Cuticular defects lead to full immunity to a major plant pathogen.

In addition to its role as a barrier, the cuticle is also a source of signals perceived by invading fungi. Cuticular breakdown products have been shown previously to be potent inducers of cutinase or developmental processes in fungal pathogens. Here the question was addressed as to whether plants themselves can perceive modifications of the cuticle. This was studied using Arabidopsis thaliana plants with altered cuticular structure. The expression of a cell wall-targeted fungal cutinase in A. thaliana was found to provide total immunity to Botrytis cinerea. The response observed in such cutinase-expressing plants is independent of signal transduction pathways involving salicylic acid, ethylene or jasmonic acid. It is accompanied by the release of a fungitoxic activity and increased expression of members of the lipid transfer protein, peroxidase and protein inhibitor gene families that provide resistance when overexpressed in wild-type plants. The same experiments were made in the bodyguard (bdg) mutant of A. thaliana. This mutant exhibits cuticular defects and remained free of symptoms after inoculation with B. cinerea. The expression of resistance was accompanied by the release of a fungitoxic activity and increased expression of the same genes as observed in cutinase-expressing plants. Structural defects of the cuticle can thus be converted into an effective multi-factorial defence, and reveal a hitherto hidden aspect of the innate immune response of plants.

PRPF mutations are associated with generalized defects in spliceosome formation and pre-mRNA splicing in patients with retinitis pigmentosa.

Proteins PRPF31, PRPF3 and PRPF8 (RP-PRPFs) are ubiquitously expressed components of the spliceosome, a macromolecular complex that processes nearly all pre-mRNAs. Although these spliceosomal proteins are conserved in eukaryotes and are essential for survival, heterozygous mutations in human RP-PRPF genes lead to retinitis pigmentosa, a hereditary disease restricted to the eye. Using cells from patients with 10 different mutations, we show that all clinically relevant RP-PRPF defects affect the stoichiometry of spliceosomal small nuclear RNAs (snRNAs), the protein composition of tri-small nuclear ribonucleoproteins and the kinetics of spliceosome assembly. These mutations cause inefficient splicing in vitro and affect constitutive splicing ex-vivo by impairing the removal of at least 9% of endogenously expressed introns. Alternative splicing choices are also affected when RP-PRPF defects are present. Furthermore, we show that the steady-state levels of snRNAs and processed pre-mRNAs are highest in the retina, indicating a particularly elevated splicing activity. Our results suggest a role for PRPFs defects in the etiology of PRPF-linked retinitis pigmentosa, which appears to be a truly systemic splicing disease. Although these mutations cause widespread and important splicing defects, they are likely tolerated by the majority of human tissues but are critical for retinal cell survival.

Mutations in NDUFB11, Encoding a Complex I Component of the Mitochondrial Respiratory Chain, Cause Microphthalmia with Linear Skin Defects Syndrome.

Microphthalmia with linear skin defects (MLS) syndrome is an X-linked male-lethal disorder also known as MIDAS (microphthalmia, dermal aplasia, and sclerocornea). Additional clinical features include neurological and cardiac abnormalities. MLS syndrome is genetically heterogeneous given that heterozygous mutations in HCCS or COX7B have been identified in MLS-affected females. Both genes encode proteins involved in the structure and function of complexes III and IV, which form the terminal segment of the mitochondrial respiratory chain (MRC). However, not all individuals with MLS syndrome carry a mutation in either HCCS or COX7B. The majority of MLS-affected females have severe skewing of X chromosome inactivation, suggesting that mutations in HCCS, COX7B, and other as-yet-unidentified X-linked gene(s) cause selective loss of cells in which the mutated X chromosome is active. By applying whole-exome sequencing and filtering for X-chromosomal variants, we identified a de novo nonsense mutation in NDUFB11 (Xp11.23) in one female individual and a heterozygous 1-bp deletion in a second individual, her asymptomatic mother, and an affected aborted fetus of the subject's mother. NDUFB11 encodes one of 30 poorly characterized supernumerary subunits of NADH:ubiquinone oxidoreductase, known as complex I (cI), the first and largest enzyme of the MRC. By shRNA-mediated NDUFB11 knockdown in HeLa cells, we demonstrate that NDUFB11 is essential for cI assembly and activity as well as cell growth and survival. These results demonstrate that X-linked genetic defects leading to the complete inactivation of complex I, III, or IV underlie MLS syndrome. Our data reveal an unexpected role of cI dysfunction in a developmental phenotype, further underscoring the existence of a group of mitochondrial diseases associated with neurocutaneous manifestations.

Lack of GDAP1 induces neuronal calcium and mitochondrial defects in a knockout mouse model of charcot-marie-tooth neuropathy.

Mutations in GDAP1, which encodes protein located in the mitochondrial outer membrane, cause axonal recessive (AR-CMT2), axonal dominant (CMT2K) and demyelinating recessive (CMT4A) forms of Charcot-Marie-Tooth (CMT) neuropathy. Loss of function recessive mutations in GDAP1 are associated with decreased mitochondrial fission activity, while dominant mutations result in impairment of mitochondrial fusion with increased production of reactive oxygen species and susceptibility to apoptotic stimuli. GDAP1 silencing in vitro reduces Ca2+ inflow through store-operated Ca2+ entry (SOCE) upon mobilization of endoplasmic reticulum (ER) Ca2+, likely in association with an abnormal distribution of the mitochondrial network. To investigate the functional consequences of lack of GDAP1 in vivo, we generated a Gdap1 knockout mouse. The affected animals presented abnormal motor behavior starting at the age of 3 months. Electrophysiological and biochemical studies confirmed the axonal nature of the neuropathy whereas histopathological studies over time showed progressive loss of motor neurons (MNs) in the anterior horn of the spinal cord and defects in neuromuscular junctions. Analyses of cultured embryonic MNs and adult dorsal root ganglia neurons from affected animals demonstrated large and defective mitochondria, changes in the ER cisternae, reduced acetylation of cytoskeletal α-tubulin and increased autophagy vesicles. Importantly, MNs showed reduced cytosolic calcium and SOCE response. The development and characterization of the GDAP1 neuropathy mice model thus revealed that some of the pathophysiological changes present in axonal recessive form of the GDAP1-related CMT might be the consequence of changes in the mitochondrial network biology and mitochondria-endoplasmic reticulum interaction leading to abnormalities in calcium homeostasis.

Age-Dependent Defects of Regulatory B Cells in Wiskott-Aldrich Syndrome Gene Knockout Mice.

The Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency characterized by recurrent infections, thrombocytopenia, eczema, and high incidence of malignancy and autoimmunity. The cellular mechanisms underlying autoimmune complications in WAS have been extensively studied; however, they remain incompletely defined. We investigated the characteristics of IL-10-producing CD19+CD1dhighCD5+ B cells (CD1dhighCD5+ Breg) obtained from Was gene knockout (WKO) mice and found that their numbers were significantly lower in these mice compared to wild type (WT) controls. Moreover, we found a significant age-dependent reduction of the percentage of IL-10-expressing cells in WKO CD1dhighCD5+ Breg cells as compared to age-matched WT control mice. CD1dhighCD5+ Breg cells from older WKO mice did not suppress the in vitro production of inflammatory cytokines from activated CD4+ T cells. Interestingly, CD1dhighCD5+ Breg cells from older WKO mice displayed a basal activated phenotype which may prevent normal cellular responses, among which is the expression of IL-10. These defects may contribute to the susceptibility to autoimmunity with age in patients with WAS.

The Combined Pedicled Anterolateral Thigh and Vastus Lateralis Flap as Filler for Complex Perineal Defects.

Extensive defects of the pelvis and genitoperineal region are a reconstructive challenge. We discuss a consecutive series of 25 reconstructions with the pedicled anterolateral thigh (ALT) flap including muscle part of the vastus lateralis (VL) in 23 patients from October 1999 to September 2012.Only surface defects larger than 100 cm and reconstructions by composite ALT + VL were included in this retrospective analysis. Of the 23 patients, 19 underwent oncologic resection, whereas 4 cases presented Fournier gangrene. Three patients did not reach 6 months of follow-up and were excluded from further data analysis. Among the remaining 20 patients (22 reconstructions), average follow-up period was 14 months (range, 10-18 months). Patient's average age was 60 years. Average size of the defect was 182 cm.Postoperative complications included 1 (4.5%) flap necrosis out of 22 raised flaps, 1 partial flap necrosis after venous congestion, and 2 cases where a complementary reconstructive procedure was performed due to remaining defect or partial flap failure. In 6 cases, peripheral wound dehiscence (27%) was treated by debridement followed by split-thickness skin graft or advancement local flaps. Defect size was significantly related to postoperative complications and increased hospital stay, especially in those patients who underwent preoperative radiotherapy. At the end of the follow-up period, a long-term and satisfactory coverage was obtained in all patients without functional deficits.This consecutive series of composite ALT + VL flap shows that, in case of extended defects, the flap provides an excellent and adjustable muscle mass, is reliable with minimal donor-site morbidity, and can even be designed as a sensate flap.

Long term trends in prevalence of neural tube defects in Europe: population based study.

STUDY QUESTION: What are the long term trends in the total (live births, fetal deaths, and terminations of pregnancy for fetal anomaly) and live birth prevalence of neural tube defects (NTD) in Europe, where many countries have issued recommendations for folic acid supplementation but a policy for mandatory folic acid fortification of food does not exist? METHODS: This was a population based, observational study using data on 11 353 cases of NTD not associated with chromosomal anomalies, including 4162 cases of anencephaly and 5776 cases of spina bifida from 28 EUROCAT (European Surveillance of Congenital Anomalies) registries covering approximately 12.5 million births in 19 countries between 1991 and 2011. The main outcome measures were total and live birth prevalence of NTD, as well as anencephaly and spina bifida, with time trends analysed using random effects Poisson regression models to account for heterogeneities across registries and splines to model non-linear time trends. SUMMARY ANSWER AND LIMITATIONS: Overall, the pooled total prevalence of NTD during the study period was 9.1 per 10 000 births. Prevalence of NTD fluctuated slightly but without an obvious downward trend, with the final estimate of the pooled total prevalence of NTD in 2011 similar to that in 1991. Estimates from Poisson models that took registry heterogeneities into account showed an annual increase of 4% (prevalence ratio 1.04, 95% confidence interval 1.01 to 1.07) in 1995-99 and a decrease of 3% per year in 1999-2003 (0.97, 0.95 to 0.99), with stable rates thereafter. The trend patterns for anencephaly and spina bifida were similar, but neither anomaly decreased substantially over time. The live birth prevalence of NTD generally decreased, especially for anencephaly. Registration problems or other data artefacts cannot be excluded as a partial explanation of the observed trends (or lack thereof) in the prevalence of NTD. WHAT THIS STUDY ADDS: In the absence of mandatory fortification, the prevalence of NTD has not decreased in Europe despite longstanding recommendations aimed at promoting peri-conceptional folic acid supplementation and existence of voluntary folic acid fortification. FUNDING, COMPETING INTERESTS, DATA SHARING: The study was funded by the European Public Health Commission, EUROCAT Joint Action 2011-2013. HD and ML received support from the European Commission DG Sanco during the conduct of this study. No additional data available.

Alzheimer's disease-like APP processing in wild-type mice identifies synaptic defects as initial steps of disease progression.

BACKGROUND: Alzheimer's disease (AD) is the most frequent form of dementia in the elderly and no effective treatment is currently available. The mechanisms triggering AD onset and progression are still imperfectly dissected. We aimed at deciphering the modifications occurring in vivo during the very early stages of AD, before the development of amyloid deposits, neurofibrillary tangles, neuronal death and inflammation. Most current AD models based on Amyloid Precursor Protein (APP) overproduction beginning from in utero, to rapidly reproduce the histological and behavioral features of the disease within a few months, are not appropriate to study the early steps of AD development. As a means to mimic in vivo amyloid APP processing closer to the human situation in AD, we used an adeno-associated virus (AAV)-based transfer of human mutant APP and Presenilin 1 (PS1) genes to the hippocampi of two-month-old C57Bl/6 J mice to express human APP, without significant overexpression and to specifically induce its amyloid processing. RESULTS: The human APP, βCTF and Aβ42/40 ratio were similar to those in hippocampal tissues from AD patients. Three months after injection the murine Tau protein was hyperphosphorylated and rapid synaptic failure occurred characterized by decreased levels of both PSD-95 and metabolites related to neuromodulation, on proton magnetic resonance spectroscopy ((1)H-MRS). Astrocytic GLT-1 transporter levels were lower and the tonic glutamatergic current was stronger on electrophysiological recordings of CA1 hippocampal region, revealing the overstimulation of extrasynaptic N-methyl D-aspartate receptor (NMDAR) which precedes the loss of long-term potentiation (LTP). These modifications were associated with early behavioral impairments in the Open-field, Y-maze and Morris Mater Maze tasks. CONCLUSIONS: Altogether, this demonstrates that an AD-like APP processing, yielding to levels of APP, βCTF and Aβ42/Aβ40 ratio similar to those observed in AD patients, are sufficient to rapidly trigger early steps of the amyloidogenic and Tau pathways in vivo. With this strategy, we identified a sequence of early events likely to account for disease onset and described a model that may facilitate efforts to decipher the factors triggering AD and to evaluate early neuroprotective strategies.

Transabdominal-pelvic-perineal (TAPP) anterolateral thigh flap: A new reconstructive technique for complex defects following extended abdominoperineal resection.

BACKGROUND: Abdominoperineal resection (APR) following radiotherapy is associated with a high rate of perineal wound complications. The anterolateral thigh (ALT) flap, combined with the vastus lateralis (VL) muscle, can cover complex perineal and pelvic anteroposterior defects. This is used for the first time transabdominally through the pelvis and the perineum (TAPP) in the infero-posterior directions; this technique has been described and illustrated in this study. METHODS: Among over 90 patients who underwent perineal reconstruction between May 2004 and June 2011, six patients presented high-grade tumours invading perineum, pelvis and sacrum, thereby resulting in a continuous anteroposterior defect. ALT + VL TAPP reconstructions were performed after extended APR and, subsequently, sacrectomy. Patients were examined retrospectively to determine demographics, operative time, complications (general and flap-related), time to complete healing and length of hospital stay. Long-term flap coverage, flap volume stability and functional and aesthetic outcomes were assessed. RESULTS: Mean operating time of the reconstruction was 290 min. No deaths occurred. One patient presented partial flap necrosis. Another patient presented a novel wound dehiscence after flap healing, due to secondary skin dissemination of the primary tumour. Following volumetric flap analysis on serial post-operative CT scans, no significant flap atrophy was observed. All flaps fully covered the defects. No late complications such as fistulas or perineal hernias occurred. Donor-site recovery was uneventful with no functional deficits. CONCLUSIONS: The use of the ALT + VL flap transabdominally is an innovative method to reconstruct exceptionally complex perineal and pelvic defects extending up to the lower back. This flap guarantees superior bulk, obliterating all pelvic dead space, with the fascia lata (FL) supporting the pelvic floor.