Highly efficient de novo mutant identification in a Sorghum bicolor TILLING population using the ComSeq approach

Screening large populations for carriers of known or de novo rare single nucleotide polymorphisms (SNPs) is required both in Targeting induced local lesions in genomes (TILLING) experiments in plants and in screening of human populations. We previously suggested an approach that combines the mathematical field of compressed sensing with next‐generation sequencing to allow such large‐scale screening. Based on pooled measurements, this method identifies multiple carriers of heterozygous or homozygous rare alleles while using only a small fraction of resources. Its rigorous mathematical foundations allow scalable and robust detection, and provide error correction and resilience to experimental noise. Here we present a large‐scale experimental demonstration of our computational approach, in which we targeted a TILLING population of 1024 Sorghum bicolor lines to detect carriers of de novo SNPs whose frequency was less than 0.1%, using only 48 pools. Subsequent validation confirmed that all detected lines were indeed carriers of the predicted mutations. This novel approach provides a highly cost‐effective and robust tool for biologists and breeders to allow identification of novel alleles and subsequent functional analysis.     

A Rapid and Improved Method to Generate Recombinant Dengue Virus Vaccine Candidates

Dengue is one of the most important mosquito-borne infections accounting for severe morbidity and mortality worldwide. Recently, the tetravalent chimeric live attenuated Dengue vaccine Dengvaxia^® was approved for use in several dengue endemic countries. In general, live attenuated vaccines (LAV) are very efficacious and offer long-lasting immunity against virus-induced disease. Rationally designed LAVs can be generated through reverse genetics technology, a method of generating infectious recombinant viruses from full length cDNA contained in bacterial plasmids. In vitro transcribed (IVT) viral RNA from these infectious clones is transfected into susceptible cells to generate recombinant virus. However, the generation of full-length dengue virus cDNA clones can be difficult due to the genetic instability of viral sequences in bacterial plasmids. To circumvent the need for a single plasmid containing a full length cDNA, in vitro ligation of two or three cDNA fragments contained in separate plasmids can be used to generate a full-length dengue viral cDNA template. However, in vitro ligation of multiple fragments often yields low quality template for IVT reactions, resulting in inconsistent low yield RNA. These technical difficulties make recombinant virus recovery less efficient. In this study, we describe a simple, rapid and efficient method of using LONG-PCR to recover recombinant chimeric Yellow fever dengue (CYD) viruses as potential dengue vaccine candidates. Using this method, we were able to efficiently generate several viable recombinant viruses without introducing any artificial mutations into the viral genomes. We believe that the techniques reported here will enable rapid and efficient recovery of recombinant flaviviruses for evaluation as vaccine candidates and, be applicable to the recovery of other RNA viruses.     

Size and charge heterogeneity of murine IgG-binding factors (IgG-BF)

Size and charge of murine IgG-binding factors (IgG-BF) were determined. Four different sources were used to produce the factors: a) cells of a T cell hybrid (T2D4) constitutively secreting IgG-BF upon incubation in serum-free medium, b) T2D4 cells incubated with mouse monoclonal IgG1 antibody in order to induce in vitro the production of isotype-specific IgG1-BF, c) T2D4 cells induced in vivo by passage as ascites in nude mice and incubated in serum-free medium, and d) in vivo alloantigen-activated T cells (ATC) incubated in serum-free medium. IgG-BF were affinity purified on Sepharose beads coated with rabbit or mouse IgG and identified by their biologic activities, i.e., inhibition of in vitro secondary IgG antibody production to SRBC and inhibition of rosette formation between Fc gamma receptor-positive spleen cells and rabbit IgG-sensitized erythrocytes. IgG-BF produced by either of these cell sources was found to be heterogeneous in both size and charge. In each case, IgG-BF activities were recovered in three fractions of apparent Mr-74,000 to 78,000, 35,000 to 40,000, and 19,000 to 23,000-and in four fractions of pI-4.7 (or 5.3, depending on experimental conditions), 6.5, 7.7, and 8.4. Moreover, IgG-BF translated in vitro from T2D4 poly A RNA by using rabbit reticulocyte lysate exhibited the same heterogeneity. Thus, IgG-BF contain different proteins exerting similar biologic activities.     

Purification of soluble recombinant human FcgammaRII (CD32).

The present study describes the methodology used to purify human recombinant low-affinity FcgammaRIIa2 produced in E. coli and to evaluate its binding to surface IgG. The recombinant molecule was purified by a two-step chromatographic procedure, including affinity chromatography using IV.3 anti-FcgammaRIIa1/2 immunosorbent, followed by gel filtration chromatography. Using this method, the purified recombinant FcgammaRIIa2 was 99% pure. It exhibited an isoeletric point of 5.2. Binding studies demonstrated a specific binding of the purified recombinant molecule to surface IgG expressed by human B cells. Thus, we have set up a method which allows to purify functional human recombinant FcgammaRIIa2 for further characterization of its biological activities.     

FcγR expressed on T-cell hybrids: Specificity,behavior and relationship with Ia antigens

The fine specificity of receptor for the Fc portion of IgG (FcγR) expressed on T-cell hybrids secreting soluble FcγR (sFcγR) which suppresses antibody production, was investigated. FcγR was found to bind IgG from mouse, human, and rabbit species. It reacted with mouse IgG1 and IgG2a but not IgG2b, and human IgG1 and IgG3 but not IgG4. Mouse IgG and their subclasses bound more avidly to FcγR than human and rabbit IgG. FcγR of T-cell hybrids was sensitive to pronase and resistant to trypsin. In kinetics experiments, the behavior of FcγR on the membrane of T-cell hybrids was analyzed and compared to that of I-region-coded antigens expressed on these hybrids. Upon incubation at 37 °C in balanced salt solution (BSS), T-cell hybrids released FcγR into the medium. The reexpression of FcγR, after pronase cleavage or shedding, was complete within 3 hr of incubation in culture medium and required protein synthesis. I-A-coded antigens, present on these hybrids, disappeared simultaneously with FcγR upon incubation of cells at 37 °C in BSS. Within 3 hr of incubation in culture medium, although the reexpression of Fc°R was complete, no Ia antigens could be detected. They were reexpressed later, as tested after 19 hr of culture. During a single growth cycle, the expression of FcγR was maximal during log phase.     

Formation of new high density glycogen-microtubule structures is induced by cardiac steroids

Cardiac steroids (CS), an important class of naturally occurring compounds, are synthesized in plants and animals. The only established receptor for CS is the ubiquitous Na(+),K(+)-ATPase, a major plasma membrane transporter. The binding of CS to Na(+),K(+)-ATPase causes the inhibition of Na(+) and K(+) transport and elicits cell-specific activation of several intracellular signaling mechanisms. It is well documented that the interaction of CS with Na(+),K(+)-ATPase is responsible for numerous changes in basic cellular physiological properties, such as electrical plasma membrane potential, cell volume, intracellular [Ca(2+)] and pH, endocytosed membrane traffic, and the transport of other solutes. In the present study we show that CS induces the formation of dark structures adjacent to the nucleus in human NT2 and ACHN cells. These structures, which are not surrounded by membranes, are clusters of glycogen and a distorted microtubule network. Formation of these clusters results from a relocation of glycogen and microtubules in the cells, two processes that are independent of one another. The molecular mechanisms underlying the formation of the clusters are mediated by the Na(+),K(+)-ATPase, ERK1/2 signaling pathway, and an additional unknown factor. Similar glycogen clusters are induced by hypoxia, suggesting that the CS-induced structural change, described in this study, may be part of a new type of cellular stress response.     

Identification of CD63 as a tissue inhibitor of metalloproteinase-1 interacting cell surface protein

This study identified CD63, a member of the tetraspanin family, as a TIMP-1 interacting protein by yeast two-hybrid screening. Immunoprecipitation and confocal microscopic analysis confirmed CD63 interactions with TIMP-1, integrin beta1, and their co-localizations on the cell surface of human breast epithelial MCF10A cells. TIMP-1 expression correlated with the level of active integrin beta1 on the cell surface independent of cell adhesion. While MCF10A cells within a three-dimensional (3D) matrigel matrix form polarized acinar-like structures, TIMP-1 overexpression disrupted breast epithelial cell polarization and inhibited caspase-mediated apoptosis in centrally located cells, necessary for the formation and maintenance of the hollow acinar-like structures. Small hairpin RNA (shRNA)-mediated CD63 downregulation effectively reduced TIMP-1 binding to the cell surface, TIMP-1 co-localization with integrin beta1, and consequently reversed TIMP-1-mediated integrin beta1 activation, cell survival signaling and apoptosis inhibition. CD63 downregulation also restored polarization and apoptosis of TIMP-1 overexpressing MCF10A cells within a 3D-matrigel matrix. Taken together, the present study identified CD63 as a cell surface binding partner for TIMP-1, regulating cell survival and polarization via TIMP-1 modulation of tetraspanin/integrin signaling complex.