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.     

A20 Deficiency in Lung Epithelial Cells Protects against Influenza A Virus Infection

A20 negatively regulates multiple inflammatory signalling pathways. We here addressed the role of A20 in club cells (also known as Clara cells) of the bronchial epithelium in their response to influenza A virus infection. Club cells provide a niche for influenza virus replication, but little is known about the functions of these cells in antiviral immunity. Using airway epithelial cell-specific A20 knockout (A20^AEC-KO) mice, we show that A20 in club cells critically controls innate immune responses upon TNF or double stranded RNA stimulation. Surprisingly, A20^AEC-KO mice are better protected against influenza A virus challenge than their wild type littermates. This phenotype is not due to decreased viral replication. Instead host innate and adaptive immune responses and lung damage are reduced in A20^AEC-KO mice. These attenuated responses correlate with a dampened cytotoxic T cell (CTL) response at later stages during infection, indicating that A20^AEC-KO mice are better equipped to tolerate Influenza A virus infection. Expression of the chemokine CCL2 (also named MCP-1) is particularly suppressed in the lungs of A20^AEC-KO mice during later stages of infection. When A20^AEC-KO mice were treated with recombinant CCL2 the protective effect was abrogated demonstrating the crucial contribution of this chemokine to the protection of A20^AEC-KO mice to Influenza A virus infection. Taken together, we propose a mechanism of action by which A20 expression in club cells controls inflammation and antiviral CTL responses in response to influenza virus infection.     

<Emphasis Type="Italic">Euphyllia paradivisa</Emphasis>, a successful mesophotic coral in the northern Gulf of Eilat/Aqaba,Red Sea

Mesophotic coral ecosystems (MCEs) host a thriving community of biota that has remained virtually unexplored. Here we report for the first time on a large population of the endangered coral species Euphyllia paradivisa from the MCEs of the Gulf of Eilat/Aqaba (GOE/A), Red Sea. The mesophotic zone in some parts of the study site harbors a specialized coral community predominantly comprising E. paradivisa (73 % of the total coral cover), distributed from 36 to 72 m depth. Here we sought to elucidate the strict distribution but high abundance of E. paradivisa in the MCEs at the GOE/A. We present 4 yr of observations and experiments that provide insight into the physiological plasticity of E. paradivisa: its low mortality rates at high light intensities, high competitive abilities, successful symbiont adaptation to the shallow-water environment, and tolerance to bleaching conditions or survival during prolonged bleaching. Despite its ability to survive under high irradiance in shallow water, E. paradivisa is not found in the shallow reef of the GOE/A. We suggest several factors that may explain the high abundance and exclusivity of E. paradivisa in the MCE: its heterotrophic capabilities; its high competition abilities; the possibility of it finding a deep-reef refuge there from fish predation; and its concomitant adaptation to this environment.     

Parental Perceptions of Quality of Life in Children on Long-Term Ventilation at Home as Compared to Enterostomy Tubes

Objective

Health related quality of life (HRQL) of children using medical technology at home is largely unknown. Our aim was to examine the HRQL in children on long-term ventilation at home (LTHV) in comparison to a cohort using an enterostomy tube.

Study Design

Participants were divided into three groups: 1) LTHV without an enterostomy tube (LTHV cohort); 2) Enterostomy tube (GT cohort); 3) LTHV with an enterostomy tube (LTHV+GT cohort). Caregivers of children ≥ 5 years and followed at SickKids, Toronto, Canada, completed three questionnaires: Health Utilities Index 2/3 (HUI2/3), Caregiver Priorities Caregiver Health Index (CPCHILD), and the Paediatric Quality of Life Inventory (PedsQL). The primary outcome was the difference in utility (HUI2/3) scores between the cohorts.

Results

One hundred and nineteen children were enrolled; 47 in the LTHV cohort, 44 in the GT cohort, and 28 in the LTHV+GT cohort. In univariate analysis, HUI2 mean (SE) scores were lowest for the GT cohort, 0.4 (0.04) followed by the LTHV+GT, 0.42 (0.05) and then the LTHV cohort, 0.7 (0.04), p = 0.001. A similar trend was seen for the HUI3 mean (SE) scores: GT cohort, 0.1 (0.06), followed by the LTHV +GT cohort, 0.2 (0.08) and then the LTHV cohort, 0.5 (0.06), p = 0.0001. Technology cohort, nursing hours and the severity of health care needs predicted HRQL as measured by the HUI2/3.

Conclusion

The HRQL of these children is low. Children on LTHV had higher HRQL than children using enterostomy tubes. Further work is needed to identify modifiable factors that can improve HRQL.     

The role of the idiotypic network in the induction of experimental systemic lupus erythematosus

Systemic lupus erythematosus (SLE) has been induced in C3H.SW mice by their immunization with a human monoclonal anti-DNA antibody that bears a common idiotype-16/6 Id. Following immunization, high levels of murine anti-16/6 and anti-anti-16/6 antibodies were detected in the sera of the immunized mice. Elevated titers of autoantibodies reacting with ssDNA, dsDNA, poly(I), poly(G), RNP, Ro, and La were also observed. The serological findings were associated with significant proteinuria, leukopenia, and elevated erythrocyte sedimentation rate. Immune complex deposition in the glomerular mesangium and sclerosis of the glomeruli were demonstrated. To study whether or not anti-idiotype antibodies are involved in the induction of the disease, a murine monoclonal antibody against the 16/6 Id was prepared and injected into C3H.SW mice. The anti-16/6 Id antibody induced experimental SLE similarly to the 16/6 Id with an accelerated kidney pathology. A study performed on different mouse strains indicated that the susceptibility to the induction of SLE by the 16/6 Id is strain dependent and directly correlates to their ability to produce anti-16/6 Id specific antibodies.     

Suppression ofSeptoria tritici by Phenazine- or Siderophore-deficient mutants ofPseudomonas

Pseudomonas aeruginosa LEC1, a soil isolate from Israel, suppressed septoria tritici blotch of wheat caused bySeptoria tritici. Tn5-751 insertion mutagenesis of strain LEC1 produced one pyoverdine-negative (pvd) and three different pyocyanine-deficient (pcy) mutants. The nonfluorescentpvd mutant, like the wild-type, inhibited growth ofS. tritici on different solid media and suppressed the formation of pycnidia on wheat leaves. In contrast, thepcy mutants had lost part of the inhibitory activity in vitro and also part of the suppressive ability in vivo. It is suggested that antibiosis plays an important role in the suppression of septoria tritici blotch of wheat.     

Characterization and biologic activities of an anti-idiotype-specific T cell line and its derived clones

Monoclonal anti-idiotypic antibodies were prepared against monoclonal antibodies (mAb103) specific to the synthetic polypeptide antigen (T,G)-A-L. A cell line was established by the stimulation of C3H.SW mouse T cells with one of the monoclonal anti-idiotypes (mAbA-6) that reacted with both mAb103 and conventional (T,G)-A-L-specific antibodies. The T cell line proliferated specifically in the presence of the homologous mAbA-6 and to a lesser degree when triggered with (T,G)-A-L. The line could help (T,G)-A-L primed B cells in the production of (T,G)-A-L-specific antibodies when stimulated in vitro with either (T,G)-A-L or mAbA-6. Clones obtained from the line were stimulated and maintained in culture in presence of mAbA-6 whereas others were stimulated and grown in the presence of (T,G)-A-L. Both types of clones proliferated only in the presence of mAbA-6 although (T,G)-A-L could inhibit efficiently and specifically the latter proliferation. A significant number of the (T,G)-A-L-stimulated clones could collaborate with (T,G)-A-L primed B cells in the presence of either (T,G)-A-L or mAbA-6 for the production of specific antibodies. Immunoblotting experiments indicated that mAbA-6 reacted with both the T cell receptor of the mAbA-6-specific T cell line and of a (T,G)-A-L-specific T cell line but not with that of a line specific to a nonrelated antigen.     

Chemotaxonomic discrimination among the fungal genera Tolypocladium, beauveria and Paecilomyces

Fungi of the genera Beauveria and Paecilomyces were found to produce cyclotetradepsipeptides, beauverolides. Production of beauverolides was not detected at the genus Tolypocladium. Analysis of beauverolides therefore provides a very simple chemotaxonomic test which seems to be suitable for fast discrimination between the genera Beauveria vs Tolypocladium and complementing morphological examination. A GC-MS study of β-hydroxy acid distribution in the beauverolide hydrolyzates revealed that all strains prdouce γ-methyl-β-hydroxy acids only. Their occurrence thus cannot be used as a taxonomic marker of different species within the genera Beauveria and Paecilomyces.