Transcriptomic Profiling Identifies Ferroptosis-Related Gene Signatures in Ischemic Muscle Satellite Cells Affected by Peripheral Artery Disease-Brief Report.

BACKGROUND: We hypothesized that transcriptomic profiling of muscle satellite cells in peripheral artery disease (PAD) would identify damage-related pathways contributing to skeletal muscle myopathy. We identified a potential role for ferroptosis-a form of programmed lytic cell death by iron-mediated lipid peroxidation-as one such pathway. Ferroptosis promotes myopathy in ischemic cardiac muscle but has an unknown role in PAD. METHODS: Muscle satellite cells from donors with PAD were obtained during surgery. cDNA libraries were processed for single-cell RNA sequencing using the 10X Genomics platform. Protein expression was confirmed based on pathways inferred by transcriptomic analysis. RESULTS: Unsupervised cluster analysis of over 25 000 cells aggregated from 8 donor samples yielded distinct cell populations grouped by a shared unique transcriptional fingerprint. Quiescent cells were diminished in ischemic muscle while myofibroblasts and apoptotic cells were prominent. Differential gene expression demonstrated a surprising increase in genes associated with iron transport and oxidative stress and a decrease in GPX4 (glutathione peroxidase 4) in ischemic PAD-derived cells. Release of the danger signal HMGB1 (high mobility group box-1) correlated with ferroptotic markers including surface transferrin receptor and were higher in ischemia. Furthermore, lipid peroxidation in muscle satellite cells was modulated by ferrostatin, a ferroptosis inhibitor. Histology confirmed iron deposition and lipofuscin, an inducer of ferroptosis in PAD-affected muscle. CONCLUSIONS: This report presents a novel finding that genes known to be involved in ferroptosis are differentially expressed in human skeletal muscle affected by PAD. Targeting ferroptosis may be a novel therapeutic strategy to reduce PAD myopathy.

Microbial communities and their correlation with flavor compound formation during the mechanized production of light-flavor Baijiu.

Light-flavor Baijiu fermentation is a typical spontaneous solid-state fermentation process fueled by a variety of microorganisms. Mechanized processes have been increasingly employed in Baijiu production to replace traditional manual operation processes, however, the microbiological and physicochemical dynamics in mechanized processes remain largely unknown. Here, we investigated the microbial community succession and flavor compound formation during a whole mechanized fermentation process of light-flavor Baijiu using the conventional dilution plating method, PacBio single-molecule real-time (SMRT) sequencing and headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry. The results showed that largely different fungal and bacterial communities were involved in the soaking and fermentation processes. A clear succession from Pantoea agglomerans to Bacillus (B.) smithii and B. coagulans in dominant bacterial species and from Cladosporium exasperatum to Saccharomyces cerevisiae and Lichtheimia ramosa in dominant fungal species occurred in the soaking processes. In the fermentation process, the most dominant bacterial species was shifted from Pantoea agglomerans to Lactobacillus (La.) acetotolerans and the most dominant fungal species were shifted from Lichtheimia ramose and Rhizopus arrhizus to Saccharomyces cerevisiae. The bacterial and fungal species positively associated with acidity and the formation of ethanol and different flavor compounds were specified. The microbial species exhibited strong co-occurrence or co-exclusion relationships were also identified. The results are helpful for the improvement of mechanized fermentation process of light-flavor Baijiu production.

Construction of Human Immune and Naive scFv Phage Display Libraries.

Antibody phage display is a widely used in vitro selection technology for the generation of human recombinant antibodies and has yielded thousands of useful antibodies for research, diagnostics, and therapy. In order to successfully generate antibodies using phage display, the basis is the construction of high-quality antibody gene libraries. Here, we describe detailed methods for the construction of such high-quality immune and naive scFv gene libraries of human origin. These protocols were used to develop human naive (e.g., HAL9/10) and immune libraries, which resulted in thousands of specific antibodies for all kinds of applications.

Computer-Aided Directed Evolution Generates Novel AAV Variants with High Transduction Efficiency.

Adeno-associated viruses (AAVs) have become safe and effective tools for therapeutic in vivo gene drug delivery. Among many AAV serotypes, AAV2 is the most well-characterized. Although many studies have been carried out on the engineering of the capsid VR-VIII region, few attempts have been made in the VR-IV region. Here, we targeted amino acid positions 442-469 of the VR-IV region and established an engineering paradigm of computer-aided directed evolution, based on training samples from previous datasets, to obtain a viral vector library with high diversity (~95,089). We further examined two variants selected from the library. The transduction efficiency of these two novel AAV variants, AAV2.A1 and AAV2.A2, in the central nervous system was 10-15 times higher than that of AAV2. This finding provides new vehicles for delivering gene drugs to the brain.

Novel Antibiotic Resistance Genes Identified by Functional Gene Library Screening in Stenotrophomonas maltophilia and Chryseobacterium spp. Bacteria of Soil Origin.

As one of the most diverse habitats of microorganisms, soil has been recognised as a reservoir of both antibiotics and the antibiotic resistance genes (ARGs). Bacteria naturally inhabiting soil or water often possess innate ARGs to counteract the chemical compounds produced by competitors living in the same environment. When such bacteria are able to cause infections in immunocompromised patients, their strong innate antibiotic resistance mechanisms make treatment difficult. We generated functional gene libraries using antibiotic-resistant Stenotrophomonas maltophilia and Chryseobacterium spp. bacteria isolated from agricultural soils in Lithuania to select for the genetic determinants responsible for their resistance. We were able to find novel variants of aminoglycoside and ß-lactam resistance genes, with ß-lactamases isolated from the Chryseobacterium spp. functional gene library, one of which is a variant of IND-like metallo-ß-lactamase (MBL) IND-17 and the other of which is a previously uncharacterised MBL we named CHM (Chryseobacterium metallo ß-lactamase). Our results indicate that soil microorganisms possess a diversity of ARG variants, which could potentially be transferred to the clinical setting.

Accurate gene consensus at low nanopore coverage.

BACKGROUND: Nanopore technologies allow high-throughput sequencing of long strands of DNA at the cost of a relatively large error rate. This limits its use in the reading of amplicon libraries in which there are only a few mutations per variant and therefore they are easily confused with the sequencing noise. Consensus calling strategies reduce the error but sacrifice part of the throughput on reading typically 30 to 100 times each member of the library. FINDINGS: In this work, we introduce SINGLe (SNPs In Nanopore reads of Gene Libraries), an error correction method to reduce the noise in nanopore reads of amplicons containing point variations. SINGLe exploits that in an amplicon library, all reads are very similar to a wild-type sequence from which it is possible to experimentally characterize the position-specific systematic sequencing error pattern. Then, it uses this information to reweight the confidence given to nucleotides that do not match the wild-type in individual variant reads and incorporates it on the consensus calculation. CONCLUSIONS: We tested SINGLe in a mutagenic library of the KlenTaq polymerase gene, where the true mutation rate was below the sequencing noise. We observed that contrary to other methods, SINGLe compensates for the systematic errors made by the basecallers. Consequently, SINGLe converges to the true sequence using as little as 5 reads per variant, fewer than the other available methods.

Optimizing the Expression of Human Dopamine Receptors in Escherichia coli.

The human dopamine receptors D2S and D3 belong to the group of G protein-coupled receptors (GPCRs) and are important drug targets. Structural analyses and development of new receptor subtype specific drugs have been impeded by low expression yields or receptor instability. Fusing the T4 lysozyme into the intracellular loop 3 improves crystallization but complicates conformational studies. To circumvent these problems, we expressed the human D2S and D3 receptors in Escherichia coli using different N- and C-terminal fusion proteins and thermostabilizing mutations. We optimized expression times and used radioligand binding assays with whole cells and membrane homogenates to evaluate KD-values and the number of receptors in the cell membrane. We show that the presence but not the type of a C-terminal fusion protein is important. Bacteria expressing receptors capable of ligand binding can be selected using FACS analysis and a fluorescently labeled ligand. Improved receptor variants can thus be generated using error-prone PCR. Subsequent analysis of clones showed the distribution of mutations over the whole gene. Repeated cycles of PCR and FACS can be applied for selecting highly expressing receptor variants with high affinity ligand binding, which in the future can be used for analytical studies.

Design of abnormal data detection system for protein gene library based on data mining technology.

In view of the shortcomings of the current abnormal data detection system of the protein gene library, such as low detection rate and high error detection rate, the abnormal data detection system of the protein gene library based on data mining technology is designed. The protein gene enters the firewall module of the system, and enters the immune module when it does not match the firewall rules; the memory detector in the immune module presents the protein gene, if the memory detector does not match the protein gene, the mature detector presents the protein gene, if the mature detector does not match the protein gene, it is determined as the normal protein gene data package, if it matches, it is considered that The abnormal data of protein gene was processed by the collaborative stimulation module, and the control module controlled by C8051F060 chip to detect the abnormal data of protein gene library. The immune module generates new protein gene sequences through an immature detector, simulates the immune mechanism of protein gene through a mature detector module, and simulates the secondary response in the abnormal data detection system of protein gene library through memory detector. The system introduces data mining technology into the detection and uses a two-level dynamic optimization algorithm to calculate the ASG similarity value of protein gene secondary structure arrangement. According to this value, the abnormal data detection of the protein gene library is realized by randomly generating protein genes, negative selection, clone selection and copying memory cells through gene expression. The experimental results show that the system can quickly detect abnormal data of the protein gene library, ensure the detection efficiency, and the detection accuracy reaches 97.1%. The system can reduce the error rate of normal protein gene detection as an abnormal protein gene.

A Novel Screening Approach for the Dissection of Cellular Regulatory Networks of NF-κB Using Arrayed CRISPR gRNA Libraries.

CRISPR/Cas9 is increasingly being used as a tool to prosecute functional genomic screens. However, it is not yet possible to apply the approach at scale across a full breadth of cell types and endpoints. In order to address this, we developed a novel and robust workflow for array-based lentiviral CRISPR/Cas9 screening. We utilized a ß-lactamase reporter gene assay to investigate mediators of TNF-α-mediated NF-κB signaling. The system was adapted for CRISPR/Cas9 through the development of a cell line stably expressing Cas9 and application of a lentiviral gRNA library comprising mixtures of four gRNAs per gene. We screened a 743-gene kinome library whereupon hits were independently ranked by percent inhibition, Z' score, strictly standardized mean difference, and T statistic. A consolidated and optimized ranking was generated using Borda-based methods. Screening data quality was above acceptable limits (Z' ≥ 0.5). In order to determine the contribution of individual gRNAs and to better understand false positives and negatives, a subset of gRNAs, against 152 genes, were profiled in singlicate format. We highlight the use of known reference genes and high-throughput, next-generation amplicon and RNA sequencing to assess screen data quality. Screening with singlicate gRNAs was more successful than screening with mixtures at identifying genes with known regulatory roles in TNF-α-mediated NF-κB signaling and was found to be superior to previous RNAi-based methods. These results add to the available data on TNF-α-mediated NF-κB signaling and establish a high-throughput functional genomic screening approach, utilizing a vector-based arrayed gRNA library, applicable across a wide variety of endpoints and cell types at a genome-wide scale.

[Genetic changes and biological potential of proliferative nodule in congenital pigmented nevus].

Objective: To study the genetic changes and biological potential of proliferative nodule in congenital melanocytic nevus. Methods: Whole-exome sequencing was carried out using the technique of next-generation sequencing (NGS) in order to detect the genomic alterations of two cases of proliferative nodules (PN) in congenital melanocytic nevi (CMN). Twelve cases of CMN and ten cases of malignant melanoma were used as benign and malignant controls, respectively. Mutated genes that possessed statistically significant difference between benign and malignant controls were listed, according to what benign and malignant statuses were classified and clustered. The heatmaps of clustering analyses were depicted using heatmap package. Fluorescence in situ hybridization (FISH) was also used to validate the above results. Results: Eighty-six common somatic gene mutations were detected in two samples of PN. Compared with CMN, PN had 52 more mutated genes. Furthermore, 22 of these 52 mutated genes were also detected in malignant melanoma samples. Two cases of PN fell between benign CMN and malignant melanoma in germline mutation clustering. Both cases of PN were positive in the FISH tests. Conclusions: The genetic changes of PN partially overlap with those of CMN and malignant melanoma. Therefore, although most of the PN manifest as a benign lesion clinically, it may have certain malignant potential at the genetic level, and warrant long-term monitoring and follow-up.

RNAi as a Tool to Study Virulence in the Pathogenic Yeast Candida glabrata.

The yeast Candida glabrata is a major opportunistic pathogen causing mucosal and systemic infections in humans. Systemic infections caused by this yeast have high mortality rates and are difficult to treat due to this yeast's intrinsic and frequently adapting antifungal resistance. To understand and treat C. glabrata infections, it is essential to investigate the molecular basis of C. glabrata virulence and resistance. We established an RNA interference (RNAi) system in C. glabrata by expressing the Dicer and Argonaute genes from Saccharomyces castellii (a budding yeast with natural RNAi). Our experiments with reporter genes and putative virulence genes showed that the introduction of RNAi resulted in 30 and 70% gene-knockdown for the construct-types antisense and hairpin, respectively. The resulting C. glabrata RNAi strain was used for the screening of a gene library for new virulence-related genes. Phenotypic profiling with a high-resolution quantification of growth identified genes involved in the maintenance of cell integrity, antifungal drugs, and ROS resistance. The genes identified by this approach are promising targets for the treatment of C. glabrata infections.

Functional expression and activity screening of all human cytochrome P450 enzymes in fission yeast.

Here, a complete set of recombinant fission yeast strains that coexpress each of the 57 human cytochrome P450 (CYP) enzymes together with their natural human electron transfer partner(s) was cloned. This strain collection was tested with two luminogenic probe substrates, and 31 human CYPs (including the orphan enzymes CYP2A7, CYP4A22 and CYP20A1) were found to metabolize at least one of these. Since other substrates are known for the remaining enzymes, all human CYPs are now shown to be active. Interestingly, CYP5A1 was found for the first time to work on a substrate other than prostaglandin H2 , and, moreover, to catalyze an aliphatic hydroxylation reaction that consumes molecular oxygen. Also, the ability of CYP11A1 to catalyze an aryl hydroxylation is another unexpected result.

Pathway and Gene Discovery from Natural Hosts and Organisms.

Information in public sequence databases on the genomes and metagenomes of microbes and plants has grown rapidly. In conjunction with technological developments in computational identification of biosynthetic gene clusters, molecular biology, synthetic biology, and analytical tools, this has revealed genes for enzymes with optimal and targeted function, as well as a rich pool of uncharacterized metabolic pathways. This chapter discusses different approaches to discovery of genes and metabolic pathways in microbes and plants in nature, such as genomic mining, transcriptome analysis, and metabolite profiling.

Construction and next-generation sequencing analysis of a large phage-displayed VNAR single-domain antibody library from six naïve nurse sharks.

Background: Shark new antigen receptor variable domain (VNAR) antibodies can bind restricted epitopes that may be inaccessible to conventional antibodies. Methods: Here, we developed a library construction method based on polymerase chain reaction (PCR)-Extension Assembly and Self-Ligation (named "EASeL") to construct a large VNAR antibody library with a size of 1.2 × 1010 from six naïve adult nurse sharks (Ginglymostoma cirratum). Results: The next-generation sequencing analysis of 1.19 million full-length VNARs revealed that this library is highly diversified because it covers all four classical VNAR types (Types I-IV) including 11% of classical Type I and 57% of classical Type II. About 30% of the total VNARs could not be categorized as any of the classical types. The high variability of complementarity determining region (CDR) 3 length and cysteine numbers are important for the diversity of VNARs. To validate the use of the shark VNAR library for antibody discovery, we isolated a panel of VNAR phage binders to cancer therapy-related antigens, including glypican-3, human epidermal growth factor receptor 2 (HER2), and programmed cell death-1 (PD1). Additionally, we identified binders to viral antigens that included the Middle East respiratory syndrome (MERS) and severe acute respiratory syndrome (SARS) spike proteins. The isolated shark single-domain antibodies including Type I and Type II VNARs were produced in Escherichia coli and validated for their antigen binding. A Type II VNAR (PE38-B6) has a high affinity (Kd = 10.1 nM) for its antigen. Conclusions: The naïve nurse shark VNAR library is a useful source for isolating single-domain antibodies to a wide range of antigens. The EASeL method may be applicable to the construction of other large diversity gene expression libraries.

Synthesis of Trinucleotide Building Blocks in Solution and on Solid Phase.

We have developed two methods, in solution and on solid phase, that give easy access to trinucleotide phosphoramidites capable of undergoing coupling reactions by the solid-phase phosphoramidite approach. The solution protocol is characterized by application of 5'-O-dimethoxytrityl (DMT) and 3'-O-tert-butyldimethylsilyl (TBDMS) as a pair of orthogonal protecting groups and 2-cyanoethyl (CE) for protection of the phosphate. Starting with suitably functionalized monomers, synthesis proceeds in the 3'- to 5'-direction, delivering the fully protected trinucleotide. The 3'-O-protecting group is cleaved followed by phosphitylation of the free 3'-OH group. The solid-phase protocol is based on standard phosphoramidite chemistry in conjunction with a dithiomethyl linkage connecting the 3'-starting nucleoside to the polymer. The disulfide bridge can be cleaved under neutral conditions for release of the trinucleotide from the support preserving all other protecting groups. © 2018 by John Wiley & Sons, Inc.

Engineering Escherichia coli to increase triacetic acid lactone (TAL) production using an optimized TAL sensor-reporter system.

Triacetic acid lactone (TAL) (4-hydroxy-6-methyl-2-pyrone) can be upgraded into a variety of higher-value products, and has potential to be developed into a renewable platform chemical through metabolic engineering. We previously developed an endogenous TAL sensor based on the regulatory protein AraC, and applied it to screen 2-pyrone synthase (2-PS) variant libraries in E. coli, resulting in the identification of variants conferring up to 20-fold improved TAL production in liquid culture. In this study, the sensor-reporter system was further optimized and used to further improve TAL production from recombinant E. coli, this time by screening a genomic overexpression library. We identified new and unpredictable gene targets (betT, ompN, and pykA), whose plasmid-based expression improved TAL yield (mg/L/OD595) up to 49% over the control strain. This work further demonstrates the utility of customized transcription factors as molecular reporters in high-throughput engineering of biocatalytic strains.

Polybacterial community analysis in human conjunctiva through 16S rRNA gene libraries.

The conjunctival sac of healthy human harbours a variety of microorganisms. When the eye is compromised, an occasional inadvertent spread happens to the adjacent tissue, resulting in bacterial ocular infections. Microbiological investigation of the conjunctival swab is one of the broadly used modality to study the aetiological agent of conjunctiva. However, most of the time such methods yield unsatisfactory results. Hence, the present study intends to identify the bacterial community in human conjunctiva of pre-operative subjects through 16S rRNA gene libraries. Out of 45 samples collected from preoperative patients undergoing cataract surgery, 36 libraries were constructed with bacterial nested-PCR-positive samples. The representative clones with unique restriction pattern were generated through Amplified Ribosomal DNA Restriction Analysis (ARDRA) which were sequenced for phylogenetic affiliation. A total of 211 representative clones were obtained which were distributed in phyla Actinobacteria, Firmicutes, α-Proteobacteria, ß-Proteobacteria, γ-Proteobacteria, Bacteroidetes, and Deinococcus-Thermus. Findings revealed the presence of polybacterial community, especially in some cases even though no bacterium or a single bacterium alone was identified through cultivable method. Remarkably, we identified 17 species which have never been reported in any ocular infections. The sequencing data reported 6 unidentified bacteria suggesting the possibility of novel organisms in the sample. Since, polybacterial community has been identified consisting of both gram positive and gram negative bacteria, a broad spectrum antibiotic therapy is advisable to the patients who are undergoing cataract surgery. Consolidated effort would significantly improve a clear understanding of the nature of microbial community in the human conjunctiva which will promote administration of appropriate antibiotic regimen and also help in the development of oligonucleotide probes to screen the predominant pathogens for early predisposition.

Preparation of trinucleotide phosphoramidites as synthons for the synthesis of gene libraries.

The preparation of protein libraries is a key issue in protein engineering and biotechnology. Such libraries can be prepared by a variety of methods, starting from the respective gene library. The challenge in gene library preparation is to achieve controlled total or partial randomization at any predefined number and position of codons of a given gene, in order to obtain a library with a maximum number of potentially successful candidates. This purpose is best achieved by the usage of trinucleotide synthons for codon-based gene synthesis. We here review the strategies for the preparation of fully protected trinucleotides, emphasizing more recent developments for their synthesis on solid phase and on soluble polymers, and their use as synthons in standard DNA synthesis.

Standardized cloning vectors for protein production and generation of large gene libraries in Escherichia coli.

Here, we modified the multiple cloning sites from commonly used expression vectors to create a new suite of cloning plasmids that simplify and speed up cloning procedures in Escherichia coli. Each of our standardized plasmids contains two BsaI restriction sites, allowing for highly efficient cloning of genes and bringing their expression under control of either a T7 (pET21a_BsaI, pET28a_BsaI, and pMAL-c5T_BsaI) or T5 promoter (pUR22 and pUR23). Another plasmid in our suite (pTNA_BsaI) allows for generation of large gene libraries containing >108 variants, which can be constitutively expressed in selection experiments using metabolic complementation of auxotrophic E. coli strains. Coupling restriction and ligation with the BsaI restriction enzyme minimizes hands-on time, while the need for only three different primers to clone a target gene into the six different vectors keeps overall cloning costs low.

Bacilli community of saline-alkaline soils from the Ararat Plain (Armenia) assessed by molecular and culture-based methods.

The bacterial community composition in the A horizon of a natural saline-alkaline soil located in Ararat Plain (Armenia) was studied using molecular and culture-based methods The sequence analysis of a 16S rRNA gene clone library and denaturing gradient gel electrophoresis (DGGE) profiles indicated dominance of Firmicutes populations. The majority of the sequences of the bacterial 16S rRNA gene library were close relatives of representatives belonging to the genera Halobacillus (41.2%), Piscibacillus (23.5%), Bacillus (23.5%) and Virgibacillus (11.8%). Eight novel moderately halophilic bacilli isolates were successfully obtained from the enriched cultures of the saline-alkaline soil samples. 16S rRNA gene sequence analyses of isolates revealed their affiliation (97.7-99.7% similarity) to representatives of the genera Bacillus, Piscibacillus and Halobacillus. All isolates were able to tolerate high concentrations of NaCl and highly alkaline conditions. This is the first study combining cultivation-independent and -dependent approaches to reveal the bacterial diversity of the saline-alkaline soils of Ararat Plain and it suggested an important role of bacilli as key microbes in biogeochemical cycles of these environments.