Salmonella Type III Secretion Effector SrfJ: A Glucosylceramidase Affecting the Lipidome and the Transcriptome of Mammalian Host Cells.

Type III secretion systems are found in many Gram-negative pathogens and symbionts of animals and plants. Salmonella enterica has two type III secretion systems associated with virulence, one involved in the invasion of host cells and another involved in maintaining an appropriate intracellular niche. SrfJ is an effector of the second type III secretion system. In this study, we explored the biochemical function of SrfJ and the consequences for mammalian host cells of the expression of this S. enterica effector. Our experiments suggest that SrfJ is a glucosylceramidase that alters the lipidome and the transcriptome of host cells, both when expressed alone in epithelial cells and when translocated into macrophages in the context of Salmonella infection. We were able to identify seventeen lipids with higher levels and six lipids with lower levels in the presence of SrfJ. Analysis of the forty-five genes, the expression of which is significantly altered by SrfJ with a fold-change threshold of two, suggests that this effector may be involved in protecting Salmonella from host immune defenses.

Mimicking the Physicochemical Properties of the Cornea: A Low-Cost Approximation Using Highly Available Biopolymers.

Corneal diseases represent a significant global health challenge, often resulting in blindness, for which penetrating keratoplasty is the clinical gold standard. However, in cases involving compromised ocular surfaces or graft failure, osteo-odonto keratoprosthesis (OOKP) emerges as a vital yet costly and complex alternative. Thus, there is an urgent need to introduce soft biomaterials that mimic the corneal tissue, considering its translation's physicochemical, biological, and economic costs. This study introduces a cross-linked mixture of economically viable biomaterials, including gelatin, chitosan, and poly-D-lysine, that mimic corneal properties. The physicochemical evaluation of certain mixtures, specifically gelatin, chitosan, and poly-D-lysine cross-linked with 0.10% glutaraldehyde, demonstrates that properties such as swelling, optical transmittance, and thermal degradation are comparable to those of native corneas. Additionally, constructs fabricated with poly-D-lysine exhibit good cytocompatibility with fibroblasts at 72 h. These findings suggest that low-cost biopolymers, particularly those incorporating poly-D-lysine, mimic specific corneal characteristics and have the potential to foster fibroblast survival. While further studies are required to reach a final corneal-mimicking solution, this study contributes to positioning low-cost reagents as possible alternatives to develop biomaterials with physicochemical properties like those of the human cornea.

Poly(ethylene imine)-chitosan carbon dots: study of its physical-chemical properties and biological in vitro performance.

Carbon dots (CDs) have been quickly extended for nanomedicine uses because of their multiple applications, such as bioimaging, sensors, and drug delivery. However, the interest in increasing their photoluminescence properties is not always accompanied by cytocompatibility. Thus, a knowledge gap exists regarding their interactions with biological systems linked to the selected formulations and synthesis methods. In this work, we have developed carbon dots (CDs) based on poly (ethylene imine) (PEI) and chitosan (CS) by using microwave irradiation, hydrothermal synthesis, and a combination of both, and further characterized them by physicochemical and biological means. Our results indicate that synthesized CDs have sizes between 1 and 5 nm, a high presence of amine groups on the surface, and increased positive ζ potential values. Further, it is established that the choice and use of different synthesis procedures can contribute to a different answer to the CDs regarding their optical and biological properties. In this regard, PEI-only CDs showed the longest photoluminescent emission lifetime, non-hemolytic activity, and high toxicity against fibroblast. On the other hand, CS-only CDs have higher PL emission, non-cytotoxicity associated with fibroblast, and high hemolytic activity. Interestingly, their combination using the proposed methodologies allow a synergic effect in their CDs properties. Therefore, this work contributes to developing and characterizing CD formulations based on PEI and CS and better understanding the CD's properties and biological interaction.

Interferon-gamma ameliorates experimental autoimmune encephalomyelitis by inducing homeostatic adaptation of microglia.

Compelling evidence has shown that interferon (IFN)-γ has dual effects in multiple sclerosis and in its animal model of experimental autoimmune encephalomyelitis (EAE), with results supporting both a pathogenic and beneficial function. However, the mechanisms whereby IFN-γ may promote neuroprotection in EAE and its effects on central nervous system (CNS)-resident cells have remained an enigma for more than 30 years. In this study, the impact of IFN-γ at the peak of EAE, its effects on CNS infiltrating myeloid cells (MC) and microglia (MG), and the underlying cellular and molecular mechanisms were investigated. IFN-γ administration resulted in disease amelioration and attenuation of neuroinflammation associated with significantly lower frequencies of CNS CD11b+ myeloid cells and less infiltration of inflammatory cells and demyelination. A significant reduction in activated MG and enhanced resting MG was determined by flow cytometry and immunohistrochemistry. Primary MC/MG cultures obtained from the spinal cord of IFN-γ-treated EAE mice that were ex vivo re-stimulated with a low dose (1 ng/ml) of IFN-γ and neuroantigen, promoted a significantly higher induction of CD4+ regulatory T (Treg) cells associated with increased transforming growth factor (TGF)-ß secretion. Additionally, IFN-γ-treated primary MC/MG cultures produced significantly lower nitrite in response to LPS challenge than control MC/MG. IFN-γ-treated EAE mice had a significantly higher frequency of CX3CR1high MC/MG and expressed lower levels of program death ligand 1 (PD-L1) than PBS-treated mice. Most CX3CR1highPD-L1lowCD11b+Ly6G- cells expressed MG markers (Tmem119, Sall2, and P2ry12), indicating that they represented an enriched MG subset (CX3CR1highPD-L1low MG). Amelioration of clinical symptoms and induction of CX3CR1highPD-L1low MG by IFN-γ were dependent on STAT-1. RNA-seq analyses revealed that in vivo treatment with IFN-γ promoted the induction of homeostatic CX3CR1highPD-L1low MG, upregulating the expression of genes associated with tolerogenic and anti-inflammatory roles and down-regulating pro-inflammatory genes. These analyses highlight the master role that IFN-γ plays in regulating microglial activity and provide new insights into the cellular and molecular mechanisms involved in the therapeutic activity of IFN-γ in EAE.

Cytocompatible drug delivery hydrogels based on carboxymethylagarose/chitosan pH-responsive polyelectrolyte complexes.

Several drugs are chemically unstable in the gastric environment and have low bioavailability restricted by intestinal absorption, which motivates the development of alternative routes for drug release, such as transdermal drug carriers for drug delivery to specific areas of the skin. Herein, novel polyelectrolyte complexes (PEC) consisting of carboxymethylagarose (CMA) and chitosan (CS) were prepared. pH-responsive CMA/CS hydrogels were obtained by mixing CMA and CS at various weight ratios. Swelling ratio was modulated by varying the CMA and CS weight ratio, and the highest swelling values were achieved for 2:1 wt% hydrogels at 25 °C and pH 6.0. PEC films were characterized by ATR-FTIR spectroscopy, TGA, DSC, and SEM. Results indicated that CMA and CS were successfully crosslinked by ionic complexation. As a model drug, diclofenac sodium (DS) was loaded in CMA/CS PECs. Association efficiency and loading capacity were ca. 69% and 79%, respectively, exhibiting 67% cumulative release after 72 h at 37 °C and pH 6.0 through Fickian diffusion mechanism. Viability assay of immortalized human keratinocyte (HaCat) cells showed ca. 100% survival in the presence of hydrogels and DS. Therefore, this work suggests that CMA/CS PECs can be applied as pH-responsive carriers for dermal drug delivery.

Generation of Random luxCDABE Transcriptional Fusions in the Genome of Salmonella enterica.

The luxCDABE operon of Photorhabdus luminescens can be used as a bioluminescent reporter to measure gene transcription nondestructively. Here we describe protocols to (1) generate random transcriptional fusions of the lux operon to genes of the Salmonella genome, (2) screen for specific fusions with constitutive expression, Salmonella pathogenicity island 1-related expression, or Salmonella pathogenicity island 2-related expression, and (3) determine the site of luxCDABE integration.