Polymerase chain reaction (PCR) is a technique for nucleic acid amplification, which has been widely used in molecular biology. Owing to the limitations such as large size, high power consumption, and complicated operation, PCR is only used in hospitals or research institutions. To meet the requirements of portable applications, we developed a fast, battery-powered, portable device for PCR amplification and end-point detection. The device consisted of a PCR thermal control system, PCR reaction chip, and fluorescence detection system. The PCR thermal control system was formed by a thermal control chip and external drive circuits. Thin-film heaters and resistance temperature detectors (RTDs) were fabricated on the thermal control chip and were regulated with external drive circuits. The average heating rate was 32 °C/s and the average cooling rate was 7.5 °C/s. The disposable reaction chips were fabricated using a silicon substrate, silicone rubber, and quartz plate. The fluorescence detection system consisted a complementary metal-oxide-semiconductor (CMOS) camera, an LED, and mirror units. The device was driven by a 24 V Li-ion battery. We amplified HPV16E6 genomic DNA using our device and achieved satisfactory results.

We developed a strategy for direct DNA amplification of single cells on a PEG-modified silica chip with 30 600 picoliter-sized microwells. HPV-positive cells in heterogeneous populations were successfully detected with high accuracy sensitivity as high as single copy.

We present an integrated microfluidic device for quantifying intracellular materials at the single-cell level.

Conventional cell-sized well arrays have advantages of high occupancy, simple operation, and low cost for capturing single-cells. However, they have insufficient space for including reagents required for cell treatment or analysis, which restricts the wide application of cell-sized well arrays as a single-cell research tool alone. Here, we present a novel dual-well array chip, which integrates capture-wells (20 μm in diameter) with reaction-wells (100 μm in diameter) and describe a flow method for convenient single-cell analysis requiring neither complicated infra-structure nor high expenditure, while enabling highly efficient single cell trapping (75.8%) with only 11.3% multi-cells. Briefly, the cells are first loaded into the dual-wells by gravity and then multi-cells in the reaction-wells are washed out by phosphate buffer saline. Next, biochemical reagents are loaded into reaction-wells using the scraping method and the chip is packed as a sandwich structure. We thereby successfully measured intracellular β-galactosidase activity of K562 cells at the single-cell level. We also used computational simulations to illustrate the working principle of dual-well structure and found out a relationship between the wall shear stress distribution and the aspect ratio of the dual-well array chip which provides theoretical guidance for designing multi-wells chip for convenient single-cell analysis. Our work produced the first dual-well chip that can simultaneously provide a high occupancy rate for single cells and sufficient space for reagents, as well as being low in cost and simple to operate. We believe that the feasibility and convenience of our method will enhance its use as a practical single-cell research tool.

Heteroresistance refers to phenotypic heterogeneity of microbial clonal populations under antibiotic stress, and it has been thought to be an allocation of a subset of “resistant” cells for surviving in higher concentrations of antibiotic. The assump-tion fits the so-called bet-hedging strategy, where a bacterial population “hedges” its “bet” on different phenotypes to be selected by unpredicted environment stresses. To test this hypothesis, we constructed a heteroresistance model by introducing a bla CTX-M-14 gene (coding for a cephalosporin hydrolase) into a sensitive Escherichia coli strain. We confirmed heteroresistance in this clone and that a subset of the cells expressed more hydrolase and formed more colonies in the presence of ceftriaxone (ex-hibited stronger “resistance”). However, subsequent single-cell-level investigation by using a microfluidic device showed that a subset of cells with a distinguishable phenotype of slowed growth and intensified hydrolase expression emerged, and they were not positively selected but increased their proportion in the population with ascending antibiotic concentrations. Therefore, heteroresistance—the gradually decreased colony-forming capability in the presence of antibiotic—was a result of a decreased growth rate rather than of selection for resistant cells. Using a mock strain without the resistance gene, we further demonstrated the existence of two nested growth-centric feedback loops that control the expression of the hydrolase and maximize population growth in various antibiotic concentrations. In conclusion, phenotypic heterogeneity is a population-based strategy beneficial for bacterial survival and propagation through task allocation and interphenotypic collaboration, and the growth rate provides a critical control for the expression of stress-related genes and an essential mechanism in responding to environmental stresses. MG1655, LB

Molecular detect methods were widely used in diagnosis of infectious diseases,genetic diseases and prenatal prediction.Nowadays,with the more and more emerging infectious diseases and the development of precision medicine,automated molecular diagnostic system has attracted more and more attention as its rapid,accurate and convenient advantage.The automated molecular diagnostic systems and the company were summarized,and the development trend in this field was prospected.

Marinobacter psychrophilus strain 20041(T) was isolated from sea-ice of the Canadian Basin. Here we report the complete sequence of the 3.9-Mb genome of this strain. The complete genome sequence will facilitate the study of the physiology and evolution of Marinobacter species.

Draconibacterium orientale strain FH5(T) isolated from a marine sediment sample from coast of Weihai, China, was a new species within the proposed new genus Draconibacterium in class Bacteroidia. Here, we present the genome sequence of D. orientale FH5(T), which contains 5,132,075 bp with a G+C content of 41.31%. The genome sequence will contribute to a better understanding of the physiology of this species.

Techniques for single fluorescent molecules detection have garnered a great amount of interests from science community in recent years largely due to their ability to reveal the individual molecular properties and behaviors which were often obscured by techniques based on ensemble average.In order to overcome diffraction-limit of optical observation, fluorescent molecules must be diluted to a very low concentration-way below normal physiological concentration.However, this may adversely impact the kinetics for the chemical or biochemical reaction under study.Zero-mode waveguides ( ZMWs ) , as a new kind of single fluorescent molecule detection device, effectively reduce the observation volume to the level of zeptoliter ( 10-21 L) , which is much lower than optical diffraction-limited volume, by utilizing of a nanoaperture structure.The zeptoliter-observation volume provides an environment where a single fluorescent molecule can be observed at normal physiological concentration.The ZMWs have been extensively used in the field of single fluorescent molecule detection due to its outstanding performance than diffraction-limited optics.Therefore we provided an overview of ZMWs, including discussions on its principle, fabrication techniques, and applications in DNA sequencing, biomembrane structure elucidation, biomacromolecule interaction studies and kinetics study of single-molecule reaction.

The chloroplast and mitochondrion of brown algae（Class Phaeophyceae of Phylum Ochrophyta） may have originated from different endosymbiosis.In this study,we carried out phylogenomic analysis to distinguish their evolutionary lineages by using algal RNA-seq datasets of the 1 000 Plants（1KP） Project and publicly available complete genomes of mitochondria and chloroplasts of Kingdom Chromista.We have found that there is a split between Class Phaeophyceae of Phylum Ochrophyta and the others（Phylum Cryptophyta and Haptophyta） in Kingdom Chromista,and identified more diversity in chloroplast genes than mitochondrial ones in their phylogenetic trees.Taxonomy resolution for Class Phaeophyceae showed that it was divided into Laminariales-Ectocarpales clade and Fucales clade,and phylogenetic positions of Kjellmaniella crassifolia,Hizikia fusifrome and Ishige okamurai were confirmed.Our analysis provided the basic phylogenetic relationships of Chromista algae,and demonstrated their potential ability to study endosymbiotic events.

The human pharyngeal microbiome, which resides at the juncture of digestive and respiratory tracts, may have an active role in the prevention of respiratory tract infections, similar to the actions of the intestinal microbiome against enteric infections. Recent studies have demonstrated that the pharyngeal microbiome comprises an abundance of bacterial species that interacts with the local epithelial and immune cells, and together, they form a unique micro-ecological system. Most of the microbial species in microbiomes are obligate symbionts constantly adapting to their unique surroundings. Indigenous commensal species are capable of both maintaining dominance and evoking host immune responses to eliminate invading species. Temporary damage to the pharyngeal microbiome due to the impaired local epithelia is also considered an important predisposing risk factor for infections. Therefore, reinforcement of microbiome homeostasis to prevent invasion of infection-prone species would provide a novel treatment strategy in addition to antibiotic treatment and vaccination. Hence continued research efforts on evaluating probiotic treatment and developing appropriate procedures are necessary to both prevent and treat respiratory infections.

ABSTRACT Heteroresistance refers to phenotypic heterogeneity of microbial clonal populations under antibiotic stress, and it has been thought to be an allocation of a subset of “resistant” cells for surviving in higher concentrations of antibiotic. The assumption fits the so-called bet-hedging strategy, where a bacterial population “hedges” its “bet” on different phenotypes to be selected by unpredicted environment stresses. To test this hypothesis, we constructed a heteroresistance model by introducing a blaCTX-M-14 gene (coding for a cephalosporin hydrolase) into a sensitive Escherichia coli strain. We confirmed heteroresistance in this clone and that a subset of the cells expressed more hydrolase and formed more colonies in the presence of ceftriaxone (exhibited stronger “resistance”). However, subsequent single-cell-level investigation by using a microfluidic device showed that a subset of cells with a distinguishable phenotype of slowed growth and intensified hydrolase expression emerged, and they were not positively selected but increased their proportion in the population with ascending antibiotic concentrations. Therefore, heteroresistance—the gradually decreased colony-forming capability in the presence of antibiotic—was a result of a decreased growth rate rather than of selection for resistant cells. Using a mock strain without the resistance gene, we further demonstrated the existence of two nested growth-centric feedback loops that control the expression of the hydrolase and maximize population growth in various antibiotic concentrations. In conclusion, phenotypic heterogeneity is a population-based strategy beneficial for bacterial survival and propagation through task allocation and interphenotypic collaboration, and the growth rate provides a critical control for the expression of stress-related genes and an essential mechanism in responding to environmental stresses. IMPORTANCE Heteroresistance is essentially phenotypic heterogeneity, where a population-based strategy is thought to be at work, being assumed to be variable cell-to-cell resistance to be selected under antibiotic stress. Exact mechanisms of heteroresistance and its roles in adaptation to antibiotic stress have yet to be fully understood at the molecular and single-cell levels. In our study, we have not been able to detect any apparent subset of “resistant” cells selected by antibiotics; on the contrary, cell populations differentiate into phenotypic subsets with variable growth statuses and hydrolase expression. The growth rate appears to be sensitive to stress intensity and plays a key role in controlling hydrolase expression at both the bulk population and single-cell levels. We have shown here, for the first time, that phenotypic heterogeneity can be beneficial to a growing bacterial population through task allocation and interphenotypic collaboration other than partitioning cells into different categories of selective advantage.

Three Gracilaria species, G. chouae, G. blodgettii, G. vermiculophylla and a close relative species, Gracilariopsis lemaneiformis which is now nominated as Gracilaria lemaneiformis, are the typically indigenous species which are important resources for the production of special proteins, phycobilisomes, special carbohydrates, and agar in China. In this study, de novo transcriptome sequencing on these four species using the next generation sequencing technology was performed for the first time. Functional annotations on assembled sequencing reads showed that the transcriptomic profiles were quite different between G. lemaneiformis and other three Gracilaria species. Comparative analysis of differential gene expression related to carbohydrate and phycobiliprotein metabolisms also showed that the expression profiles of these essential genes were different in four species. The genes encoding allophycocyanin, phycocyanin and phycoerythrin were further examined in four species and their deduced amino acid sequences were used for phylogenetic analysis to confirm that G. lemaneiformis had close relationship to genus Gracilaria, as well as that within genus Gracilaria, G. chouae had closer relationship to G. vermiculophylla rather than to G. blodgettii. The de novo transcriptome study on four species provided a valuable genomic resource for further understanding and analysis on biological and evolutionary study among marine algae.

Brown algae（Chromista,Ochrophyta,Phaeophyceae） are a large group of multicellular algae that play important roles in the ocean’s ecosystem and biodiversity.However,poor molecular bases for studying their phylogenetic evolutions and novel metabolic characteristics have hampered progress in the field.In this study,we sequenced the de novo transcriptome of 18 major species of brown algae in China,covering six orders and seven families,using the high-throughput sequencing platform Illumina HiSeq 2000.From the transcriptome data of these 18 species and publicly available genome data of Ectocarpus siliculosus and Phaeodactylum tricornutum,we identified 108 nuclear-generated orthologous genes and clarified the phylogenetic relationships among these brown algae based on a multigene method.These brown algae could be separated into two clades: Clade Ishigeales-Dictyotales and Clade Ectocarpales-Laminariales-Desmarestiale-Fucales.The former was at the base of the phylogenetic tree,indicating its early divergence,while the latter was divided into two branches,with Order Fucales diverging from Orders Ectocarpales,Laminariales,and Desmarestiale.In our analysis of taxonomy-contentious species,Sargassum fusiforme and Saccharina sculpera were found to be closely related to genera Sargassum and Saccharina,respectively,while Petalonia fascia showed possible relation to genus Scytosiphon.The study provided molecular evidence for the phylogenetic taxonomy of brown algae.

The common carp, Cyprinus carpio, is one of the most important cyprinid species and globally accounts for 10% of freshwater aquaculture production. Here we present a draft genome of domesticated C. carpio (strain Songpu), whose current assembly contains 52,610 protein-coding genes and approximately 92.3% coverage of its paleotetraploidized genome (2n = 100). The latest round of whole-genome duplication has been estimated to have occurred approximately 8.2 million years ago. Genome resequencing of 33 representative individuals from worldwide populations demonstrates a single origin for C. carpio in 2 subspecies (C. carpio Haematopterus and C. carpio carpio). Integrative genomic and transcriptomic analyses were used to identify loci potentially associated with traits including scaling patterns and skin color. In combination with the high-resolution genetic map, the draft genome paves the way for better molecular studies and improved genome-assisted breeding of C. carpio and other closely related species.

DNA sequencing using reversible terminators, as one sequencing by synthesis strategy, has garnered a great deal of interest due to its popular application in the second-generation high-throughput DNA sequencing technology. In this review, we provided its history of development, classification, and working mechanism of this technology. We also outlined the screening strategies for DNA polymerases to accommodate the reversible terminators as substrates during polymerization; particularly, we introduced the "REAP" method developed by us. At the end of this review, we discussed current limitations of this approach and provided potential solutions to extend its application.