Using an in-vitro biofilm model to assess the virulence potential of Bacterial Vaginosis or non-Bacterial Vaginosis Gardnerella vaginalis isolates

Gardnerella vaginalis is the most common species found in bacterial vaginosis (BV). However, it is also present in a significant proportion of healthy women and G. vaginalis vaginal colonization does not always lead to BV. In an effort to better understand the differences between G. vaginalis isolated from women with a positive (BV) versus a negative (non-BV) diagnosis of BV, we compared the virulence potential of 7 BV and 7 non-BV G. vaginalis isolates and assessed the virulence factors related to biofilm formation, namely: initial adhesion and cytotoxic effect, biofilm accumulation, susceptibility to antibiotics, and transcript levels of the known vaginolysin, and sialidase genes. Furthermore, we also determined the ability of G. vaginalis to displace lactobacilli previously adhered to HeLa cells. Our results showed that non-BV strains were less virulent than BV strains, as suggested by the lower cytotoxicity and initial adhesion to Hela cells. Significant differences in expression of known virulence genes were also detected, further suggesting a higher virulence potential of the BV associated G. vaginalis. Importantly, we demonstrated that BV associated G. vaginalis were able to displace pre-coated vaginal protective lactobacilli and we hypothesize this to be a trigger for BV development.European Union funds (FEDER/COMPETE) and by national funds (FCT) under the project with reference FCOMP-01-0124-FEDER-008991 (PTDC/BIA-MIC/098228/2008). FCT Strategic Project of UID/BIO/04469/2013 unit the project NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte(ON.2 – O Novo Norte), QREN, FEDER, and the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462). FCT individual fellowship SFRH/BD/93963/2013

Direct Visualization by Cryo-EM of the Mycobacterial Capsular Layer: A Labile Structure Containing ESX-1-Secreted Proteins

The cell envelope of mycobacteria, a group of Gram positive bacteria, is composed of a plasma membrane and a Gram-negative-like outer membrane containing mycolic acids. In addition, the surface of the mycobacteria is coated with an ill-characterized layer of extractable, non-covalently linked glycans, lipids and proteins, collectively known as the capsule, whose occurrence is a matter of debate. By using plunge freezing cryo-electron microscopy technique, we were able to show that pathogenic mycobacteria produce a thick capsule, only present when the cells were grown under unperturbed conditions and easily removed by mild detergents. This detergent-labile capsule layer contains arabinomannan, α-glucan and oligomannosyl-capped glycolipids. Further immunogenic and proteomic analyses revealed that Mycobacterium marinum capsule contains high amounts of proteins that are secreted via the ESX-1 pathway. Finally, cell infection experiments demonstrated the importance of the capsule for binding to cells and dampening of pro-inflammatory cytokine response. Together, these results show a direct visualization of the mycobacterial capsular layer as a labile structure that contains ESX-1-secreted proteins

Biallelic MED27 variants lead to variable ponto-cerebello-lental degeneration with movement disorders.

MED27 is a subunit of the Mediator multiprotein complex, which is involved in transcriptional regulation. Biallelic MED27 variants have recently been suggested to be responsible for an autosomal recessive neurodevelopmental disorder with spasticity, cataracts and cerebellar hypoplasia. We further delineate the clinical phenotype of MED27-related disease by characterizing the clinical and radiological features of 57 affected individuals from 30 unrelated families with biallelic MED27 variants. Using exome sequencing and extensive international genetic data sharing, 39 unpublished affected individuals from 18 independent families with biallelic missense variants in MED27 have been identified (29 females, mean age at last follow-up 17 ± 12.4 years, range 0.1-45). Follow-up and hitherto unreported clinical features were obtained from the published 12 families. Brain MRI scans from 34 cases were reviewed. MED27-related disease manifests as a broad phenotypic continuum ranging from developmental and epileptic-dyskinetic encephalopathy to variable neurodevelopmental disorder with movement abnormalities. It is characterized by mild to profound global developmental delay/intellectual disability (100%), bilateral cataracts (89%), infantile hypotonia (74%), microcephaly (62%), gait ataxia (63%), dystonia (61%), variably combined with epilepsy (50%), limb spasticity (51%), facial dysmorphism (38%) and death before reaching adulthood (16%). Brain MRI revealed cerebellar atrophy (100%), white matter volume loss (76.4%), pontine hypoplasia (47.2%) and basal ganglia atrophy with signal alterations (44.4%). Previously unreported 39 affected individuals had seven homozygous pathogenic missense MED27 variants, five of which were recurrent. An emerging genotype-phenotype correlation was observed. This study provides a comprehensive clinical-radiological description of MED27-related disease, establishes genotype-phenotype and clinical-radiological correlations and suggests a differential diagnosis with syndromes of cerebello-lental neurodegeneration and other subtypes of 'neuro-MEDopathies'

Understanding deformation with high angular resolution electron backscatter diffraction (HR-EBSD)

High angular resolution electron backscatter diffraction (HR-EBSD) affords an increase in angular resolution, as compared to 'conventional' Hough transform based EBSD, of two orders of magnitude, enabling measurements of relative misorientations of 1 x 10−4 rads (~ 0.006°) and changes in (deviatoric) lattice strain with a precision of 1 x 10−4. This is achieved through direct comparison of two or more diffraction patterns using sophisticated cross-correlation based image analysis routines. Image shifts between zone axes in the two-correlated diffraction pattern are measured with sub-pixel precision and this realises the ability to measure changes in interplanar angles and lattice orientation with a high degree of sensitivity. These shifts are linked to strains and lattice rotations through simple geometry. In this manuscript, we outline the basis of the technique and two case studies that highlight its potential to tackle real materials science challenges, such as deformation patterning in polycrystalline alloys

AstroEBSD: exploring new space in pattern indexing with methods launched from an astronomical approach

Electron backscatter diffraction (EBSD) is a technique used to measure crystallographic features in the scanning electron microscope. The technique is highly automated and readily accessible in many laboratories. EBSD pattern indexing is conventionally performed with raw electron backscatter patterns. These patterns are software processed to locate the band centres (and sometimes edges) from which the crystallographic index of each band is determined. Once a consistent index for many bands is obtained, the crystal orientation with respect to a reference sample and detector orientation can be determined and presented. Unfortunately, because of challenges related to crystal symmetry, there are limited available pattern-indexing approaches and this has probably hampered open development of the technique. In this article, a new method of pattern indexing is presented, based upon a method with which satellites locate themselves in the night sky, and its effectiveness is systematically demonstrated using dynamical simulations and real experimental patterns. The benefit of releasing this new algorithm as open-source software is demonstrated when this indexing process is utilized, together with dynamical solutions, to provide some of the first accuracy assessments of an indexing solution. In disclosing a new indexing algorithm, and software processing toolkit, the authors hope to open up EBSD developments to more users. The software code and example data are released alongside this article for third party developments

AstroEBSD: exploring new space in pattern indexing with methods launched from an astronomical approach

Electron backscatter diffraction (EBSD) is a technique used to measure crystallographic features in the scanning electron microscope. The technique is highly automated and readily accessible in many laboratories. EBSD pattern indexing is conventionally performed with raw electron backscatter patterns. These patterns are software processed to locate the band centres (and sometimes edges) from which the crystallographic index of each band is determined. Once a consistent index for many bands is obtained, the crystal orientation with respect to a reference sample and detector orientation can be determined and presented. Unfortunately, because of challenges related to crystal symmetry, there are limited available pattern-indexing approaches and this has probably hampered open development of the technique. In this article, a new method of pattern indexing is presented, based upon a method with which satellites locate themselves in the night sky, and its effectiveness is systematically demonstrated using dynamical simulations and real experimental patterns. The benefit of releasing this new algorithm as open-source software is demonstrated when this indexing process is utilized, together with dynamical solutions, to provide some of the first accuracy assessments of an indexing solution. In disclosing a new indexing algorithm, and software processing toolkit, the authors hope to open up EBSD developments to more users. The software code and example data are released alongside this article for third party developments