Antigen Recognition By Autoreactive Cd4+ Thymocytes Drives Homeostasis Of The Thymic Medulla

The thymic medulla is dedicated for purging the T-cell receptor (TCR) repertoire of self-reactive specificities. Medullary thymic epithelial cells (mTECs) play a pivotal role in this process because they express numerous peripheral tissue-restricted self-antigens. Although it is well known that medulla formation depends on the development of single-positive (SP) thymocytes, the mechanisms underlying this requirement are incompletely understood. We demonstrate here that conventional SP CD4⁺ thymocytes bearing autoreactive TCRs drive a homeostatic process that fine-tunes medullary plasticity in adult mice by governing the expansion and patterning of the medulla. This process exhibits strict dependence on TCR-reactivity with self-antigens expressed by mTECs, as well as engagement of the CD28-CD80/CD86 costimulatory axis. These interactions induce the expression of lymphotoxin α in autoreactive CD4⁺ thymocytes and RANK in mTECs. Lymphotoxin in turn drives mTEC development in synergy with RANKL and CD40L. Our results show that Ag-dependent interactions between autoreactive CD4⁺ thymocytes and mTECs fine-tune homeostasis of the medulla by completing the signaling axes implicated in mTEC expansion and medullary organization.     

Mutations in Lama1 Disrupt Retinal Vascular Development and Inner Limiting Membrane Formation

The Neuromutagenesis Facility at the Jackson Laboratory generated a mouse model of retinal vasculopathy, nmf223, which is characterized clinically by vitreal fibroplasia and vessel tortuosity. nmf223 homozygotes also have reduced electroretinogram responses, which are coupled histologically with a thinning of the inner nuclear layer. The nmf223 locus was mapped to chromosome 17, and a missense mutation was identified in Lama1 that leads to the substitution of cysteine for a tyrosine at amino acid 265 of laminin α1, a basement membrane protein. Despite normal localization of laminin α1 and other components of the inner limiting membrane, a reduced integrity of this structure was suggested by ectopic cells and blood vessels within the vitreous. Immunohistochemical characterization of nmf223 homozygous retinas demonstrated the abnormal migration of retinal astrocytes into the vitreous along with the persistence of hyaloid vasculature. The Y265C mutation significantly reduced laminin N-terminal domain (LN) interactions in a bacterial two-hybrid system. Therefore, this mutation could affect interactions between laminin α1 and other laminin chains. To expand upon these findings, a Lama1 null mutant, Lama1^tm1.1Olf, was generated that exhibits a similar but more severe retinal phenotype than that seen in nmf223 homozygotes. The increased severity of the Lama1 null mutant phenotype is probably due to the complete loss of the inner limiting membrane in these mice. This first report of viable Lama1 mouse mutants emphasizes the importance of this gene in retinal development. The data presented herein suggest that hypomorphic mutations in human LAMA1 could lead to retinal disease.     

Tracing hotspots of soil erosion in high mountain environments: how forensic science based on plant eDNA can lead the way. An opinion

Plant and Soil - High mountain environments are among the most fragile on Earth. Due to anthropogenic disturbances and the exposure to extreme weather events, the rates of soil erosion have...     

Mapping of Bacterial Biofilm Local Mechanics by Magnetic Microparticle Actuation

Most bacteria live in the form of adherent communities forming three-dimensional material anchored to artificial or biological surfaces, with profound impact on many human activities. Biofilms are recognized as complex systems but their physical properties have been mainly studied from a macroscopic perspective. To determine biofilm local mechanical properties, reveal their potential heterogeneity, and investigate their relation to molecular traits, we have developed a seemingly new microrheology approach based on magnetic particle infiltration in growing biofilms. Using magnetic tweezers, we achieved what was, to our knowledge, the first three-dimensional mapping of the viscoelastic parameters on biofilms formed by the bacterium Escherichia coli. We demonstrate that its mechanical profile may exhibit elastic compliance values spread over three orders of magnitude in a given biofilm. We also prove that heterogeneity strongly depends on external conditions such as growth shear stress. Using strains genetically engineered to produce well-characterized cell surface adhesins, we show that the mechanical profile of biofilm is exquisitely sensitive to the expression of different surface appendages such as F pilus or curli. These results provide a quantitative view of local mechanical properties within intact biofilms and open up an additional avenue for elucidating the emergence and fate of the different microenvironments within these living materials.     

Computational Modelling of Metastasis Development in Renal Cell Carcinoma

The biology of the metastatic colonization process remains a poorly understood phenomenon. To improve our knowledge of its dynamics, we conducted a modelling study based on multi-modal data from an orthotopic murine experimental system of metastatic renal cell carcinoma. The standard theory of metastatic colonization usually assumes that secondary tumours, once established at a distant site, grow independently from each other and from the primary tumour. Using a mathematical model that translates this assumption into equations, we challenged this theory against our data that included: 1) dynamics of primary tumour cells in the kidney and metastatic cells in the lungs, retrieved by green fluorescent protein tracking, and 2) magnetic resonance images (MRI) informing on the number and size of macroscopic lesions. Critically, when calibrated on the growth of the primary tumour and total metastatic burden, the predicted theoretical size distributions were not in agreement with the MRI observations. Moreover, tumour expansion only based on proliferation was not able to explain the volume increase of the metastatic lesions. These findings strongly suggested rejection of the standard theory, demonstrating that the time development of the size distribution of metastases could not be explained by independent growth of metastatic foci. This led us to investigate the effect of spatial interactions between merging metastatic tumours on the dynamics of the global metastatic burden. We derived a mathematical model of spatial tumour growth, confronted it with experimental data of single metastatic tumour growth, and used it to provide insights on the dynamics of multiple tumours growing in close vicinity. Together, our results have implications for theories of the metastatic process and suggest that global dynamics of metastasis development is dependent on spatial interactions between metastatic lesions.     

Improving Quality Control of Contagious Caprine Pleuropneumonia Vaccine with Tandem Mass Spectrometry

Vaccines to protect livestock against contagious caprine pleuropneumonia (CCPP) consist of inactivated, adjuvanted antigens. Quality control of these vaccines is challenging as total protein quantification provides no indication of protein identity or purity, and culture is not an option. Here, a tandem mass spectrometry approach is used to identify the mycoplasma antigen contained in reference samples and in commercial CCPP vaccines. By the same approach, the relative amounts of mycoplasma antigen and residual proteins originating from the production medium are determined. Mass spectrometry allows easy and rapid identification of the peptides present in the vaccine samples. Alongside the most probable mycoplasma species effectively present in the vaccines, a very high proportion of peptides from medium constituents are detected in the commercial vaccines tested.     

Design and evaluation of analogues of the bacterial cell-wall peptidoglycan motif L-Lys-D-Ala-D-Ala for use in a vancomycin biosensor

Four small molecular receptors of vancomycin have been designed to make part of a novel biosensor device based on the FTIR-ATR detection: N-Boc (2a) or N-Ac (2b)-6-aminocaproyl-D-Ala-D-Ala and N-Boc (3a) or N-Ac (3b)-6-aminocaproyl-D-Ala-d-Ser. Using an original microbiological approach to assess the competition of compounds with the natural target of vancomycin in bacteria, EC(50) values of 6.3-8.0 x 10(-5)M (2a-b) and 7.1-9.3 x 10(-4)M (3a-b) were determined. Vancomycin:2b complex was characterized by MS.     

Improved mass spectrometric proteomic profiling of the secretome of rat vascular endothelial cells

Serum albumin contamination of cells cultured in vitro significantly impedes the mass spectrometric analysis of proteins secreted by the cells. Here we report a novel washing and culturing technique for rat vascular endothelial cells that considerably reduces the concentration of the commonly used additive for cell culture, bovine serum albumin (BSA), in the secretome of these cells. Cells are rinsed stringently and cultured for 24 h in serum-free media without appreciably impeding cell growth or viability. The percentage of BSA scans identified by tandem mass spectrometry (LC-MS/MS) in stringently rinsed cells (average 13.2%) was significantly lower than either the moderately rinsed or no rinse cell treatments (average 35.2% and 45.2% respectively). Furthermore, the stringent wash treatment allowed the confident identification of a larger portion of the secretome of rat endothelial cells by LC-MS/MS.