Differential effects of coconut versus soy oil on gut microbiota composition and predicted metabolic function in adult mice

Background: Animal studies show that high fat (HF) diet-induced gut microbiota contributes to the development of obesity. Oil composition of high-fat diet affects metabolic inflammation differently with deleterious effects by saturated fat. The aim of the present study was to examine the diversity and metabolic capacity of the cecal bacterial community in C57BL/6 N mice administered two different diets, enriched respectively with coconut oil (HFC, high in saturated fat) or soy oil (HFS, high in polyunsaturated fat). The relative impact of each hypercaloric diet was evaluated after 2 and 8 weeks of feeding, and compared with that of a low-fat, control diet (LF). Results: The HFC diet induced the same body weight gain and fat storage as the HFS diet, but produced higher plasma cholesterol levels after 8 weeks of treatment. At the same time point, the cecal microbiota of HFC diet-fed mice was characterized by an increased relative abundance of Allobaculum, Anaerofustis, F16, Lactobacillus reuteri and Deltaproteobacteria, and a decreased relative abundance of Akkermansia muciniphila compared to HFS mice. Comparison of cecal microbiota of high-fat fed mice versus control mice indicated major changes that were shared between the HFC and the HFS diet, including the increase in Lactobacillus plantarum, Lutispora, and Syntrophomonas, while some other shifts were specifically associated to either coconut or soy oil. Prediction of bacterial gene functions showed that the cecal microbiota of HFC mice was depleted of pathways involved in fatty acid metabolism, amino acid metabolism, xenobiotic degradation and metabolism of terpenoids and polyketides compared to mice on HFS diet. Correlation analysis revealed remarkable relationships between compositional changes in the cecal microbiota and alterations in the metabolic and transcriptomic phenotypes of high-fat fed mice. Conclusions: The study highlights significant differences in cecal microbiota composition and predictive functions of mice consuming a diet enriched in coconut vs soy oil. The correlations established between specific bacterial taxa and various traits linked to host lipid metabolism and energy storage give insights into the role and functioning of the gut microbiota that may contribute to diet-induced metabolic disorders

Neutralizing antibodies to Omicron after the fourth SARS-CoV-2 mRNA vaccine dose in immunocompromised patients highlight the need of additional boosters

IntroductionImmunocompromised patients have been shown to have an impaired immune response to COVID-19 vaccines.MethodsHere we compared the B-cell, T-cell and neutralizing antibody response to WT and Omicron BA.2 SARS-CoV-2 virus after the fourth dose of mRNA COVID-19 vaccines in patients with hematological malignancies (HM, n=71), solid tumors (ST, n=39) and immune-rheumatological (IR, n=25) diseases. The humoral and T-cell responses to SARS-CoV-2 vaccination were analyzed by quantifying the anti-RBD antibodies, their neutralization activity and the IFN-γ released after spike specific stimulation.ResultsWe show that the T-cell response is similarly boosted by the fourth dose across the different subgroups, while the antibody response is improved only in patients not receiving B-cell targeted therapies, independent on the pathology. However, 9% of patients with anti-RBD antibodies did not have neutralizing antibodies to either virus variants, while an additional 5.7% did not have neutralizing antibodies to Omicron BA.2, making these patients particularly vulnerable to SARS-CoV-2 infection. The increment of neutralizing antibodies was very similar towards Omicron BA.2 and WT virus after the third or fourth dose of vaccine, suggesting that there is no preferential skewing towards either virus variant with the booster dose. The only limited step is the amount of antibodies that are elicited after vaccination, thus increasing the probability of developing neutralizing antibodies to both variants of virus.DiscussionThese data support the recommendation of additional booster doses in frail patients to enhance the development of a B-cell response directed against Omicron and/or to enhance the T-cell response in patients treated with anti-CD20

Modelling human choices: MADeM and decision‑making

Research supported by FAPESP 2015/50122-0 and DFG-GRTK 1740/2. RP and AR are also part of the Research, Innovation and Dissemination Center for Neuromathematics FAPESP grant (2013/07699-0). RP is supported by a FAPESP scholarship (2013/25667-8). ACR is partially supported by a CNPq fellowship (grant 306251/2014-0)

Optimization of Gene-Targeting in Cell Culture to Improve Animal Production

Il gene-targeting in colture cellulari associato alla tecnica del trasferimento nucleare oggi rappresenta il sistema d'elezione nella creazione di animali transgenici. Purtroppo la ricombinazione omologa (HR) è poco efficiente soprattutto in cellule somatiche. La positive-negative selection (PNS) è la tecnica di arricchimento usata per geni non attivamente trascritti nel tipo cellulare utilizzato. In questo lavoro abbiamo scelto come locus bersaglio la b-lattoglobulina bovina e testato tre nuove cassette di selezione negativa, che non codificando per antibiotico-resistenze, determinano condizioni di coltura meno tossiche.Gene-targeting of cultured cells combined with nuclear transfer currently is the most effective procedure to produce transgenic livestock. Nevertheless homologous recombination (HR) is a low frequency event in mammalian cells, above all in somatic cells. Positive-negative selection (PNS) is the enrichment strategy to target genes that are not actively transcribed in the cell type of choice. In this work we chose to target the bovine b-lactoglobulin gene and we tested three new negative selection cassettes in bovine fibroblasts. Such new targeting vectors allow a single selective drug employ and produce less toxic culture conditions

Correction of a Recessive Genetic Defect by CRISPR-Cas9-Mediated Endogenous Repair

CRISPR-Cas9 technology is a relatively recently developed tool for easy and efficient targeting of DNA. However, its efficiency for the repair of a mutated sequence is low. Moreover, most CRISPR-based gene correction approaches require the use of an exogenous template. Here, we investigated whether we could use the CRISPR-Cas9 system and the autologous repair machinery to correct human recessive genetic disorders having two different mutations in two alleles (compound heterozygotes). We reasoned that by targeting an intronic sequence located between the two mutations, we could generate at least one normal allele via the repair of induced double-strand breaks through either gene conversion or mitotic crossover. In particular, using a simple hypoxanthine-guanine phosphoribosyltransferase (Hprt)-based system, we show we can form a normal and functional Hprt gene. Thus, we give proof of principle that homology-directed recombination can be exploited in compound heterozygote cells to correct a genetic defect without exogenous templates

Cell fusion in the liver, revisited

There is wide agreement that cell fusion is a physiological process in cells in mammalian bone, muscle and placenta. In other organs, such as the cerebellum, cell fusion is controversial. The liver contains a considerable number of polyploid cells: They are commonly believed to originate by genome endoreplication, although the contribution of cell fusion to polyploidization has not been excluded. Here, we address the topic of cell fusion in the liver from a historical point of view. We discuss experimental evidence clearly supporting the hypothesis that cell fusion occurs in the liver, specifically when bone marrow cells were injected into mice and shown to rescue genetic hepatic degenerative defects. Those experiments-carried out in the latter half of the last century-were initially interpreted to show "transdifferentiation", but are now believed to demonstrate fusion between donor macrophages and host hepatocytes, raising the possibility that physiologically polyploid cells, such as hepatocytes, could originate, at least partially, through homotypic cell fusion. In support of the homotypic cell fusion hypothesis, we present new data generated using a chimera-based model, a much simpler model than those previously used. Cell fusion as a road to polyploidization in the liver has not been extensively investigated, and its contribution to a variety of conditions, such as viral infections, carcinogenesis and aging, remains unclear

A pre-screening FISH-based method to detect CRISPR/Cas9 off-targets in mouse embryonic stem cells

The clustered regularly interspaced short palindromic repeat (CRISPR)/associated 9 (Cas9) technology has been recently added to the tools allowing efficient and easy DNA targeting, representing a very promising approach to gene engineering. Using the CRISPR/Cas9 system we have driven the integration of exogenous DNA sequences to the X-linked Hprt gene of mouse embryonic stem cells. We show here that a simple fluorescence in situ hybridization (FISH)-based strategy allows the detection and the frequency evaluation of non-specific integrations of a given plasmid. FISH analysis revealed that these integrations do not match the software predicted off-target loci. We conclude that the frequency of these CRISPR-mediated off-target DNA cuts is negligible, since, due to the occurrence of spontaneous double-strand breaks, we observed more aspecific plasmid integrations than those corresponding to predicted off-target sites

Comparison of expression vectors in Lactobacillus reuteri strains

Synthesis of heterologous proteins in lactobacilli is strongly influenced by the promoter selected for the expression. In addition, the activity of the promoters themselves may vary among different bacterial hosts. Three different promoters were investigated for their capability to drive EGFP expression in L. lactis spp. cremoris MG1363, in Lactobacillus reuteri DSM 20016T and in five Lactobacillus reuteri strains isolated from chicken crops. The promoters of L. acidophilus Surface Layer Protein gene (slp), L. acidophilus lactate dehydrogenase gene (ldhL) and enterococcal rRNA adenine N-6-methyltransferase gene (ermB) were fused to the coding sequence of EGFP and inserted into the backbone of pTRKH3 shuttle vector (pTRKH3-slpGFP, pTRKH3-ldhGFP, pTRKH3-ermGFP). Besides conventional analytical methods, a new quick fluorimetric approach was set up to quantify the EGFP fluorescence in transformed clones using the QubitTM fluorometer. ermB proved to be the most effective promoter in L. reuteri isolates producing 3.90 x 10-7 g of fluorescent EGFP (ml x ODstationary culture)-1. In the same conditions ldhL promoter produced 2.66 x 10-7 g of fluorescent EGFP (ml x ODstationary culture)-1. Even though the slp promoter was efficient in L. lactis spp. cremoris MG1363, it was nearly inactive both in L. reuteri DSM 20016T and in L. reuteri isolates

Fusion between cancer cells and macrophages occurs in a murine model of spontaneous neu+ breast cancer without increasing its metastatic potential

Cell fusion between neoplastic and normal cells has been suggested to play a role in the acquisition of a malignant phenotype. Several studies have pointed to the macrophage as the normal partner in this fusion, suggesting that the fused cells could acquire new invasive properties and become able to disseminate to distant organs. However, this conclusion is mainly based on studies with transplantable cell lines. We tested the occurrence of cell fusion in the MMTV-neu model of mouse mammary carcinoma. In the first approach, we generated aggregation chimeras between GFP/ neu and RFP/neu embryos. Tumor cells would display both fluorescent proteins only if cell fusion with normal cells occurred. In addition, if cell fusion conferred a growth/dissemination advantage, cells with both markers should be detectable in lung metastases at increased frequency. We confirmed that fused cells are present at low but consistent levels in primary neoplasms and that the macrophage is the normal partner in the fusion events. Similar results were obtained using a second approach in which bone marrow from mice carrying the Cre transgene was transplanted into MMTV-neu/LoxP-tdTomato transgenic animals, in which the Tomato gene is activated only in the presence of CRE recombinase. However, no fused cells were detected in lung metastases in either model. We conclude that fusion between macrophages and tumor cells does not confer a selective advantage in our spontaneous model of breast cancer, although these data do not rule out a possible role in models in which an inflammation environment is prominent