C/EBP homologous protein promotes Sonic Hedgehog secretion from type II alveolar epithelial cells and activates Hedgehog signaling pathway of fibroblast in pulmonary fibrosis.

BACKGROUND: Endoplasmic reticulum (ER) stress is involved in the pathological process of pulmonary fibrosis, including IPF. It affects a broad scope of cellular types during pulmonary fibrosis but the role in epithelial-mesenchymal crosstalk has not been fully defined. The present study aimed to investigate the effects of Shh secretion by ER  stress-challenged type II alveolar epithelial cells (AECII) on fibroblast and pulmonary fibrosis. METHODS: Conditioned medium (CM) from tunicamycin (TM)-treated AECII was collected and incubated with fibroblast. Short hairpin RNA (shRNA) was used for RNA interference of C/EBP homologous protein (CHOP). The effects of CHOP and HH signaling were evaluated by TM administration under the background of bleomycin-induced pulmonary fibrosis in mice. RESULTS: Both expression of CHOP and Shh in AECII, and HH signaling in mesenchyme were upregulated in IPF lung. TM-induced Shh secretion from AECII activates HH signaling and promotes pro-fibrotic effects of fibroblast. Interfering CHOP expression reduced ER stress-induced Shh secretion and alleviated pulmonary fibrosis in mice. CONCLUSIONS: Our work identified a novel mechanism by which ER stress is involved in pulmonary fibrosis. Inhibition of ER stress or CHOP in epithelial cells alleviated pulmonary fibrosis by suppressing Shh/HH signaling pathway of fibroblasts.

SENP1 regulates the transformation of lung resident mesenchymal stem cells and is associated with idiopathic pulmonary fibrosis progression.

BACKGROUND: Lung resident mesenchymal stem cells (LR-MSCs) play an important role in idiopathic pulmonary fibrosis (IPF) by transforming into myofibroblasts, thereby losing their repair ability. Evidence suggests that key proteins of multiple signaling pathways are involved in myofibroblast differentiation of LR-MSCs, such as ß-Catenin and GLI family zinc finger 1 (GLI1). These proteins are regulated by SUMO (small ubiquitin-like modifier) modification, which is a post-translational modification that promotes protein degradation, while Sumo specific protein 1 (SENP1)-mediated deSUMOylation produces the opposite biological effects. Therefore, we speculated that SENP1 might be a potential target for treating pulmonary fibrosis by preventing the myofibroblast differentiation of LR-MSCs. METHODS: LR-MSCs were isolated from mice by using immunomagnetic beads. The extracted LR-MSCs were identified by flow cytometric analysis and multilineage differentiation assays. Lentivirus packaged shRNA silenced the expression of SENP1 in vitro and vivo. The silencing efficacy of SENP1 was verified by real-time quantitative PCR. The effect of down-regulated SENP1 on the myofibroblast differentiation of LR-MSCs was assessed by Immunofluorescence and Western blot. Immunoprecipitation was used to clarify that SENP1 was a key target for regulating the activity of multiple signaling pathways in the direction of LR-MSCs differentiation. LR-MSCs resident in the lung was analyzed with in vivo imaging system. HE and Masson staining was used to evaluate the therapeutic effect of LR-MSCs with SENP1 down-regulation on the lung of BLM mice. RESULTS: In this study, we found that the myofibroblast differentiation of LR-MSCs in IPF lung tissue was accompanied by enhanced SENP1-mediated deSUMOylation. The expression of SENP1 increased in LR-MSCs transition of bleomycin (BLM)-induced lung fibrosis. Interfering with expression of SENP1 inhibited the transformation of LR-MSCs into myofibroblasts in vitro and in vivo and restored their therapeutic effect in BLM lung fibrosis. In addition, activation of the WNT/ß-Catenin and Hedgehog/GLI signaling pathways depends on SENP1-mediated deSUMOylation. CONCLUSIONS: SENP1 might be a potential target to restore the repair function of LR-MSCs and treat pulmonary fibrosis. Video Abstract.

Rational Optimization of Raman-Activated Cell Ejection and Sequencing for Bacteria.

In Raman-activated cell ejection and sequencing (RACE-Seq), success rate and sequence coverage have generally been low for shotgun sequencing of individual post-RACE cells. Here we quantitatively evaluated the influence of cell lysis condition, nucleic acid amplification condition, and parameters of Raman measurement on RACE-Seq performance. Variations in laser energy input during Raman signal acquisition, but not duration of alkaline lysate lysis, temperature, or measurement under dry or aqueous conditions, are vital to the success of multiple displacement amplification (MDA). In fact, laser irradiation is reversely linked to MDA product quality. However, introduction of oils prior to MDA, by mitigating such negative effects of Raman irradiation, elevates genome coverage of post-RACE Escherichia coli cells from <20% to ∼50%, while greatly improving the success rate of RACE-Seq for soil microbiota. Our findings provide a practical solution for enhancing RACE-Seq performance and pinpoint protection of cells from laser irradiation as a priority in method development.

Relation between sex hormones and leucocyte telomere length in men with idiopathic pulmonary fibrosis.

BACKGROUND AND OBJECTIVE: IPF is an ageing-related lung disorder featuring progressive lung scarring. IPF patients are frequently identified with short telomeres but coding mutations in telomerase can only explain a minority of cases. Sex hormones regulate telomerase activity in vitro and levels of sex hormones are related to LTL. The objective of this study was to explore whether sex hormones were associated with LTL, whether they interacted with genetic variants in telomerase and whether polymorphisms in the exon of androgen metabolism genes were associated with plasma testosterone concentrations in male IPF patients. METHODS: A case-control study was performed on 101 male IPF subjects and 51 age-matched healthy controls. Early morning plasma sex hormones were quantified, and whole-exome sequencing was used to identify rare protein-altering variants of telomerase and SNP in the exon of androgen metabolism genes. LTL was analysed by PCR and expressed as a T/S ratio. RESULTS: LTL, testosterone and DHT were decreased significantly in the IPF group. After adjustments for age and variant status in telomerase-related genes, only testosterone was positively associated with LTL (P = 0.001). No significant interaction (P = 0.661) was observed between rare protein-altering variants of telomerase and testosterone. No coding SNP in androgen metabolism genes were significantly associated with testosterone concentrations. CONCLUSION: Plasma testosterone is associated with LTL independent of age or rare protein-altering variants of telomerase. No genetic variations of androgen-related pathway genes are associated with androgen concentrations. Further studies are warranted to examine whether hormonal interventions might retard telomere loss in male IPF patients.

The Association Between MUC5B Mutations and Clinical Outcome in Patients with Rheumatoid Arthritis-Associated Interstitial Lung Disease: A Retrospective Exploratory Study in China.

BACKGROUND Patients with rheumatoid arthritis (RA) who develop interstitial lung disease (RA-ILD), show features of usual interstitial pneumonia (UIP) on high-resolution computed tomography (HRCT). This retrospective exploratory clinical study aimed to investigate the association between mutations in the MUC5B gene and clinical outcome in patients with RA, with or without RA-ILD, using whole-exome sequencing (WES). MATERIAL AND METHODS WES was performed using peripheral blood samples for mutations in the MUC5B gene in 51 patients diagnosed with RA without ILD, and 45 patients with RA-ILD. The cumulative incidence in acute exacerbations of RA-ILD and variables associated with acute exacerbations of RA-ILD were analyzed. RESULTS In patients with RA-ILD, the main genetic variants of MUC5B were identified, with an odds ratio (OR) of 3.410 (p=0.013). Nine patients with RA without ILD (17.6%) and 19 patients with RA-ILD (42.2%) expressed MUC5B variants. Patients with RA-ILD carrying MUC5B variants had a significantly increased duration of RA-ILD (p=0.03) and showed a UIP pattern on lung HRCT (p=0.01). Acute exacerbations of RA-ILD occurred in 25 patients during follow-up, including 13 patients with mutant MUC5B and 12 patients with wildtype MUC5B. Univariate analysis showed that MUC5B mutations (p=0.043), older age of onset of RA (p=0.041), increased serum anti-citrullinated protein antibodies (ACPAs) (p=0.033), and a UIP imaging pattern on HRCT (p=0.015) were significantly correlated with acute exacerbations of RA-ILD. However, these findings were not supported by multivariate analysis (p=0.065). CONCLUSIONS The carrier status of MUC5B variants was an indicator of reduced prognosis and increased exacerbations of RA-ILD.

Raman-activated cell sorting and metagenomic sequencing revealing carbon-fixing bacteria in the ocean.

It is of great significance to understand CO2 fixation in the oceans. Using single cell Raman spectra (SCRS) as biochemical profiles, Raman activated cell ejection (RACE) was able to link phenotypes and genotypes of cells. Here, we show that mini-metagenomic sequences from RACE can be used as a reference to reconstruct nearly complete genomes of key functional bacteria by binning shotgun metagenomic sequencing data. By applying this approach to 13 C bicarbonate spiked seawater from euphotic zone of the Yellow Sea of China, the dominant bacteria Synechococcus spp. and Pelagibacter spp. were revealed and both of them contain carotenoid and were able to incorporate 13 C into the cells at the same time. Genetic analysis of the reconstructed genomes suggests that both Synechococcus spp. and Pelagibacter spp. contained all genes necessary for carotenoid synthesis, light energy harvesting and CO2 fixation. Interestingly, the reconstructed genome indicates that Pelagibacter spp. harbored intact sets of genes for ß-carotene (precursor of retional), proteorhodopsin synthesis and anaplerotic CO2 fixation. This novel approach shines light on the role of marine 'microbial dark matter' in global carbon cycling, by linking yet-to-be-cultured Synechococcus spp. and Pelagibacter spp. to carbon fixation and flow activities in situ.

Molecular identification and transcriptional regulation of porcine IFIT2 gene.

IFN-induced protein with tetratricopeptide repeats 2 (IFIT2) plays important roles in host defense against viral infection as revealed by studies in humans and mice. However, little is known on porcine IFIT2 (pIFIT2). Here, we performed molecular cloning, expression profile, and transcriptional regulation analysis of pIFIT2. pIFIT2 gene, located on chromosome 14, is composed of two exons and have a complete coding sequence of 1407 bp. The encoded polypeptide, 468 aa in length, has three tetratricopeptide repeat motifs. pIFIT2 gene was unevenly distributed in all eleven tissues studied with the most abundance in spleen. Poly(I:C) treatment notably strongly upregulated the mRNA level and promoter activity of pIFIT2 gene. Upstream sequence of 1759 bp from the start codon which was assigned +1 here has promoter activity, and deltaEF1 acts as transcription repressor through binding to sequences at position - 1774 to - 1764. Minimal promoter region exists within nucleotide position - 162 and - 126. Two adjacent interferon-stimulated response elements (ISREs) and two nuclear factor (NF)-κB binding sites were identified within position - 310 and - 126. The ISRE elements act alone and in synergy with the one closer to start codon having more strength, so do the NF-κB binding sites. Synergistic effect was also found between the ISRE and NF-κB binding sites. Additionally, a third ISRE element was identified within position - 1661 to - 1579. These findings will contribute to clarifying the antiviral effect and underlying mechanisms of pIFIT2.

Generation of Chimeric RNAs by cis-splicing of adjacent genes (cis-SAGe) in mammals.

Chimeric RNA molecules, possessing exons from two or more independent genes, are traditionally believed to be produced by chromosome rearrangement. However, recent studies revealed that cis-splicing of adjacent genes (cis- SAGe) is one of the major mechanisms underlying the formation of chimeric RNAs. cis-SAGe refers to intergenic splicing of directly adjacent genes with the same transcriptional orientation, resulting in read-through transcripts, termed chimeric RNAs, which contain sequences from two or more parental genes. cis-SAGe was first identified in tumor cells, since then its potential in carcinogenesis has attracted extensive attention. More and more scientists are focusing on it. With the development of research, cis-SAGe was found to be ubiquitous in various normal tissues, and might make a crucial contribution to the formation of novel genes in the evolution of genomes. In this review, we summarize the splicing pattern, expression characteristics, possible mechanisms, and significance of cis-SAGe in mammals. This review will be helpful for general understanding of the current status and development tendency of cis-SAGe.

Choreography of Transcriptomes and Lipidomes of Nannochloropsis Reveals the Mechanisms of Oil Synthesis in Microalgae.

To reveal the molecular mechanisms of oleaginousness in microalgae, transcriptomic and lipidomic dynamics of the oleaginous microalga Nannochloropsis oceanica IMET1 under nitrogen-replete (N+) and N-depleted (N-) conditions were simultaneously tracked. At the transcript level, enhanced triacylglycerol (TAG) synthesis under N- conditions primarily involved upregulation of seven putative diacylglycerol acyltransferase (DGAT) genes and downregulation of six other DGAT genes, with a simultaneous elevation of the other Kennedy pathway genes. Under N- conditions, despite downregulation of most de novo fatty acid synthesis genes, the pathways that shunt carbon precursors from protein and carbohydrate metabolic pathways into glycerolipid synthesis were stimulated at the transcript level. In particular, the genes involved in supplying carbon precursors and energy for de novo fatty acid synthesis, including those encoding components of the pyruvate dehydrogenase complex (PDHC), glycolysis, and PDHC bypass, and suites of specific transporters, were substantially upregulated under N- conditions, resulting in increased overall TAG production. Moreover, genes involved in the citric acid cycle and ß-oxidation in mitochondria were greatly enhanced to utilize the carbon skeletons derived from membrane lipids and proteins to produce additional TAG or its precursors. This temporal and spatial regulation model of oil accumulation in microalgae provides a basis for improving our understanding of TAG synthesis in microalgae and will also enable more rational genetic engineering of TAG production.

In-Situ Fabrication of MOF-Derived Co-Co Layered Double Hydroxide Hollow Nanocages/Graphene Composite: A Novel Electrode Material with Superior Electrochemical Performance.

Rational design of a transition metal layered double hydroxide (LDH) and graphene composite is vitally important for designing high-performance supercapacitor electrodes. Although various methods are performed, the realization of high-performance is still impeded by the agglomeration of graphene and layered double hydroxide. Here, metal-organic framework derived cobalt-cobalt layered double hydroxide (Co-Co LDH) hollow nanocages, uniformly deposited on graphene nanosheets, are fabricated through facile in situ co-deposition and thermal ion-exchange reaction. Electrochemical investigation reveals that Co-Co LDH/15 mg graphene is rather outstanding, which delivers high specific capacitance of 1205 F g-1 , excellent rate capability (60.3 % capacitance retention is obtained after the current density increased 6.67 times), and cycling stability. The excellent performance of electrode is also confirmed by assembling an asymmetric supercapacitor, which delivers high energy density of 49.5 Wh kg-1 as well as the maximum power density of 7000 W kg-1 . The Co-Co LDH/graphene composite proves a promising concept for constructing hierarchical structure materials in the future.

Nannochloropsis genomes reveal evolution of microalgal oleaginous traits.

Oleaginous microalgae are promising feedstock for biofuels, yet the genetic diversity, origin and evolution of oleaginous traits remain largely unknown. Here we present a detailed phylogenomic analysis of five oleaginous Nannochloropsis species (a total of six strains) and one time-series transcriptome dataset for triacylglycerol (TAG) synthesis on one representative strain. Despite small genome sizes, high coding potential and relative paucity of mobile elements, the genomes feature small cores of ca. 2,700 protein-coding genes and a large pan-genome of >38,000 genes. The six genomes share key oleaginous traits, such as the enrichment of selected lipid biosynthesis genes and certain glycoside hydrolase genes that potentially shift carbon flux from chrysolaminaran to TAG synthesis. The eleven type II diacylglycerol acyltransferase genes (DGAT-2) in every strain, each expressed during TAG synthesis, likely originated from three ancient genomes, including the secondary endosymbiosis host and the engulfed green and red algae. Horizontal gene transfers were inferred in most lipid synthesis nodes with expanded gene doses and many glycoside hydrolase genes. Thus multiple genome pooling and horizontal genetic exchange, together with selective inheritance of lipid synthesis genes and species-specific gene loss, have led to the enormous genetic apparatus for oleaginousness and the wide genomic divergence among present-day Nannochloropsis. These findings have important implications in the screening and genetic engineering of microalgae for biofuels.

Transcriptomics Analysis on Excellent Meat Quality Traits of Skeletal Muscles of the Chinese Indigenous Min Pig Compared with the Large White Breed.

The Min pig (Sus scrofa) is a well-known indigenous breed in China. One of its main advantages over European breeds is its high meat quality. Additionally, different cuts of pig also show some different traits of meat quality. To explore the underlying mechanism responsible for the differences of meat quality between different breeds or cuts, the longissimus dorsi muscle (LM) and the biceps femoris muscle (BF) from Min and Large White pigs were investigated using transcriptome analysis. The gene expression profiling identified 1371 differentially expressed genes (DEGs) between LM muscles from Min and Large White pigs, and 114 DEGs between LM and BF muscles from the same Min pigs. Gene Ontology (GO) enrichment of biological functions and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the gene products were mainly involved in the IRS1/Akt/FoxO1 signaling pathway, adenosine 5'-monophosphate-activated protein kinase (AMPK) cascade effects, lipid metabolism and amino acid metabolism pathway. Such pathways contributed to fatty acid metabolism, intramuscular fat deposition, and skeletal muscle growth in Min pig. These results give an insight into the mechanisms underlying the formation of skeletal muscle and provide candidate genes for improving meat quality. It will contribute to improving meat quality of pigs through molecular breeding.

Genomic Foundation of Starch-to-Lipid Switch in Oleaginous Chlorella spp.

The ability to rapidly switch the intracellular energy storage form from starch to lipids is an advantageous trait for microalgae feedstock. To probe this mechanism, we sequenced the 56.8-Mbp genome of Chlorella pyrenoidosa FACHB-9, an industrial production strain for protein, starch, and lipids. The genome exhibits positive selection and gene family expansion in lipid and carbohydrate metabolism and genes related to cell cycle and stress response. Moreover, 10 lipid metabolism genes might be originated from bacteria via horizontal gene transfer. Transcriptomic dynamics tracked via messenger RNA sequencing over six time points during metabolic switch from starch-rich heterotrophy to lipid-rich photoautotrophy revealed that under heterotrophy, genes most strongly expressed were from the tricarboxylic acid cycle, respiratory chain, oxidative phosphorylation, gluconeogenesis, glyoxylate cycle, and amino acid metabolisms, whereas those most down-regulated were from fatty acid and oxidative pentose phosphate metabolism. The shift from heterotrophy into photoautotrophy highlights up-regulation of genes from carbon fixation, photosynthesis, fatty acid biosynthesis, the oxidative pentose phosphate pathway, and starch catabolism, which resulted in a marked redirection of metabolism, where the primary carbon source of glycine is no longer supplied to cell building blocks by the tricarboxylic acid cycle and gluconeogenesis, whereas carbon skeletons from photosynthesis and starch degradation may be directly channeled into fatty acid and protein biosynthesis. By establishing the first genetic transformation in industrial oleaginous C. pyrenoidosa, we further showed that overexpression of an NAD(H) kinase from Arabidopsis (Arabidopsis thaliana) increased cellular lipid content by 110.4%, yet without reducing growth rate. These findings provide a foundation for exploiting the metabolic switch in microalgae for improved photosynthetic production of food and fuels.

Facile synthesis of exfoliated Co-Al LDH-carbon nanotube composites with high performance as supercapacitor electrodes.

A novel sandwich-like structured Co-Al LDH-Carbon Nanotube (CNT) composite has been successfully synthesized by the elegant combination between exfoliated Co-Al LDH nanosheets and modified CNTs, which was achieved through an electrostatic assembly method. It is worth noting that the negatively charged CNTs, sandwiched between the positively charged nanosheets via the electrostatic force, can not only expand the area of contact of electrolyte ions but also highly improve the conductivity. The as-prepared Co-Al LDHs-CNTs composite exhibited a high specific capacitance of 884 F g(-1) and a good cycle stability over 2000 cycles. Therefore, such a facile synthetic route to fabricate the layered structure composite may open a new strategy to prepare other composites with largely enhanced electrochemical properties, which can be of great promise in energy storage device application.

Cycloalkane degradation by an uncultivated novel genus of Gammaproteobacteria derived from China's marginal seas.

The consumption of cycloalkanes is prevalent in low-temperature marine environments, likely influenced by psychrophilic microorganisms. Despite their significance, the primary active species responsible for marine cycloalkane degradation remain largely unidentified due to cultivation challenges. In this study, we provide compelling evidence indicating that the uncultured genus C1-B045 of Gammaproteobacteria is a pivotal participant in cycloalkane decomposition within China's marginal seas. Notably, the relative abundance of C1-B045 surged from 15.9% in the methylcyclohexane (MCH)-consuming starter culture to as high as 97.5% in MCH-utilizing extinction cultures following successive dilution-to-extinction and incubation cycles. We used stable isotope probing, Raman-activated gravity-driven encapsulation, and 16 S rRNA gene sequencing to link cycloalkane-metabolizing phenotype to genotype at the single-cell level. By annotating key enzymes (e.g., alkane monooxygenase, cyclohexanone monooxygenase, and 6-hexanolactone hydrolase) involved in MCH metabolism within C1-B045's representative metagenome-assembled genome, we developed a putative MCH degradation pathway.

Nannochloropsis plastid and mitochondrial phylogenomes reveal organelle diversification mechanism and intragenus phylotyping strategy in microalgae.

BACKGROUND: Microalgae are promising feedstock for production of lipids, sugars, bioactive compounds and in particular biofuels, yet development of sensitive and reliable phylotyping strategies for microalgae has been hindered by the paucity of phylogenetically closely-related finished genomes. RESULTS: Using the oleaginous eustigmatophyte Nannochloropsis as a model, we assessed current intragenus phylotyping strategies by producing the complete plastid (pt) and mitochondrial (mt) genomes of seven strains from six Nannochloropsis species. Genes on the pt and mt genomes have been highly conserved in content, size and order, strongly negatively selected and evolving at a rate 33% and 66% of nuclear genomes respectively. Pt genome diversification was driven by asymmetric evolution of two inverted repeats (IRa and IRb): psbV and clpC in IRb are highly conserved whereas their counterparts in IRa exhibit three lineage-associated types of structural polymorphism via duplication or disruption of whole or partial genes. In the mt genomes, however, a single evolution hotspot varies in copy-number of a 3.5 Kb-long, cox1-harboring repeat. The organelle markers (e.g., cox1, cox2, psbA, rbcL and rrn16_mt) and nuclear markers (e.g., ITS2 and 18S) that are widely used for phylogenetic analysis obtained a divergent phylogeny for the seven strains, largely due to low SNP density. A new strategy for intragenus phylotyping of microalgae was thus proposed that includes (i) twelve sequence markers that are of higher sensitivity than ITS2 for interspecies phylogenetic analysis, (ii) multi-locus sequence typing based on rps11_mt-nad4, rps3_mt and cox2-rrn16_mt for intraspecies phylogenetic reconstruction and (iii) several SSR loci for identification of strains within a given species. CONCLUSION: This first comprehensive dataset of organelle genomes for a microalgal genus enabled exhaustive assessment and searches of all candidate phylogenetic markers on the organelle genomes. A new strategy for intragenus phylotyping of microalgae was proposed which might be generally applicable to other microalgal genera and should serve as a valuable tool in the expanding algal biotechnology industry.

Medium optimization, molecular characterization, and bioactivity of exopolysaccharides from Pleurotus eryngii.

An optimal medium for exopolysaccharides (EPS) production was obtained through one-factor-at-a-time method and response surface methodology. Under optimal culture medium, the maximum EPS concentration in shake flask was 5.16 g/l. Two groups of EPSs (designated as Fr-I and Fr-II) were obtained from the culture filtrates by size exclusion chromatography/multiangle laser light scattering, and the weight average molar masses (M w) of Fr-I and Fr-II were determined to be 4.098 × 10(4) and 1.114 × 10(4) g/mol, respectively. The molecular confirmation of Fr-I was revealed to be a rigid rod form in aqueous solution. Moreover, monosaccharide composition and characteristic groups were investigated by GC and Fourier transform infrared, respectively. Finally, pharmacology experiment in vitro indicated EPS Fr-II of Pleurotus eryngii exhibited higher antioxidant and antitumor abilities than Fr-I, which might be attributed to the different molecular weights and chemical compositions in the EPS fraction.

Sequence and expression regulation of the BCL2L2 gene in pigs.

B cell lymphoma-2-like 2 (BCL2L2), an important regulator of apoptosis, plays vital roles in several physiological processes, as revealed by studies in humans and mice. However, reports on pig BCL2L2 are few, and the encoding gene has not been identified experimentally. This study was designed to clone the porcine BCL2L2 gene and its alternative splicing (AS) transcripts using molecular biological techniques and to analyze the regulatory mechanisms underlying transcription and translation. The BCL2L2 cDNA (V1) was 807 bp in length and encoded a polypeptide of 193 aa containing four BCL-2 homology domains. A total of nine AS transcripts were obtained, among which V2 and V3 differed from V1 in the 5' untranslated region (UTR). The core promoter was mapped to a range of -1102 to -759 bp (the first nucleotide of the start codon was designated as +1). There were several functional cis-elements, including one SP1 and two C/EBPα binding sites at around -759 bp. AS in the 5' UTR is involved in the regulation of gene expression, as revealed by dual-luciferase reporter and western blot analysis, and the secondary structure of the 5' UTRs may be the reason for the differential expression of V1-3. At the same time, an upstream open reading frame (ORF) existed in each of the three 5' UTRs, was found to repress the expression of the main ORF. Additionally, the roles of porcine BCL2L2 in cell proliferation and apoptosis were preliminarily analyzed. The results will contribute to further characterizing the role of BCL2L2.

Optical-based microbubble for on-demand droplet release from static droplet array (SDA) for dispensing one droplet into one tube.

Static droplet array (SDA) is a pivotal tool for high-capacity screening assays, yet extraction and collection the target droplets that contain unique analytes or cells from the SDA remains one major technical bottleneck that limits its broader application. Here we present an optical-based on-demand droplet release (OODR) system by incorporating a 1064 nm laser-responsive indium tin oxide (ITO) layer into a chamber array-based droplet microfluidic chip. By focusing the 1064 nm laser onto the ITO layer, microbubbles can be created via local heating to selectively push-out the droplets from the chamber. Then the released droplet is readily exported in a one-droplet-one-tube (ODOT) manner by the inherent capillary force into pipette tip. Releasing of the droplets containing fluorescein sodium demonstrated ∼100% successful rate (9 out of 6400 droplets were successfully released) and low residual (only ∼5% of the droplet volume remains in the chamber). White or fluorescence image-based releasing of single-cell-droplets directly after cell loading or multi-cells-droplets derived from on-chip single-cell cultivation for both E. coli and yeast cells further demonstrated the wide applicability of OODR. The present system is user-friendly and has the potential to be applied in various high-throughput screening assays, including single molecule/cell analysis, drug screening, and phenotype-based cell sorting.

Improved single-cell genome amplification by a high-efficiency phi29 DNA polymerase.

Single-cell genomic whole genome amplification (WGA) is a crucial step in single-cell sequencing, yet its low amplification efficiency, incomplete and uneven genome amplification still hinder the throughput and efficiency of single-cell sequencing workflows. Here we introduce a process called Improved Single-cell Genome Amplification (iSGA), in which the whole single-cell sequencing cycle is completed in a high-efficient and high-coverage manner, through phi29 DNA polymerase engineering and process engineering. By establishing a disulfide bond of F137C-A377C, the amplification ability of the enzyme was improved to that of single-cell. By further protein engineering and process engineering, a supreme enzyme named HotJa Phi29 DNA Polymerase was developed and showed significantly better coverage (99.75%) at a higher temperature (40°C). High single-cell genome amplification ability and high coverage (93.59%) were also achieved for commercial probiotic samples. iSGA is more efficient and robust than the wild-type phi29 DNA polymerase, and it is 2.03-fold more efficient and 10.89-fold cheaper than the commercial Thermo Scientific EquiPhi29 DNA Polymerase. These advantages promise its broad applications in large-scale single-cell sequencing.