HMOX1-overexpressing mesenchymal stem cell-derived exosomes facilitate diabetic wound healing by promoting angiogenesis and fibroblast function.

Many scholars have suggested that exosomes (Exos) can carry active molecules to induce angiogenesis and thus accelerate diabetic wound healing. Heme oxygenase-1 (HO-1) encoded by the gene HMOX1 promotes wound healing in DM by enhancing angiogenesis. Nevertheless, whether HMOX1 regulates wound healing in DM through mesenchymal stem cell-derived exosomes (MSC-Exos) remains to be further explored. The primary isolated- and cultured-cells expressed MSC-specific marker proteins, and had low immunogenicity and multi-differentiation potential, which means that MSCs were successfully isolated in this study. Notably, HO-1 protein expression was significantly higher in Exo-HMOX1 than in Exos, indicating that HMOX1 could be delivered to Exos as an MSCs-secreted protein. After verifying the -Exo structure, fibroblasts, keratinocytes, and human umbilical vein endothelial cells (HUVECs) were incubated with Exo-HMOX1 or Exo, and the findings displayed that Exo-HMOX1 introduction promoted the proliferation and migration of fibroblasts, keratinocytes and the angiogenic ability of HUVECs in vitro study. After establishing diabetic wound model mice, PBS, Exo, and Exo-HMOX1 were subcutaneously injected into multiple sites on the 1st, 3rd, 7th, and 14th day, DM injected with Exo-HMOX1 showed faster wound healing, re-epithelialization, collagen deposition, and angiogenesis than those in PBS and Exo groups in vitro study. In summary, Exo-HMOX1 could enhance the activity of fibroblasts, keratinocytes, and HUVEC, and accelerate wound healing by promoting angiogenesis in DM.

Self-Healing, High-Strength, and Antimicrobial Polysiloxane Based on Amino Acid Hydrogen Bond.

Recent years have witnessed the rapid development of self-healing and recyclable materials because they can extend the life of the material. For polysiloxane materials, exploring polysiloxanes with high-strength and self-healing properties remains a challenge. In this work, a high-strength and self-healing polysiloxane containing N-acetyl-L-cysteine (NACL) side groups is prepared. The NACL is used to form strong hydrogen bonds to build a self-healing network. Molecular simulations help explain the reasons and processes for the repair of modified polysiloxanes. On the one hand, the obtained modified polysiloxanes have good self-healing properties. The self-healing efficiency of modified polysiloxane can reach 96.9%. As the number of NACL increases, the tensile strength of the modified polysiloxane increases. For PMVS-30%NACL, the tensile strength can reach 4.36 MPa, and the strain can reach 586%. On the other hand, modified polysiloxane has an apparent inhibitory effect on Staphylococcus aureus. With the increase in the number of NACL, the antibacterial effect of modified polysiloxane is more obvious. Furthermore, NACL is a bio-based amino acid with excellent biocompatibility. This work expands the idea of designing and synthesizing high-strength polysiloxanes with antibacterial properties. It has great potential in the field of polysiloxane antimicrobial coatings.

cano-wgMLST_BacCompare: A Bacterial Genome Analysis Platform for Epidemiological Investigation and Comparative Genomic Analysis.

With the decreasing cost of next-generation sequencing, whole-genome sequence-based bacterial genome comparisons are expected to become a mainstream process in the public health domain. Extended multilocus sequence typing (MLST) methods are becoming increasingly popular for use in comparing bacterial genetic relatedness in epidemiological investigations. Several extended MLST schemes based on biological signatures have been reported. Among them, whole-genome MLST (wgMLST) has gradually become one of the most widely used approaches for bacterial strain typing. In addition to using bacterial typing, many researchers aim to identify differences in the genes of compared strains. Because these differences might provide insights into critical bacterial functions, such as virulence and pathogenicity, researchers usually study these genes that differ between strains. Hence, we designed a web service tool based on wgMLST-constructed tree topology coupled with the feature selection method to create the "canonical wgMLST (cano-wgMLST) tree." The genes that differ between strains are shown at each split of the tree, thereby directly providing information for performing a comparative genomic analysis for each strain pair. We demonstrated that this web service tool could be operated efficiently on two datasets consisting of 22 Campylobacter jejuni isolates and 59 S. Heidelberg isolates, respectively. We imposed this tool on a designated web server, cano-wgMLST_BacCompare, to enable users to create a wgMLST tree and canonical wgMLST tree automatically from their uploaded bacterial genomes for not only epidemiological investigation but also comparative genomic analysis. Additionally, detailed information on how to use this service is provided. The cano-wgMLST_BacCompare is available at http://baccompare.imst.nsysu.edu.tw.

The Cano-eMLST Program: An Approach for the Calculation of Canonical Extended Multi-Locus Sequence Typing, Making Comparison of Genetic Differences Among Bunches of Bacterial Strains.

Extended multi-locus sequence typing (eMLST) methods have become popular in the field of genomic epidemiology. Before eMLST methods can be applied in epidemiological investigations, the selection of a suitable scheme is critical. The core genome scheme (cgMLST) has become the most popular eMLST approach for strain typing in the epidemiological domain. In addition to strain typing, many public health researchers and clinical microbiologists wish to investigate which genes cause genetic differences between compared strains. Therefore, a tool that can be used to extract canonical genes with an eMLST scheme would be particularly useful. In this study, we present cano-eMLST, a well-designed program that applies a feature-selection methodology to create a canonical locus combination with discriminatory power by traversing a genetic relatedness tree based on a user-selected scheme. The cano-eMLST program is provided mainly to help infectious disease laboratory researchers identify potential factors related to bacterial pathogenesis. The core program (tree-traversing approach) of cano-eMLST is implemented in Perl and Python. All the necessary dependencies and environmental settings are provided in the encapsulated version (VirtualBox or VMware) and self-installation version (all use source code and libraries).

Engineering the ribosomal DNA in a megabase synthetic chromosome.

We designed and synthesized a 976,067-base pair linear chromosome, synXII, based on native chromosome XII in Saccharomyces cerevisiae SynXII was assembled using a two-step method, specified by successive megachunk integration and meiotic recombination-mediated assembly, producing a functional chromosome in S. cerevisiae. Minor growth defect "bugs" detected in synXII, caused by deletion of tRNA genes, were rescued by introducing an ectopic copy of a single tRNA gene. The ribosomal gene cluster (rDNA) on synXII was left intact during the assembly process and subsequently replaced by a modified rDNA unit used to regenerate rDNA at three distinct chromosomal locations. The signature sequences within rDNA, which can be used to determine species identity, were swapped to generate a Saccharomyces synXII strain that would be identified as Saccharomyces bayanus by standard DNA barcoding procedures.

Acute exacerbation of hepatitis C in hepatocellular carcinoma patients receiving chemotherapy.

Acute hepatitis C exacerbations can occur in cancer patients carrying hepatitis C virus (HCV) when receiving systemic chemotherapy. However, clinical studies evaluating these complications remain rare due to the lack of clinically proven effective and tolerable anti-HCV treatments at late cancer stages. Furthermore, no data were available regarding hepatitis C exacerbation in advanced hepatocellular carcinoma (HCC) patients receiving chemotherapy. To address this issue, 48 patients with HCV-related advanced HCC, who underwent systemic chemotherapy using 5- fluorouracil, cisplatin, and mitoxantrone from 2008 to 2014 were analyzed. Nine patients developed acute hepatitis exacerbations defined by HCV-RNA elevation ≥10-fold and alanine transaminase (ALT) elevation ≥5-fold of the upper normal limit. Six were genotype 1b and 3 were genotype 2. Three patterns of clinical courses were observed including single episode of exacerbation (n = 5), fluctuated flares (n = 3), and delayed exacerbation (n = 1). Hepatic failure developed in five patients. Patients with acute exacerbations were less likely to have pretreatment ascites (11.1% vs. 53.8%; P = 0.028) and displayed a lower baseline ALT (44.1 ± 28.5 U/L vs. 72.6 ± 19.2 U/L; P = 0.007). Paradoxically, despite a high risk of hepatic failure, occurrence of hepatitis C exacerbation was associated with a favorable overall survival (P = 0.027; 22.8 vs. 5.4 months). In conclusion, hepatitis C exacerbation can occur in HCC patients receiving chemotherapy, leading to liver failure. However, the flare was associated with a better overall survival, possibly due to its association with a better baseline liver function. J. Med. Virol. 89:153-160, 2017. © 2016 Wiley Periodicals, Inc.

EcoExpress-Highly Efficient Construction and Expression of Multicomponent Protein Complexes in Escherichia coli.

The bacterium Escherichia coli remains the leading host for protein expression in large quantity for the purpose of crystallization or other biochemical studies. However, expression of multicomponent protein complexes remains a challenge, and is often laborious and time-consuming. Here we developed a method named EcoExpress, which allows efficient construction of plasmids to express individual protein with user-defined epitope-tag, followed by one-pot assembly of a single vector to express the entire protein complex for copurification. A versatile set of vectors was designed to provide various choices to control the expression of a protein with different promoters, and to accept different number of components for coexpression. Using EcoExpress, we demonstrated that each subunit within a protein complex could be expressed individually or simultaneously, and the entire complex could be copurified. In addition, to overcome the decreased assembly efficiency with the increasing number of components, a novel oligonucleotides blocking method was designed and tested. EcoExpress provides the scientific community with a toolbox to rapidly investigate the function of protein complexes.

YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae.

It is a routine task in metabolic engineering to introduce multicomponent pathways into a heterologous host for production of metabolites. However, this process sometimes may take weeks to months due to the lack of standardized genetic tools. Here, we present a method for the design and construction of biological parts based on the native genes and regulatory elements in Saccharomyces cerevisiae. We have developed highly efficient protocols (termed YeastFab Assembly) to synthesize these genetic elements as standardized biological parts, which can be used to assemble transcriptional units in a single-tube reaction. In addition, standardized characterization assays are developed using reporter constructs to calibrate the function of promoters. Furthermore, the assembled transcription units can be either assayed individually or applied to construct multi-gene metabolic pathways, which targets a genomic locus or a receiving plasmid effectively, through a simple in vitro reaction. Finally, using ß-carotene biosynthesis pathway as an example, we demonstrate that our method allows us not only to construct and test a metabolic pathway in several days, but also to optimize the production through combinatorial assembly of a pathway using hundreds of regulatory biological parts.

Combining radiofrequency ablation and ethanol injection may achieve comparable long-term outcomes in larger hepatocellular carcinoma (3.1-4 cm) and in high-risk locations.

Radiofrequency ablation (RFA) is more effective for hepatocellular carcinoma (HCC) < 3 cm. Combining percutaneous ethanol injection and RFA for HCC can increase ablation; however, the long-term outcome remains unknown. The aim of this study was to compare long-term outcomes between patients with HCC of 2-3 cm versus 3.1-4 cm and in high-risk versus non-high-risk locations after combination therapy. The primary endpoint was overall survival and the secondary endpoint was local tumor progression (LTP). Fifty-four consecutive patients with 72 tumors were enrolled. Twenty-two (30.6%) tumors and 60 (83.3%) tumors were of 3.1-4 cm and in high-risk locations, respectively. Primary technique effectiveness was comparable between HCC of 2-3 cm versus 3.1-4 cm (98% vs. 95.5%, p = 0.521), and HCC in non-high risk and high-risk locations (100% vs. 96.7%, p = 1.000). The cumulative survival rates at 1 year, 3 years, and 5 years were 90.3%, 78.9%, and 60.3%, respectively, in patients with HCC of 2-3 cm; 95.0%, 84.4%, and 69.3% in HCC of 3.1-4.0 cm (p = 0.397); 90.0%, 71.1%, and 71.1% in patients with HCC in non-high-risk locations; and 92.7%, 81.6%, and 65.4% in high-risk locations (p = 0.979). The cumulative LTP rates at 1 year, 3 years, and 5 years were 10.2%, 32.6%, and 32.6%, respectively, in all HCCs; 12.6%, 33.9%, and 33.9% in HCC of 2-3 cm; 4.8%, 29.5%, and 29.5% in HCC of 3.1-4 cm (p = 0.616); 16.7%, 50.0%, and 50.0% in patients with HCC in non-high-risk locations; and 8.8%, 29.9%, and 29.9% in patients with HCC in high-risk locations (p = 0.283). The cumulative survival and LTP rates were not significantly different among the various subgroups. Combining RFA and percutaneous ethanol injection achieved comparable long-term outcomes in HCCs of 2-3 cm versus 3.1-4.0 cm and in high-risk versus non-high-risk locations. A randomized controlled or cohort studies with larger sample size are warranted.

Evaluation of the radial deformability of poly(dG)-poly(dC) DNA and G4-DNA using vibrating scanning polarization force microscopy.

Poly(dG)-poly(dC) DNA and G4-DNA are two types of synthetic molecules that are regarded as promising candidates for molecular nanodevices. In this work, vibrating scanning polarization force microscopy (VSPFM) was employed to study the radial deformability of these two molecules coadsorbed on a Ni(2+)-modified mica surface. The values of the radial compressive elastic modulus of these two types of molecules were found to be similar (approximately 5-10 MPa) when the external loading force was between approximately 0.04 and approximately 0.12 nN. However, G4-DNA molecules possessed higher stiffness than poly(dG)-poly(dC) DNA (approximately 20-40 vs approximately 10-20 MPa) when the loading force was larger than approximately 0.12 nN. The results will aid us in understanding these molecule's mechanical performances.

[Isothermal unidirectional elongation method of gene synthesis].

Gene synthesis is gaining popularity as a versatile means of cloning genes. The prevailing synthesis methods are PCR-based and ligase-based gene synthesis. Both of them are not independent of the sequence content of the target gene. In the present study, a new gene synthesis method-isothermal unidirectional elongation method (IUEM) is proposed, which involves the cooperation of three enzymes, and is independent or partially independent of the target gene sequence by the specially designed oligonucleotides that form hairpins. The synthesis process is isothermal and serial in IUEM. Several factors that affect the elongation were studied, and two DNA segments of length 254 bp and 300 bp were synthesized with IUEM. The synthesized segments could work as building blocks for the synthesis of longer DNA sequences.