Salivary Diagnosis of Dental Caries: A Systematic Review.

The activity of dental caries, combined with its multifactorial etiology, alters salivary molecule composition. The present systematic review was developed to answer the following question: "Are salivary biomarkers reliable for diagnosis of dental caries?". Following the "Preferred Reporting Item for Systematic Reviews and Meta-analysis" (PRISMA) guidelines, the review was conducted using multiple database research (Medline, Web of Science, and Scopus). Studies performed on healthy subjects with and without dental caries and providing detailed information concerning the clinical diagnosis of caries (Decayed, Missing, Filled Teeth-DMFT and International Caries Detection and Assessment System-ICDAS criteria) were included. The quality assessment was performed following a modified version of the Joanna Briggs Institute Prevalence Critical Appraisal Checklist. The protocol was registered in the International Prospective Register of Systematic Reviews (PROSPERO, ID: CRD42022304505). Sixteen papers were included in the review. All studies reported statistically significant differences in the concentration of salivary molecules between subjects with and without caries (p < 0.05). Proteins were the most investigated molecules, in particular alpha-amylase and mucins. Some studies present a risk of bias, such as identifying confounding factors and clearly defining the source population. Nevertheless, the 16 papers were judged to be of moderate to high quality. There is evidence that some salivary compounds studied in this review could play an important diagnostic role for dental caries, such as salivary mucins, glycoproteins (sCD14), interleukins (IL-2RA, 4,-13), urease, carbonic anhydrase VI, and urea.

The Association between Salivary Metabolites and Gingival Bleeding Score in Healthy Subjects: A Pilot Study.

Periodontal diseases, including gingivitis and periodontitis, are among the most prevalent diseases in humans. Gingivitis is the mildest form of periodontal disease, characterized by inflammation of the gingiva caused by the accumulation of dental plaque. Salivary diagnostics are becoming increasingly popular due to the variation in saliva composition in response to pathological processes. We used a metabolomics approach to investigate whether a specific saliva metabolic composition could indicate preclinical stage of gingivitis. 1H-NMR spectroscopy was used to obtain the salivary metabolite profiles of 20 healthy subjects. Univariate/multivariate statistical analysis evaluated the whole saliva metabolite composition, and the Full-Mouth Bleeding Score (FMBS) was employed as a classification parameter. Identifying a signature of specific salivary metabolites could distinguish the subjects with high FMBS scores but still within the normal range. This set of metabolites may be due to the enzymatic activities of oral bacteria and be associated with the early stages of gingival inflammation. Although this analysis is to be considered exploratory, it seems feasible to establish an FMBS threshold that distinguishes between the absence and presence of early inflammatory alterations at the salivary level.

Human Serum and Salivary Metabolomes: Diversity and Closeness.

Saliva, which contains molecular information that may reflect an individual's health status, has become a valuable tool for discovering biomarkers of oral and general diseases. Due to the high vascularization of the salivary glands, there is a molecular exchange between blood and saliva. However, the composition of saliva is complex and influenced by multiple factors. This study aimed to investigate the possible relationships between the salivary and serum metabolomes to gain a comprehensive view of the metabolic phenotype under physiological conditions. Using 1H-NMR spectroscopy, we obtained the serum metabolite profiles of 20 healthy young individuals and compared them with the metabolomes of parotid, submandibular/sublingual, and whole-saliva samples collected concurrently from the same individuals using multivariate and univariate statistical analysis. Our results show that serum is more concentrated and less variable for most of the shared metabolites than the three saliva types. While we found moderate to strong correlations between serum and saliva concentrations of specific metabolites, saliva is not simply an ultrafiltrate of blood. The intense oral metabolism prevents very strong correlations between serum and salivary concentrations. This study contributes to a better understanding of salivary metabolic composition, which is crucial for utilizing saliva in laboratory diagnostics.

Plant Defense Elicitation by the Hydrophobin Cerato-Ulmin and Correlation with Its Structural Features.

Cerato-ulmin (CU) is a 75-amino-acid-long protein that belongs to the hydrophobin family. It self-assembles at hydrophobic-hydrophilic interfaces, forming films that reverse the wettability properties of the bound surface: a capability that may confer selective advantages to the fungus in colonizing and infecting elm trees. Here, we show for the first time that CU can elicit a defense reaction (induction of phytoalexin synthesis and ROS production) in non-host plants (Arabidopsis) and exerts its eliciting capacity more efficiently when in its soluble monomeric form. We identified two hydrophobic clusters on the protein's loops endowed with dynamical and physical properties compatible with the possibility of reversibly interconverting between a disordered conformation and a ß-strand-rich conformation when interacting with hydrophilic or hydrophobic surfaces. We propose that the plasticity of those loops may be part of the molecular mechanism that governs the protein defense elicitation capability.

Sample optimization for saliva 1H-NMR metabolic profiling.

Nuclear Magnetic Resonance (NMR) based metabolomic analysis of whole saliva has provided potential diagnostic biomarkers for numerous human diseases contributing to a better understanding of their mechanisms. However, a comprehensive interpretation of the significance of metabolites in whole, parotid, and submandibular/sublingual saliva subtypes is still missing. Precision and reproducibility of sample preparation is an essential step. Here, we present a simple and efficient protocol for saliva 1H-NMR metabolic profiling. This procedure has been specifically designed and optimized for the identification and quantification of low concentration metabolites (as low as 1.1 µM) and is suitable for all the saliva subtypes.

Frataxin Structure and Function.

Mammalian frataxin is a small mitochondrial protein involved in iron sulfur cluster assembly. Frataxin deficiency causes the neurodegenerative disease Friedreich's Ataxia. Valuable knowledge has been gained on the structural dynamics of frataxin, metal-ion-protein interactions, as well as on the effect of mutations on protein conformation, stability and internal motions. Additionally, laborious studies concerning the enzymatic reactions involved have allowed for understanding the capability of frataxin to modulate Fe-S cluster assembly function. Remarkably, frataxin biological function depends on its interaction with some proteins to form a supercomplex, among them NFS1 desulfurase and ISCU, the scaffolding protein. By combining multiple experimental tools including high resolution techniques like NMR and X-ray, but also SAXS, crosslinking and mass-spectrometry, it was possible to build a reliable model of the structure of the desulfurase supercomplex NFS1/ACP-ISD11/ISCU/frataxin. In this chapter, we explore these issues showing how the scientific view concerning frataxin structure-function relationships has evolved over the last years.

Insights on the conformational dynamics of human frataxin through modifications of loop-1.

Human frataxin (FXN) is a highly conserved mitochondrial protein involved in iron homeostasis and activation of the iron-sulfur cluster assembly. FXN deficiency causes the neurodegenerative disease Friedreich's Ataxia. Here, we investigated the effect of alterations in loop-1, a stretch presumably essential for FXN function, on the conformational stability and dynamics of the native state. We generated four loop-1 variants, carrying substitutions, insertions and deletions. All of them were stable and well-folded proteins. Fast local motions (ps-ns) and slower long-range conformational dynamics (µs-ms) were altered in some mutants as judged by NMR. Particularly, loop-1 modifications impact on the dynamics of a distant region that includes residues from the ß-sheet, helix α1 and the C-terminal. Remarkably, all the mutants retain the ability to activate cysteine desulfurase, even when two of them exhibit a strong decrease in iron binding, revealing a differential sensitivity of these functional features to loop-1 perturbation. Consequently, we found that even for a small and relatively rigid protein, engineering a loop segment enables to alter conformational dynamics through a long-range effect, preserving the native-state structure and important aspects of function.

Nuclear factor (erythroid-derived 2)-like 2 (NRF2) drug discovery: Biochemical toolbox to develop NRF2 activators by reversible binding of Kelch-like ECH-associated protein 1 (KEAP1).

Mechanisms that activate innate antioxidant responses, as a way to mitigate oxidative stress at the site of action, hold much therapeutic potential in diseases, such as Parkinson's disease, Alzheimer's disease and Huntington's disease, where the use of antioxidants as monotherapy has not yielded positive results. The nuclear factor NRF2 is a transcription factor whose activity upregulates the expression of cell detoxifying enzymes in response to oxidative stress. NRF2 levels are modulated by KEAP1, a sensor of oxidative stress. KEAP1 binds NRF2 and facilitates its ubiquitination and subsequent degradation. Recently, compounds that reversibly disrupt the NRF2-KEAP1 interaction have been described, opening the field to a new era of safer NRF2 activators. This paper describes a set of new, robust and informative biochemical assays that enable the selection and optimization of non-covalent KEAP1 binders. These include a time-resolved fluorescence resonance energy transfer (TR-FRET) primary assay with high modularity and robustness, a surface plasmon resonance (SPR) based KEAP1 direct binding assay that enables the quantification and analysis of full kinetic binding parameters and finally a 1H-15N heteronuclear single quantum coherence (HSQC) NMR assay suited to study the interaction surface of KEAP1 with residue-specific information to validate the interaction of ligands in the KEAP1 binding site.

Monomeric nature of dengue virus NS3 helicase and thermodynamic analysis of the interaction with single-stranded RNA.

Dengue virus nonstructural protein 3 (NS3) is a multifunctional protein formed by a superfamily-2 RNA helicase linked to a protease domain. In this work, we report results from in vitro experiments designed to determine the oligomeric state of dengue virus NS3 helicase (NS3h) and to characterize fundamental properties of the interaction with single-stranded (ss)RNA. Pulsed field gradient-NMR spectroscopy was used to determine the effective hydrodynamic radius of NS3h, which was constant over a wide range of protein concentrations in the absence and presence of ssRNA. Size exclusion chromatography-static light scattering experiments showed that NS3h eluted as a monomeric molecule even in the presence of ssRNA. Binding of NS3h to ssRNA was studied by quantitative fluorescence titrations using fluorescein-labeled and unlabeled ssRNA oligonucleotides of different lengths, and the effect of the fluorescein label on the interaction parameters was also analyzed. Experimental results were well described by a statistical thermodynamic model based on the theory of non-specific interactions of large ligands to a one-dimensional lattice. We found that binding of NS3h to ssRNA oligonucleotides and to poly(A) is characterized by minimum and occluded binding site sizes both of 10 nucleotides and by a weak positive cooperativity between adjacent proteins.

Minute time scale prolyl isomerization governs antibody recognition of an intrinsically disordered immunodominant epitope.

Conformational rearrangements in antibody·antigen recognition are essential events where kinetic discrimination of isomers expands the universe of combinations. We investigated the interaction mechanism of a monoclonal antibody, M1, raised against E7 from human papillomavirus, a prototypic viral oncoprotein and a model intrinsically disordered protein. The mapped 12-amino acid immunodominant epitope lies within a "hinge" region between the N-terminal intrinsically disordered and the C-terminal globular domains. Kinetic experiments show that despite being within an intrinsically disordered region, the hinge E7 epitope has at least two populations separated by a high energy barrier. Nuclear magnetic resonance traced the origin of this barrier to a very slow (t(1/2)∼4 min) trans-cis prolyl isomerization event involving changes in secondary structure. The less populated (10%) cis isomer is the binding-competent species, thus requiring the 90% of molecules in the trans configuration to isomerize before binding. The association rate for the cis isomer approaches 6 × 10(7) M(-1) s(-1), a ceiling for antigen-antibody interactions. Mutagenesis experiments showed that Pro-41 in E7Ep was required for both binding and isomerization. After a slow postbinding unimolecular rearrangement, a consolidated complex with K(D) = 1.2 × 10(-7) M is reached. Our results suggest that presentation of this viral epitope by the antigen-presenting cells would have to be "locked" in the cis conformation, in opposition to the most populated trans isomer, in order to select the specific antibody clone that goes through affinity and kinetic maturation.

Solution and crystal structure of BA42, a protein from the Antarctic bacterium Bizionia argentinensis comprised of a stand-alone TPM domain.

The structure of the BA42 protein belonging to the Antarctic flavobacterium Bizionia argentinensis was determined by nuclear magnetic resonance and X-ray crystallography. This is the first structure of a member of the PF04536 family comprised of a stand-alone TPM domain. The structure reveals a new topological variant of the four ß-strands constituting the central ß-sheet of the αßα architecture and a double metal binding site stabilizing a pair of crossing loops, not observed in previous structures of proteins belonging to this family. BA42 shows differences in structure and dynamics in the presence or absence of bound metals. The affinity for divalent metal ions is close to that observed in proteins that modulate their activity as a function of metal concentration, anticipating a possible role for BA42.

Ligand binding promiscuity of human liver fatty acid binding protein: structural and dynamic insights from an interaction study with glycocholate and oleate.

Human liver fatty acid binding protein (hL-FABP) has been reported to act as an intracellular shuttle of lipid molecules, thus playing a central role in systemic metabolic homeostasis. The involvement of hL-FABP in the transport of bile salts has been postulated but scarcely investigated. Here we describe a thorough NMR investigation of glycocholate (GCA) binding to hL-FABP. The protein molecule bound a single molecule of GCA, in contrast to the 1:2 stoichiometry observed with fatty acids. GCA was found to occupy the large internal cavity of hL-FABP, without requiring major conformational rearrangement of the protein backbone; rather, this led to increased stability, similar to that estimated for the hL-FABP:oleate complex. Fast-timescale dynamics appeared not to be significantly perturbed in the presence of ligands. Slow motions (unlike for other proteins of the family) were retained or enhanced upon binding, consistent with a requirement for structural plasticity for promiscuous recognition.

Structural insights on the selective interaction of the histidine-rich piscidin antimicrobial peptide Of-Pis1 with membranes.

Of-Pis1 is a potent piscidin antimicrobial peptide (AMP), recently isolated from rock bream (Oplegnathus fasciatus). This rich in histidines and glycines 24-amino acid peptide displays high and broad antimicrobial activity and no significant hemolytic toxicity against human erythrocytes, suggesting low toxicity. To better understand the mechanism of action of Of-Pis1 and its potential selectivity, using NMR and CD spectroscopies, we studied the interaction with eukaryotic and procaryotic membranes and membrane models. Anionic sodium dodecyl sulfate (SDS) and lipopolysaccharide (LPS) micelles were used to mimic procaryotic membranes, while zwitterionic dodecyl phosphocholine (DPC) was used as eukaryotic membrane surrogate. In an aqueous environment, Of-Pis1 adopts a flexible random coil conformation. In DPC and SDS instead, the N-terminal region of Of-Pis1 forms an amphipathic α-helix with the non-polar face in close contact with the micelles. Slower solvent exchange and higher pKas of the histidine residues in SDS than in DPC suggest that Of-Pis1 interacts more tightly with SDS. Of-Pis1 also binds tightly and structurally perturbs LPS micelles. Of-Pis1 interacts with both Escherichia coli and mammalian cell membranes, but only in the presence of Escherichia coli membranes it populates the helical conformation. Furthermore, ligand-based NMR experiments support a tighter and more specific interaction with bacterial than with eukaryotic membranes. Overall, these data clearly show the selective interaction of this broadly active AMP with bacterial over eukaryotic membranes. The conformational information is discussed in terms of Of-Pis1 amino acid sequence and composition to provide insights useful to design more potent and selective AMPs.

Metabolic characterisation of transglutaminase 2 inhibitor effects in breast cancer cell lines.

Transglutaminase 2 (TG2), which mediates post-translational modifications of multiple intracellular enzymes, is involved in the pathogenesis and progression of cancer. We used 1 H-NMR metabolomics to study the effects of AA9, a novel TG2 inhibitor, on two breast cancer cell lines with distinct phenotypes, MCF-7 and MDA-MB-231. AA9 can promote apoptosis in both cell lines, but it is particularly effective in MD-MB-231, inhibiting transamidation reactions and decreasing cell migration and invasiveness. This metabolomics study provides evidence of a major effect of AA9 on MDA-MB-231 cells, impacting glutamate and aspartate metabolism, rather than on MCF-7 cells, characterised by choline and O-phosphocholine decrease. Interestingly, AA9 treatment induces myo-inositol alteration in both cell lines, indicating action on phosphatidylinositol metabolism, likely modulated by the G protein activity of TG2 on phospholipase C. Considering the metabolic deregulations that characterise various breast cancer subtypes, the existence of a metabolic pathway affected by AA9 further points to TG2 as a promising hot spot. The metabolomics approach provides a powerful tool to monitor the effectiveness of inhibitors and better understand the role of TG2 in cancer.

Fatty acid acylated peptide therapeutics: discovery of omega-n oxidation of the lipid chain as a novel metabolic pathway in preclinical species.

We recently described C18 fatty acid acylated peptides as a new class of potent long-lasting single-chain RXFP1 agonists that displayed relaxin-like activities in vivo. Early pharmacokinetics and toxicological studies of these stearic acid acylated peptides revealed a relevant oxidative metabolism occurring in dog and minipig, and also seen at a lower extent in monkey and rat. Mass spectrometry combined to NMR spectroscopy studies revealed that the oxidation occurred, unexpectedly, on the stearic acid chain at ω-1, ω-2 and ω-3 positions. Structure-metabolism relationship studies on acylated analogues with different fatty acids lengths (C15-C20) showed that the extent of oxidation was higher with longer chains. The oxidized metabolites could be generated in vitro using liver microsomes and engineered bacterial CYPs. These systems were correlating poorly with in vivo metabolism observed across species; however, the results suggest that this biotransformation pathway might be catalyzed by some unknown CYP enzymes.

The structure of the elicitor Cerato-platanin (CP), the first member of the CP fungal protein family, reveals a double ψß-barrel fold and carbohydrate binding.

Cerato-platanin (CP) is a secretion protein produced by the fungal pathogen Ceratocystis platani, the causal agent of the plane canker disease and the first member of the CP family. CP is considered a pathogen-associated molecular pattern because it induces various defense responses in the host, including production of phytoalexins and cell death. Although much is known about the properties of CP and related proteins as elicitors of plant defense mechanisms, its biochemical activity and host target(s) remain elusive. Here, we present the three-dimensional structure of CP. The protein, which exhibits a remarkable pH and thermal stability, has a double ψß-barrel fold quite similar to those found in expansins, endoglucanases, and the plant defense protein barwin. Interestingly, although CP lacks lytic activity against a variety of carbohydrates, it binds oligosaccharides. We identified the CP region responsible for binding as a shallow surface located at one side of the ß-barrel. Chemical shift perturbation of the protein amide protons, induced by oligo-N-acetylglucosamines of various size, showed that all the residues involved in oligosaccharide binding are conserved among the members of the CP family. Overall, the results suggest that CP might be involved in polysaccharide recognition and that the double ψß-barrel fold is widespread in distantly related organisms, where it is often involved in host-microbe interactions.

Onset psychosis and PTSS after severe respiratory symptomatology caused by SARS-CoV-2 infection: a case report.

Covid-19 is an infective respiratory illness caused by a novel virus, which might present different degrees of severity: from mild or even asymptomatic carriers to severe pneumonia, requiring intubation and intensive care unit (ICU) management. SARS-CoV-2 may cause also central nervous system involvement, including psychiatric manifestations. Some cases of psychosis apparently covid-related have been reported since the start of the pandemic; we will briefly review some of them here, then we will report a case concerning a patient with emerging psychosis during the disease caused by the virus. Our case describes a man with no prior personal or familiar psychiatric history, who developed delusion and a post-traumatic stress symptoms (PTSS) which required hospitalization in a psychiatric unit. The patient was treated with antipsychotic medications and underwent a brief follow-up.

Metabolic Plasticity of Candida albicans in Response to Different Environmental Conditions.

The ubiquitous commensal Candida albicans, part of the human microbiota, is an opportunistic pathogen able to cause a wide range of diseases, from cutaneous mycoses to life-threatening infections in immunocompromised patients. Candida albicans adapts to different environments and survives long-time starvation. The ability to switch from yeast to hyphal morphology under specific environmental conditions is associated with its virulence. Using hydrogen nuclear magnetic resonance spectroscopy, we profiled the intracellular and extracellular metabolome of C. albicans kept in water, yeast extract-peptone-dextrose (YPD), and M199 media, at selected temperatures. Experiments were carried out in hypoxia to mimic a condition present in most colonized niches and fungal infection sites. Comparison of the intracellular metabolites measured in YPD and M199 at 37 °C highlighted differences in specific metabolic pathways: (i) alanine, aspartate, glutamate metabolism, (ii) arginine and proline metabolism, (iii) glycerolipid metabolism, attributable to the diverse composition of the media. Moreover, we hypothesized that the subtle differences in the M199 metabolome, observed at 30 °C and 37 °C, are suggestive of modifications propaedeutic to a subsequent transition from yeast to hyphal form. The analysis of the metabolites' profiles of C. albicans allows envisaging a molecular model to better describe its ability to sense and adapt to environmental conditions.

Oligomerization, albumin binding and catabolism of therapeutic peptides in the subcutaneous compartment: An investigation on lipidated GLP-1 analogs.

Lipidation, a common strategy to improve half-life of therapeutic peptides, affects their tendency to oligomerize, their interaction with plasmatic proteins, and their catabolism. In this work, we have leveraged the use of NMR and SPR spectroscopy to elucidate oligomerization propensity and albumin interaction of different analogs of the two marketed lipidated GLP-1 agonists liraglutide and semaglutide. As most lipidated therapeutic peptides are administered by subcutaneous injection, we have also assessed in vitro their catabolism in the SC tissue using the LC-HRMS-based SCiMetPep assay. We observed that oligomerization had a shielding effect against catabolism. At the same time, binding to albumin may provide only limited protection from proteolysis due to the higher unbound peptide fraction present in the subcutaneous compartment with respect to the plasma. Finally, identification of catabolites in rat plasma after SC dosing of semaglutide showed a good correlation with the in vitro data, with Tyr19-Leu20 being the major cleavage site. Early characterization of the complex interplay between oligomerization, albumin binding, and catabolism at the injection site is essential for the synthesis of lipidated peptides with good pharmacokinetic profiles.

A Multidisciplinary Approach Establishes a Link between Transglutaminase 2 and the Kv10.1 Voltage-Dependent K+ Channel in Breast Cancer.

Since the multifunctionality of transglutaminase 2 (TG2) includes extra- and intracellular functions, we investigated the effects of intracellular administration of TG2 inhibitors in three breast cancer cell lines, MDA-MB-231, MDA-MB-436 and MDA-MB-468, which are representative of different triple-negative phenotypes, using a patch-clamp technique. The first cell line has a highly voltage-dependent a membrane current, which is low in the second and almost absent in the third one. While applying a voltage protocol to responsive single cells, injection of TG2 inhibitors triggered a significant decrease of the current in MDA-MB-231 that we attributed to voltage-dependent K+ channels using the specific inhibitors 4-aminopyridine and astemizole. Since the Kv10.1 channel plays a dominant role as a marker of cell migration and survival in breast cancer, we investigated its relationship with TG2 by immunoprecipitation. Our data reveal their physical interaction affects membrane currents in MDA-MB-231 but not in the less sensitive MDA-MB-436 cells. We further correlated the efficacy of TG2 inhibition with metabolic changes in the supernatants of treated cells, resulting in increased concentration of methyl- and dimethylamines, representing possible response markers. In conclusion, our findings highlight the interference of TG2 inhibitors with the Kv10.1 channel as a potential therapeutic tool depending on the specific features of cancer cells.