The role of peptidoglycan in chlamydial cell division: towards resolving the chlamydial anomaly.

Chlamydiales are obligate intracellular bacteria including some important pathogens causing trachoma, genital tract infections and pneumonia, among others. They share an atypical division mechanism, which is independent of an FtsZ homologue. However, they divide by binary fission, in a process inhibited by penicillin derivatives, causing the formation of an aberrant form of the bacteria, which is able to survive in the presence of the antibiotic. The paradox of penicillin sensitivity of chlamydial cells in the absence of detectable peptidoglycan (PG) was dubbed the chlamydial anomaly, since no PG modified by enzymes (Pbps) that are the usual target of penicillin could be detected in Chlamydiales. We review here the recent advances in this field with the first direct and indirect evidences of PG-like material in both Chlamydiaceae and Chlamydia-related bacteria. Moreover, PG biosynthesis is required for proper localization of the newly described septal proteins RodZ and NlpD. Taken together, these new results set the stage for a better understanding of the role of PG and septal proteins in the division mechanism of Chlamydiales and illuminate the long-standing chlamydial anomaly. Moreover, understanding the chlamydial division mechanism is critical for the development of new antibiotics for the treatment of chlamydial chronic infections.

A founder CEP120 mutation in Jeune asphyxiating thoracic dystrophy expands the role of centriolar proteins in skeletal ciliopathies.

Jeune asphyxiating thoracic dystrophy (JATD) is a skeletal dysplasia characterized by a small thoracic cage and a range of skeletal and extra-skeletal anomalies. JATD is genetically heterogeneous with at least nine genes identified, all encoding ciliary proteins, hence the classification of JATD as a skeletal ciliopathy. Consistent with the observation that the heterogeneous molecular basis of JATD has not been fully determined yet, we have identified two consanguineous Saudi families segregating JATD who share a single identical ancestral homozygous haplotype among the affected members. Whole-exome sequencing revealed a single novel variant within the disease haplotype in CEP120, which encodes a core centriolar protein. Subsequent targeted sequencing of CEP120 in Saudi and European JATD cohorts identified two additional families with the same missense mutation. Combining the four families in linkage analysis confirmed a significant genome-wide linkage signal at the CEP120 locus. This missense change alters a highly conserved amino acid within CEP120 (p.Ala199Pro). In addition, we show marked reduction of cilia and abnormal number of centrioles in fibroblasts from one affected individual. Inhibition of the CEP120 ortholog in zebrafish produced pleiotropic phenotypes characteristic of cilia defects including abnormal body curvature, hydrocephalus, otolith defects and abnormal renal, head and craniofacial development. We also demonstrate that in CEP120 morphants, cilia are shortened in the neural tube and disorganized in the pronephros. These results are consistent with aberrant CEP120 being implicated in the pathogenesis of JATD and expand the role of centriolar proteins in skeletal ciliopathies.

Contribution of femoral and proximal sciatic nerve branches to the sensory innervation of hindlimb digits in the rat

The present study was performed to investigate the possibility of 'aberrant' innervation of the tips of the hindlimb digits in the rat, i.e., from other sources than the femoral and the main sciatic branches (tibial, peroneal, sural). Cutaneous injections of fluorescent tracers in the digits were combined with either selective nerve transections to restrict afferent routes followed by detection of labeled neurons in dorsal root ganglia (DRGs), or by a delayed application of a second tracer to afferent nerves under study to detect double labeled neurons in DRGs. The results show that the tips of the digits were represented in DRGs L3-6. The femoral nerve afferents from digits 1 and 2 projected primarily to DRG L3 and to a smaller extent to DRG L4. A small number of neurons from primarily medial digits 1 and 2, but also from lateral digits 3-5, were found to project to DRGs L4 and L5 via a proximal branch that leaves the sciatic nerve near the sciatic notch and runs distally in the posterior part of the thigh, here called the musculocutaneous nerve of the hindlimb. We also have some evidence indicating innervation of the tips of the digits from the posterior cutaneous nerve of the thigh. Aberrant innervation such as that described here might contribute to remaining and perhaps abnormal sensibility after nerve injury and is of interest for the interpretation of results in experimental studies of collateral and regenerative sprouting after such injury

APP processing and b-amyloid deposition in sporadic Creutzfeldt-Jakob patients is dependent on Dab1.

Alzheimer"s disease and prion pathologies (e.g., Creutzfeldt-Jakob disease (CJD)) display profound neural lesions associated with aberrant protein processing and extracellular amyloid deposits. Dab1 has been implicated in the regulation of Amyloid Precursor Protein (APP), but a direct link between human prion diseases and Dab1/APP interactions has not been published. Here we examined this putative relationship in seventeen cases of sporadic CJD (sCJD) post mortem. Biochemical analyses of brain tissue revealed two groups, which also correlated with PrPsc types 1 and 2. One group, with PrPsc type 1 showed increased Dab1 phosphorylation, and lower CTF production with an absence of A deposition. The second sCJD group, which carried PrPsc type 2, showed lower levels of Dab1 phosphorylation and CTF production, and A deposition. Thus, the present observations suggest a correlation between Dab1-phosphorylation, A deposition and PrPsc type in sCJD.

Regulation of recombination at yeast nuclear pores controls repair and triplet repeat stability.

Secondary structure-forming DNA sequences such as CAG repeats interfere with replication and repair, provoking fork stalling, chromosome fragility, and recombination. In budding yeast, we found that expanded CAG repeats are more likely than unexpanded repeats to localize to the nuclear periphery. This positioning is transient, occurs in late S phase, requires replication, and is associated with decreased subnuclear mobility of the locus. In contrast to persistent double-stranded breaks, expanded CAG repeats at the nuclear envelope associate with pores but not with the inner nuclear membrane protein Mps3. Relocation requires Nup84 and the Slx5/8 SUMO-dependent ubiquitin ligase but not Rad51, Mec1, or Tel1. Importantly, the presence of the Nup84 pore subcomplex and Slx5/8 suppresses CAG repeat fragility and instability. Repeat instability in nup84, slx5, or slx8 mutant cells arises through aberrant homologous recombination and is distinct from instability arising from the loss of ligase 4-dependent end-joining. Genetic and physical analysis of Rad52 sumoylation and binding at the CAG tract suggests that Slx5/8 targets sumoylated Rad52 for degradation at the pore to facilitate recovery from acute replication stress by promoting replication fork restart. We thereby confirmed that the relocation of damage to nuclear pores plays an important role in a naturally occurring repair process.

Mutations in LONP1, a mitochondrial matrix protease, cause CODAS syndrome.

Cerebral, ocular, dental, auricular, skeletal anomalies (CODAS) syndrome (MIM 600373) was first described and named by Shehib et al, in 1991 in a single patient. The anomalies referred to in the acronym are as follows: cerebral-developmental delay, ocular-cataracts, dental-aberrant cusp morphology and delayed eruption, auricular-malformations of the external ear, and skeletal-spondyloepiphyseal dysplasia. This distinctive constellation of anatomical findings should allow easy recognition but despite this only four apparently sporadic patients have been reported in the last 20 years indicating that the full phenotype is indeed very rare with perhaps milder or a typical presentations that are allelic but without sufficient phenotypic resemblance to permit clinical diagnosis. We performed exome sequencing in three patients (an isolated case and a brother and sister sib pair) with classical features of CODAS. Sanger sequencing was used to confirm results as well as for mutation discovery in a further four unrelated patients ascertained via their skeletal features. Compound heterozygous or homozygous mutations in LONP1 were found in all (8 separate mutations; 6 missense, 1 nonsense, 1 small in-frame deletion) thus establishing the genetic basis of CODAS and the pattern of inheritance (autosomal recessive). LONP1 encodes an enzyme of bacterial ancestry that participates in protein turnover within the mitochondrial matrix. The mutations cluster at the ATP-binding and proteolytic domains of the enzyme. Biallelic inheritance and clustering of mutations confirm dysfunction of LONP1 activity as the molecular basis of CODAS but the pathogenesis remains to be explored.

Sustained CTL activation by murine pulmonary epithelial cells promotes the development of COPD-like disease

Chronic obstructive pulmonary disease (COPD) is a lethal progressive lung disease culminating in permanent airway obstruction and alveolar enlargement. Previous studies suggest CTL involvement in COPD progression; however, their precise role remains unknown. Here, we investigated whether the CTL activation receptor NK cell group 2D (NKG2D) contributes to the development of COPD. Using primary murine lung epithelium isolated from mice chronically exposed to cigarette smoke and cultured epithelial cells exposed to cigarette smoke extract in vitro, we demonstrated induced expression of the NKG2D ligand retinoic acid early tran - script 1 (RAET1)as well as NKG2D-mediated cytotoxicity. Furthermore, a genetic model of inducible RAET1 expression on mouse pulmonary epithelial cells yielded a severe emphysematous phenotype characterized by epithelial apoptosis and increased CTL activation, which was reversed by blocking NKG2D activation. We also assessed whether NKG2D ligand expression corresponded with pulmonary disease in human patients by staining airway and peripheral lung tissues from never smokers, smokers with normal lung function, and current and former smokers with COPD. NKG2D ligand expression was independent of NKG2D receptor expression in COPD patients, demonstrating that ligand expression is the limiting factor in CTL activation. These results demonstrate that aberrant, persistent NKG2D ligand expression in the pulmonary epithelium contributes to the development of COPD pathologies.

Spatial radial maze procedures and setups to dissociate local and distal relational spatial frameworks in humans.

BACKGROUND: Radial maze tasks have been used to assess optimal foraging and spatial abilities in rodents. The spatial performance was based on a capacity to rely on a configuration of local and distant cues. We adapted maze procedures assessing the relative weight of local cues and distant landmarks for arm choice in humans. NEW METHOD: The procedure allowed testing memory of places in four experimental setups: a fingertip texture-groove maze, a tactile screen maze, a virtual radial maze and a walking size maze. During training, the four reinforced positions remained fixed relative to local and distal cues. During subsequent conflict trials, these frameworks were made conflictive in the prediction of reward locations. RESULTS: Three experiments showed that the relative weight of local and distal relational cues is affected by different factors such as cues' nature, visual access to the environment, real vs. virtual environment, and gender. A fourth experiment illustrated how a walking maze can be used with people suffering intellectual disability. COMPARISON WITH EXISTING METHODS: In our procedure, long-term (reference) and short-term (working) memory can be assessed. It is the first radial task adapted to human that enables dissociating local and distal cues, to provides an indication as to their relative salience. Our mazes are moveable and easily used in limited spaces. Tasks are performed with realistic and spontaneous though controlled exploratory movements. CONCLUSION: Our tasks enabled highlighting the use of different strategies. In a clinical perspective, considering the use of compensatory strategies should orient towards adapted behavioural rehabilitation.

Global virtual teamwork. Building and communicating trust through technology

Tutkimuksen ensisijaisena tavoitteena oli tarkastella luottamuksen rakentumista virtuaalitiimissä. Keskeistä tarkastelussa olivat luottamuksen lähteiden löytäminen, suhteen rakentuminen sekä teknologiavälitteinen kommunikaatio. Myös käytännön keinoja ja sovelluksia etsittiin. Tässä tutkimuksessa luottamus nähtiin tärkeänä yhteistyön mahdollistajana sekä keskeisenä elementtinä ihmisten välisten suhteiden rakentumisessa. Tämä tutkimus oli empiirinen ja kuvaileva tapaustutkimus. Tutkimuksessa kvalitatiivista aineistoa kerättiin pääasiassa web-pohjaisen kyselyn sekä puhelinhaastattelun avulla. Aineistonkeruu toteutettiin siis pääasiassa virtuaalisesti. Saatu aineisto analysoitiin teemoittelun avulla. Tässä työssä teemoja etsittiin tekstistä pääasiassa teoriasta johdettujen oletusten perusteella. Tutkimuksen tuloksena oli, että luottamusta rakentavia mekanismeja ovat, karkeasti luokiteltuna, yhteiset päämäärät ja vastuut, kommunikaatio, sosiaalinen kanssakäyminen ja informaation jakaminen, toisten huomioiminen ja henkilökohtaiset ominaisuudet. Mekanismit eivät suuresti eronneet luottamuksen rakentumisen mekanismeista perinteisessä kontekstissa. Virtuaalitiimityön alkuvaiheessa luottamus pohjautui käsityksille toisten tiimin jäsenten kyvykkyydestä. Myös institutionaalinen identifioituminen loi pohjaa luottamukselle alkuvaiheessa. Muuten luottamus rakentui vähän kerrassaan tehtävään liittyvän kommunikaation ja sosiaalisen kommunikaation kautta. Tekojen merkitys korostui erityisesti ajan myötä. Työssä esitettiin myös käytännön keinoja luottamuksen rakentamiseksi. Olemassa olevien teknologioiden havaittiin tukevan hyvin suhteen rakentumista tiedon jakamiseen ja sen varastoimiseen liittyvissä tehtävissä. Sen sijaan vuorovaikutuksen näkökulmasta tuen ei nähty olevan yhtä kattavaa. Kaiken kaikkiaan kuitenkin parannuksella sosiaalisissa suhteissa voitaneen saada enemmän aikaan kuin parannuksilla teknologian suhteen.

A signaling mechanism coupling netrin-1/deleted in colorectal cancer chemoattraction to SNARE-mediated exocytosis in axonal growth cones

Directed cell migration and axonal guidance are essential steps in neural development. Both processes are controlled by specific guidance cues that activate the signaling cascades that ultimately control cytoskeletal dynamics. Another essential step in migration and axonal guidance is the regulation of plasmalemma turnover and exocytosis in leading edges and growth cones. However, the cross talk mechanisms linking guidance receptors and membrane exocytosis are not understood. Netrin-1 is a chemoattractive cue required for the formation of commissural pathways. Here, we show that the Netrin-1 receptor deleted in colorectal cancer (DCC) forms a protein complex with the t-SNARE (target SNARE) protein Syntaxin-1 (Sytx1). This interaction is Netrin-1 dependent both in vitro and in vivo, and requires specific Sytx1 and DCC domains. Blockade of Sytx1 function by using botulinum toxins abolished Netrin-1-dependent chemoattraction of axons in mouse neuronal cultures. Similar loss-of-function experiments in the chicken spinal cord in vivo using dominant-negative Sytx1 constructs or RNAi led to defects in commissural axon pathfinding reminiscent to those described in Netrin-1 and DCC loss-of-function models. We also show that Netrin-1 elicits exocytosis at growth cones in a Sytx1-dependent manner. Moreover, we demonstrate that the Sytx1/DCC complex associates with the v-SNARE (vesicle SNARE) tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) and that knockdown of TI-VAMP in the commissural pathway in the spinal cord results in aberrant axonal guidance phenotypes. Our data provide evidence of a new signaling mechanism that couples chemotropic Netrin-1/DCC axonal guidance and Sytx1/TI-VAMP SNARE proteins regulating membrane turnover and exocytosis.

A signaling mechanism coupling netrin-1/deleted in colorectal cancer chemoattraction to SNARE-mediated exocytosis in axonal growth cones

Directed cell migration and axonal guidance are essential steps in neural development. Both processes are controlled by specific guidance cues that activate the signaling cascades that ultimately control cytoskeletal dynamics. Another essential step in migration and axonal guidance is the regulation of plasmalemma turnover and exocytosis in leading edges and growth cones. However, the cross talk mechanisms linking guidance receptors and membrane exocytosis are not understood. Netrin-1 is a chemoattractive cue required for the formation of commissural pathways. Here, we show that the Netrin-1 receptor deleted in colorectal cancer (DCC) forms a protein complex with the t-SNARE (target SNARE) protein Syntaxin-1 (Sytx1). This interaction is Netrin-1 dependent both in vitro and in vivo, and requires specific Sytx1 and DCC domains. Blockade of Sytx1 function by using botulinum toxins abolished Netrin-1-dependent chemoattraction of axons in mouse neuronal cultures. Similar loss-of-function experiments in the chicken spinal cord in vivo using dominant-negative Sytx1 constructs or RNAi led to defects in commissural axon pathfinding reminiscent to those described in Netrin-1 and DCC loss-of-function models. We also show that Netrin-1 elicits exocytosis at growth cones in a Sytx1-dependent manner. Moreover, we demonstrate that the Sytx1/DCC complex associates with the v-SNARE (vesicle SNARE) tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) and that knockdown of TI-VAMP in the commissural pathway in the spinal cord results in aberrant axonal guidance phenotypes. Our data provide evidence of a new signaling mechanism that couples chemotropic Netrin-1/DCC axonal guidance and Sytx1/TI-VAMP SNARE proteins regulating membrane turnover and exocytosis.

A signaling mechanism coupling netrin-1/deleted in colorectal cancer chemoattraction to SNARE-mediated exocytosis in axonal growth cones

Directed cell migration and axonal guidance are essential steps in neural development. Both processes are controlled by specific guidance cues that activate the signaling cascades that ultimately control cytoskeletal dynamics. Another essential step in migration and axonal guidance is the regulation of plasmalemma turnover and exocytosis in leading edges and growth cones. However, the cross talk mechanisms linking guidance receptors and membrane exocytosis are not understood. Netrin-1 is a chemoattractive cue required for the formation of commissural pathways. Here, we show that the Netrin-1 receptor deleted in colorectal cancer (DCC) forms a protein complex with the t-SNARE (target SNARE) protein Syntaxin-1 (Sytx1). This interaction is Netrin-1 dependent both in vitro and in vivo, and requires specific Sytx1 and DCC domains. Blockade of Sytx1 function by using botulinum toxins abolished Netrin-1-dependent chemoattraction of axons in mouse neuronal cultures. Similar loss-of-function experiments in the chicken spinal cord in vivo using dominant-negative Sytx1 constructs or RNAi led to defects in commissural axon pathfinding reminiscent to those described in Netrin-1 and DCC loss-of-function models. We also show that Netrin-1 elicits exocytosis at growth cones in a Sytx1-dependent manner. Moreover, we demonstrate that the Sytx1/DCC complex associates with the v-SNARE (vesicle SNARE) tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) and that knockdown of TI-VAMP in the commissural pathway in the spinal cord results in aberrant axonal guidance phenotypes. Our data provide evidence of a new signaling mechanism that couples chemotropic Netrin-1/DCC axonal guidance and Sytx1/TI-VAMP SNARE proteins regulating membrane turnover and exocytosis.

Occurrence of the "applause sign" in patients with amyotrophic lateral sclerosis.

The applause sign was originally described as a quick bedside test to discriminate progressive supranuclear palsy (PSP) (positive applause sign, PAS) from Parkinson's disease (PD) and frontotemporal dementia (FTD) (negative applause sign). However, recent research demonstrated that the test is positive not only in a subset of patients with PD and FTD, but also in other neurodegenerative diseases. We tested 22 patients with amyotrophic lateral sclerosis (ALS) together with 22 healthy sex- and age-matched controls for the occurrence of PAS. Furthermore, we performed neuropsychological testing with the EXIT-25 battery to correlate PAS with neuropsychological deficits, especially frontal lobe dysfunction. Five ALS patients (23%) and none of the controls displayed PAS (p≤0.05). The occurrence of PAS in ALS patients was not correlated with pathologic EXIT-25 scores or subtests for aberrant motor behaviour. We describe for the first time the occurrence of the applause sign in ALS and provide additional evidence that PAS is not specific for Parkinsonian disorders. Although its occurrence has been related to aberrant motor behaviour due to frontal involvement, in our study PAS did not correlate with executive dysfunction as tested by the EXIT-25 test battery, or with subtests of aberrant motor behaviour.

Epigenetic Activation of SOX11 in Lymphoid Neoplasms by Histone Modifications

Recent studies have shown aberrant expression of SOX11 in various types of aggressive B-cell neoplasms. To elucidate the molecular mechanisms leading to such deregulation, we performed a comprehensive SOX11 gene expression and epigenetic study in stem cells, normal hematopoietic cells and different lymphoid neoplasms. We observed that SOX11 expression is associated with unmethylated DNA and presence of activating histone marks (H3K9/14Ac and H3K4me3) in embryonic stem cells and some aggressive B-cell neoplasms. In contrast, adult stem cells, normal hematopoietic cells and other lymphoid neoplasms do not express SOX11. Such repression was associated with silencing histone marks H3K9me2 and H3K27me3. The SOX11 promoter of non-malignant cells was consistently unmethylated whereas lymphoid neoplasms with silenced SOX11 tended to acquire DNA hypermethylation. SOX11 silencing in cell lines was reversed by the histone deacetylase inhibitor SAHA but not by the DNA methyltransferase inhibitor AZA. These data indicate that, although DNA hypermethylation of SOX11 is frequent in lymphoid neoplasms, it seems to be functionally inert, as SOX11 is already silenced in the hematopoietic system. In contrast, the pathogenic role of SOX11 is associated with its de novo expression in some aggressive lymphoid malignancies, which is mediated by a shift from inactivating to activating histone modifications.

Reversible Reprogramming of Circulating Memory T Follicular Helper Cell Function during Chronic HIV Infection.

Despite the overwhelming benefits of antiretroviral therapy (ART) in curtailing viral load in HIV-infected individuals, ART does not fully restore cellular and humoral immunity. HIV-infected individuals under ART show reduced responses to vaccination and infections and are unable to mount an effective antiviral immune response upon ART cessation. Many factors contribute to these defects, including persistent inflammation, especially in lymphoid tissues, where T follicular helper (Tfh) cells instruct and help B cells launch an effective humoral immune response. In this study we investigated the phenotype and function of circulating memory Tfh cells as a surrogate of Tfh cells in lymph nodes and found significant impairment of this cell population in chronically HIV-infected individuals, leading to reduced B cell responses. We further show that these aberrant memory Tfh cells exhibit an IL-2-responsive gene signature and are more polarized toward a Th1 phenotype. Treatment of functional memory Tfh cells with IL-2 was able to recapitulate the detrimental reprogramming. Importantly, this defect was reversible, as interfering with the IL-2 signaling pathway helped reverse the abnormal differentiation and improved Ab responses. Thus, reversible reprogramming of memory Tfh cells in HIV-infected individuals could be used to enhance Ab responses. Altered microenvironmental conditions in lymphoid tissues leading to altered Tfh cell differentiation could provide one explanation for the poor responsiveness of HIV-infected individuals to new Ags. This explanation has important implications for the development of therapeutic interventions to enhance HIV- and vaccine-mediated Ab responses in patients under ART.