Population-Based Analysis of the Immunoglobulin G Response to Different COVID-19 Vaccines in Brazil.

(1) Background: COVID-19 vaccination in Brazil has been performed mostly with CoronaVac (Sinovac), ChAdOx1-S (AstraZeneca-University of Oxford) and BNT162b2 (Pfizer-BioNTech) vaccines. The titers of IgG antibodies reactive to the SARS-CoV-2 spike protein correlate with vaccine efficacy. Studies comparing vaccine immunogenicity in a real-world scenario are lacking. (2) Methods: We performed a population-based study to analyze the immunoglobulin G response to different COVID-19 vaccines. Citizens older than 18 years (n = 2376) provided personal data, a self-declaration of any previous COVID-19 positive tests and information regarding COVID-19 vaccination: the vaccine popular name and the date of each dose. Blood samples were collected and the levels of IgG reactive to SARS-CoV-2 antigens were determined and compared between different vaccine groups. (3) Results: The seroconversion for anti-spike IgG achieved > 95% by February 2022 and maintained stable until June 2022. Higher anti-spike IgG titers were detected in individuals vaccinated with BNT162b2, followed by ChAdOx1-S and CoronaVac. The anti-spike IgG response was negatively correlated with age and interval after the second dose for the BNT162b2 vaccine. Natural infections boosted anti-spike IgG in those individuals who completed primary vaccination with ChAdOx1-S and CoronaVac, but not with BNT162b2. The levels of anti-spike IgG increased with the number of vaccine doses administered. The application of BNT162b2 as a 3rd booster dose resulted in high anti-spike IgG antibody titers, despite the type of vaccine used during primary vaccination. (4) Conclusions: Our data confirmed the effectiveness of the Brazilian vaccination program. Of the vaccines used in Brazil, BNT162b2 performed better to elicit anti-spike protein IgG after primary vaccination and as a booster dose and thus should be recommended as a booster whenever available. A continuous COVID-19 vaccination program will be required to sustain anti-spike IgG antibodies in the population.

Synthesis of C6-amino agarose and evaluation of its antibacterial activity.

In this paper, the biologically inert agarose was selectively modified at C6 of ß-d-Galp units to produce an amino derivative with antibacterial property. The synthetic route involved the preparation of tosyl and azido agarose intermediates. All the polysaccharide derivatives were characterized by mono- and bidimensional 1H and 13C NMR and FT-IR analysis. A water-soluble amino polymer (Mw = 39,000 g mol-1, DSamino = 0.50) was produced by partial acid hydrolysis showing bactericidal and bacteriostatic activity against P. aeruginosa (ATCC 9027), S. aureus (ATCC 6538), and E. coli (ATCC 25922), with MIC values lower than 2.5 mg mL-1 and MBC values ranging from 2.5 to 5.0 mg mL-1.

Chemical structure of native and modified sulfated heterorhamnans from the green seaweed Gayralia brasiliensis and their cytotoxic effect on U87MG human glioma cells.

A water-soluble sulfated heterorhamnan (Gb1) was isolated from the green seaweed Gayralia brasiliensis and purified by ultrafiltration, yielding a homogeneous polysaccharide (Gb1r). Both fractions contained rhamnose, xylose, galacturonic and glucuronic acids, galactose, and glucose. Chemical and spectroscopic methods allowed the determination of Gb1 and Gb1r chemical structure. Their backbones were constituted by 3-, 2-, and 2,3-linked rhamnosyl units (1:0.49:0.13 and 1:0.58:0.17, respectively), which are unsulfated (13.5 and 14.6%), disulfated (16.6 and 17.8%) or monosulfated at C-2 (8 and 8.6%) and C-4 (24.5 and 23.4%). Gb1 was oversulfated giving rise to Gb1-OS, which presented ~2.5-fold higher content of disulfated rhamnosyl units than Gb1, as determined by methylation analyses and NMR spectroscopy. Gb1 and Gb1-OS potently reduced the viability of U87MG human glioblastoma cells. Gb1 caused cell cycle arrest in the G1 phase, increased annexin V-stained cells, and no DNA fragmentation, while Gb1-OS increased the percentage of cells in the S and G2 phases and the levels of fragmented DNA and cells double-stained with annexin V/propidium iodide, suggesting an apoptosis mechanism. The results suggest that the different effects of Gb1 and Gb1-OS were related to differences in the sulfate content and position of these groups along the polysaccharide chains.

Semi-synthesis of N-alkyl-kappa-carrageenan derivatives and evaluation of their antibacterial activity.

In this article, we describe the semi-synthesis of N-alkyl-kappa-carrageenan derivatives and their antibacterial activity against Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 8739), and Pseudomonas aeruginosa (ATCC 9027). Kappa-carrageenan was submitted to partial acid hydrolysis promoting the selective cleavage of α-glycosidic bonds involving 3,6-anhydro-α-D-Galp units, giving rise to reducing low-molecular weight polysaccharide fragments, which were reacted with alkylamines of varying chain lengths by reductive amination. The carrageenan derivatives were characterized by HPSEC-MALLS-RID and 1D and 2D 1H and 13C NMR spectroscopy. The antibacterial activity of N-alkyl-kappa-carrageenan derivatives was compared with N-alkyl-(1-deoxylactitol-1-yl)-amines using a microdilution test, which indicated that inhibitory activity was dependent on the degree of substitution by hydrophobic groups at the polysaccharide structure. Comparing the effect of different N-alkyl chains, those with longer chains showed higher activity.

Efficacy and safety of oral phosphodiesterase 5 inhibitors for erectile dysfunction: a network meta-analysis and multicriteria decision analysis.

PURPOSE: To quantitatively assess the benefit-risk ratio on the efficacy and safety of all phosphodiesterase type 5 inhibitors (PDE5i) in men with erectile dysfunction. METHODS: A systematic review with network meta-analysis, surface under the cumulative ranking analysis and stochastic multicriteria acceptability analyses were performed. Searches were conducted in Pubmed, Scopus, Web of Science without limits for time-frame or language. Randomized controlled trials evaluating the efficacy or safety of any PDE5i compared to a placebo or to other PDE5i in males with erectile disfunction were included. RESULTS: Overall, 184 articles representing 179 randomized controlled trials (50,620 patients) were included. All PDE5i were significantly more efficient than placebo. Sildenafil 25 mg was statistically superior to all interventions in enhancing IIEF (with a 98% probability of being the most effective treatment), followed by sildenafil 50 mg (80% of probability). Taladafil 10 mg and 20 mg also presented good profiles (73% and 76%, respectively). Avanafil and lodenafil were less effective interventions. Mirodenafil 150 mg was the treatment that caused more adverse events, especially flushing and headaches. Sildenafil 100 mg was more related to visual disorders, while vardenafil and udenafil were more prone to cause nasal congestion. CONCLUSION: Sildenafil at low doses and tadalafil should be the first therapeutic options. Avanafil, lodenafil and mirodenafil use are hardly justified given the lack of expressive efficacy or high rates of adverse events.

Conformational analysis of ulvans from Ulva fasciata and their anticoagulant polycarboxylic derivatives.

This study investigates conformational aspects of ulvans (F2) and their polycarboxyl derivatives obtained through periodate-chlorite oxidation (C3) followed by DEAE-Sephacel fractioning (C3b and C3c). Size exclusion chromatography coupled with laser light scattering and viscometric detection, in addition to circular dichroism (CD) and molecular modeling analyses, suggested that F2 had a compact sphere conformation with a helical motif as secondary structure. In contrast, all the polycarboxyl ulvans showed a random coil conformation, although C3c (NaSO3- 21.0%; COO- 1.81 mmol·g-1; Mw 18 kg·mol-1) had a more rigid and constrained backbone than C3 (NaSO3- 21.0%; COO- 1.81 mmol·g-1; Mw 49 kg·mol-1), largely due to its higher sulfate and carboxyl content. Despite the higher ionic character of C3c, its anticoagulant activity (ACA), determined by activated partial thromboplastin time (APTT) assay, was not improved compared to that of C3. Moreover, C3b (NaSO3- 14.1%; COO- 1.23 mmol·g-1; Mw 8.1 kg·mol-1) had higher activity than F2 (NaSO3- 20.6.%; COO- 0.36 mmol·g-1; Mw 123 kg·mol-1), even with its lower sulfate content and molar mass. These results suggest that the ACA of polycarboxyl ulvans relies on carboxyl and sulfate content and may depend, in addition, on a proper flexible conformation.

Colorimetric microdilution assay: Validation of a standard method for determination of MIC, IC50%, and IC90% of antimicrobial compounds.

The emergence of multiresistant bacteria directly impacts on the search for new compounds with antimicrobial activity, and it is important the improvement of new techniques are able to determine the minimum inhibitory concentration (MIC) of antimicrobial compounds. The microdilution technique is widely used for saving culture media, reagents and compounds to be tested. However, the literature does not describe a colorimetric method capable of correlating absorbance with concentration of viable microorganisms (CFU mL-1). Therefore, the novelty of this work was the standardization and validation of a colorimetric and quantitative method capable of determining the MIC of several compounds with antimicrobial activity and the conversion of absorbance values to CFU mL-1. The conditions carried out for the method were: the use of 0.125% (w/v) 2,3,5-triphenyltetrazolium chloride (TTC) solution added after 22 h of incubation at 35 °C, followed by 2 more hours of incubation and subsequent reading in a spectrophotometer. The tested microorganisms were: Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 8739), Pseudomonas aeruginosa (ATCC 9027) and Candida albicans (ATCC 10231). The method was validated and showed linearity (R2 > 0.95), precision (RSD <26%), accuracy (75% to 122%) and robustness (p > 0.05). The validated parameters ensured the harmonization of methodology to determine not only MIC as well as inhibitory concentrations of 50% (IC50%) and 90% (IC90%) of the antimicrobial compounds.

Effects of carboxyl group on the anticoagulant activity of oxidized carrageenans.

In this paper, carrageenans having distinct sulfation patterns (κ-, ι-, ι/ν-, θ- and λ-carrageenans), were fully or partially oxidized at C-6 of the ß-d-Galp units using 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and trichloroisocyanuric acid (TCCA) in bicarbonate buffer. The modified carrageenans were characterized by mono- and bidimensional 1H and 13C NMR spectroscopy. The influence of the sulfate and carboxyl groups onto anticoagulant activity was evaluated using Activated Partial Thromboplastin Time (aPTT) in vitro assay. The results showed a synergic effect of the carboxyl groups on the anticoagulant activity, which was dependent on the regiochemistry of the sulfate groups in the polysaccharide backbone. Sulfate groups at C2 of the ß-d-GalAp units appeared to positively influence the anticoagulant effect in comparison to C4-sulfate samples. Also, the partially oxidized κ-carrageenan derivative (κLO) showed better anticoagulant effect than the fully oxidized carrageenan (κHO).

Monitoring of κ-carrageenan depolymerization by capillary electrophoresis and semisynthesis of oligosaccharide alditols.

Different hydrolysis conditions to produce κ-carrageenan oligosaccharide alditols were studied and the depolymerization process monitored by capillary electrophoresis (CE). Semisynthesis, ion-exchange and exclusion chromatography were used to obtain and isolate sulfated di-, tetra- and hexasaccharide alditols, the last being fully characterized for the first time. Those derivatives were used as standards to validate a new quantitative CE analytical method which was used to compare two different partial hydrolysis methodologies: an acid hydrolysis followed by reduction and a one-pot reductive hydrolysis using 4-methylmorpholine borane. The resulting depolymerization profiles were quite different from each other. Optimal hydrolysis conditions to produce high yields of specific sulfated oligosaccharides as well as particular mixtures of oligosaccharide alditols were determined. Moreover, using the novel CE method, we were able to distinguish up to eight different oligosaccharides in the hydrolysate mixtures.

Modification of ulvans via periodate-chlorite oxidation: Chemical characterization and anticoagulant activity.

Native (F2) and carboxyl-reduced (R) ulvans from Ulva fasciata were sequentially oxidized with periodate-chlorite affording the polycarboxyl ulvans C1, C2 and C3 (1.20, 1.41 and 1.81 mmol g-1 of COOH, respectively; 19.7, 21.3 and 21.0% of NaSO3, respectively) and R-C3 (1.86 mmol g-1 of COOH; NaSO3 = 22.7%), respectively. APTT assay (polysaccharide fractions at 150 µg mL-1) showed clotting time of 45.6 s for F2 fraction. For polycarboxyl ulvans C1, C2, C3 and R-C3 the clotting times were 101.0, 122.2, 222.0 and 227.0 s, respectively. Comparison of the APTT assay results using ulvans chemically modified by carboxyl-reduction, desulfation, periodate oxidation and/or chlorite oxidation showed the anticoagulant activity of polycarboxyl ulvans is dependent of the sulfate groups present in the native polymer. In addition, the increase of the anticoagulant activity was accompanied by the increasing of the carboxyl groups and the content of this acidic substituent seems to be more important than its positioning.

Synthesis and Pharmacological Evaluation of Heterocyclic Carboxamides: Positive Allosteric Modulators of the M1 Muscarinic Acetylcholine Receptor with Weak Agonist Activity and Diverse Modulatory Profiles.

Targeting allosteric sites at M1 muscarinic acetylcholine receptors is a promising strategy for the treatment of Alzheimer's disease. Positive allosteric modulators not only may potentiate binding and/or signaling of the endogenous agonist acetylcholine (ACh) but also may possess direct agonist activity (thus referred to as PAM-agonists). Recent studies suggest that PAM-agonists with robust intrinsic efficacy are more likely to produce adverse effects in vivo. Herein we present the synthesis and pharmacological evaluation of a series of pyrrole-3-carboxamides with a diverse range of allosteric profiles. We proposed structural modifications at top, core, or pendant moieties of a prototypical molecule. Although generally there was a correlation between the degree of agonist activity and the modulatory potency of the PAMs, some derivatives displayed weak intrinsic efficacy yet maintained strong allosteric modulation. We also identified molecules with the ability to potentiate mainly the affinity or both affinity and efficacy of ACh.

Aqueous semisynthesis of C-glycoside glycamines from agarose.

Agarose was herein employed as starting material to produce primary, secondary and tertiary C-glycoside glycamines, including mono- and disaccharide structures. The semisynthetic approach utilized was generally based on polysaccharide-controlled hydrolysis followed by reductive amination. All reactions were conducted in aqueous media and without the need of hydroxyl group protection. We were able to identify optimal conditions for the reductive amination of agar hydrolysis products and to overcome the major difficulties related to this kind of reaction, also extending it to reducing anhydrosugars. The excess of ammonium acetate, methyl- or dimethylamine, and the use of a diluted basic (pH 11) reaction media were identified as important aspects to achieve improved yields, as well as to decrease the amount of byproducts commonly related to reductive amination of carbohydrates. This strategy allowed the transposition of the 3,6-anhydro-α-L-galactopyranose unit (naturally present in the agarose structure) to all glycamines synthesized, constituting an amino-substituted C-threofuranoside moiety, which is closely related to (+)-muscarine.

Photodynamic effect of meso-(aryl)porphyrins and meso-(1-methyl-4-pyridinium)porphyrins on HaCaT keratinocytes.

Sixteen porphyrins, including neutral, anionic and cationic meso-(aryl)porphyrins and meso-(1-methyl-4-pyridinium)porphyrins were herein evaluated in terms of their photosensitizing properties against HaCaT keratinocytes. After an initial screening, the cationic porphyrins were studied in more details, by both determining their log POW and performing PDT assays in lower porphyrin concentrations. Porphyrins presenting two or more adjacent positively charged groups, directly linked to the macrocycle meso positions, appeared to be the most effective photosensitizers. The present study also included the dicationic 5,10-diphenyl-15,20-di(1-methylpyridinium-4-yl)porphyrin (14b), which has previously shown promising results on a psoriasis-like in vivo model. Overall results indicated that the beneficial effect related to porphyrins on psoriasis can be related to the decreasing of keratinocyte viability. Furthermore, some of the cationic porphyrins studied appeared as candidates to be utilized as photosensitizers for psoriasis treatment.

Sulfated heterorhamnans from the green seaweed Gayralia oxysperma: partial depolymerization, chemical structure and antitumor activity.

Sulfated heterorhamnans produced by Gayralia oxysperma were utilized for the preparation of two homogeneous and highly sulfated Smith-degraded products (M(w) of 109 and 251 kDa), which were constituted principally by 3-linked α-L-rhamnosyl units 2- or 4-sulfate and 2-linked α-L-rhamnosyl units 4- or 3,4-sulfate, in different percentages. The homogeneous products and the crude extracts containing the sulfated heterorhamnans showed cytotoxic effect against U87MG cells. These sulfated polysaccharides induced an increase in the number of cells in G1 phase with concomitant increase of the mRNA levels of p53 and p21. The presence of 2-linked disulfated rhamnose residues together with the molecular weight could be important factors to be correlated with the inhibitory effect on human glioblastoma cells.

Selective sulfation of carrageenans and the influence of sulfate regiochemistry on anticoagulant properties.

Sulfated polysaccharides are recognized for their broad range of biological activities, including anticoagulant properties. The positions occupied by the sulfate groups are often related to the level of the inherent biological activity. Herein the naturally sulfated galactans, kappa-, iota- and theta-carrageenan, were additionally sulfated by regioselective means. The anticoagulant activity of the resulting samples was then studied using the aPTT in vitro assay. The influence of sulfate regiochemistry on the anticoagulant activity was evaluated. From kappa-carrageenan three rare polysaccharides were synthesized, one of them involved a synthetic route with an amphiphilic polysaccharide intermediate containing pivaloyl groups. Iota- and theta-carrageenan were utilized in a selective C6 sulfation at ß-D-Galp units to produce different structures comprising trisulfated diads. All the samples were characterized by NMR (1D and 2D). The resulting aPPT measurements suggested that sulfation at C2 of 3,6-anhydro-α-D-Galp and C6 of ß-D-Galp increased the anticoagulant activity.

Chemical structure of the complex pyruvylated and sulfated agaran from the red seaweed Palisada flagellifera (Ceramiales, Rhodophyta).

A homogeneous agaran fraction from Palisada flagellifera (Laurencia complex, Rhodomelaceae, Ceramiales) was obtained by aqueous room-temperature extraction, followed by ion-exchange chromatography. This galactan presents a highly complex structure with at least 18 different types of derivatives. The A units were found mostly pyruvylated, 2-sulfated (∼34%), and 6-methylated (∼34%), with the latter partially 2- and 2,4-sulfated. Minor amounts of ß-D-galactopyranosyl units 2-, 6- and 2,6-sulfated, 6-glycosylated, and non-substituted are also present. The B-units are L-sugars composed predominantly of their cyclized derivatives, 3,6-anhydrogalactose and 3,6-anhydro-2-O-methylgalactose (∼56%). The former are linked to ß-D-galactosyl (6-methyl) (6-glycosylated) units, as well as to 4,6-O-(1-carboxyethylidene)-ß-D-galactose 2-sulfate in the proportion of 3:1.8, respectively. A significant amount (∼18%) of the α-L-galactopyranosyl units are linked to pyruvylated ß-D-galactose 2-sulfate residues. An important part of the B-units (20%) is represented by α-L-galactose 6-sulfate substituted on C-3 by xylosyl, galactosyl and/or 2,3-di-O-methylgalactose units or sulfate groups that preclude their cyclization to 3,6-anhydrogalactosyl derivative. The precursor units are present in relatively low percentages. Kinetic studies suggest that in P. flagellifera agaran the cyclizable units are linked to 6-O-methyl-ß-D-galactosyl and/or ß-D-galactosyl units (6-glycosylated). The structural complexity of this polysaccharide is increased by the presence of 2- and 3,6-sulfated α-L-galactoses, with the latter additionally 2-O-methylated. Therefore, the major subfraction obtained from the cold extract contains structurally complex sulfated, methylated, and pyruvylated agaran.

ß-D-(1→4), ß-D-(1→3) 'mixed linkage' xylans from red seaweeds of the order Nemaliales and Palmariales.

Xylans from five seaweeds belonging to the order Nemaliales (Galaxaura marginata, Galaxaura obtusata, Tricleocarpacylindrica, Tricleocarpa fragilis, and Scinaia halliae) and one of the order Palmariales (Palmaria palmata) collected on the Brazilian coasts were extracted with hot water and purified from acid xylomannans and/or xylogalactans through Cetavlon precipitation of the acid polysaccharides. The ß-D-(1→4), ß-D-(1→3) 'mixed linkage' structures were determined using methylation analysis and 1D and 2D NMR spectroscopy. The presence of large sequences of ß-(1→4)-linked units suggests transient aggregates of ribbon- or helical-ordered structures that would explain the low optical rotations.

Production of agaro- and carra-oligosaccharides by partial acid hydrolysis of galactans

Agaro- and carra-oligosaccharides were produced by partial acid hydrolysis of commercial agarose and kappa-carrageenan. Di- and tetrasaccharides were purified by gel filtration chromatography and characterized by NMR (1D and 2D) spectroscopy and ESIMS. The following oligosaccharides were obtained: agarobiose, agarotetraose, kappa-carrabiose and kappa-carratetraose. Agarobiose and agarotetraose were used as standards to develop a high performance size exclusion chromatography (HPSEC) method which was utilized to study the hydrolysis rate of agarose and oligosaccharide production. Six hours of hydrolysis (0.1 M TFA, 65 ºC) produced mainly di- and tetrasaccharides. The methodology for oligosaccharide production and evaluation developed in the present work shows good potential for the production of bioactive oligosaccharides.

ESI-MS differential fragmentation of positional isomers of sulfated oligosaccharides derived from carrageenans and agarans.

We have prepared a number of isomeric red seaweed galactan-derivative sulfated oligosaccharides to determine whether there were diagnostic differences among the isomeric mass spectra obtained using ESI CID MS/MS (triple quadrupole instrument). Fragmentation of the single or multicharged molecular ions from di-, tetra-, and hexasaccharides indicated that the relative positioning of the sulfate groups and type of monosaccharide unit affect the rate of cleavage of the glycosidic bonds. We also performed a comparative [M-Na](-) fragmentation study of positional isomers of sulfated disaccharides that present all four monosulfation possibilities on the galactopyranosidic ring. In this case, negative-ion ESI CID MS/MS approach gave diagnostic product ions from cross-ring cleavages along with the same main B(1) ion (from sulfated Galp), at m/z 241, for all isomers. The isomeric disaccharides were also submitted to increased spray energy conditions inducing in-source fragmentation; preformed B(1) ions were then fragmented to give similar product ions as those found in [M-Na](-) analysis. Evaluation of the relative abundances mainly for cross-ring fragment ions at m/z 138, 139, 151, 153 allowed clear distinction among the members of the disaccharide series. The different ratios for m/z 151/153 ions were consistent with the predominance of m/z 153 being related to the cases when the bond involved in the cleavage process links a sulfated carbon. A quadrupole ion trap instrument (MS(n) analysis) was also utilized to compare the results obtained with the triple quadrupole instrument.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis of oligosaccharides and oligosaccharide alditols obtained by hydrolysis of agaroses and carrageenans, two important types of red seaweed polysaccharides.

MALDI-TOF mass spectrometry analyses of several oligosaccharides (aldoses) and oligosaccharide alditols derived from agaroses, kappa- and iota-carrageenans using different matrices (2,5-dihydroxybenzoic acid, nor-harmane, ferulic acid, and the ionic liquid matrices 2,5-dihydroxybenzoic acid-n-butylamine and ferulic acid-n-butylamine) were conducted. These carbohydrates were selected as model compounds to study the MALDI prompt and post-source decay (PSD) fragmentation processes of both families of oligosaccharides. Sulfated alditols showed in the negative-ion mode the molecular ion as [M-Na](-) together with the species yielded by their prompt fragmentation (mainly desulfation) while the sulfated oligosaccharides (aldoses) showed mainly glycosidic prompt fragmentation (glycosidic C-cleavages and desulfation). Non-sulfated aldoses and alditols, which could only be analyzed in positive-ion mode ([M+Na](+)), did not suffer any prompt fragmentation. The former yielded cross-ring fragmentation in the PSD mode. Best results were obtained by using 2,5-dihydroxybenzoic acid and/or nor-harmane as matrices for all the compounds studied.