Small-Molecule Screen Identifies De Novo Nucleotide Synthesis as a Vulnerability of Cells Lacking SIRT3

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Early detection of bacterial diseases in apple plants by analysis of volatile organic compounds profiles and use of electronic nose

DNA‐based protocols are the standard methods for the diagnosis of infected plant material. Nevertheless, these methods are time‐consuming and require trained personnel, with an efficacy depending on the sampling procedure. In comparison, recognition methods based on volatile compounds emissions are less precise, but allow a non‐destructive mass screening of bulk samples, and may be implemented to steer molecular diagnosis. In this study, the analysis of volatile compounds is used for the discrimination of fire blight (Erwinia amylovora) and blossom blight (Pseudomonas syringae pv. syringae) on apple propagation material. Possible marker compounds were identified by gas chromatography–mass spectroscopy (GC‐MS) and proton transfer reaction‐time of flight‐mass spectroscopy (PTR‐ToF‐MS). In addition, two commercial electronic noses were used for diagnosis. After a preliminary validation in vitro, a diagnostic protocol was successfully developed to scale up to real nursery conditions on cold stored, asymptomatic dormant plants.     

Chemical-Induced Read-Through at Premature Termination Codons Determined by a Rapid Dual-Fluorescence System Based on S. cerevisiae

Nonsense mutations generate in-frame stop codons in mRNA leading to a premature arrest of translation. Functional consequences of premature termination codons (PTCs) include the synthesis of truncated proteins with loss of protein function causing severe inherited or acquired diseases. A therapeutic approach has been recently developed that is based on the use of chemical agents with the ability to suppress PTCs (read-through) restoring the synthesis of a functional full-length protein. Research interest for compounds able to induce read-through requires an efficient high throughput large scale screening system. We present a rapid, sensitive and quantitative method based on a dual-fluorescence reporter expressed in the yeast Saccharomyces cerevisiae to monitor and quantitate read-through at PTCs. We have shown that our novel system works equally well in detecting read-through at all three PTCs UGA, UAG and UAA.     

Three-dimensional structure determination of an anti-2-phenyloxazolone antibody: the role of somatic mutation and heavy/light chain pairing in the maturation of an immune response.

The three-dimensional structure of the Fab fragment of an anti-2-phenyloxazolone monoclonal antibody (NQ10/12.5) in its native and complexed forms has been determined at 2.8 and 3.0 A resolution, respectively. Identification of hapten-contacting residues has allowed us to evaluate the contribution of individual somatic point mutations to maturation of the immune response. In particular, amino acid residues 34 and 36 of the light chain, which are frequently mutated in antibodies with increased affinity for 2-phenyloxazolone, are shown to interact directly with the hapten. We propose that the strict maintenance of certain amino acid sequences at the potentially highly variable VL-JL and VH-D-JH junctions observed among anti-2-phenyloxazolone antibodies is due largely to structural constraints related to antigen recognition. Finally, the three-dimensional model of NQ10/12.5, which uses the typical light chain of primary response anti-2-phenyloxazolone antibodies but a different heavy chain, allows an understanding of how, by preserving key contact residues, a given heavy chain may be replaced by another, apparently unrelated one, without loss of hapten binding activity and why the V kappa Ox1 germline gene is so frequently selected amongst the other known members of this family.     

Laparoscopy and laparotomy combined with bone marrow biopsy in staging Hodgkin's disease.

The relative merits of laparoscopy with liver and spleen biopsy and staging laparotomy were studied in 91 unselected patients with Hodgkin''s disease. Laparoscopy with liver and spleen biopsy were combined with needle biopsy of the bone marrow and laparotomy was combined with open bone marrow biopsy. In 65 untreated patients six out of seven with liver or marrow disease, or both, were shown to have extranodal lymphomas in these sites by laparoscopy plus needle marrow biopsy. Among 26 patients who had been treated this finding occurred in six out of 10 patients. Spleen biopsies during laparoscopy detected infiltration by lymphoma in 14 out of 37 (38%) patients with diseases spleens. Morbidity was higher after laparotomy than after laparoscopy. Laparoscopy produced abdominal bleeding secondary to splenic biopsy in two patients. All patients with Hodgkin''s disease should be subjected to laparoscopy plus needle marrow biopsy before undergoing laparotomy.     

Mechanisms maintaining antibody-induced enhancement of allografts. II. Mediation of specific suppression by short lived CD4+ T cells

In DA rats grafted with PVG hearts, the injection of 1 ml of Wistar-Furth x DA)F1 anti-PVG serum on the day of grafting prevents rejection and induces a state of specific unresponsiveness. An adoptive transfer assay was used to test the capacity of T cell subsets, taken from rats given enhancing serum, to either restore rejection or to transfer unresponsiveness to syngeneic hosts irradiated with 9 Gy and grafted with donor (PVG) or third party (Wistar-Furth) hearts. W3/25+ (CD4+) cells from these animals retained some capacity to restore rejection until 50 days posttransplant, after which they invariably failed to restore PVG graft rejection but retained the capacity to effect Wistar-Furth rejection. At this time CD4+ cells were also capable of inhibiting naive but not specifically sensitized CD4+ cells capacity to restore PVG graft rejection in irradiated hosts. The development of CD4+ suppressor cells was concurrent with the appearance of clinically evident unresponsiveness in the host. MRC Ox8+ (CD8+) cells from enhanced rats when mixed with naive CD4+ cells delayed rejection in adoptive recipients but did not reestablish unresponsiveness. Paradoxically, the CD4+ cells that transfer unresponsiveness to the adoptive host proliferate such as normal cells in MLC to both donor and third party alloantigen. Unfractionated cells, CD4+ or CD8+ cells did not proliferate to relevant idiotype in vitro. The CD4+ cells after 3 days in culture, with either alloantigen or idiotype-bearing stimulator cells, lost their capacity to suppress in the adoptive transfer assay. The maintenance of specific unresponsiveness was thus shown to be due to a CD4+ suppressor T cell whose function was lost in culture, and therefore could not be detected in MLC or idiotype assays.     

Domain swapping of a llama VHH domain builds a crystal-wide beta-sheet structure

Among mammals, camelids have a unique immunological system since they produce functional antibodies devoid of light chains and CH1 domains. To bind antigens, whether they are proteins or haptens, camelids use the single domain VH from their heavy chain (VHH). We report here on such a llama VHH domain (VHH-R9) which was raised against a hapten, the RR6 red dye. This VHH possesses the shortest complementarity determining region 3 (CDR3) among all the known VHH sequences and nevertheless binds RR6 efficiently with a K(d) value of 83 nM. However, the crystal structure of VHH-R9 exhibits a striking feature: its CDR3 and its last beta-strand (beta9) do not follow the immunoglobulin VH domain fold, but instead extend out of the VHH molecular boundary and associate with a symmetry-related molecule. The two monomers thus form a domain-swapped dimer which establishes further contacts with symmetry-related molecules and build a crystal-wide beta-sheet structure. The driving force of the dimer formation is probably the strain induced by the short CDR3 together with the cleavage of the first seven residues.     

Scapulothoracic motion and muscle activity during the raising and lowering phases of an overhead reaching task

Scapulothoracic muscle activity is essential for normal scapulothoracic motion. While previous research has furthered the understanding of scapulothoracic motion and muscle activity during the raising phase of motion, a gap exists with respect to the lowering phase. The purpose of this study was to compare scapulothoracic motion and scapulothoracic muscle activity between the raising and lowering phases of an overhead reaching task. Twenty healthy subjects volunteered to participate in the study. Three-dimensional scapulothoracic motion was collected using an electromagnetic device. Surface electromyography (EMG) was used to assess muscle activity from the upper trapezius, lower trapezius, and serratus anterior muscles. Overall scapulothoracic motion was similar for the raising and lowering phases of the overhead reaching task. However, significantly lower EMG amplitude values existed during the lowering phase across all muscles. Less muscle activity during the lowering phase may reflect differing neuromuscular control strategies between arm raising and lowering. These findings suggest that scapulothoracic muscle activation levels during eccentric contractions may be closer to an activation threshold below which their ability to control scapulothoracic motion may be compromised subsequently leading to altered scapulothoracic motion (scapular dyskinesis). This provides a possible explanation for why scapular dyskinesis is more notable during the lowering phase of motion.