Rapid Prototyping of Polymeric Nanopillars by 3D Direct Laser Writing for Controlling Cell Behavior

Mammalian cells have been widely shown to respond to nano-and microtopography that mimics the extracellular matrix. Synthetic nano-and micron-sized structures are therefore of great interest in the field of tissue engineering, where polymers are particularly attractive due to excellent biocompatibility and versatile fabrication methods. Ordered arrays of polymeric pillars provide a controlled topographical environment to study and manipulate cells, but processing methods are typically either optimized for the nano-or microscale. Here, we demonstrate polymeric nanopillar (NP) fabrication using 3D direct laser writing (3D DLW), which offers a rapid prototyping across both size regimes. The NPs are interfaced with NIH3T3 cells and the effect of tuning geometrical parameters of the NP array is investigated. Cells are found to adhere on a wide range of geometries, but the interface depends on NP density and length. The Cell Interface with Nanostructure Arrays (CINA) model is successfully extended to predict the type of interface formed on different NP geometries, which is found to correlate with the efficiency of cell alignment along the NPs. The combination of the CINA model with the highly versatile 3D DLW fabrication thus holds the promise of improved design of polymeric NP arrays for controlling cell growth

DMTs and Covid-19 severity in MS: a pooled analysis from Italy and France

We evaluated the effect of DMTs on Covid-19 severity in patients with MS, with a pooled-analysis of two large cohorts from Italy and France. The association of baseline characteristics and DMTs with Covid-19 severity was assessed by multivariate ordinal-logistic models and pooled by a fixed-effect meta-analysis. 1066 patients with MS from Italy and 721 from France were included. In the multivariate model, anti-CD20 therapies were significantly associated (OR = 2.05, 95%CI = 1.39–3.02, p < 0.001) with Covid-19 severity, whereas interferon indicated a decreased risk (OR = 0.42, 95%CI = 0.18–0.99, p = 0.047). This pooled-analysis confirms an increased risk of severe Covid-19 in patients on anti-CD20 therapies and supports the protective role of interferon

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Micro gas chromatography based on mems technology for the analysisof volatile species in planetary environments

International audienceGas chromatography is used since the Mars Viking missions in the 70’s to characterize the nature and amount of volatile chemical compounds present in planetary atmospheres, soils or rocks. This technique allows to separate the gaseous compound injected in the instrument for their subsequent detection either by a physical sensor, or a spectrometer giving information about the structure of the volatile. This pre-separation is precious to proceed to the identification of individual species present in a complex mixture. Moreover, it is a unique method to separate and quantify enantiomers of organic molecules which is a key information in astrobiology to assess the potential for such molecules to be related to a biotic or a pre-biotic process. Finally, the potential of this technique is proven by its current use in the Curiosity rover at the Mars surface, as it allowed to demonstrate the presence of organic material endogenous to Mars for the first time ever [1].But despite its efficiency, this instrumentation is based on laboratory technologies and requires for resources which are limited (e.g. carrier gas), making it a resources consuming instrumentation. That prevents it to be considered for small and light scientific payloads. This is one among reasons why our team initiated a research and technology action with the aim to miniaturize this type of instrumentation. This work relies on the use of micro-electro mechanical systems and their integration into a complete chromatographic system with the aim to gain one order of magnitude in term of resources required to make it work.In this communication we will present the different components that were developed for this project and their tested performances which show the potential for this system to be used in future in situ exploration space probes

Gas chromatography based on Micro Electro Mechanical Systems (MEMS)

International audienceFor more than 40 years, gas chromatography (GC) is used in space exploration for analysing the chemical composition of planetary environments in situ. It has been shown to be efficient for measuring the composition of volatile compounds present in planetary atmospheres (e.g. Venera and Pioneer missions to Venus) or surfaces (e.g. Viking and Mars Science Laboratory mission to Mars). Moreover, this instrumentation has a strong potential to help for seeking organic material of interest for astrobiology, and in particular organic molecules that could be related to a prebiotic chemistry or the emergence of life. However, through time, space missions are more and more constraining for instruments in terms of resources available (e.g. energy, mass). Moreover, small scientific payloads should be used in the future in order to send micro probes in planetary environments at a low cost, or in environments difficult from access today for a regular space probes (e.g. Icy satellites). For these reasons, and as GC is a chemical analyser of high interest for planetary sciences, our team started the development of an ultra-miniaturized gas chromatograph for space application using the Micro Electro Mechanical System technology that emerges today on Earth thanks to the progresses made in the fluidic MEMS. With this aim, we started studying and adapting to space instrumentation different MEMS components capable to reproduce the three main functionalities of a GC instrument, i.e. injection, separation, and detection. From this first set of components, we developed a prototype of MEMS GC in order to learn more about the integration of this type of components, and to obtain a first estimation of the performances of such a system. We showed through this work that the system operates well and that the performances obtained are correct from an analytical point of view. We present in this contribution, the main results of this work that we continue to strengthen the hardware and to improve its analytical performances