Single-cell analyses of circulating tumor cells

Circulating tumor cells(CTCs) are a population of tumor cells mediating metastasis, which results in most of the cancer related deaths. The number of CTCs in the peripheral blood of patients is rare, and many platforms have been launched for detection and enrichment of CTCs. Enumeration of CTCs has already been used as a prognosis marker predicting the survival rate of cancer patients. Yet CTCs should be more potential. Studies on CTCs at single cell level may help revealing the underlying mechanism of tumorigenesis and metastasis. Though far from developed, this area of study holds much promise in providing new clinical application and deep understanding towards metastasis and cancer development.

A race to uncover a panoramic view of primary liver cancer

Introduction Primary liver cancer,the second most common cause of cancer related death worldwide1,presents ethnic,etiological,sex,and geographical diversity2(Figure 1A).At the histological level,liver cancer includes two major types:hepatocellular carcinoma(HCC,about 80%)and cholangiocarcinoma(CCA,about 15%).Many etiological factors contribute to HCC development,such as hepatitis

Frequency-domain diagonal extension imaging

The pixel size of a charge-coupled device(CCD)camera plays a major role in the image resolution,and the square pixels are attributed to the physical anisotropy of the sampling frequency.We synthesize the high sampling frequency directions from multiple frames acquired with different angles to enhance the resolution by 1.4×over conventional CCD orthogonal sampling.To directly demonstrate the improvement of frequency-domain diagonal extension(FDDE)microscopy,lens-free microscopy is used,as its resolution is dominantly determined by the pixel size.We demonstrate the resolution enhancement with a mouse skin histological specimen and a clinical blood smear sample.Further,FDDE is extended to lens-based photography with an ISO 12233 resolution target.This method paves a new way for enhancing the image resolution for a variety of imaging techniques in which the resolution is primarily limited by the sampling pixel size,for example,microscopy,photography,and spectroscopy.

Photoblinkingpolymer dots shine in super-resolution fluorescence imaging

Subject Code:B03With the support by the National Natural Science Foundation of China,a collaborative study by the research groups led by Prof.Sun Yujie(孙育杰)from the State Key Laboratory of Membrane Biology,Biodynamic Optical Imaging Center(BIOPIC),School of Life Sciences,Peking University and Prof.

Temporal and spatial stability of the EM/PM molecular subtypes in adult diffuse glioma

Detailed characterizations of genomic alterations have not identified subtype-specific vulnerabilities in adult gliomas. Mapping gliomas into developmental programs may uncover new vulnerabilities that are not strictly related to genomic alterations. After identifying conserved gene modules co-expressed with EGFR or PDGFRA (EM or PM), we recently proposed an EM/PM classification scheme for adult gliomas in a histological subtype- and grade-independent manner. By using cohorts of bulk samples, paired primary and recurrent samples, multi-region samples from the same glioma, single-cell RNA-seq samples, and clinical samples, we here demonstrate the temporal and spatial stability of the EM and PM subtypes. The EM and PM subtypes, which progress in a subtype-specific mode, are robustly maintained in paired longitudinal samples. Elevated activities of cell proliferation, genomic instability and microenvironment, rather than subtype switching, mark recurrent gliomas. Within individual gliomas, the EM/PM subtype was preserved across regions and single cells. Malignant cells in the EM and PM gliomas were correlated to neural stem cell and oligodendrocyte progenitor cell compartment, respectively. Thus, while genetic makeup may change during progression and/or within different tumor areas, adult gliomas evolve within a neurodevelopmental framework of the EM and PM molecular subtypes. The dysregulated developmental pathways embedded in these molecular subtypes may contain subtype-specific vulnerabilities.

An integrated microfluidic device for long-term culture of isolated single mammalian cells

We developed an integrated microfluidic chip for long-term culture of isolated single cells. This polydimethylsiloxane (PDMS) based device could accurately seed each single cell into different culture chambers, and isolate one chamber from each other with monolithically integrated pneumatic valves. We optimized the culture conditions, including the frequency of medium replacement and the introduction of conditioned medium, to keep the single cells alive for 4 days. We cultured a few hundred cells in a separated chamber on the same chip to continuously supply the conditioned medium into the culture chambers for single cells. This approach greatly facilitated the growth of single cells, and created a suitable microenvironment for observing cells' autonomous process in situ without the interference of other adjacent cells. This single cell colony assay is expandable to higher throughput, fitting the needs in the studies of drug screening and stem cell differentiation.

Transcriptome analyses of insect cells to facilitate baculovirus-insect expression

The High Five cell line （BTI-TN-5B1-4） isolated from the cabbage looper, Trichoplusia ni is an insect cell line widely used for baculovirus-mediated recombinant protein expression. Despite its widespread application in industry and academic laboratories, the genomic background of this cell line remains unclear. Here we sequenced the transcriptome of High Five cells and assembled 25,234 transcripts. Codon usage analysis showed that High Five cells have a robust codon usage capacity and therefore suit for expressing proteins of both eukaryotic- and prokaryotic-origin. Genes involved in glycosylation were profiled in our study, providing guidance for engineering glycosylated proteins in the insect cells. We also predicted signal peptides for transcripts with high expression abundance in both High Five and Sf21 cell lines, and these results have important implications for optimizing the expression level of some secretory and membrane proteins.

Generation of tooth-like structures from integration-free human urine induced pluripotent stem cells

Background:Tooth is vital not only for a good smile,but also good health.Yet,we lose tooth regularly due to accidents or diseases.An ideal solution to this problem is to regenerate tooth with patients’own cells.Here we describe the generation of tooth-like structures from integration-free human urine induced pluripotent stem cells(ifhU-iPSCs).Results:We first differentiated ifhU-iPSCs to epithelial sheets,which were then recombined with E14.5 mouse dental mesenchymes.Tooth-like structures were recovered from these recombinants in 3 weeks with success rate up to 30%for 8 different iPSC lines,comparable to H1 hESC.We further detected that ifhU-iPSC derived epithelial sheets differentiated into enamel-secreting ameloblasts in the tooth-like structures,possessing physical properties such as elastic modulus and hardness found in the regular human tooth.Conclusion:Our results demonstrate that ifhU-iPSCs can be used to regenerate patient specific dental tissues or even tooth for further drug screening or regenerative therapies.

Bacterial persistence

Although bacterial persistence was first observed in 1944,its underlying mechanism is just beginning to be understood and many fundamental questions remain.In this review,we summarize studies in order to chart the full map of bacterial persistence.Because persistence significantly contributes to disease recalcitrance,we also elucidate the probable relationships between bacterial persistence and prolonged chronic infections,with some comments on future research directions and therapeutic strategies.

Cocaine Detection in Blood Serum Using Aptamer Biosensor on Gold Nanoparticles and Progressive Dilution

To fight against the illegal usage of cocaine it is necessary to develop various analytical methodologies. We report here an aptamer-based biosensor for the determination of cocaine using gold nanoparticles as the fluorescence quencher. By employing the progressive dilution （PD） strategy, simultaneous qualitative and quantitative analysis of cocaine in blood serum was achieved without pretreatment of the sample. The method described in this paper of- fers significant improvement in the detection accuracy and can be used to quantify cocaine levels in complex bio- logical samples such as serum with a simple procedure.

C13C4.5/Spinster, an evolutionarily conserved protein that regulates fertility in C. elegans through a lysosome-mediated lipid metabolism process

Lipid droplets, which are conserved across almost all species, are cytoplasmic organelles used to store neutral lipids. Identification of lipid droplet regulators will be conducive to resolving obesity and other fat-associated diseases. In this paper, we selected 11 candidates that might be associated with lipid metabolism in Caenorhabditis elegans . Using a BODIPY 493/503-based flow cytometry screen, 6 negative and 3 positive regulators of fat content were identified. We selected one negative regulator of lipid content, C13C4.5 , for future study. C13C4.5 was mainly expressed in the worm intestine. We found that this gene was important for maintaining the metabolism of lipid droplets. Biochemical results revealed that 50% of triacylglycerol (TAG) was lost in C13C4.5 knockout worms. Stimulated Raman scattering (SRS) signals in C13C4.5 mutants showed only 49.6% of the fat content in the proximal intestinal region and 86.3% in the distal intestinal region compared with wild type animals. The mean values of lipid droplet size and intensity in C13C4.5 knockout animals were found to be significantly decreased compared with those in wild type worms. The LMP-1-labeled membrane structures in worm intestines were also enlarged in C13C4.5 mutant animals. Finally, fertility defects were found in C13C4.5 ( ok2087 ) mutants. Taken together, these results indicate that C13C4.5 may regulate the fertility of C. elegans by changing the size and fat content of lipid droplets by interfering with lysosomal morphology and function.

MR-seq:measuring a single cell's transcriptome repeatedly by RNA-seq

We described a novel single-cell RNA-seq technique called MR-seq(measure a single-cell transcriptome repeatedly),which permits statistically assessing the technical variation and identifying the differentially expressed genes between just two single cells by measuring each single cell twice. We demonstrated that MR-seq gave sensitivity and reproducibility similar to the standard single-cell RNA-seq and increased the positive predicate value. Application of MR-seq to early mouse embryos identified hundreds of candidate intra-embryonic heterogeneous genes among mouse 2-,4-and 8-cell stage embryos. MR-seq should be useful for detecting differentially expressed genes among a small number of cells.

Distinct lung microbial community states in patients with pulmonary tuberculosis

An improved understanding of the lung microbiome may lead to better strategies to diagnose, treat, and prevent pulmonary tuberculosis (PTB). However, the characteristics of the lung microbiomes of patients with TB remain largely undefined. In this study, 163 bronchoalveolar lavage (BAL) samples were collected from 163 sputum-negative suspected PTB patients. Furthermore, 12 paired BAL samples were obtained from 12 Mycobacterium tuberculosis-positive (MTB+) patients before and after negative conversion following a two-month anti-TB treatment. The V3–V4 region of the 16S ribosomal RNA (rRNA) gene was used to characterize the microbial composition of the lungs. The results showed that the prevalence of MTB in the BAL samples was 42.9% (70/163) among the sputum-negative patients. The α-diversity of lung microbiota was significantly less diverse in MTB+ patients compared with Mycobacterium tuberculosis-negative (MTB–) patients. There was a significant difference in β-diversity between MTB+ and MTB– patients. MTB+ patients were enriched with Anoxybacillus, while MTB– patients were enriched with Prevotella, Alloprevotella, Veillonella, and Gemella. There was no significant difference between the Anoxybacillus detection rates of MTB+ and MTB– patients. The paired comparison between the BAL samples from MTB+ patients and their negative conversion showed that BAL negative-conversion microbiota had a higher α-diversity. In conclusion, distinct features of airway microbiota could be identified between samples from patients with and without MTB. Our results imply links between lung microbiota and different clinical groups of active PTB.

Illuminating the structure and dynamics of chromatin by fluorescence labeling

BACKGROUND： Visualization of chromosomal loci location and dynamics is crucial for understanding many fundamental intra-nuclear processes such as DNA transcription, replication, and repair. OBJECTIVE： Here, we will describe the development of fluorescence labeling methods for chromatin imaging, including traditional as well as emerging chromatin labeling techniques in both fixed and live cells. We will also discuss current issues and provide a perspective on future developments and applications of the chromatin labeling technology. METHODS： A systematic literature search was performed using the PubMed. Studies published over the past 50 years were considered for review. More than 100 articles were cited in this review. RESULTS： Taking into account sensitivity, specificity, and spatiotemporal resolution, fluorescence labeling and imaging has been the most prevalent approach for chromatin visualization. Among all the fluorescent labeling tools, the adoption ofgenome editing tools, such as TALE and CRISPR, have great potential for the labeling and imaging of chromatin. CONCLUSION： Although a number of chromatin labeling techniques are available for both fixed and live cells, much more effort is still clearly required to develop fluorescence labeling methods capable of targeting arbitrary sequences non-intrusively to allow long-term, multiplexing, and high-throughput imaging of genomic loci and chromatin structures. The emerging technological advances will outline a next-generation effort toward the comprehensive delineation of chromatin at single-cell level with single-molecule resolution.

Terminal transfer amplification and sequencing for high-efficiency and lowbias copy number profiling of fragmented DNA samples

Dear Editor,Since its invention,next generation sequencing(NGS)has greatly facilitated biomedical research and clinical diagnosis(Sikkema-Raddatz et al.,2013).Continuous dropping of the cost further accelerated the adaptation of sequencing as a standard analytical tool,from identification of drug candidates(Walker et al.,2015)to deciphering the complex biological systems(McConnell et al.,2013).

Single cell RNA-seqrevealed landscape of infiltrating T cells in HBV+ liver cancer

Subject Code:H16With the support by the National Natural Science Foundation of China,a collaborative study by the research groups led by Prof.Zhang Zemin(张泽民)from the Biodynamic Optical Imaging Center(BIOPIC),Peking University,Prof.Peng Jirun(彭吉润)from Beijing Shijitan Hospital,Capital