Effect of Non-Nutritive Sweeteners on the Gut Microbiota.

The human gut microbiota, a complex community of microorganisms living in the digestive tract, consists of more than 1500 species distributed in more than 50 different phyla, with 99% of bacteria coming from about 30-40 species. The colon alone, which contains the largest population of the diverse human microbiota, can harbor up to 100 trillion bacteria. The gut microbiota is essential in maintaining normal gut physiology and health. Therefore, its disruption in humans is often associated with various pathological conditions. Different factors can influence the composition and function of the gut microbiota, including host genetics, age, antibiotic treatments, environment, and diet. The diet has a marked effect, impacting the gut microbiota composition, beneficially or detrimentally, by altering some bacterial species and adjusting the metabolites produced in the gut environment. With the widespread use of non-nutritive sweeteners (NNS) in the diet, recent investigations have focused on their effect on the gut microbiota as a mediator of the potential impact generated by gastrointestinal-related disturbances, such as insulin resistance, obesity, and inflammation. We summarized the results from pre-clinical and clinical studies published over the last ten years that examined the single effects of the most consumed NNS: aspartame, acesulfame-K, sucralose, and saccharin. Pre-clinical studies have given conflicting results for various reasons, including the administration method and the differences in metabolism of the same NNS among the different animal species. A dysbiotic effect of NNS was observed in some human trials, but many other randomized controlled trials reported a lack of significant impacts on gut microbiota composition. These studies differed in the number of subjects involved, their dietary habits, and their lifestyle; all factors related to the baseline composition of gut microbiota and their response to NNS. The scientific community still has no unanimous consensus on the appropriate outcomes and biomarkers that can accurately define the effects of NNS on the gut microbiota.

Super-resolution study of PIAS SUMO E3-ligases in hippocampal and cortical neurons.

The SUMOylation machinery is a regulator of neuronal activity and synaptic plasticity. It is composed of SUMO isoforms and specialized enzymes named E1, E2 and E3 SUMO ligases. Recent studies have highlighted how SUMO isoforms and E2 enzymes localize with synaptic markers to support previous functional studies but less information is available on E3 ligases. PIAS proteins - belonging to the protein inhibitor of activated STAT (PIAS) SUMO E3-ligase family - are the best-characterized SUMO E3-ligases and have been linked to the formation of spatial memory in rodents. Whether however they exert their function co-localizing with synaptic markers is still unclear. In this study, we applied for the first time structured illumination microscopy (SIM) to PIAS ligases to investigate the co-localization of PIAS1 and PIAS3 with synaptic markers in hippocampal and cortical murine neurons. The results indicate partial co-localization of PIAS1 and PIAS3 with synaptic markers in hippocampal neurons and much rarer occurrence in cortical neurons. This is in line with previous super-resolution reports describing the co-localization with synaptic markers of other components of the SUMOylation machinery.

Super-Resolution Imaging to Study Co-Localization of Proteins and Synaptic Markers in Primary Neurons.

Synapses are the functional elements of neurons and their defects or losses are at the basis of several neurodegenerative and neurological disorders. Imaging studies are widely used to investigate their function and plasticity in physiological and pathological conditions. Because of their size and structure, localization studies of proteins require high-resolution imaging techniques. In this protocol, we describe a procedure to study in primary neurons the co-localization of target proteins with synaptic markers at a super-resolution level using structured illumination microscopy (SIM). SIM is a patterned-light illumination technique that doubles the spatial resolution of wide-field microscopy, reaching a detail of around 100 nm. The protocol indicates the required controls and settings for robust co-localization studies and an overview of the statistical methods to analyze the imaging data properly.

Neuronal Localization of SENP Proteins with Super Resolution Microscopy.

SUMOylation of proteins plays a key role in modulating neuronal function. For this reason, the balance between protein SUMOylation and deSUMOylation requires fine regulation to guarantee the homeostasis of neural tissue. While extensive research has been carried out on the localization and function of small ubiquitin-related modifier (SUMO) variants in neurons, less attention has been paid to the SUMO-specific isopeptidases that constitute the human SUMO-specific isopeptidase (SENP)/Ubiquitin-Specific Protease (ULP) cysteine protease family (SENP1-3 and SENP5-7). Here, for the first time, we studied the localization of SENP1, SENP6, and SENP7 in cultured hippocampal primary neurons at a super resolution detail level, with structured illumination microscopy (SIM). We found that the deSUMOylases partially colocalize with pre- and post-synaptic markers such as synaptophysin and drebrin. Thus, further confirming the presence with synaptic markers of the negative regulators of the SUMOylation machinery.