Speeding-up the Determination of Protein-Ligand Affinities by STD NMR: The Reduced Data Set STD NMR Approach (rd-STD NMR).

STD NMR spectroscopy is a powerful ligand-observed NMR tool for screening and characterizing the interactions of small molecules and low molecular weight fragments with a given macromolecule, identifying the main intermolecular contacts in the bound state. It is also a powerful analytical technique for the accurate determination of protein-ligand dissociation constants (KD) of medium-to-weak affinity, of interest in the pharmaceutical industry. However, accurate KD determination and epitope mapping requires a long series of experiments at increasing saturation times to carry out a full analysis using the so-called STD NMR build-up curve approach and apply the "initial slopes approximation". Here, we have developed a new protocol to bypass this important limitation, which allows us to obtain initial slopes by using just two saturation times and, hence, to very quickly determine precise protein-ligand dissociation constants by STD NMR.

Periostin in the relation between periodontal disease and atherosclerotic coronary artery disease: A pilot randomized clinical study.

OBJECTIVE: The aim of this study was to analyze the effects of periodontal treatment on markers of atherosclerotic coronary artery disease and circulating levels of periostin. BACKGROUND: Periostin is necessary for periodontal stability, but it is highly present in atherosclerotic plaques. Treatment of periodontal disease, with low levels of local periostin, is thought to reduce systemic levels of periostin. Thus, this may contribute to cardiovascular health. METHODS: A pilot randomized controlled clinical trial was designed to include patients with severe periodontal disease and history of atherosclerotic coronary artery disease. Samples of gingival crevicular fluid (GCF) and serum were collected before and after periodontal treatment by periodontal surgery or non-surgical therapy. The levels of several markers of inflammation and cardiovascular damage were evaluated including CRP, IFN-γ, IL-1ß, IL-10, MIP-1α, periostin, and TNF-α in GCF and CRP, Fibrinogen, IFN-γ, IL-1ß, IL-6, IL-10, L-Selectin, MIP-1α, Periostin, TNF-α, and vWF in serum. RESULTS: A total of 22 patients with an average of 56 years old were recruited for participating in this study. Twenty of them were male. Most of them (82%) had suffered an acute myocardial event and underwent surgery for placing 1, 2, or 3 stents in the coronary arteries more than 6 months ago but less than 1 year. The treatment of periodontal disease resulted in an overall improvement of all periodontal parameters. Regarding the evaluation of GCF and serum, a significant increase of periostin in the GCF was observed after periodontal surgery. In contrast, although other markers in GCF and serum improved, no significant correlations were found. CONCLUSION: Treatment of periodontal disease through periodontal surgery induces a local and transient increase in the levels of periostin in the gingival crevicular fluid. The effects on systemic markers of inflammation and cardiovascular function have not been confirmed.

Unravelling the Inflammatory Processes in the Early Stages of Diabetic Nephropathy and the Potential Effect of (Ss)-DS-ONJ.

Inflammatory processes play a central role in the pathogenesis of diabetic nephropathy (DN) in the early stages of the disease. The authors demonstrate that the glycolipid mimetic (Ss)-DS-ONJ is able to abolish inflammation via the induction of autophagy flux and provokes the inhibition of inflammasome complex in ex vivo and in vitro models, using adult kidney explants from BB rats. The contribution of (Ss)-DS-ONJ to reducing inflammatory events is mediated by the inhibition of classical stress kinase pathways and the blocking of inflammasome complex activation. The (Ss)-DS-ONJ treatment is able to inhibit the epithelial-to-mesenchymal transition (EMT) progression, but only when the IL18 levels are reduced by the treatment. These findings suggest that (Ss)-DS-ONJ could be a novel, and multifactorial treatment for DN.

Synthesis of sp2-Iminosugar Selenoglycolipids as Multitarget Drug Candidates with Antiproliferative, Leishmanicidal and Anti-Inflammatory Properties.

sp2-Iminosugar glycolipids (sp2-IGLs) represent a consolidated family of glycoconjugate mimetics encompassing a monosaccharide-like glycone moiety with a pseudoamide-type nitrogen replacing the endocyclic oxygen atom of carbohydrates and an axially-oriented lipid chain anchored at the pseudoanomeric position. The combination of these structural features makes them promising candidates for the treatment of a variety of conditions, spanning from cancer and inflammatory disorders to parasite infections. The exacerbated anomeric effect associated to the putative sp2-hybridized N-atom imparts chemical and enzymatic stability to sp2-IGLs and warrants total α-anomeric stereoselectivity in the key glycoconjugation step. A variety of O-, N-, C- and S-pseudoglycosides, differing in glycone configurational patterns and lipid nature, have been previously prepared and evaluated. Here we expand the chemical space of sp2-IGLs by reporting the synthesis of α-d-gluco-configured analogs with a bicyclic (5N,6O-oxomethylidene)nojirimycin (ONJ) core incorporating selenium at the glycosidic position. Structure-activity relationship studies in three different scenarios, namely cancer, Leishmaniasis and inflammation, convey that the therapeutic potential of the sp2-IGLs is highly dependent, not only on the length of the lipid chain (linear aliphatic C12 vs. C8), but also on the nature of the glycosidic atom (nitrogen vs. sulfur vs. selenium). The ensemble of results highlights the α-dodecylseleno-ONJ-glycoside as a promising multitarget drug candidate.

Stereoselective Synthesis of Iminosugar 2-Deoxy(thio)glycosides from Bicyclic Iminoglycal Carbamates Promoted by Cerium(IV) Ammonium Nitrate and Cooperative Brønsted Acid-Type Organocatalysis.

The first examples of iminosugar-type 2-deoxy(thio)glycoside mimetics are reported. The key step is the activation of a bicyclic iminoglycal carbamate to generate a highly reactive acyliminium cation. Cerium(IV) ammonium nitrate efficiently promoted the formation of 2-deoxy S-glycosides in the presence of thiols, probably by in situ generation of catalytic HNO3, with complete α-stereoselectivity. Cooperative phosphoric acid/Schreiner's thiourea organocatalysis proved better suited for generating 2-deoxy O-glycosides, significantly broadening the scope of the approach.

Thiol-ene "Click" Synthesis and Pharmacological Evaluation of C-Glycoside sp2-Iminosugar Glycolipids.

The unique stereoelectronic properties of sp2-iminosugars enable their participation in glycosylation reactions, thereby behaving as true carbohydrate chemical mimics. Among sp2-iminosugar conjugates, the sp2-iminosugar glycolipids (sp2-IGLs) have shown a variety of interesting pharmacological properties ranging from glycosidase inhibition to antiproliferative, antiparasitic, and anti-inflammatory activities. Developing strategies compatible with molecular diversity-oriented strategies for structure-activity relationship studies was therefore highly wanted. Here we show that a reaction sequence consisting in stereoselective C-allylation followed by thiol-ene "click" coupling provides a very convenient access to α-C-glycoside sp2-IGLs. Both the glycone moiety and the aglycone tail can be modified by using sp2-iminosugar precursors with different configurational profiles (d-gluco or d-galacto in this work) and varied thiols, as well as by oxidation of the sulfide adducts (to the corresponding sulfones in this work). A series of derivatives was prepared in this manner and their glycosidase inhibitory, antiproliferative and antileishmanial activities were evaluated in different settings. The results confirm that the inhibition of glycosidases, particularly α-glucosidase, and the antitumor/leishmanicidal activities are unrelated. The data are also consistent with the two later activities arising from the ability of the sp2-IGLs to interfere in the immune system response in a cell line and cell context dependent manner.

sp2 -Iminosugar α-glucosidase inhibitor 1-C-octyl-2-oxa-3-oxocastanospermine specifically affected breast cancer cell migration through Stim1, ß1-integrin, and FAK signaling pathways.

Aberrant glycosylation changes on many glycoproteins are often related to cancer progression and metastasis. sp2 -Iminosugar-type castanospermine analogues, inhibitors of α-glucosidases, have been reported to exhibit antitumor activity. However, their effects on cell migration and the underlying molecular mechanism are not fully understood. Here, we investigated the effect of the pseudo-C-octyl glycoside 2-oxa-3-oxocastanospermine derivatives (CO-OCS) on breast cancer cells (MCF-7 and MDA-MB-231 cells), and MCF-10A mammary normal cell lines. We showed that CO-OCS treatment results in the drastic decrease of breast cancer cell migration without affecting cell proliferation. Furthermore, CO-OCS significantly reduced both the expression of ß1-integrin, which is a crucial interacting partner of Focal Adhesion Kinase (FAK), and the phosphorylation rates of FAK and ERK1/2. CO-OCS also drastically reduced Ca2+ entry through Store Operated Channels (SOC). Orai1 and Stim1, two N-glycosylated proteins, are involved in Store-Operated Calcium Entry (SOCE), and are essential for breast tumor cell migration. Our results showed that CO-OCS decreased the expression, at the protein level, of Stim1 without affecting that of Orai1. Moreover, cell migration and SOCE were attenuated by CO-OCS as well as when Stim1 was silenced. In contrast, in MCF-10A cells, CO-OCS slightly reduced cell migration, but was without effect on gene expression of Stim1, Orai1, ß1-integrin, or FAK and ERK1/2 activation. Our results provide strong evidence for a significant effect of CO-OCS on breast cancer cell migration and support that this effect was associated with ß1-integrin, Stim1, and FAK signaling pathways.

H-classic: a new method to identify classic articles in Implant Dentistry, Periodontics, and Oral Surgery.

INTRODUCTION: The study of classic papers permits analysis of the past, present, and future of a specific area of knowledge. This type of analysis is becoming more frequent and more sophisticated. Our objective was to use the H-classics method, based on the h-index, to analyze classic papers in Implant Dentistry, Periodontics, and Oral Surgery (ID, P, and OS). MATERIAL AND METHODS: First, an electronic search of documents related to ID, P, and OS was conducted in journals indexed in Journal Citation Reports (JCR) 2014 within the category 'Dentistry, Oral Surgery & Medicine'. Second, Web of Knowledge databases were searched using Mesh terms related to ID, P, and OS. Finally, the H-classics method was applied to select the classic articles in these disciplines, collecting data on associated research areas, document type, country, institutions, and authors. RESULTS: Of 267,611 documents related to ID, P, and OS retrieved from JCR journals (2014), 248 were selected as H-classics. They were published in 35 journals between 1953 and 2009, most frequently in the Journal of Clinical Periodontology (18.95%), the Journal of Periodontology (18.54%), International Journal of Oral and Maxillofacial Implants (9.27%), and Clinical Oral Implant Research (6.04%). These classic articles derived from the USA in 49.59% of cases and from Europe in 47.58%, while the most frequent host institution was the University of Gothenburg (17.74%) and the most frequent authors were J. Lindhe (10.48%) and S. Socransky (8.06%). CONCLUSION: The H-classics approach offers an objective method to identify core knowledge in clinical disciplines such as ID, P, and OS.

Synthesis of multibranched australine derivatives from reducing castanospermine analogues through the Amadori rearrangement of gem-diamine intermediates: selective inhibitors of ß-glucosidase.

A practical one-pot synthesis of bi- and triantennated australine analogues from a pivotal sp(2)-iminosugar-type reducing castanospermine precursor is reported. The transformation involves a gem-diamine intermediate that undergoes the indolizidine → pyrrolizidine Amadori-type rearrangement and proceeds under strict control of the generalized anomeric effect to afford a single diastereomer. The final compounds behave as selective competitive inhibitors of ß-glucosidase and are promising candidates as pharmacological chaperones for Gaucher disease.

Investigations on the oxygen dependence of a 2-oxoglutarate histone demethylase.

Histone N(ϵ)-methyl lysine demethylases are important in epigenetic regulation. KDM4E (histone lysine demethylase 4E) is a representative member of the large Fe(II)/2-oxoglutarate- dependent family of human histone demethylases. In the present study we report kinetic studies on the reaction of KDM4E with O2. Steady-state assays showed that KDM4E has a graded response to O2 over a physiologically relevant range of O2 concentrations. Pre-steady state assays implied that KDM4E reacts slowly with O2 and that there are variations in the reaction kinetics which are dependent on the methylation status of the substrate. The results demonstrate the potential for histone demethylase activity to be regulated by oxygen availability.

Comparison of the MIPS technique versus linear incision with tissue preservation in osseointegrated implants in the Ponto model®.

BACKGROUND AND OBJECTIVES: The surgery of osseointegrated implants has undergone different modifications over the years with the aim of achieving better results and facilitating the surgical technique. Today the most commonly used technique is the linear incision with tissue preservation and placement of the abutment and implant. The long-term success of this technique has served as the basis for the development of the so-called minimally invasive surgical approach (MIPS). This study compares the short-, medium- and long-term results between the classic linear incision technique and the MIPS technique. MATERIAL AND METHODS: A prospective study was conducted on patients who had an osseointegrated implant placed between February 2016 and February 2020. A total of 59 surgeries were performed, 32 surgeries according to the linear incision technique with tissue preservation and 27 with MIPS technique. Outcomes were evaluated at one week, one month and one year. RESULTS: Statistically significant differences were achieved between the 2 groups at one week after surgery. Eighty per cent of the MIPS patients had Holgers grades 0-1 compared to 35% of the linear technique patients (p = 0.001). No statistically significant differences were observed at one month (p = 0.457) and one year (p = 0.228). One case with grade 4 was recorded which resulted in implant extrusion one month after surgery with the MIPS technique. A new osseointegrated implant was placed 2 months after the fall using the same MIPS technique with good results. We were also able to verify that the duration of surgery was much shorter with the MIPS technique and better tolerated in terms of postoperative discomfort by the patient. CONCLUSIONS: In our experience, the MIPS technique is the technique of choice for surgery of osseointegrated Ponto model implants as it is simpler, faster and presents fewer problems in the immediate postoperative period, with similar long-term postoperative results.

A selenoureido-iminoglycolipid transported by zeolitic-imidazolate framework nanoparticles: a novel antioxidant therapeutic approach.

A selenium-containing metal-organic framework with remarkable antioxidant capacity and ROS-scavenging activity was constructed by a controlled de novo encapsulation approach of a glycoconjugate mimetic, specifically a sp2-iminoglycolipid bearing a selenoureido fragment (DSeU), within a zeolitic-imidazolate framework exoskeleton. Biocompatible and homogeneous nanosized particles of ∼70 nm (DSeU@ZIF8) were obtained, which could be efficiently internalized in cells, overcoming the poor solubility in biological media and limited bioavailability of glycolipids. The ZIF-particle served as nanocarrier for the intracellular delivery of the selenocompound to cells, promoted by the acidic pH inside endosomes/lysosomes. As demonstrated by in vitro studies, the designed DSeU@ZIF8 nanoparticles displayed a high antioxidant activity at low doses; lower intracellular ROS levels were observed upon the uptake of DSeU@ZIF8 by human endothelial cells. Even more interesting was the finding that these DSeU@ZIF8 particles were able to reverse to a certain level the oxidative stress induced in cells by pre-treatment with an oxidizing agent. This possibility of modulating the oxidative stress in living cells may have important implications in the treatment of diverse pathological complications that are generally accompanied with elevated ROS levels.

Fluoro-labelled sp2-iminoglycolipids with immunomodulatory properties.

The unique electronic properties of the fluorine atom make its strategic incorporation into a bioactive compound a very useful tool in the design of drugs with optimized pharmacological properties. In the field of the carbohydrates, its selective installation at C2 position has proven particularly interesting, some 2-deoxy-2-fluorosugar derivatives being currently in the market. We have now transferred this feature into immunoregulatory glycolipid mimetics that contain a sp2-iminosugar moiety, namely sp2-iminoglycolipids (sp2-IGLs). The synthesis of two epimeric series of 2-deoxy-2-fluoro-sp2-IGLs, structurally related to nojirimycin and mannonojirimycin, has been accomplished by sequential Selectfluor-mediated fluorination and thioglycosidation of sp2-iminoglycals. Exclusively the α-anomer is obtained regardless of the configurational profile of the sp2-IGL (d-gluco or d-manno), highlighting the overwhelming anomeric effect in these prototypes. Notably, the combination of a fluorine atom at C2 and an α-oriented sulfonyl dodecyl lipid moiety in compound 11 led to remarkable anti-proliferative properties, featuring similar GI50 values than the chemotherapy drug Cisplatin against several tumor cell lines and better selectivity. The biochemical data further evidence a strong reduction of the number of tumor cell colonies and apoptosis induction. Mechanistic investigations revealed that this fluoro-sp2-IGL induces the non-canonical activation mode of the mitogen-activated protein kinase signaling pathway, causing p38α autoactivation under an inflammatory context.

Efficient transfection of hepatocytes mediated by mRNA complexed to galactosylated cyclodextrins.

In this study, we aimed at specific targeting of polycationic amphiphilic cyclodextrins (paCDs) to HepG2 cells via the asialoglycoprotein receptor (ASGPr). The transfection efficiencies of paCDs modified with galactose moieties were evaluated. In preliminary experiments, attempts to transfect HepG2 cells with pDNA complexed with different modified paCDs resulted in very low transfection levels. In additional series of experiments, we found out that nucleic acid/cyclodextrin complexes (CDplexes) were efficiently taken up by the cells and that photochemical internalization, which facilitates release from endosomes, did not improve transfection. Further experiments showed that pDNA can be readily released from the CDplexes when exposed to negatively charged vesicles. These observations imply that the lack of transfection cannot be attributed to a lack of internalization, release of CDplexes from the endosomal compartment, or release of free pDNA from the CDplexes. This in turn suggests that the nuclear entry of the pDNA represents the main limiting factor in the transfection process. To verify that HepG2 cells were transfected with targeted CDplexes containing mRNA, which does not require entry into the nucleus for being translated. With mRNA encoding the green fluorescent protein, fractions of GFP-positive cells of up to 31% were obtained. The results confirmed that the galactosylated complexes are specifically internalized via the ASGPr.

sp2-Iminosugar O-, S-, and N-glycosides as conformational mimics of α-linked disaccharides; implications for glycosidase inhibition.

The synthesis of mimics of the α(1→6)- and α(1→4)-linked disaccharides isomaltose and maltose featuring a bicyclic sp(2)-iminosugar nonreducing moiety O-, S-, or N-linked to a glucopyranoside residue is reported. The strong generalized anomeric effect operating in sp(2)-iminosugars determines the α-stereochemical outcome of the glycosylation reactions, independent of the presence or not of participating protecting groups and of the nature of the heteroatom. It also imparts chemical stability to the resulting aminoacetal, aminothioacetal, or gem-diamine functionalities. All the three isomaltose mimics behave as potent and very selective inhibitors of isomaltase and maltase, two α-glucosidases that bind the parent disaccharides either as substrate or inhibitor. In contrast, large differences in the inhibitory properties were observed among the maltose mimics, with the O-linked derivative being a more potent inhibitor than the N-linked analogue; the S-linked pseudodisaccharide did not inhibit either of the two target enzymes. A comparative conformational analysis based on NMR and molecular modelling revealed remarkable differences in the flexibility about the glycosidic linkage as a function of the nature of the linking atom in this series. Thus, the N-pseudodisaccharide is more rigid than the O-linked derivative, which exhibits conformational properties very similar to those of the natural maltose. The analogous pseudothiomaltoside is much more flexible than the N- or O-linked derivatives, and can access a broader area of the conformational space, which probably implies a strong entropic penalty upon binding to the enzymes. Together, the present results illustrate the importance of taking conformational aspects into consideration in the design of functional oligosaccharide mimetics.

Predictive factors for maxillary sinus augmentation outcomes: a case series analysis.

AIM: To determine the influence of different local and systemic factors on histologic, histomorphometric, and radiographic outcomes after maxillary sinus augmentation. MATERIALS AND METHODS: Fifty-two sinus augmentation procedures were performed. Grafting material consisted of a mixture of anorganic bovine bone (ABB) and autogenous bone. After 6 months, bone core biopsies were harvested from implant sites for histologic and histomorphometric analyses. Data regarding age, gender, type of edentulism, alcohol consumption, smoking habits, and history of periodontal disease were recorded and statistically analyzed. RESULTS: Histomorphometric analyses revealed the presence of 35.75% ± 16.42% of vital bone, 40.56% ± 16.23% of nonmineralized tissue, and 23.69% ± 18.23% of residual ABB particles. Radiographic vertical bone resorption inversely correlated with residual ABB. A significant difference in bone resorption patterns was observed for completely edentulous patients and for those with a history of periodontitis. Tobacco and alcohol negatively influenced vital bone formation after sinus augmentation. Implant and prostheses survival after 2 years of functional loading was not directly affected by patient's individual habits. CONCLUSION: Certain patient-related variables such as history of periodontitis, type of edentulism, or smoking/drinking habits play an important role in bone graft maturation after maxillary sinus floor elevation.

Mannobioside biomimetics that trigger DC-SIGN binding selectivity.

Selective DC-SIGN targeting vs. langerin might lead to anti-infective agents, given their counteracting effects upon infection by some pathogens. Here we show that multivalent sp2-iminosugar-containing mannobioside analogs can achieve total DC-SIGN selectivity by levering the canonic binding mode towards high-mannose oligosaccharide ligands, behaving as factual biomimics.

Dental implant migration in grafted maxillary sinus.

BACKGROUND: Migration of oral implants through a previously consolidated graft is an extremely rare event. We reported 2 cases of migration at least 6 months postimplantation in sinus grafted in 2 stages, consequently over 6-month graft maturation period. CASE DESCRIPTION: Sinus elevations and implants placement in 2 patients were performed. Clinical and radiographic follow-up after the prosthetic rehabilitation showed displacement of 1 implant in each patient through the histopathologically verified stable grafts. CLINICAL IMPLICATIONS: Implants migration through composite graft after maxillary sinus elevation was reported. Reasons for this to occur might be due to the following: fail to achieve implant primary stability, Schneiderian membrane perforation, or graft resorption due to increased osteoclastic activity.

Hyaluronic acid reduces inflammation and crevicular fluid IL-1ß concentrations in peri-implantitis: a randomized controlled clinical trial.

PURPOSE: This study investigated the effects of hyaluronic acid (HA) on peri-implant clinical variables and crevicular concentrations of the proinflammatory biomarkers interleukin (IL)-1ß and tumor necrosis factor (TNF)-α in patients with peri-implantitis. METHODS: A randomized controlled trial was conducted in peri-implantitis patients. Patients were randomized to receive a 0.8% HA gel (test group), an excipient-based gel (control group 1), or no gel (control group 2). Clinical periodontal variables and marginal bone loss after 0, 45, and 90 days of treatment were assessed. IL-1ß and TNF-α levels in crevicular fluid were measured by enzyme-linked immunosorbent assays at baseline and after 45 days of treatment. Clustering analysis was performed, considering the possibility of multiple implants in a single patient. RESULTS: Sixty-one patients with 100 dental implants were assigned to the test group, control group 1, or control group 2. Probing pocket depth (PPD) was significantly lower in the test group than in both control groups at 45 days (control 1: 95% CI, -1.66, -0.40 mm; control 2: 95% CI, -1.07, -0.01 mm) and 90 days (control 1: 95% CI, -1.72, -0.54 mm; control 2: 95% CI, -1.13, -0.15 mm). There was a trend towards less bleeding on probing in the test group than in control group 2 at 90 days (P=0.07). Implants with a PPD ≥5 mm showed higher levels of IL-1ß in the control group 2 at 45 days than in the test group (P=0.04). CONCLUSIONS: This study demonstrates for the first time that the topical application of a HA gel in the peri-implant pocket and around implants with peri-implantitis may reduce inflammation and crevicular fluid IL-1ß levels. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03157193.