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Vaccination against an airborne pathogen is very effective if it induces also the development of mucosal antibodies that can protect against infection. The mRNA-based vaccine-encoding SARS-CoV-2 full-length spike protein (BNT162b2, Pfizer/BioNTech) protects also against infection despite being administered systemically. Here, we show that upon vaccination, cognate IgG molecules are also found in the saliva and are more abundant in SARS-CoV-2 previously exposed subjects, paralleling the development of plasma IgG. The antibodies titer declines at 3 months from vaccination. We identified a concentration of specific IgG in the plasma above which the relevant IgG can be detected in the saliva. Regarding IgA antibodies, we found only protease-susceptible IgA1 antibodies in plasma while they were present at very low levels in the saliva over the course of vaccination of SARS-CoV-2-naïve subjects. Thus, in response to BNT162b2 vaccine, plasma IgG can permeate into mucosal sites and participate in viral protection. It is not clear why IgA1 are detected in low amount, they may be proteolytically cleaved.

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 (

Metastasis is facilitated by the formation of a "premetastatic niche," which is fostered by primary tumor-derived factors. Colorectal cancer (CRC) metastasizes mainly to the liver. We show that the premetastatic niche in the liver is induced by bacteria dissemination from primary CRC. We report that tumor-resident bacteria Escherichia coli disrupt the gut vascular barrier (GVB), an anatomical structure controlling bacterial dissemination along the gut-liver axis, depending on the virulence regulator VirF. Upon GVB impairment, bacteria disseminate to the liver, boost the formation of a premetastatic niche, and favor the recruitment of metastatic cells. In training and validation cohorts of CRC patients, we find that the increased levels of PV-1, a marker of impaired GVB, is associated with liver bacteria dissemination and metachronous distant metastases. Thus, PV-1 is a prognostic marker for CRC distant recurrence and vascular impairment, leading to liver metastases. Copyright © 2021 Elsevier Inc. All rights reserved.

Influenza A virus is the principal agent responsible of the respiratory tract's infections in humans. Every year, highly pathogenic and infectious strains with new antigenic assets appear, making ineffective vaccines so far developed. The discovery of RNA interference (RNAi) opened the way to the progress of new promising drugs against Influenza A virus and also to the introduction of disease resistance traits in genetically modified animals. In this paper, we show that Madin-Darby Canine Kidney (MDCK) cell line expressing short hairpin RNAs (shRNAs) cassette, designed on a specific conserved region of the nucleoprotein (NP) viral genome, can strongly inhibit the viral replication of four viral strains sharing the target sequence, reducing the viral mRNA respectively to 2.5×10(-4), 7.5×10(-5), 1.7×10(-3), 1.9×10(-4) compared to the control, as assessed by real-time PCR. Moreover, we demonstrate that during the challenge with a viral strain bearing a single mismatch on the target sequence, although a weaker inhibition is observed, viral mRNA is still lowered down to 1.2×10(-3) folds in the shRNA-expressing clone compared to the control, indicating a broad potential use of this approach. In addition, we developed a highly predictive and fast screening test of siRNA sequences based on dual-luciferase assay, useful for the in vitro prediction of the potential effect of viral inhibition. In conclusion, these findings reveal new siRNA sequences able to inhibit Influenza A virus replication and provide a basis for the development of siRNAs as prophylaxis and therapy for influenza infection both in humans and animals.

At weaning, piglets are exposed to many stressors, such as separation from the sow, mixing with other litters, end of lactational immunity, and a change in their environment and gut microbiota. The sudden change of feeding regime after weaning causes morphological and histological changes in the small intestine which are critical for the immature digestive system. Sixteen female piglets were studied to assess the effect of sorbic acid supplementation on the small intestine tissue transcriptome. At weaning day (T0, piglet age 28 days), four piglets were sacrificed and ileal tissue samples collected. The remaining 12 piglets were weighed and randomly assigned to different postweaning (T5, piglet age 33 days) diets. Diet A (n = 6) contained 5 g/kg of sorbic acid. In diet B (n = 6), the organic acids were replaced by barley flour. Total RNA was isolated and then hybridized to CombiMatrix CustomArray™ 90-K platform microarrays, screening about 30 K genes. Even though diet had no detectable effect on the transcriptome during the first 5 days after weaning, results highlighted some of the response mechanisms to the stress of weaning occurring in the piglet gut. A total of 205 differentially expressed genes were used for functional analysis using the bioinformatics tools BLAST2GO, Ingenuity Pathway Analysis 8.0, and Dynamic Impact Approach (DIA). Bioinformatic analysis revealed that apoptosis, RIG-I-like, and NOD-like receptor signaling were altered as a result of weaning. Interferons and caspases gene families were the most activated after weaning in response to piglets to multiple stressors. Results suggest that immune and inflammatory responses were activated and likely are a cause of small intestine atrophy as revealed by a decrease in villus height and villus/crypt ratio.

Mice fed long-term high-fat diets (HFD) are an established model for human metabolic disorders, such as obesity and diabetes. However, also the effects of short-term HFD feeding should be investigated to understand which are the first events that trigger the onset of a pre-disease condition, the so-called metabolic syndrome, that increases the risk of developing clinical diseases. In this study, C57BL/6N mice were fed a control diet (CTR) or a HFD for 1 (T1) or 2 weeks (T2). Metabolic and histological effects were examined. Cecum transcriptomes of HFD and CTR mice were compared at T2 by microarray analysis. Differentially expressed genes were validated by real-time PCR in the cecum and in the liver. After 2 weeks of diet administration, HFD mice showed an altered expression pattern in only seven genes, four of which are involved in the circadian clock regulatory pathway. Real-time PCR confirmed microarray results of the cecum and revealed the same trend of clock gene expression changes in the liver. These findings suggest that clock genes may play an important role in early controlling gut output systems in response to HFD in mice and that their expression change may also represent an early signaling of the development of an intestinal pro-inflammatory status.

The 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 enhanced green fluorescent protein (EGFP) expression in Lactococcus lactis spp. cremoris MG1363, in Lactobacillus reuteri DSM 20016(T) and in five L. reuteri strains isolated from chicken crops. The promoters of the Lactobacillus 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 the 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 Qubit() fluorometer. ermB proved to be the most effective promoter in L. reuteri isolates, producing 3.90 x 10(-7) g of fluorescent EGFP (mL OD(stationary culture))(-1). Under the same conditions, the ldhL promoter produced 2.66 x 10(-7) g of fluorescent EGFP (mL OD(stationary culture))(-1). Even though the slp promoter was efficient in L. lactis spp. cremoris MG1363, it was nearly inactive both in L. reuteri DSM 20016(T) and in L. reuteri isolates.

The gut microbiota has been shown to be involved in host energy homeostasis and diet-induced metabolic disorders. To gain insight into the relationships among diet, microbiota and the host, we evaluated the effects of a high-fat (HF) diet on the gut bacterial community in weaning mice. C57BL/6 mice were fed either a control diet or a diet enriched with soy oil for 1 and 2 weeks. Administration of the HF diet caused an increase in plasma total cholesterol levels, while no significant differences in body weight gain were observed between the two diets. Denaturing gradient gel electrophoresis (DGGE) profiles indicated considerable variations in the caecal microbial communities of mice on the HF diet, as compared with controls. Two DGGE bands with reduced intensities in HF-fed mice were identified as representing Lactobacillus gasseri and an uncultured Bacteroides species, whereas a band of increased intensity was identified as representing a Clostridium populeti-related species upon sequencing. Quantitative real-time PCR confirmed a statistically significant 1-log decrease in L. gasseri cell numbers after HF feeding, and revealed a significantly lower level of Bifidobacterium spp. in the control groups after 1 and 2 weeks compared with that in the HF groups. These alterations of intestinal microbiota were not associated with caecum inflammation, as assessed by histological analysis. The observed shifts of specific bacterial populations within the gut may represent an early consequence of increased dietary fat.

The zona-free method of SCNT designed for bovine and pig cloning (Booth et al. 2001; Vajta et al. 2001; Oback et al. 2003) was successfully used for horse (Galli et al. 2003). Although simple and efficient in farm animals, its application in the mouse met several problems (Ribas et al. 2005, 2006). The aim of our work was to produce cloned mice using HM1 embryonic stem (ES)cells adapting a zona-free method. Seven- to 24-week-old superovulated B6D2F1 female mice were used as oocytes donors. Cumulus cells were removed by 0.3% hyaluronidase and the zona pellucida by 0.5% pronase in KSOM-HEPES (KSOM-H) 1 h later (Ribas et al. 2006) or immediately after hyaluronidase treatment at 37°C. The HM1 ES cells were cultured in KnockOut DMEM supplemented with leukemia inhibitory factor and 15% fetal bovine serum with or without 2i (Ying et al. 2008) and were synchronized at M phase by 3 ng mL–1 nocodazole for 3 h before fusion. Only spherical cells were selected for NT. Metaphase II chromosome spindle complexes were removed by micromanipulation in KSOM-H medium with 5 μg mL–1 cytochalasin B. Lectin-treated enucleated oocytes were attached to the donor cells in KSOM-H with nocodazole and fused by 2 pulses of 1.3 kV cm–1 DC for 30 μs in 0.3 M mannitol medium. Following 10- to 15-min incubation in KSOM-H, the fusion was assessed and repeated if the constructs were nonfused. Cloned embryos were activated in 1 mM SrCl2 in Ca2+-free KSOM medium for 2 to 2.5 or 5 to 6 h and cultured in 20-μL KSOM droplets using the well-of-the-well (WOW) method (Vajta et al. 2000) under mineral oil at 37°C and 5% CO2. Day 4 compacted morulae and blastocysts were surgically transferred into the uterus of Day-2.5 pseudopregnant recipients that were sacrificed on Day 19.5 to examine fetal development. The donor mice age was important for oocyte survival: ~16% of oocytes of 7- to 10-week-old mice lysed before or during fusion in 33% of experiments (n experiments = 15), whereas oocytes of older mice were not sensitive to enzymatic treatment and electric impulses even after 3 fusion rounds (n = 19). The time of pronase treatment did not affect oocyte survival, whereas extending the time between hyaluronidase treatment and enucleation revealed self-activation in ~25% of oocytes. The fusion efficiency of ES cells was significantly lower compared with serum-starved fibroblasts (61%, n = 623 v. 100%, n = 80). The duration of SrCl2 treatment did not affect embryo development (cleavage: 82% v. 84%; Day 4 blastocysts: 49% v. 52%). ES cell culture with 2i increased Day 4 blastocyst development (60.7% v. 50.4%; P = 0.07), and their ability to implant (52.6% v. 38.2%; P = 0.06). Moreover, only NT embryos derived from 2i-ES cells developed to term (8.2%, n = 5; P = 0.08), and produced live fetuses (4.9%, n = 3). In light of these results, the fusion of ES cells remains the critical step in the mouse zona-free protocol.Partially supported by grant Superpig from Regione Lombardia.

We investigated the effect of the 52 MAR (matrix attachment region) sequence of chicken lysozyme gene as a possible insulator for the expression of our transgenes in somatic cells to be used for nuclear transfer. With the preliminary purpose to demonstrate a possible positive effect (position or copy number) on the long-term combined expression during in vitro culture, we have created a bicistronic ubiquitous expression vector with (MAR+) or without (MAR–) MAR. The main structure of our constructs is composed of the pCAGGS promoter driving the expression of a reporter gene (enhanced green fluorescent protein, EGFP) followed by a loxable selection cassette (loxP-PGKneo). The MAR region was inserted before the selection cassette. After KpnI digestion, the resulting linearized vectors were purified and subsequently used to transfect adult porcine fibroblast cell lines using the Nucleofector system (Amaxa, Cologne, Germany). Fibroblasts were cultured in DMEM/M199 medium (1:1) + 10% FCS supplemented with 5 ng mL–1 of basic fibroblast growth factor (bFGF). In every experiment, 1 � 106 cells were transfected with 2.5 µg of linearized plasmid and selected for 3 weeks with medium supplemented with 400 µg mL–1 of Geneticin (G418 sulfate, Calbiochem, La Jolla, CA). On Day 8 of G418 selection, we analyzed 150 colonies for each treatment, using fluorescence microscopy with fluorescein isothiocyanate filters. Colonies were classified according to size (large) and cell morphology (small cells without signs of aging). In addition, colonies were classified for uniform GFP expression (uniform), patchy GFP expression (variegated), and no GFP expression (negative). Resistant colonies derived from MAR+ and MAR– vectors, respectively, had 36 (24%), 42 (28%), and 56 (37%) v. 79 (53%), 58 (39%), and 29 (19%) uniform, variegated, and negative GFP. Differences were significant for variegated and negative in MAR+ v. MAR– (chi square, P < 0.05). Thirty-six MAR+ and 42 MAR– colonies uniformly expressing GFP were transferred to 24-well plates and subjected to G418 selection until Day 22, when 7 MAR+ and 15 MAR– clones were still growing in culture. Four of seven MAR+ (57%) and 7/15 MAR– (47%) uniformly expressed high levels of GFP. In conclusion, we found that significantly fewer colonies expressed GFP with the MAR+ vector; however, within the GFP-expressing clones, expression was more uniform. Therefore, we did not find a beneficial effect of MAR sequences on expression in somatic cells during in vitro culture; however, further work is needed to investigate their effect after nuclear transfer and/or on the next generation of cloned transgenic animals.