating ribosome and the role of ribosome hibernation in the treatment of antimicrobial infection.

Small design from big alignment: engineering proteins with multiple sequence alignment as the starting point.

Multiple sequence alignment (MSA) is a fundamental way to gain information that cannot be obtained from the analysis of any individual sequence included in the alignment. It provides ways to investigate the relationship between sequence and function from a perspective of evolution. Thus, the MSA of proteins can be employed as a reference for protein engineering. In this paper, we reviewed the recent advances to highlight how protein engineering was benefited from the MSA of proteins. These methods include (1) engineering the thermostability or solubility of proteins by making it closer to the consensus sequence of the alignment through introducing site mutations; (2) structure-based engineering proteins with comparative modeling; (3) creating paleoenzymes featured with high thermostability and promiscuity by constructing the ancestral sequences derived from multiple sequence alignment; and (4) incorporating site-mutations targeting the evolutionarily coupled sites identified from multiple sequence alignment.

KCNQ1 gain-of-function mutation in familial atrial fibrillation.

Atrial fibrillation (AF) is a common cardiac arrhythmia whose molecular etiology is poorly understood. We studied a family with hereditary persistent AF and identified the causative mutation (S140G) in the KCNQ1 (KvLQT1) gene on chromosome 11p15.5. The KCNQ1 gene encodes the pore-forming alpha subunit of the cardiac I(Ks) channel (KCNQ1/KCNE1), the KCNQ1/KCNE2 and the KCNQ1/KCNE3 potassium channels. Functional analysis of the S140G mutant revealed a gain-of-function effect on the KCNQ1/KCNE1 and the KCNQ1/KCNE2 currents, which contrasts with the dominant negative or loss-of-function effects of the KCNQ1 mutations previously identified in patients with long QT syndrome. Thus, the S140G mutation is likely to initiate and maintain AF by reducing action potential duration and effective refractory period in atrial myocytes.