Tn5 transposase and tagmentation procedures for massively scaled sequencing projects.

Massively parallel DNA sequencing of thousands of samples in a single machine-run is now possible, but the preparation of the individual sequencing libraries is expensive and time-consuming. Tagmentation-based library construction, using the Tn5 transposase, is efficient for generating sequencing libraries but currently relies on undisclosed reagents, which severely limits development of novel applications and the execution of large-scale projects. Here, we present simple and robust procedures for Tn5 transposase production and optimized reaction conditions for tagmentation-based sequencing library construction. We further show how molecular crowding agents both modulate library lengths and enable efficient tagmentation from subpicogram amounts of cDNA. The comparison of single-cell RNA-sequencing libraries generated using produced and commercial Tn5 demonstrated equal performances in terms of gene detection and library characteristics. Finally, because naked Tn5 can be annealed to any oligonucleotide of choice, for example, molecular barcodes in single-cell assays or methylated oligonucleotides for bisulfite sequencing, custom Tn5 production and tagmentation enable innovation in sequencing-based applications.

Full-length RNA-seq from single cells using Smart-seq2.

Emerging methods for the accurate quantification of gene expression in individual cells hold promise for revealing the extent, function and origins of cell-to-cell variability. Different high-throughput methods for single-cell RNA-seq have been introduced that vary in coverage, sensitivity and multiplexing ability. We recently introduced Smart-seq for transcriptome analysis from single cells, and we subsequently optimized the method for improved sensitivity, accuracy and full-length coverage across transcripts. Here we present a detailed protocol for Smart-seq2 that allows the generation of full-length cDNA and sequencing libraries by using standard reagents. The entire protocol takes ∼2 d from cell picking to having a final library ready for sequencing; sequencing will require an additional 1-3 d depending on the strategy and sequencer. The current limitations are the lack of strand specificity and the inability to detect nonpolyadenylated (polyA(-)) RNA.

Smart-seq2 for sensitive full-length transcriptome profiling in single cells.

Single-cell gene expression analyses hold promise for characterizing cellular heterogeneity, but current methods compromise on either the coverage, the sensitivity or the throughput. Here, we introduce Smart-seq2 with improved reverse transcription, template switching and preamplification to increase both yield and length of cDNA libraries generated from individual cells. Smart-seq2 transcriptome libraries have improved detection, coverage, bias and accuracy compared to Smart-seq libraries and are generated with off-the-shelf reagents at lower cost.

An update on viral association of human cancers.

Up to now, seven viruses that infect humans have been identified as oncogenic and are closely associated with different human cancers. Most of them encode oncogenes whose products play important roles in the development of cancers in the context of environmental and genetic factors; others may act via indirect mechanisms. The transforming activities of the human oncogenic viruses have much in common with the well-studied tumorigenic processes elicited by the acutely transforming murine retroviruses. Many of these mechanisms have been elucidated for or are represented in the successive steps leading to the efficient in vitro immortalization by the lymphotropic herpesvirus Epstein-Barr virus, although the establishment of malignancy in vivo takes longer. The development of cancer is a complicated process involving multiple factors, from the host and the environment. Although any one of these etiologic factors may exert an effect on the carcinogenic process, vaccination against the viral pathogen in several cases has shown efficacy in preventing the spread of the virus and, in turn, the development of the associated cancers. Modern laboratory techniques can be expected to facilitate the identification of new emerging viruses whose association with malignancies is suggested by epidemiologic and clinical data.

Expression, purification and functional analysis of an odorant binding protein AaegOBP22 from Aedes aegypti.

Mosquitoes that act as disease vectors rely upon olfactory cues for host-seeking, mating, blood feeding and oviposition. To reduce the risk of infection in humans, one of the approaches focuses on mosquitoes' semiochemical system in the effort to disrupt undesirable host-insect interaction. Odorant binding proteins (OBPs) play a key role in mosquitoes' semiochemical system. Here, we report the successful expression, purification of an odorant binding protein AaegOBP22 from Aedes aegypti in heterologous system. Protein purification methods were set up by Strep-Tactin affinity binding and size-exclusion chromatography. Analysis by SDS-PAGE and mass spectrum revealed the protein's purity and molecular weight. Circular dichroism spectra showed the AaegOBP22 secondary structure had a pH dependent conformational change. The protein functions of AaegOBP22 were tested by fluorescent probe 1-NPN binding assays and ligands competitive binding assays. The results show AaegOBP22 proteins have characteristics of selective binding with various ligands.

Epstein-Barr virus infection leads to partial phenotypic reversion of terminally differentiated malignant B cells.

The B cell lymphomas associated with Epstein-Barr virus (EBV) are not limited to any specific stage of B cell differentiation but covers widely different B cell phenotypes. In vitro infection of the virus negative tumors with a recombinant EBV strain has provided important insights into virus-tumor interaction. Here, we investigated the interaction between EBV and terminally differentiated tumor derived B cells, namely multiple myeloma (MM). The in vitro EBV infected MM expressed restricted viral latency. Acquisition of the virus was accompanied by a partial reprogramming to a mature B cell phenotype. Thus, the plasma cell markers syndecan-1 (CD138), Blimp1 and MUM1 were downregulated, while expression of HLADR, CIITA and TCL1, which are normally not expressed in plasmacytoid cells, was upregulated. The silenced transcription factor gene encoding Pax5 and its target BLNK were activated. Significantly, the free lambda light chains secreted in the medium were reduced in EBV infected MM clones. Collectively, these results suggest that the restricted EBV latency can cause at least partial phenotypic reversion of terminally differentiated B tumor cells. We suggest that the restricted EBV latent gene expression may not only be the consequence but the cause of the mature B cell phenotype, actively participating in the virus persistence.

The K15 protein of Kaposi's sarcoma-associated herpesvirus recruits the endocytic regulator intersectin 2 through a selective SH3 domain interaction.

Kaposi's sarcoma-associated herpesvirus, also known as human herpesvirus 8, is closely associated with several cancers including Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. The rightmost end of the KSHV genome encodes a protein, K15, with multiple membrane-spanning segments and an intracellular carboxy-terminal tail that contains several conserved motifs with the potential to recruit interaction domains (i.e., SH2, SH3, TRAF) of host cell proteins. K15 has been implicated in downregulating B cell receptor (BCR) signaling through its conserved motifs and may thereby play a role in maintaining viral latency and/or preventing apoptosis of the infected B cells. However, K15's mode of action is largely unknown. We have used mass spectrometry, domain and peptide arrays, and surface plasmon resonance to identify binding partners for a conserved proline-rich sequence (PPLP) in the K15 cytoplasmic tail. We show that the PPLP motif selectively binds the SH3-C domain of an endocytic adaptor protein, Intersectin 2 (ITSN2). This interaction can be observed both in vitro and in cells, where K15 and ITSN2 colocalize to discrete compartments within the B cell. The ability of K15 to associate with ITSN2 suggests a new role for the K15 viral protein in intracellular protein trafficking.

Activity profiling of deubiquitinating enzymes in cervical carcinoma biopsies and cell lines.

Ubiquitin specific proteases (USPs) regulate the production and recycling of ubiquitin and are thereby critically involved in the control of cell growth, differentiation, and apoptosis. Increasing evidence implicates deregulation of USPs in malignant transformation but there is very little information on the overall and specific activity of USPs in normal and tumor tissues. We have used a chemistry-based functional proteomics approach to profile the activities of individual USPs in biopsies of human papillomavirus (HPV) carrying cervical carcinoma and adjacent normal tissue. To assess the contribution of HPV proteins, USP activity was also compared in HPV positive and negative cervical carcinoma cell lines and HPV E6/E7 immortalized human keratinocytes. The activity of the C-terminal hydrolases UCH-L3 and UCH37 was upregulated in the majority of tumor tissues compared to the adjacent normal tissues. UCH-L1 activity was lower in a significant proportion of the tumors but to a less extent in advanced tumors. In accordance with the relatively low UCH-L1 activity in tumor biopsies, UCH-L1 was detected only in one out of eight cervical carcinoma lines. UCH-L1, UCH-L3, USP7, and USP9X activity was upregulated following HPV E6/E7 immortalization of keratinocytes, suggesting a role of these enzymes in growth transformation.

Responses of mice immunized with a DNA vaccine encoding carcinoembryonic antigen (CEA).

C57BL/6 and BALB/c mice were immunized with two different plasmids (p91023B and pKCEA66) encoding different forms of the tumor-associated antigen carcinoembryonic antigen (CEA). The wild type form of CEA (p91023B), which is expressed at the cell surface, induces stronger anti-CEA IgG response after DNA-plasmid immunizations than the modified intracellular form of CEA (pKCEA66), which was designed to mount strong cellular responses. Boosting with recombinant CEA (rCEA) increased the anti-CEA IgG response significantly. In the tumor protection model used, where SCID mice are challenged with human tumor cells mixed with splenocytes from immunized mice, both innate and specific immune responses are responsible for the protective effect.

Expression of CD27-CD70 on early B cell progenitors in the bone marrow: implication for diagnosis and therapy of childhood ALL.

OBJECTIVE: CD27, a member of the TNF receptor family, plays an important role in lymphoid proliferation, differentiation and apoptosis. This study addresses the expression of CD27 and its ligand, CD70, in children with acute lymphoblastic leukemia (ALL) and the possible role of this receptor-ligand pair in the pathogenesis of ALL. PATIENTS AND METHODS: Expression of CD27 and CD70 was evaluated with three-color flow cytometry in blood and bone marrow (BM) samples in children with ALL and controls. The biological role of these molecules on leukemic cell proliferation was studied in an in vitro culture system. RESULTS: The expression of the membrane bound CD27, as well as membrane bound CD70, on CD19(+) cells in the BM was significantly increased in ALL children compared to the expression found in the controls. Importantly, a substantial reduction in the in vitro proliferation of leukemic cells could be observed when the leukemic cells were cultured in presence of a blocking anti-human CD70 monoclonal antibody. The level of soluble CD27 (sCD27) in serum was also investigated and found to be significantly elevated in leukemic children as compared to healthy children. CONCLUSION: The high expression of CD27 and CD70 on ALL cells may represent an amplification of the normal CD27-CD70 expression present on early B cell progenitors. Our finding suggests that interference with CD27-CD70 interaction may represent novel treatment opportunities in ALL. Further studies are required to pin-point the role of this receptor-ligand pair in normal and malignant hematopoiesis.

WW domains provide a platform for the assembly of multiprotein networks.

WW domains are protein modules that mediate protein-protein interactions through recognition of proline-rich peptide motifs and phosphorylated serine/threonine-proline sites. To pursue the functional properties of WW domains, we employed mass spectrometry to identify 148 proteins that associate with 10 human WW domains. Many of these proteins represent novel WW domain-binding partners and are components of multiprotein complexes involved in molecular processes, such as transcription, RNA processing, and cytoskeletal regulation. We validated one complex in detail, showing that WW domains of the AIP4 E3 protein-ubiquitin ligase bind directly to a PPXY motif in the p68 subunit of pre-mRNA cleavage and polyadenylation factor Im in a manner that promotes p68 ubiquitylation. The tested WW domains fall into three broad groups on the basis of hierarchical clustering with respect to their associated proteins; each such cluster of bound proteins displayed a distinct set of WW domain-binding motifs. We also found that separate WW domains from the same protein or closely related proteins can have different specificities for protein ligands and also demonstrated that a single polypeptide can bind multiple classes of WW domains through separate proline-rich motifs. These data suggest that WW domains provide a versatile platform to link individual proteins into physiologically important networks.

The Epstein-Barr virus protein, latent membrane protein 2A, co-opts tyrosine kinases used by the T cell receptor.

Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis and is associated with several human malignancies. The EBV protein latent membrane protein 2A (LMP2A) promotes viral latency in memory B cells by interfering with B cell receptor signaling and provides a survival signal for mature B cells that have lost expression of surface immunoglobulin. The latter function has suggested that LMP2A may enhance the survival of EBV-positive tumors. EBV is associated with several T cell malignancies and, since LMP2A has been detected in several of these disorders, we examined the ability of LMP2A to transmit signals and interfere with T cell receptor signaling in T cells. We show that LMP2A is tyrosine-phosphorylated in Jurkat TAg T cells, which requires expression of the Src family tyrosine kinases, Lck and Fyn. Lck and Fyn are recruited to the tyrosine-phosphorylated Tyr112 site in LMP2A, whereas phosphorylation of an ITAM motif in LMP2A creates a binding site for the ZAP-70/Syk tyrosine kinases. LMP2A also associates through its two PPPPY motifs with AIP4, a NEDD4 family E3 ubiquitin ligase; this interaction results in ubiquitylation of LMP2A and serves to regulate the stability of LMP2A and LMP2A-kinase complexes. Furthermore, stable expression of LMP2A in Jurkat T cells down-regulated T cell receptor levels and attenuated T cell receptor signaling. Thus, through recruiting tyrosine kinases involved in T cell receptor activation, LMP2A may provide a survival signal for EBV-positive T cell tumors, whereas LMP2A-associated NEDD4 E3 ligases probably titer the strength of this signal.

RBSP3 (HYA22) is a tumor suppressor gene implicated in major epithelial malignancies.

Chromosome 3p21.3 region is frequently (>90%) deleted in lung and other major human carcinomas. We subdivided 3p21.3 into LUCA and AP20 subregions and discovered frequent homozygous deletions (10-18%) in both subregions. This finding strongly implies that they harbor multiple tumor suppressor genes involved in the origin and/or development of major epithelial cancers. In this study, we performed an initial analysis of RBSP3/HYA22, a candidate tumor suppressor genes located in the AP20 region. Two sequence splice variants of RBSP3/HYA22 (A and B) were identified, and we provide evidence for their tumor suppressor function. By sequence analysis RBSP3/HYA22 belongs to a gene family of small C-terminal domain phosphatases that may control the RNA polymerase II transcription machinery. Expression of the gene was drastically (>20-fold) decreased in 11 of 12 analyzed carcinoma cell lines and in three of eight tumor biopsies. We report missense and nonsense mutations in tumors where RBSP3/HYA22 was expressed, growth suppression with regulated transgenes in culture, suppression of tumor formation in severe combined immunodeficient mice, and dephosphorylation of ppRB by RBSP3/HYA22, presumably leading to a block of the cell cycle at the G1/S boundary.

Restriction site tagged (RST) microarrays: a novel technique to study the species composition of complex microbial systems.

We have developed a new type of microarray, restriction site tagged (RST), for example NotI, microarrays. In this approach only sequences surrounding specific restriction sites (i.e. NotI linking clones) were used for generating microarrays. DNA was labeled using a new procedure, NotI representation, where only sequences surrounding NotI sites were labeled. Due to these modifications, the sensitivity of RST microarrays increases several hundred-fold compared to that of ordinary genomic microarrays. In a pilot experiment we have produced NotI microarrays from Gram-positive and Gram-negative bacteria and have shown that even closely related Escherichia coli strains can be easily discriminated using this technique. For example, two E.coli strains, K12 and R2, differ by less than 0.1% in their 16S rRNA sequences and thus the 16S rRNA sequence would not easily discriminate between these strains. However, these strains showed distinctly different hybridization patterns with NotI microarrays. The same technique can be adapted to other restriction enzymes as well. This type of microarray opens the possibility not only for studies of the normal flora of the gut but also for any problem where quantitative and qualitative analysis of microbial (or large viral) genomes is needed.

Signal sequence deletion and fusion to tetanus toxoid epitope augment antitumor immune responses to a human carcinoembryonic antigen (CEA) plasmid DNA vaccine in a murine test system.

Carcinoembryonic antigen (CEA, CEACAM5) is expressed on several human carcinomas including colon cancer. CEA contains signal peptides that target the protein through the endoplasmic reticulum and to the cell membrane. We constructed a plasmid DNA vaccine encoding a truncated CEA (deltaCEA), devoid of its signal peptides, and demonstrated that it was retained inside the cell, while full-length CEA (wtCEA) was expressed on the membrane. We hypothesized that intracellular retention of deltaCEA would enhance MHC class I presentation of CEA peptides, thus favoring cellular immune responses. In addition, a promiscuous T-helper epitope (Q830-L844 of tetanus toxoid) was fused to the N-terminal of the truncated CEA gene (tetdeltaCEA). C57BL/6 mice immunized with DNA encoding wtCEA or tetdeltaCEA developed both humoral and cellular immune responses to CEA. SCID mice transplanted with spleen cells from tetdeltaCEA but not wtCEA-immunized C57BL/6 mice showed strong suppression of tumor growth after inoculation of human CEA-expressing colon carcinoma cells. Immune spleen cell populations depleted for either B, T or both B and T cells were active, indicating that effector cells might also reside in other populations. The present approach to manipulating antigen presentation may open new possibilities for immunotherapy against colon and other CEA-secreting carcinomas.

Traveling-wave pattern generator controls movement and organization of sensory feedback in a spinal cord model.

A traveling wave in a two-dimensional spinal cord model constitutes a stable pattern generator for quadruped gaits. In the context of the somatotopic organization of the spinal cord, this pattern generator is sufficient to generate stable locomotive limb trajectories. The elastic properties of muscles alone, providing linear negative feedback, are sufficient to stabilize stance and locomotion in the presence of perturbative forces. We further show that such a pattern generator is capable of organizing sensory processing in the spinal cord. A single-layer perceptron was trained to associate the sensory feedback from the limb (coding force, length, and change of length for each muscle) with the two-dimensional activity profile of the traveling wave. This resulted in a well-defined spatial organization of the connections within the spinal network along a rostrocaudal axis. The spinal network driven by peripheral afferents alone supported autonomous locomotion in the positive feedback mode, whereas in the negative feedback mode stance was stabilized in response to perturbations. Systematic variation of a parameter representing the effect of gamma-motor neurons on muscle spindle activity in our model led to a corresponding shift of limb position during stance and locomotion, resulting in a systematic displacement alteration of foot positions.

Emergence of coherent traveling waves controlling quadruped gaits in a two-dimensional spinal cord model.

The concerted and self-organizing behavior of spinal cord segments in generating locomotor patterns is modulated by afferent sensory information and controlled by descending pathways from the brainstem, cerebellum, or cortex. The purpose of this study was to define a minimal set of parameters that could control a similar self-organizing behavior in a two-dimensional neural network. When we implemented synaptic depression and active membrane repolarization as two properties of the neurons, the two-dimensional neural network generated traveling waves. Their wavelength and angle of propagation could be independently controlled by two parameters that modulated excitatory premotor neurons and inhibitory commissural neurons. It is further demonstrated that the selection of wave parameters corresponds to the selection of quadruped gaits.

NotI passporting to identify species composition of complex microbial systems.

We describe here a new method for large-scale scanning of microbial genomes on a quantitative and qualitative basis. To achieve this aim we propose to create NotI passports: databases containing NotI tags. We demonstrated that these tags comprising 19 bp of sequence information could be successfully generated using DNA isolated from intestinal or fecal samples. Such NotI passports allow the discrimination between closely related bacterial species and even strains. This procedure for generating restriction site tagged sequences (RSTS) is called passporting and can be adapted to any other rare cutting restriction enzyme. A comparison of 1312 tags from available sequenced Escherichia coli genomes, generated with the NotI, PmeI and SbfI restriction enzymes, revealed only 219 tags that were not unique. None of these tags matched human or rodent sequences. Therefore the approach allows analysis of complex microbial mixtures such as in human gut and identification with high accuracy of a particular bacterial strain on a quantitative and qualitative basis.

Human cell lines engineered for tetracycline-regulated expression of tumor suppressor candidate genes from a frequently affected chromosomal region, 3p21.

BACKGROUND: We modified a tetracycline-regulated system that can control the activity of individual genes quantitatively and reversibly in transgenic mammals. Despite these advances, there remained one problem in the intensive use of the tet-system: the limited range of acceptor cell lines, expressing a tetracycline-controlled transcriptional activator (tTA). This study describes in detail new vectors and a unifying strategy to generate tTA-expressing cell lines. METHOD: Two retroviral vectors pLNCtTA-hCMV and pLNCtTA-EF1alpha coding for the tTA were used to engineer cell lines to constitutively express tTA. New expression vectors pETE-Hyg and pETE-Bsd were also created that replicate in episomal form in human cells and facilitate tetracycline-regulated expression of targeted genes. RESULTS: The primate-tropic retroviruses efficiently delivered the regulatory tTA gene into 12 selected human cancer cell lines. Two candidate tumor suppressor genes from the human 3p21-p22 region MAPKAPK3 (3pK) and MLH1 were cloned into the episomal vector and transfected into engineered A9 and KRC/Y cells. The transfectants were subcutaneously grown in SCID mice, and the expression of the transgene was successfully controlled in vivo by tetracycline administered ad libitum in drinking water. The experiments demonstrated that both transgenes did not antagonize the tumorous growth of these cells. CONCLUSIONS: New retroviral and episomal vectors appear particularly suited for tight regulation of genes that cause suppression of cell growth. The generated cell lines can be used in various applications to study the effect of an inducible transgene in human cancer cells.

HHV-6 A- or B-specific P41 antigens do not reveal virus variant-specific IgG or IgM responses in human serum.

The etiology of multiple sclerosis (MS) remains unknown, but there are indications of a role of human herpesvirus 6 (HHV-6), especially variant A, in the pathogenesis. Higher serum antibody reactivity against an HHV-6 early protein, p41, has been found in MS cases than in controls. The antigen, however, was purified from infected cells with a monoclonal antibody also reactive with a protein (p38) likely to be of cellular origin. To avoid serological crossreactivity with the cellular protein, recombinant p41 proteins from HHV-6A strain GS and HHV-6B strain Z29 were expressed as glutathione-S-transferase fusion proteins (p41-GST), and used as antigens in an enzyme-linked immunosorbent assay (ELISA). p41 variant specific monoclonal antibodies reacted strongly with the respective recombinant proteins. Serum IgM and IgG reactivities with the recombinant p41 antigens were analysed in patients with manifest MS, patients with optic neuritis, patients with other neurological diseases, and in one group of healthy controls. All sera were HHV-6 IgG seropositive by immunofluorescence. The serum IgM or IgG reactivities against the recombinant p41 antigens did not differ significantly between the groups, and the reactivities against the variant A and B antigens were identical. In many samples, the reactivity was very low. The results indicate that p41 is not an optimal target for HHV-6 serology studies, and that the data obtained with the p41 antigen prepared from infected cells (possibly including also p38) should be interpreted with caution.