Potential clinical application of microRNAs in bladder cancer.

Bladder cancer (BC) is the tenth most prevalent malignancy globally, presenting significant clinical and societal challenges because of its high incidence, rapid progression, and frequent recurrence. Presently, cystoscopy and urine cytology serve as the established diagnostic methods for BC. However, their efficacy is limited by invasive nature and low sensitivity. Therefore, the development of highly specific biomarkers and effective non-invasive detection strategies is imperative for achieving a precise and timely diagnosis of BC, as well as for facilitating an optimal tumor treatment and an improved prognosis. microRNAs (miRNAs), short noncoding RNA molecules spanning around 20-25 nucleotides, are implicated in the regulation of diverse carcinogenic pathways. Substantially altered miRNAs form robust functional regulatory networks that exert a notable influence on the tumorigenesis and progression of BC. Investigations into aberrant miRNAs derived from blood, urine, or extracellular vesicles indicate their potential roles as diagnostic biomarkers and prognostic indicators in BC, enabling miRNAs to monitor the progression and predict the recurrence of the disease. Simultaneously, the investigation centered on miRNA as a potential therapeutic agent presents a novel approach for the treatment of BC. This review provides a comprehensive analysis of the biological roles of miRNAs in tumorigenesis and progression, and systematically summarizes the potential as diagnosis and prognosis biomarkers as well as therapeutic targets for BC. Additionally, we evaluate the progress made in laboratory techniques within this field and discuss the prospects.

Amplification-Free Analysis of Bladder Cancer MicroRNAs on Wrinkled Silica Nanoparticles with DNA-Functionalized Quantum Dots.

Bladder cancer (BC) occurrence and progression are accompanied by alterations in microRNAs (miRNAs) expression levels. Simultaneous detection of multiple miRNAs contributes to the accuracy and reliability of the BC diagnosis. In this work, wrinkled silica nanoparticles (WSNs) were applied as the microreactor for multiplex miRNAs analysis without enzymes or nucleic acid amplification. Conjugated on the surface of WSNs, the S9.6 antibody was adopted as the universal module for binding DNA/miRNA duplexes, regardless of their sequence. Furthermore, single-stranded DNA (ssDNA) was labeled with quantum dots (QDs) for identifying a given miRNA to form QDs-ssDNA/miRNA, which enabled the specific capture of the corresponding QDs on the wrinkled surface of WSNs. Based on the detection of fluorescence signals that were ultimately focused on WSNs, target miRNAs could be sensitively identified to a femtomolar level (5 fM) with a wide dynamic range of up to 6 orders of magnitude. The proposed strategy achieved high specificity to obviously distinguish single-base mutation sequences and possessed multiplex assay capability. Moreover, the assay exhibited excellent practicability in the multiplex detection of miRNAs in clinical serum specimens.

Metabolism mechanism of glycosaminoglycans by the gut microbiota: Bacteroides and lactic acid bacteria: A review.

Glycosaminoglycans (GAGs), as a class of biopolymers, play pivotal roles in various biological metabolisms such as cell signaling, tissue development, cell apoptosis, immune modulation, and growth factor activity. They are mainly present in the colon in free forms, which are essential for maintaining the host's health by regulating the colonization and proliferation of gut microbiota. Therefore, it is important to explain the specific members of the gut microbiota for GAGs' degradation and their enzymatic machinery in vivo. This review provides an outline of GAGs-utilizing entities in the Bacteroides, highlighting their polysaccharide utilization loci (PULs) and the enzymatic machinery involved in chondroitin sulfate (CS) and heparin (Hep)/heparan sulfate (HS). While there are some variations in GAGs' degradation among different genera, we analyze the reputed GAGs' utilization clusters in lactic acid bacteria (LAB), based on recent studies on GAGs' degradation. The enzymatic machinery involved in Hep/HS and CS metabolism within LAB is also discussed. Thus, to elucidate the precise mechanisms utilizing GAGs by diverse gut microbiota will augment our understanding of their effects on human health and contribute to potential therapeutic strategies for diseases.

CRISPR-Cas systems of lactic acid bacteria and applications in food science.

CRISPR-Cas (Clustered regularly interspaced short palindromic repeats-CRISPR associated proteins) systems are widely distributed in lactic acid bacteria (LAB), contributing to their RNA-mediated adaptive defense immunity. The CRISPR-Cas-based genetic tools have exhibited powerful capability. It has been highly utilized in different organisms, accelerating the development of life science. The review summarized the components, adaptive immunity mechanisms, and classification of CRISPR-Cas systems; analyzed the distribution and characteristics of CRISPR-Cas system in LAB. The review focuses on the development of CRISPR-Cas-based genetic tools in LAB for providing latest development and future trend. The diverse and broad applications of CRISPR-Cas systems in food/probiotic industry are introduced. LAB harbor a plenty of CRISPR-Cas systems, which contribute to generate safer and more robust strains with increased resistance against bacteriophage and prevent the dissemination of plasmids carrying antibiotic-resistance markers. Furthermore, the CRISPR-Cas system from LAB could be used to exploit novel, flexible, programmable genome editing tools of native host and other organisms, resolving the limitation of genetic operation of some LAB species, increasing the important biological functions of probiotics, improving the adaptation of probiotics in complex environments, and inhibiting the growth of foodborne pathogens. The development of the genetic tools based on CRISPR-Cas system in LAB, especially the endogenous CRISPR-Cas system, will open new avenues for precise regulation, rational design, and flexible application of LAB.

New progress in the identifying regulatory factors of exopolysaccharide synthesis in lactic acid bacteria.

The exopolysaccharides (EPSs) of lactic acid bacteria (LAB) have presented various bioactivities and beneficial characteristics, rendering their vast commercial value and attracting a broad interest of researchers. The diversity of EPS structures contributes to the changes of EPS functions. However, the low yield of EPS of LAB has severely limited these biopolymers' comprehensive studies and applications in different areas, such as functional food, health and medicine fields. The clarification of biosynthesis mechanism of EPS will accelerate the synthesis and reconstruction of EPS. In recent years, with the development of new genetic manipulation techniques, there has been significant progress in the EPS biosynthesis mechanisms in LAB. In this review, the structure of LAB-derived EPSs, the EPS biosynthesis basic pathways in LAB, the EPS biosynthetic gene cluster, and the regulation mechanism of EPS biosynthesis will be summarized. It will focus on the latest progress in EPS biosynthesis regulation of LAB and provide prospects for future related developments.

Magnetic Bead Spherical Nucleic Acid Microstructure for Reliable DNA Preservation and Repeated DNA Reading.

DNA is an attractive medium for long-term data storage because of its density, ease of copying, sustainability, and longevity. Recent advances have focused on the development of new encoding algorithms, automation, and sequencing technologies. Despite progress in these subareas, the most challenging hurdle in the deployment of DNA storage remains the reliability of preservation and the repeatability of reading. Herein, we report the construction of a magnetic bead spherical nucleic acid (MB-SNA) composite microstructure and its use as a cost-effective platform for reliable DNA preservation and repeated reading. MB-SNA has an inner core of silica@γ-Fe2O3@silica microbeads and an outer spherical shell of double-stranded DNA (dsDNA) with a density as high as 34 pmol/cm2. For MB-SNA, each strand of dsDNA stored a piece of data, and the high-density packing of dsDNA achieved high-capacity storage. MB-SNA was advantageous in terms of reliable preservation over free DNA. By accelerated aging tests, the data of MB-SNA is demonstrated to be readable after 0.23 million years of preservation at -18 °C and 50% relative humidity. Moreover, MB-SNA facilitated repeated reading by facile PCR-magnetic separation. After 10 cycles of PCR access, the retention rate of dsDNA for MB-SNA is demonstrated to be as high as 93%, and the accuracy of sequencing is more than 98%. In addition, MB-SNA makes cost-effective DNA storage feasible. By serial dilution, the physical limit for MB-SNA to achieve accurate reading is probed to be as low as two microstructures.

An optical keypad lock with high resettability based on a quantum dot-porphyrin FRET nanodevice.

Due to their appealing properties, nanomaterials have become ideal candidates for the implementation of computing systems. Herein, an optical keypad lock based on a Förster resonance energy transfer (FRET) nanodevice is developed. The nanodevice is composed of a green-emission quantum dot with a thick silica shell (gQD@SiO2) and peripheric blue-emission quantum dots with ultrathin silica spacer (bQD@SiO2), on which 5,10,15,20-tetrakis(4-sulfophenyl)porphyrin (TSPP) is covalently linked. The nanodevice outputs dual emission-based ratiometric fluorescence, depending on the FRET efficiency of bQD-porphyrin pairs, which is highly sensitive to the metalation of TSPP: values are 59.7%, 44.8%, and 10.1% for bQD-Zn(ii)TSPP, bQD-TSPP, and bQD-Fe(iii)TSPP pairs, respectively. As such, by using the competitive chelation-induced transmetalation of TSPP, the nanodevice is capable of implementing a 3-input keypad lock that is unlocked only by the correct input order of Zn(ii) chelator, iron ions, and UV light. Interestingly, the reversible transmetalation of TSPP permits the reset (lock) operation of the keypad lock with the correct input order of ascorbic acid, Zn(ii), and UV light. Application of the nanodevice is exemplified by the construction of paper and cellular keypad locks, respectively, both of which feature signal readability and/or high resettability, showing high potential for personal information identification and bio-encryption applications.

Inulin increases the EPS biosynthesis of Lactobacillus delbrueckii ssp. bulgaricus LDB-C1.

OBJECTIVE: Its eps gene cluster, the antioxidant activity and monosaccharide composition of exopolysaccharides, the expression levels of related genes at different fermentations were analyzed for clarifying the exopolysaccharide biosynthesis mechanism of Lactobacillus delbrueckii subsp. bulgaricus LDB-C1. RESULTS: The comparison analysis of eps gene clusters indicated that the gene clusters present diversity and strain specificity. The crude exopolysaccharides from LDB-C1 exhibited a good antioxidant activity. Compared with glucose, fructose, galactose, and fructooligosaccharide, inulin significantly improved the exopolysaccharide biosynthesis. The structures of EPSs were significantly different under different carbohydrate fermentation conditions. Inulin obviously increased the expressions of most EPS biosynthesis related genes at fermentation 4 h. CONCLUSION: Inulin accelerated the beginning of the exopolysaccharide production in LDB-C1, and the enzymes promoted by inulin was beneficial for the accumulation of exopolysaccharide at the whole fermentation process.

Comparative transcriptome analysis for the biosynthesis of antioxidant exopolysaccharide in Streptococcus thermophilus CS6.

BACKGROUND: Food grade Streptococcus thermophilus produces biological exopolysaccharides (EPSs) with great potential with respect to catering for higher health-promoting demands; however, how S. thermophilus regulates the biosynthesis of EPS is not completely understood, decelerating the application of these polymers. In our previous study, maltose, soy peptone and initial pH were three key factors of enhancing EPS yield in S. thermophilus CS6. Therefore, we aimed to investigate the regulating mechanisms of EPS biosynthesis in S. thermophilus CS6 via the method of comparative transcriptome and differential carbohydrate metabolism. RESULTS: Soy peptone addition (58.6 g L-1 ) and a moderate pH (6.5) contributed to a high bacterial biomass and a high EPS yield (407 mg L-1 ). Maltose, soy peptone and initial pH greatly influenced lactose utilization in CS6. Soy peptone addition induced a high accumulation of mannose and arabinose in intracellular CS6, differential monosaccharide composition (mannose, glucose and arabinose) in EPS and high radical [2,2-diphenyl-1-picrylhydrazyl, superoxide and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] scavenging activities. Carbohydrate transportation, sugar activation and eps cluster-associated genes were differentially expressed to regulate EPS biosynthesis. Correlation analysis indicated high production of EPSs depended on high expression of lacS, galPMKUTE, pgm, gt2-5&4-1 and epsLM. CONCLUSION: The production of antioxidant EPS in S. thermophilus CS6 depended on the regulation of galactose metabolism cluster and eps cluster. The present study recommends a new approach for enhancing EPS production by transcriptomic regulation for further food and health application of EPS. © 2022 Society of Chemical Industry.

Simultaneous Detection of Bladder Cancer Exosomal MicroRNAs Based on Inorganic Nanoflare and DNAzyme Walker.

Bladder cancer (BC) is one of the most common cancers in the world, with high morbidity and mortality. It is essential to develop a non-invasive, highly accurate, and simple method for BC diagnosis. This work proposed a fluorescent biosensor based on inorganic nanoflares combined with a DNAzyme walker for the simultaneous detection of BC exosomal microRNAs (miRNAs). This biosensor was constructed on the Au nanoparticle (AuNP) modified with the carbon dot (CD)-labeled substrates and DNAzyme strands (AuNP@CDs inorganic nanoflares-DNAzyme, APCD). In the presence of target miRNAs, DNAzyme was activated and then cleaved the CD-labeled substrates and automatically walked along the AuNP, allowing fluorescence recovery. Due to the structure and functional composition, the APCD biosensors demonstrated high sensitivity and specificity, with the reached limit of detection for a single miRNA at the femtomolar level and wide linear range from 50 fM to 10 nM. Furthermore, the simultaneous analysis of BC-related exosomal miR-133b and miR-135b in clinical serum specimens was achieved and consistent with qRT-PCR, suggesting it is a potential method for the diagnosis of BC and other cancers.

The Carbohydrate Metabolism of Lactiplantibacillus plantarum.

Lactiplantibacillus plantarum has a strong carbohydrate utilization ability. This characteristic plays an important role in its gastrointestinal tract colonization and probiotic effects. L. plantarum LP-F1 presents a high carbohydrate utilization capacity. The genome analysis of 165 L. plantarum strains indicated the species has a plenty of carbohydrate metabolism genes, presenting a strain specificity. Furthermore, two-component systems (TCSs) analysis revealed that the species has more TCSs than other lactic acid bacteria, and the distribution of TCS also shows the strain specificity. In order to clarify the sugar metabolism mechanism under different carbohydrate fermentation conditions, the expressions of 27 carbohydrate metabolism genes, catabolite control protein A (CcpA) gene ccpA, and TCSs genes were analyzed by quantitative real-time PCR technology. The correlation analysis between the expressions of regulatory genes and sugar metabolism genes showed that some regulatory genes were correlated with most of the sugar metabolism genes, suggesting that some TCSs might be involved in the regulation of sugar metabolism.

Structure, physicochemical characterization, and antioxidant activity of the highly arabinose-branched exopolysaccharide EPS-M2 from Streptococcus thermophilus CS6.

Streptococcus thermophilus CS6 could produce the high exopolysaccharide (EPS) level in optimized skimmed milk medium. However, physicochemical properties and structure of these polymers have not been fully characterized. In this study, two purified fractions (EPS-M1 and EPS-M2) exhibited good rheology, thermostability and antioxidant activity. Further monosaccharide composition, molecular weight and NMR analysis indicated EPS-M2 was composed of galactose, arabinose and glucose (5:2.5:1) with an average molecular weight of 2.22 × 104 Da and its suggested repeating unit was →6)-[α-L-Araf-(1 â†’ 3)]-ß-D-Galp-(1 â†’ 4)-ß-D-Galp-(1 â†’ 6)-[α-L-Araf-(1 â†’ 5)-{α-L-Araf-(1 â†’ 3)}-α-L-Araf-(1 â†’ 3)]-ß-D-Galp-(1 â†’ 4)-ß-D-Galp-(1 â†’ 6)-[ß-D-Galp-(1 â†’ 5)-α-L-Araf-(1 â†’ 5)-α-L-Araf-(1 â†’ 3)]-ß-D-Galp-(1 â†’ 6)-[ß-D-Galp-(1 â†’ 5)-α-L-Araf-(1 â†’ 5)-{α-L-Araf-(1 â†’ 3)}-α-L-Araf-(1 â†’ 3)]-ß-D-Galp-(1→. High EPS production relied on the expression of eps gene cluster and key enzymes of nucleotide sugar metabolism. Overall, EPS-M2 from a potential functional starter S. thermophilus CS6 provided opportunities for natural thickener, stabilizer, and antioxidant agent exploration in the food industry.

Safety and robustness aspects analysis of Lactobacillus delbrueckii ssp. bulgaricus LDB-C1 based on the genome analysis and biological tests.

Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) is a microaerophylic anaerobe, which is widely used in the production of yogurt, cheese, and other fermented dairy products. L. bulgaricus and its partner Streptococcus thermophilus were used as starter cultures of yogurt in the world for thousands of years. In our previous study, L. bulgaricus LDB-C1 was obtained from traditional fermented milk, and possessed some characteristics like high exopolysaccharide yield and good fermentation performance. The analysis of its CRISPR-Cas system, antibiotic resistance, virulence factors, and mobile elements, was performed to reveal the stability of the strain LDB-C1. It was found that LDB-C1 contains a plenty of spacers in the CRISPR region, indicating it might have better performance against the infection of phages and plasmids. Furthermore, the acquired or transmittable antibiotic resistance/virulence factor genes were absent in the tested L. bulgaricus strain LDB-C1.

Exosome-transmitted microRNA-133b inhibited bladder cancer proliferation by upregulating dual-specificity protein phosphatase 1.

Bladder Cancer (BC) is the ninth most common tumor in the world and one of the most common malignant tumors of the urinary system. Some studies reported that miR-133b expression is reduced in BC, but whether it plays a role in the development of BC and its mechanism is unclear. microRNAs can be packaged into exosomes to mediate communication between tumor cells, affecting their proliferation and apoptosis. The objective of this study was to investigate the effect of exosomal miR-133b on BC proliferation and its molecular mechanism. Firstly, the expression of miR-133b was evaluated in BC and adjacent normal tissues, as well as in serum exosomes of BC patients and healthy controls. Then the delivery and internalization of exosomes in cells was observed through fluorescence localization. Cell viability and apoptosis were assessed in BC cells transfected with mimics and incubated with exosomes. The role of exosomal miR-133b was also analyzed in nude mice transplant tumors. Furthermore, the target gene of miR-133b was predicted through bioinformatics. The level of miR-133b was significantly decreased in BC tissues and in exosomes from serum of patients, which was correlated with poor overall survival in TCGA. Exosomal miR-133b could be obtained using BC cells after transfection with miR-133b mimics. The miR-133b expression increased after incubation with exosomal miR-133b, which lead to the inhibition of viability and increase of apoptosis in BC cells. Exosomal miR-133b could suppress tumor growth in vivo. In addition, we found that exosomal miR-133b may play a role in suppressing BC proliferation by upregulating dual-specificity protein phosphatase 1 (DUSP1). These findings may offer promise for new therapeutic directions of BC.

Six long noncoding RNAs as potentially biomarkers involved in competitive endogenous RNA of hepatocellular carcinoma.

To investigate lncRNAs acting as competing endogenous RNAs (ceRNAs) involved in oncogenesis and progression of HCC. Different expressed lncRNAs, microRNAs, and mRNAs (DElncRNAs, DEmiRNAs, DEmRNAs), downloaded from The Cancer Genome Atlas (TCGA) database, were identified by edgeR package. CeRNA network was constructed based on miRcode, TargetScan, and miRTarBase. Target DEmRNAs were annotated by KEGG pathway and GO analysis. Negatively correlated lncRNA-miRNA pairs were analyzed by Pearson correlation coefficient, simultaneously, overall survival (OS) were evaluated. The expression of these lncRNAs were examined in HCC cell lines and tissues through qRT-PCR. 1070 DElncRNAs, 147 DEmiRNAs and 1993 DEmRNAs were acquired. CeRNA network was successfully established, including 27 lncRNAs, 5 miRNAs, and 30 mRNAs significantly correlated with OS. The DEmRNAs were significantly enriched in "Cell Cycle" and "pathways in cancer". Six lncRNAs and 2 miRNAs were negatively correlated. These lncRNAs were validated by qRT-PCR. These observations will provide a novel perspective to elucidate HCC pathogenesis.

Optimization of electrotransformation (ETF) conditions in lactic acid bacteria (LAB).

Lactic acid bacteria (LAB) play important roles in the food industry, animal husbandry and medicine which are closely related to human life. Modern gene engineering technology is the major means to reveal gene functions, study metabolic pathways and metabolomes of strains and improve the properties of strains. However, up to now, the molecular technologies that can be applied to LAB are still very scarce. One of the main reasons for this phenomenon is the low efficiency of transformation, furthermore, the transformation protocols developed are strain specific. At present, the most common method in the transformation of LAB is electrotransformation (ETF), which has relatively high transformation efficiency, convenient operation and good reproducibility. In the process of ETF, many factors may be involved in the regulation of the ETF efficiency of the strains, including the characteristics of the strains, the properties of exogenous plasmids, the parameters of electric pulse, the application of cell wall weakening agents, the composition of washing and electroporation buffers, the resuspending culture media and heat treatment. In recent years, other methods have also been utilized to boost the transformation efficiency of strains, such as combined chemical-physical methods and bacterial conjugation. Various experimental parameters and alternative technologies to ETF which may impact the transformation efficiency are summarized in this paper. This review describes some meaningful factors for the development of transformation systems, and provides direction and foundations for the optimization and establishment of novel transformation strategies of LAB.

Genome Analysis and Physiological Characterization of Four Streptococcus thermophilus Strains Isolated From Chinese Traditional Fermented Milk.

Streptococcus thermophilus plays important roles in the dairy industry and is widely used as a dairy starter in the production of fermented dairy products. The genomes of S. thermophilus strains CS5, CS9, CS18, and CS20 from fermented milk in China were sequenced and used for biodiversity analysis. In the present study, the phylogenetic analysis of all 34 S. thermophilus genomes publicly available including these four strains reveals that the phylogenetic reconstruction does not match geographic distribution as strains isolated from the same continent are not even clustered on the nearby branches. The core and variable genes were also identified, which vary among strains from 0 to 202. CS9 strain contained 127 unique genes from a variety of distantly related species. It was speculated that CS9 had undergone horizontal gene transfer (HGT) during the long evolutionary process. The safety evaluation of these four strains indicated that none of them contains antibiotic resistance genes and that they are all sensitive to multiple antibiotics. In addition, the strains do not contain any pathogenic virulence factors or plasmids and thus can be considered safe. Furthermore, these strains were investigated in terms of their technological properties including milk acidification, exopolysaccharide (EPS) and γ-aminobutyric acid (GABA) production, and in vitro survival capacity in the gastrointestinal tract. CS9 possesses a special eps gene cluster containing significant traces of HGT, while the eps gene clusters of CS5, CS18, and CS20 are almost the same. The monosaccharide compositional analysis indicated that crude EPS-CS5, EPS-CS9, EPS-CS18, and EPS-CS20 contain similar monosaccharide compositions with different ratios. Furthermore, CS9 was one of a few GABA-producing strains that could ferment glutamate to produce GABA, which is beneficial for improving the acid tolerance of the strain. CS18 has the most potential for the production of fermented food among these four strains because of its fast growth rate, rapid acidifying capacity, and stronger acid and bile salt resistance capacity. This study focused on the genome analysis of the four new S. thermophilus strains to investigate the diversity of strains and provides a reference for selecting excellent strains by use of the genome data.

Production of Gamma-Aminobutyric Acid from Lactic Acid Bacteria: A Systematic Review.

Gamma-aminobutyric acid (GABA) is widely distributed in nature and considered a potent bioactive compound with numerous and important physiological functions, such as anti-hypertensive and antidepressant activities. There is an ever-growing demand for GABA production in recent years. Lactic acid bacteria (LAB) are one of the most important GABA producers because of their food-grade nature and potential of producing GABA-rich functional foods directly. In this paper, the GABA-producing LAB species, the biosynthesis pathway of GABA by LAB, and the research progress of glutamate decarboxylase (GAD), the key enzyme of GABA biosynthesis, were reviewed. Furthermore, GABA production enhancement strategies are reviewed, from optimization of culture conditions and genetic engineering to physiology-oriented engineering approaches and co-culture methods. The advances in both the molecular mechanisms of GABA biosynthesis and the technologies of synthetic biology and genetic engineering will promote GABA production of LAB to meet people's demand for GABA. The aim of the review is to provide an insight of microbial engineering for improved production of GABA by LAB in the future.

Characterization and comparison of CRISPR Loci in Streptococcus thermophilus.

Clustered regularly interspaced short palindromic repeats (CRISPR) consists of a series of regular repeat-spacer sequences. It can not only act as a natural immune system in most prokaryotes, but also be utilized as the tool of newly developed genome modification and evolutionary researches. Streptococcus thermophilus is an important model organism for the study and application of CRISPR systems. In present study, the occurrence and diversity of CRISPR-Cas systems in the genomes of S. thermophilus were investigated including 4 new sequenced strains CS5, CS9, CS18, CS20, and other 23 strains downloaded from NCBI website. 66 CRISPR/Cas systems were identified among these 27 strains and could divided into four subsystems according to the arrangement of Cas proteins, notably I-E, II-A, II-C and III-A. Overall, 26 type II-C systems, 18 type II-A systems, 13 type III-A systems, 9 type I-E systems were identified. It was mentioned that CS20 contained two type II-C systems which had not been identified in the other 26 S. thermophilus strains. Overall, 1,080 spacers were analyzed and blasted. Sequence identity searches of spacers implied that most spacers derived from partial sequences of exogenous DNA, including various bacteriophages and plasmids. Of note, a large number of novel spacers were found in this study, indicating the unique phage environment they have undergone, especially CS20 strain. In addition, the analysis of the cas1 and cas9 genes revealed the genetic relationship among CRISPR-Cas system in these strains. Furthermore, the analysis of CRISPR spacers also indicated protospacer adjacent motif (PAM) sequences. Summary of PAM sequences could lay the foundations for the application of S. thermophilus CRISPR-Cas system. Our results suggested CS5 and CS18 can be used as model strains in the research of CRISPR-Cas system, and CS20 might have greater application potential in gene editing.

Quantum Dot Based Fluorescent Traffic Light Nanoprobe for Specific Imaging of Avidin-Type Biotin Receptor and Differentiation of Cancer Cells.

Sensitive and specific visualization of cell surface biotin receptors (BRs) a class of clinically important biomarkers, remains a challenge. In this work, a dual-emission ratiometric fluorescent nanoprobe is developed for specific imaging of cell surface avidin, a subtype of BRs. The nanoprobe comprises a dual-emission quantum dot nanohybrid, wherein a silica-encapsulated red-emitting QD (rQD@SiO2) is used as the "core" and green-emitting QDs (gQDs) are used as "satellites", which are further decorated with a new "love-hate"-type BR ligand, a phenanthroline-biotin conjugate with an amino linker. The nanoprobe shows intense rQD emission but quenched gQD emission by the BR ligand. Upon imaging, the rQD emission stays constant and the gQD emission is restored as cell surface avidin accrues. Accordingly, the overlaid fluorescence color collected from red and green emission changes from red to yellow and then to green. We refer to such a color change as a traffic light pattern and the nanoprobe as a fluorescent traffic light nanoprobe. We demonstrate the application of our fluorescent traffic light nanoprobe to characterize cancer cells. By the traffic light pattern, cervical carcinoma and normal cells, as well as different-type cancer cells including BR-negative colon cancer cells, BR-positive hepatoma carcinoma cells, breast cancer cells, and their subtypes, have been visually differentiated. We further demonstrate a use of our nanoprobe to distinguish the G2 phase from other stages in a cell cycle. These applications provide new insights into visualizing cell surface biomarkers with remarkable imaging resolution and accuracy.