Microbiota-gut-brain axis and ketogenic diet: how close are we to tackling epilepsy?

The microbiota-gut-brain axis refers to the intricate bidirectional communication between commensal microorganisms residing in the digestive tract and the central nervous system, along neuroendocrine, metabolic, immune, and inflammatory pathways. This axis has been suggested to play a role in several neurological disorders, such as Parkinson's disease, Alzheimer's disease, multiple sclerosis, and epilepsy, paving the way for microbiome-based intervention strategies for the mitigation and treatment of symptoms. Epilepsy is a multifaceted neurological condition affecting more than 50 million individuals worldwide, 30% of whom do not respond to conventional pharmacological therapies. Among the first-hand microbiota modulation strategies, nutritional interventions represent an easily applicable option in both clinical and home settings. In this narrative review, we summarize the mechanisms underlying the microbiota-gut-brain axis involvement in epilepsy, discuss the impact of antiepileptic drugs on the gut microbiome, and then the impact of a particular dietary pattern, the ketogenic diet, on the microbiota-gut-brain axis in epileptic patients. The investigation of the microbiota response to non-pharmacological therapies is an ever-expanding field with the potential to allow the design of increasingly accessible and successful intervention strategies.

Gut microbiota resilience and recovery after anticancer chemotherapy.

Although research on the role of the gut microbiota (GM) in human health has sharply increased in recent years, what a "healthy" gut microbiota is and how it responds to major stressors is still difficult to establish. In particular, anticancer chemotherapy is known to have a drastic impact on the microbiota structure, potentially hampering its recovery with serious long-term consequences for patients' health. However, the distinguishing features of gut microbiota recovery and non-recovery processes are not yet known. In this narrative review, we first investigated how gut microbiota layouts are affected by anticancer chemotherapy and identified potential gut microbial recovery signatures. Then, we discussed microbiome-based intervention strategies aimed at promoting resilience, i.e., the rapid and complete recovery of a healthy gut microbial network associated with a better prognosis after such high-impact pharmacological treatments.

Bifidobacterium in anticancer immunochemotherapy: friend or foe?

The gut microbiome has received a crescendo of attention in recent years due to myriad influences on human pathophysiology, including cancer. Anticancer therapy research is constantly looking for new hints to improve response to therapy while reducing the risk of relapse. In this scenario, Bifidobacterium, which inhabits the gut microbial ecosystem (especially that of children) and is considered a health-associated microbe, has emerged as a key target to assist anticancer treatments for a better prognosis. However, some researchers have recently hypothesized an unfavorable role of Bifidobacterium spp. in anticancer immunochemotherapy, leading to some confusion in the field. This narrative review summarizes the current knowledge on the role of Bifidobacterium spp. in relation to anticancer treatments, discussing the pros and cons of its presence in the gut microbiome of cancer patients. The current intervention strategies based on the administration of probiotic strains of Bifidobacterium are then discussed. Finally, the need to conduct further studies, especially functional ones, is underlined to provide robust experimental evidence, especially on the underlying molecular mechanisms, and thus resolve the controversies on this microbe for the long-term success of immunochemotherapy.

Tetra- and Hexavalent Siglec-8 Ligands Modulate Immune Cell Activation.

Carbohydrate-binding proteins are generally characterized by poor affinities for their natural glycan ligands, predominantly due to the shallow and solvent-exposed binding sites. To overcome this drawback, nature has exploited multivalency to strengthen the binding by establishing multiple interactions simultaneously. The development of oligovalent structures frequently proved to be successful, not only for proteins with multiple binding sites, but also for proteins that possess a single recognition domain. Herein we present the syntheses of a number of oligovalent ligands for Siglec-8, a monomeric I-type lectin found on eosinophils and mast cells, alongside the thermodynamic characterization of their binding. While the enthalpic contribution of each binding epitope was within a narrow range to that of the monomeric ligand, the entropy penalty increased steadily with growing valency. Additionally, we observed a successful agonistic binding of the tetra- and hexavalent and, to an even larger extent, multivalent ligands to Siglec-8 on immune cells and modulation of immune cell activation. Thus, triggering a biological effect is not restricted to multivalent ligands but could be induced by low oligovalent ligands as well, whereas a monovalent ligand, despite binding with similar affinity, showed an antagonistic effect.

A comparative absorption study of sucrosomial® orodispersible vitamin D3 supplementation vs. a reference chewable tablet and soft gel capsule vitamin D3 in improving circulatory 25(OH)D levels in healthy adults with vitamin D deficiency-Results from a prospective randomized clinical trial.

Background: Vitamin D (Vit D) deficiency (VDD), associated with diverse health conditions, is commonly treated with Vit D3 supplements. However, the gastrointestinal (GI) absorption of Vit D3 in different formulations has not been well studied. Objective: We aimed to compare the absorption of an innovative phospholipids-sucrester matrix biodelivery vehicle-based (sucrosomial®) orodispersible Vit D3 preparation against a reference chewable tablet and soft gel capsule (SGC) Vit D3 formulations in Vit D-deficient healthy adults. Methods: In study 1, 25 subjects were randomized to receive a weekly single dose of 200,000 IU of sucrosomial® Vit D3 (n = 12) or chewable tablet Vit D3 (n = 13) for 3 weeks. In study 2, 20 subjects were randomized to receive a single dose of 200,000 IU every other week of sucrosomial® Vit D3 (n = 10) or SGC Vit D3 (n = 10) for 6 weeks. Circulatory 25-hydroxyvitamin D3 [25(OH)D] levels were reassessed after 2, 3, and 6 weeks in study 1 and after 4 and 6 weeks in study 2. Results: In study 1, after 2 weeks, circulatory 25(OH)D levels increased significantly in both Vit D3 treatment groups (p < 0.0001) but improved markedly in the sucrosomial® Vit D3 group, with no further considerable change after 3 and 6 weeks in both groups. Overall, at all three follow-ups, sucrosomial® Vit D3 treatment achieved significantly higher and sustained 25(OH)D levels (p < 0.001). In study 2, after 4 weeks, both Vit D3 treatment groups showed significant improvement in circulatory 25(OH)D levels (p < 0.0001) but substantially higher in the sucrosomial® group with statistically significant differences between the two treatment groups (p = 0.02). At the 6-week follow-up, only subjects in the sucrosomial® Vit D3 group showed a further increase in circulatory 25(OH)D levels (p = 0.049), but no further significant changes in the levels of the SGC Vit D3 group (p = 0.062), showing a statistically significant difference between the two treatment groups (p = 0.002). The Vit D3 treatment was well tolerated by all participants, and no treatment-emergent effects or serious adverse events were reported. Conclusion: Our results suggest that the sucrosomial® Vit D3 preparation absorbs efficiently in the GI system, achieving adequately higher and sustained circulatory Vit D levels in VDD, and thus can effectively contribute to the body protection against VDD-associated health conditions. Clinical trial registration: clinicaltrials.gov, identifier: NCT05706259.

Polyphenol and Tannin Nutraceuticals and Their Metabolites: How the Human Gut Microbiota Influences Their Properties.

Nutraceuticals have been receiving increasing attention in the last few years due to their potential role as adjuvants against non-communicable chronic diseases (cardiovascular disease, diabetes, cancer, etc.). However, a limited number of studies have been performed to evaluate the bioavailability of such compounds, and it is generally reported that a substantial elevation of their plasma concentration can only be achieved when they are consumed at pharmacological levels. Even so, positive effects have been reported associated with an average dietary consumption of several nutraceutical classes, meaning that the primary compound might not be solely responsible for all the biological effects. The in vivo activities of such biomolecules might be carried out by metabolites derived from gut microbiota fermentative transformation. This review discusses the structure and properties of phenolic nutraceuticals (i.e., polyphenols and tannins) and the putative role of the human gut microbiota in influencing the beneficial effects of such compounds.

Pharmacomicrobiomics in Anticancer Therapies: Why the Gut Microbiota Should Be Pointed Out.

Anticancer treatments have shown a variable therapeutic outcome that may be partly attributable to the activity of the gut microbiota on the pathology and/or therapies. In recent years, microbiota-drug interactions have been extensively investigated, but most of the underlying molecular mechanisms still remain unclear. In this review, we discuss the relationship between the gut microbiota and some of the most commonly used drugs in oncological diseases. Different strategies for manipulating the gut microbiota layout (i.e., prebiotics, probiotics, antibiotics, and fecal microbiota transplantation) are then explored in order to optimize clinical outcomes in cancer patients. Anticancer technologies that exploit tumor-associated bacteria to target tumors and biotransform drugs are also briefly discussed. In the field of pharmacomicrobiomics, multi-omics strategies coupled with machine and deep learning are urgently needed to bring to light the interaction among gut microbiota, drugs, and host for the development of truly personalized precision therapies.

A Potent Mimetic of the Siglec-8 Ligand 6'-Sulfo-Sialyl Lewisx.

Siglecs are members of the immunoglobulin gene family containing sialic acid binding N-terminal domains. Among them, Siglec-8 is expressed on various cell types of the immune system such as eosinophils, mast cells and weakly on basophils. Cross-linking of Siglec-8 with monoclonal antibodies triggers apoptosis in eosinophils and inhibits degranulation of mast cells, making Siglec-8 a promising target for the treatment of eosinophil- and mast cell-associated diseases such as asthma. The tetrasaccharide 6'-sulfo-sialyl Lewisx has been identified as a specific Siglec-8 ligand in glycan array screening. Here, we describe an extended study enlightening the pharmacophores of 6'-sulfo-sialyl Lewisx and the successful development of a high-affinity mimetic. Retaining the neuraminic acid core, the introduction of a carbocyclic mimetic of the Gal moiety and a sulfonamide substituent in the 9-position gave a 20-fold improved binding affinity. Finally, the residence time, which usually is the Achilles tendon of carbohydrate/lectin interactions, could be improved.

Optimised Synthesis of the Bacterial Magic Spot (p)ppGpp Chemosensor PyDPA.

Guanosine penta- or tetraphosphate (pppGpp or ppGpp, respectively) is a nucleotide signalling molecule with a marked effect on bacterial physiology during stress. Its accumulation slows down cell metabolism and replication, supposedly leading to the formation of the antibiotic-tolerant persister phenotype. A specifically tailored fluorescent chemosensor, PyDPA, allows the detection of (p)ppGpp in solution with high selectivity, relative to that of other nucleotides. Herein, an optimised synthetic approach is presented that improves the overall yield from 9 to 67 % over 7 steps. The simplicity and robustness of this approach will allow groups investigating the many facets of (p)ppGpp easy access to this probe.

Animal models are reliably mimicking human diseases? A morphological study that compares animal with human NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is a clinical-pathological syndrome that includes a wide spectrum of morphological alterations. In research, animal models are crucial in evaluating not only the pathogenesis of NAFLD and its progression, but also the therapeutic effects of various agents. Investigations on the ultrastructural features of NAFLD in humans are not copious, due to the difficulty to obtain human samples and to the long time of NAFLD to evolve. Translational comparative studies on the reliability of animal models in representing the histopathologic picture as seen in humans are missing. To overcome this lack of investigations, we compared the ultrastructural NAFLD features of an animal model versus human. Sprague-Dawley rats were fed with a high fat diet (HFD) for 1-4 weeks, while control rats were fed with a standard diet. Human specimens were collected from patients with diagnosed fatty liver disease, undergoing liver biopsies or surgery. Rat and human samples were examined by light microscopy and by transmission and high resolution scanning electron microscopy. The present work demonstrated that NAFLD in animal model and in human, share overlapping ultrastructural features. In conclusion, animal HFD represent an appropriate tool in studying the pathogenesis of NAFLD.

Morphological evidence that pentagastrin regulates secretion in the human parotid gland.

Salivary secretion is principally regulated by autonomic nerves. However, recent evidence from in vivo animal experiments suggests that gastrointestinal peptide hormones can also influence saliva production. The aim of the present study was to define the secretagogue activity of the gastrin-analogue pentagastrin in human salivary glands. For this purpose, parotid tissues were exposed to pentagastrin in vitro. Morphological techniques were used to evaluate modifications to serous acinar cells associated with secretion. Using a variant of the osmium maceration method, high resolution scanning electron microscopy allowed assessment of the morphology of the cytoplasmic aspect of the plasmalemma to demonstrate secretory activity. To quantify responses to pentagastrin, we recorded morphometric data on microvilli, microbuds, and protrusions. Dose-dependent morphological changes were observed, whereas protein concentration increased in the incubate. The use of selective receptor antagonists showed pentagastrin to act principally via cholecystokinin-A receptors. The morphological responses observed following exposure to pentagastrin differed from those elicited following exposure to the pan-muscarinic agonist carbachol. This study provides the first demonstration of a direct secretory action of gastrointestinal peptides on salivary glands in humans.

In vitro release of lysozyme from gelatin microspheres: effect of cross-linking agents and thermoreversible gel as suspending medium.

This study was aimed to characterize the microstructure and the performance of gelatin microspheres (GMs) cross-linked by two different cross-linkers viz. d-glucose and glutaraldehyde. New formulations were obtained, suspending the GMs in a thermoreversible Pluronic F127 (PF127) liquid-crystalline gel. Lysozyme was used as a model biomacromolecular drug to evaluate release features. Both types of cross-linked GMs were prepared by thermal gelation method. The lysozyme-loaded microspheres were characterized by scanning electron microscopy (SEM) for size distribution, shape, and surface texture. SEM revealed that both types of lysozyme-loaded GMs were spherical in shape and that the surface of glutaraldehyde cross-linked GMs was smoother than that of the glucose cross-linked GMs. The degree of cross-linking of microspheres was investigated using ATR-FTIR technique. The prepared GMs were suspended in 20% w/v aqueous PF127 gel for which the usual sol-gel transition temperature of 22 °C did not change in the presence of GMs, as indicated by rheological measurements. SAXS study of the PF127 gel confirmed the occurrence of a discrete cubic liquid-crystalline phase of the Fm3m type whose lattice parameter slightly decreased as a result of GMs addition. The in vitro release of lysozyme from both types of cross-linked GMs was successfully controlled when they were suspended in PF127 gel, thus suggesting the potential use of this new combined formulation as a drug-depot system.

The evolution of anatomical illustration and wax modelling in Italy from the 16th to early 19th centuries.

Although the contribution to anatomical illustration by Vesalius and his followers has received much attention, less credit has been given to Veslingius and particularly Fabricius. By 1600, Fabricius had amassed more than 300 paintings that together made the Tabulae Pictae, a great atlas of anatomy that was highly admired by his contemporaries. Many of his new observations were incorporated into subsequent books, including those by Casserius, Spighelius, Harvey and Veslingius. Also of importance were the Tabulae by Eustachius (1552), which, although only published in 1714, greatly influenced anatomical wax modelling. In 1742, Pope Benedict XIV established a Museum of Anatomy in Bologna, entrusting to Ercole Lelli the creation of several anatomical preparations in wax. Felice Fontana realised that the production of a large number of models by the casting method would make cadaveric specimens superfluous for anatomical teaching and in 1771 he asked the Grand Duke to fund a wax-modelling workshop in Florence as part of the Natural History Museum, later known as La Specola. Fontana engaged Giuseppe Ferrini as his first modeller and then the 19-year-old Clemente Susini who, by his death in 1814, had superintended the production of, or personally made, more than 2000 models. In 1780, the Austrian Emperor Joseph II visited La Specola and ordered a great number of models for his Josephinum museum; these were made by Fontana with the help of Clemente Susini and supervised by the anatomist Paolo Mascagni. It is, however, in Cagliari that some of Susini's greatest waxes are to be found. These were made when he was free of Fontana's influence and were based on dissections made by Francesco Antonio Boi (University of Cagliari). Their distinctive anatomical features include the emphasis given to nerves and the absence of lymphatics in the brain, a mistake made on earlier waxes. The refined technical perfection of the anatomical details demonstrates the closeness of the cooperation between Susini and Boi, whereas the expressiveness of the faces and the harmony of colours make the models of Cagliari masterpieces of figurative art.

Mitochondria of human Leydig cells as seen by high resolution scanning electron microscopy.

The three-dimensional ultrastructure of over 1000 mitochondria in human Leydig cells (from twelve sexually mature patients) was examined by high resolution scanning electron microscopy (HRSEM) of osmium-macerated specimens, as well as by transmission electron microscopy of conventional ultrathin sections. The stereo-pair imaging of the osmium-macerated specimens by HRSEM is also very useful for investigating the three-dimensional structure of cytoplasmic membranous organelles with great clarity. The mitochondria, which mainly are elongated (although some are ovate), possess cristae that are almost exclusively tubular and that occasionally display constrictions and terminal bulbules. Lamelliform cristae are quite rare. Occasionally, the tubular cristae are joined together to form a simple network. Classic crista junctions could not be identified with certainty, although the base of the tubular cristae might correspond functionally to such junctions. As a whole, in line with the identical and common embryological origin of adrenal cortex and gonads, mitochondria of human Leydig cell closely resemble those of steroidogenic cells of human suprarenal cortex treated by the same maceration method.

Histatin-induced alterations in Candida albicans: a microscopic and submicroscopic comparison.

Despite the numerous studies performed in an attempt to clarify the issue, the mechanism of action of salivary histatins remains unclear. The aim of the present study was to correlate histatin-induced morphological changes in Candida albicans by fluorescence microscopy (FM), transmission electron microscopy (TEM), and high resolution scanning electron microscopy (HRSEM). Each of the fluorescent dyes used by FM (i.e., tetramethylrhodamine methyl ester perchlorate for mitochondrial potential, Lysotracker for lysosome acidic compartment, and 4',6-diamino-2-phenylindole dihydrochloride for DNA) exhibited a specific staining in control cells. Following histatin treatment, we observed a recurring staining pattern, corresponding to fluorescence concentration along the cell periphery, suggesting a loss of dye specificity. To assess histatin-induced cytoplasmic modifications, ultrastructural analysis was then carried out. After treatments with histatins, TEM revealed characteristic intracellular modifications including: vacuole overgrowth, nuclear disappearance, loss of organelle identity, as well as the appearance of electron-dense membranes, likely of mitochondrial origin. Additionally, structures resembling autophagosomes were occasionally observed. By HRSEM, mitochondrial swelling was invariably the first sign of a histatin-induced effect. Other modifications included intracellular membrane disarrangement, organelles in disarray, and a large central cavity with deformed bodies displaced to the cell periphery, similar to what was detected by TEM. In summary, our study illustrates the occurrence of ultrastructural modifications following administration of histatins. Observations made with FM, TEM, and HRSEM provided different views of the same signs, demonstrating a definite action of histatins on C. albicans morphology. The possible functional meanings of these morphological results is discussed in light of the most recent biochemical data on histatin fungicidal activity.

Structure of cristae in cardiac mitochondria of aged rat.

Interfibrillar mitochondria (IFM) of the heart in aged Fischer 344 rats show a biochemical defect which might be reflected in their morphology. We examined by high resolution scanning electron microscopy over 5500 mitochondria to determine if a concomitant structural alteration existed. This methodology provides a means of examining mitochondrial cristae in three dimensions. Cristae of in situ subsarcolemmal mitochondria (SSM) and of IFM in both 6- and 24-month-old Fischer rats are predominantly lamelliform. When isolated, these organelles, whether of SSM or IFM origin, display enhanced heterogeneity, but they have similar crista morphology irrespective of the age of the rat. Crista configuration does not play a major role in age-related cardiac mitochondrial defects.

Cytomorphological study on human submandibular gland following treatment with secretagogue drugs.

Using specimens of human submandibular glands, we have investigated in vitro the morphological modifications induced by clozapine, a dibenzodiazepine derivative that is used in psychotic patients and that provokes hypersalivation, a side-effect of therapy. The effects of the drug, used alone or in combination with carbachol, have been compared with those observed after treatment with drugs acting on specific receptors. To quantify the response to stimulation, we have calculated (with statistical methods) the number of microvilli and microbuds (corresponding to pits seen in images obtained by transmission electron microscopy) per square micrometre of the cytoplasmic surface of the intercellular canaliculi luminal membrane in images obtained by high-resolution scanning electron microscopy. Clozapine, when directly acting on human submandibular specimens, induces a small secretory response in serous cells; this is partially decreased by muscarinic and adrenergic antagonists and by combined incubation with carbachol, thus confirming its behaviour as a partial agonist to muscarinic receptors. We also suggests that the drug acts on the nerve terminals contained within the glandular specimens.