The protein product of the het-s heterokaryon incompatibility gene of the fungus Podospora anserina behaves as a prion analog

The het-s locus of Podospora anserina is a heterokaryon incompatibility locus. The coexpression of the antagonistic het-s and het-S alleles triggers a lethal reaction that prevents the formation of viable heterokaryons. Strains that contain the het-s allele can display two different phenotypes, [Het-s] or [Het-s*], according to their reactivity in incompatibility. The detection in these phenotypically distinct strains of a protein expressed from the het-s gene indicates that the difference in reactivity depends on a posttranslational difference between two forms of the polypeptide encoded by the het-s gene. This posttranslational modification does not affect the electrophoretic mobility of the protein in SDS/PAGE. Several results suggest a similarity of behavior between the protein encoded by the het-s gene and prions. The [Het-s] character can propagate in [Het-s*] strains as an infectious agent, producing a [Het-s*] → [Het-s] transition, independently of protein synthesis. Expression of the [Het-s] character requires a functional het-s gene. The protein present in [Het-s] strains is more resistant to proteinase K than that present in [Het-s*] mycelium. Furthermore, overexpression of the het-s gene increases the frequency of the transition from [Het-s*] to [Het-s]. We propose that this transition is the consequence of a self-propagating conformational modification of the protein mediated by the formation of complexes between the two different forms of the polypeptide.     

Inhibition of Pseudomonas aeruginosa biofilm formation on wound dressings

Chronic nonhealing skin wounds often contain bacterial biofilms that prevent normal wound healing and closure and present challenges to the use of conventional wound dressings. We investigated inhibition of Pseudomonas aeruginosa biofilm formation, a common pathogen of chronic skin wounds, on a commercially available biological wound dressing. Building on prior reports, we examined whether the amino acid tryptophan would inhibit P. aeruginosa biofilm formation on the three‐dimensional surface of the biological dressing. Bacterial biomass and biofilm polysaccharides were quantified using crystal violet staining or an enzyme linked lectin, respectively. Bacterial cells and biofilm matrix adherent to the wound dressing were visualized through scanning electron microscopy. d ‐/l ‐tryptophan inhibited P. aeruginosa biofilm formation on the wound dressing in a dose dependent manner and was not directly cytotoxic to immortalized human keratinocytes although there was some reduction in cellular metabolism or enzymatic activity. More importantly, d ‐/l ‐tryptophan did not impair wound healing in a splinted skin wound murine model. Furthermore, wound closure was improved when d ‐/l ‐tryptophan treated wound dressing with P. aeruginosa biofilms were compared with untreated dressings. These findings indicate that tryptophan may prove useful for integration into wound dressings to inhibit biofilm formation and promote wound healing.     

Inflammatory signaling regulates embryonic hematopoietic stem and progenitor cell production

Identifying signaling pathways that regulate hematopoietic stem and progenitor cell (HSPC) formation in the embryo will guide efforts to produce and expand HSPCs ex vivo. Here we show that sterile tonic inflammatory signaling regulates embryonic HSPC formation. Expression profiling of progenitors with lymphoid potential and hematopoietic stem cells (HSCs) from aorta/gonad/mesonephros (AGM) regions of midgestation mouse embryos revealed a robust innate immune/inflammatory signature. Mouse embryos lacking interferon γ (IFN-γ) or IFN-α signaling and zebrafish morphants lacking IFN-γ and IFN-ϕ activity had significantly fewer AGM HSPCs. Conversely, knockdown of IFN regulatory factor 2 (IRF2), a negative regulator of IFN signaling, increased expression of IFN target genes and HSPC production in zebrafish. Chromatin immunoprecipitation (ChIP) combined with sequencing (ChIP-seq) and expression analyses demonstrated that IRF2-occupied genes identified in human fetal liver CD34⁺ HSPCs are actively transcribed in human and mouse HSPCs. Furthermore, we demonstrate that the primitive myeloid population contributes to the local inflammatory response to impact the scale of HSPC production in the AGM region. Thus, sterile inflammatory signaling is an evolutionarily conserved pathway regulating the production of HSPCs during embryonic development.     

Streamlined ion torrent PGM-based diagnostics: BRCA1 and BRCA2 genes as a model

To meet challenges in terms of throughput and turnaround time, many diagnostic laboratories are shifting from Sanger sequencing to higher throughput next-generation sequencing (NGS) platforms. Bearing in mind that the performance and quality criteria expected from NGS in diagnostic or research settings are strikingly different, we have developed an Ion Torrent''s PGM-based routine diagnostic procedure for BRCA1/2 sequencing. The procedure was first tested on a training set of 62 control samples, and then blindly validated on 77 samples in parallel with our routine technique. The training set was composed of difficult cases, for example, insertions and/or deletions of various sizes, large-scale rearrangements and, obviously, mutations occurring in homopolymer regions. We also compared two bioinformatic solutions in this diagnostic context, an in-house academic pipeline and the commercially available NextGene software (Softgenetics). NextGene analysis provided higher sensitivity, as four previously undetected single-nucleotide variations were found. Regarding specificity, an average of 1.5 confirmatory Sanger sequencings per patient was needed for complete BRCA1/2 screening. Large-scale rearrangements were identified by two distinct analyses, that is, bioinformatics and fragment analysis with electrophoresis profile comparison. Turnaround time was enhanced, as a series of 30 patients were sequenced by one technician, making the results available for the clinician in 10 working days following blood sampling. BRCA1/2 genes are a good model, representative of the difficulties commonly encountered in diagnostic settings, which is why we believe our findings are of interest for the whole community, and the pipeline described can be adapted by any user of PGM for diagnostic purposes.     

Activation and Deactivation of the Polymerization of Ethylene over rac‐EtInd2ZrCl2 and (nBuCp)2ZrCl2 on an Activating Silica Support

A study is carried out in order to better understand the kinetic behavior of two different metallocene precursors supported on an activating silica support, and, in particular, to attempt to reduce the significance of catalyst deactivation through the use of different alkylating agents. It is observed that it is difficult to prevent the deactivation of the rac‐EtInd₂ZrCl₂ sites on the activating support, and, furthermore, the deactivation is accompanied by the disa­ppearance of sites producing the highest‐molecular‐weight polymer. On the contrary, with the (nBuCp)₂ZrCl₂ precursor, it is possible to manipulate the addition of either tri‐isobutylaluminum (TIBA) or triethylaluminum (TEA) to significantly reduce the deactivation of the catalyst on the activating supports. Finally, the molecular‐weight distribution seems to depend much more on the intrinsic properties of the active sites than on how the active sites are treated.

     

Prevalence and risk factors of cataract after chemotherapy with or without central nervous system irradiation for childhood acute lymphoblastic leukaemia: an LEA study

Corticosteroid and central nervous system (CNS) irradiation can induce cataract in childhood lymphoblastic leukaemia survivors. Few prospective studies with systematic ophthalmological evaluation have been published. Cataract was prospectively assessed by serial slip lamp tests in 517 patients. All had acute lymphoblastic leukaemia, all had been treated by chemotherapy with or without CNS irradiation, and none had received haematopoietic stem cell transplantation. Median ages at last evaluation and follow‐up duration from leukaemia diagnosis were 16·8 and 10·9 years, respectively. Cataract was observed in 21/517 patients (4·1%). Cumulative incidence was 4·5 ± 1·2% at 15 years and reached 26 ± 8·1% at 25 years. CNS irradiation was the only risk factor: prevalence was 11·1% in patients who had received irradiation and 2·8% in those who did not. We did not detect any steroid dose effect: cumulative dose was 5133 and 5190 mg/m² in patients with and without cataract, respectively. Cataract occurrence did not significantly impact quality of life. We conclude that, in the range of steroid dose reported here, the cataract risk proves very low 15 years after treatment without CNS irradiation but an even more prolonged follow‐up is required because of potential very late occurrence.     

The Ser/Thr protein kinase AfsK regulates polar growth and hyphal branching in the filamentous bacteria Streptomyces

In cells that exhibit apical growth, mechanisms that regulate cell polarity are crucial for determination of cellular shape and for the adaptation of growth to intrinsic and extrinsic cues. Broadly conserved pathways control cell polarity in eukaryotes, but less is known about polarly growing prokaryotes. An evolutionarily ancient form of apical growth is found in the filamentous bacteria Streptomyces, and is directed by a polarisome-like complex involving the essential protein DivIVA. We report here that this bacterial polarization machinery is regulated by a eukaryotic-type Ser/Thr protein kinase, AfsK, which localizes to hyphal tips and phosphorylates DivIVA. During normal growth, AfsK regulates hyphal branching by modulating branch-site selection and some aspect of the underlying polarisome-splitting mechanism that controls branching of Streptomyces hyphae. Further, AfsK is activated by signals generated by the arrest of cell wall synthesis and directly communicates this to the polarisome by hyperphosphorylating DivIVA. Induction of high levels of DivIVA phosphorylation by using a constitutively active mutant AfsK causes disassembly of apical polarisomes, followed by establishment of multiple hyphal branches elsewhere in the cell, revealing a profound impact of this kinase on growth polarity. The function of AfsK is reminiscent of the phoshorylation of polarity proteins and polarisome components by Ser/Thr protein kinases in eukaryotes.     

Urgent endoscopy in severe non-variceal upper gastrointestinal hemorrhage: does the Glasgow-Blatchford score help endoscopists?

Abstract Objective. The Glasgow-Blatchford score (GBS) has been validated to select severe patients with non-variceal upper gastrointestinal hemorrhage (UGIH). The aim was to compare the yield of the triage based on the GBS with an endoscopist' decision to perform an urgent upper gastrointestinal endoscopy (UGIE) in newly admitted patients and inpatients with UGIH in the setting of an endoscopy on-duty service in 13 tertiary care centers. Material and methods. During a 6-month period, GBS and patient data were collected for all patients with non-variceal UGIH for whom an UGIE was requested in emergency. If patients experienced severe endoscopic lesion, surgery or death, they were categorized as patients who had been at need for urgent UGIE. Results. The 102 UGIH patients included (mean age 62, men 73%) had a median GBS of 12 (range 0-21), significantly lower for new patients compared with inpatients (11, range 0-21 vs. 14, range 2-21, respectively, p = 0.001). If triage for urgent UGIE had followed the GBS, no more patients would have had an urgent UGIE compared with what endoscopists performed (99/102 (97%) vs. 92/102 (90%), respectively, p = 0.09). Sensitivity for the detection of patients who needed an UGIE was no different with the GBS than endoscopists (98% vs. 98%, respectively, p = 0.10) and both showed insufficient specificity (4% and 19%, respectively). Conclusions. The GBS does not detect more patients at need for urgent UGIE than on-duty endoscopists. Both methods lead to numerous unjustified UGIEs. A score that would equally help endoscopists in their decision to intervene urgently is still warranted.