Enzymatic synthesis and characterization of different families of chitooligosaccharides and their bioactive properties

Chitooligosaccharides (COS) are homo- or hetero-oligomers of D-glucosamine (GlcN) and N-acetyl-D-glucosamine (GlcNAc) that can be obtained by chitosan or chitin hydrolysis. Their enzymatic production is preferred over other methodologies (physical, chemical, etc.) due to the mild conditions required, the fewer amounts of waste and its efficiency to control product composition. By properly selecting the enzyme (chitinase, chitosanase or nonspecific enzymes) and the substrate properties (degree of deacetylation, molecular weight, etc.), it is possible to direct the synthesis towards any of the three COS types: fully acetylated (faCOS), partially acetylated (paCOS) and fully deacetylated (fdCOS). In this article, we review the main strategies to steer the COS production towards a specific group. The chemical characterization of COS by advanced techniques, e.g., high-performance anion-exchange chromatography with pulsed amperometric detection (HPAECPAD) and MALDI-TOF mass spectrometry, is critical for structure-function studies. The scaling of processes to synthesize specific COS mixtures is difficult due to the low solubility of chitin/chitosan, the heterogeneity of the reaction mixtures, and high amounts of salts. Enzyme immobilization can help to minimize such hurdles. The main bioactive properties of COS are herein reviewed. Finally, the anti-inflammatory activity of three COS mixtures was assayed in murine macrophages after stimulation with lipopolysaccharidesThis work was supported by grants from the EU EMFF-Blue Economy-2018 (FISH4FISH- 863697 project), the Spanish Ministry of Economy and Competitiveness (Grants BIO2016-76601- C3-1,2-R), the Spanish Ministry of Science and Innovation (Grants PID2019-105838RB-C31/C32), Fundación Ramón Areces (XIX Call of Research Grants in Life and Material Sciences) and by an institutional grant from Fundación Ramón Areces to the Centro de Biología Molecula

HLA association with the susceptibility to anti-synthetase syndrome

Objective: To investigate the human leukocyte antigen (HLA) association with anti-synthetase syndrome (ASSD). Methods: We conducted the largest immunogenetic HLA-DRB1 and HLA-B study to date in a homogeneous cohort of 168 Caucasian patients with ASSD and 486 ethnically matched healthy controls by sequencing-based-typing. Results: A statistically significant increase of HLA-DRB1*03:01 and HLA-B*08:01 alleles in patients with ASSD compared to healthy controls was disclosed (26.2% versus 12.2%, P = 1.56E–09, odds ratio–OR [95% confidence interval–CI] = 2.54 [1.84–3.50] and 21.4% versus 5.5%, P = 18.95E–18, OR [95% CI] = 4.73 [3.18–7.05]; respectively). Additionally, HLA-DRB1*07:01 allele was significantly decreased in patients with ASSD compared to controls (9.2% versus 17.5%, P = 0.0003, OR [95% CI] = 0.48 [0.31–0.72]). Moreover, a statistically significant increase of HLA-DRB1*03:01 allele in anti-Jo-1 positive compared to anti-Jo-1 negative patients with ASSD was observed (31.8% versus 15.5%, P = 0.001, OR [95% CI] = 2.54 [1.39–4.81]). Similar findings were observed when HLA carrier frequencies were assessed. The HLA-DRB1*03:01 association with anti-Jo-1 was unrelated to smoking history. No HLA differences in patients with ASSD stratified according to the presence/absence of the most representative non-anti-Jo-1 anti-synthetase autoantibodies (anti-PL-12 and anti-PL-7), arthritis, myositis or interstitial lung disease were observed. Conclusions: Our results support the association of the HLA complex with the susceptibility to ASSD

Hydroxychloroquine is associated with a lower risk of polyautoimmunity: data from the RELESSER Registry

OBJECTIVES: This article estimates the frequency of polyautoimmunity and associated factors in a large retrospective cohort of patients with SLE. METHODS: RELESSER (Spanish Society of Rheumatology Lupus Registry) is a nationwide multicentre, hospital-based registry of SLE patients. This is a cross-sectional study. The main variable was polyautoimmunity, which was defined as the co-occurrence of SLE and another autoimmune disease, such as autoimmune thyroiditis, RA, scleroderma, inflammatory myopathy and MCTD. We also recorded the presence of multiple autoimmune syndrome, secondary SS, secondary APS and a family history of autoimmune disease. Multiple logistic regression analysis was performed to investigate possible risk factors for polyautoimmunity. RESULTS: Of the 3679 patients who fulfilled the criteria for SLE, 502 (13.6%) had polyautoimmunity. The most frequent types were autoimmune thyroiditis (7.9%), other systemic autoimmune diseases (6.2%), secondary SS (14.1%) and secondary APS (13.7%). Multiple autoimmune syndrome accounted for 10.2% of all cases of polyautoimmunity. A family history was recorded in 11.8%. According to the multivariate analysis, the factors associated with polyautoimmunity were female sex [odds ratio (95% CI), 1.72 (1.07, 2.72)], RP [1.63 (1.29, 2.05)], interstitial lung disease [3.35 (1.84, 6.01)], Jaccoud arthropathy [1.92 (1.40, 2.63)], anti-Ro/SSA and/or anti-La/SSB autoantibodies [2.03 (1.55, 2.67)], anti-RNP antibodies [1.48 (1.16, 1.90)], MTX [1.67 (1.26, 2.18)] and antimalarial drugs [0.50 (0.38, 0.67)]. CONCLUSION: Patients with SLE frequently present polyautoimmunity. We observed clinical and analytical characteristics associated with polyautoimmunity. Our finding that antimalarial drugs protected against polyautoimmunity should be verified in future studies

Levan versus fructooligosaccharide synthesis using the levansucrase from Zymomonas mobilis: effect of reaction conditions

This work addresses the effect of sucrose concentration and temperature on the three activities displayed by the levansucrase from Zymomonas mobilis: formation of levan (polymerization), production of short-chain fructooligosaccharides (FOS), and sucrose hydrolysis. Of the conditions tested, levan formation reached the highest value at 4ºC and 100 g/L sucrose. The increase of temperature (40ºC) and sucrose concentration (600 g/L) caused a significant decrease of the levan concentration and a higher production of FOS. However, an increase of the temperature also caused an enhancement of the undesired hydrolytic activity. Several inulin-type FOS (1-kestose, nystose, 1F-fructosylnystose), neoFOS (blastose, neokestose, neonystose) and levan-type FOS (6-kestose, 6,6-nystose) were synthesized by levansucrase. The latter compound was purified and characterized by mass spectrometry and 2D NMR. Using 600 g/L sucrose at 40°C, the maximum yield of FOS was reached at 85% sucrose conversion; at this point, the reaction mixture contained (in weight basis) 31% glucose, 14% fructose, 15% sucrose and 40% FOS (including a small contribution of levan).This work was supported by a grant from the Spanish Ministry of Economy and Competitiveness (BIO2013-48779-C4-1-R). We thank the support of COST-Action CM1303 on Systems Biocatalysts. We thank Amano Enzyme Inc. (Japan) for the kind donation of the levansucrase. We thank Beneo Iberica for the generous supply of an inulin sample (Raftiline). P. S-M. thanks the Spanish Ministry of Education for FPU Grant.Peer reviewe

Continuous Packed Bed Reactor with Immobilized β-Galactosidase for Production of Galactooligosaccharides (GOS)

The β-galactosidase from Bacillus circulans was covalently attached to aldehyde-activated (glyoxal) agarose beads and assayed for the continuous production of galactooligosaccharides (GOS) in a packed-bed reactor (PBR). The immobilization was fast (1 h) and the activity of the resulting biocatalyst was 97.4 U/g measured with o-nitrophenyl-β-d-galactopyranoside (ONPG). The biocatalyst showed excellent operational stability in 14 successive 20 min reaction cycles at 45 °C in a batch reactor. A continuous process for GOS synthesis was operated for 213 h at 0.2 mL/min and 45 °C using 100 g/L of lactose as a feed solution. The efficiency of the PBR slightly decreased with time; however, the maximum GOS concentration (24.2 g/L) was obtained after 48 h of operation, which corresponded to 48.6% lactose conversion and thus to maximum transgalactosylation activity. HPAEC-PAD analysis showed that the two major GOS were the trisaccharide Gal-β(1→4)-Gal-β(1→4)-Glc and the tetrasaccharide Gal-β(1→4)-Gal-β(1→4)-Gal-β(1→4)-Glc. The PBR was also assessed in the production of GOS from milk as a feed solution. The stability of the bioreactor was satisfactory during the first 8 h of operation; after that, a decrease in the flow rate was observed, probably due to partial clogging of the column. This work represents a step forward in the continuous production of GOS employing fixed-bed reactors with immobilized β-galactosidases

lmmunocytochemical distribution of serotonin and neuropeptide Y, NPY in mouse adrenal gland

By the use of imrnunocytochetnicd staining methods. we studied the morphology and distribution of SHT and NPY immunoreactive cells and fibres in the mouse adrenal gland. The 5HT-immunoreactive cells were numerous and widely localized in the medullar tissue. These cells were arranged in three cellular types with regard to their morphological and immunocytochemical features. One of them showed cells with polygonal shape, being intensified like the typical medullary chromaffin cells. These imniunoreactive cells were observed arranged in medullar islets. The second SHT-inimi~noreactive celular type was constituted by cells with polygonal shape and strong immunoreactivity. The third one was formed by cells with irnmunoreactive prolongations. We found some islets of chromaffin nonirnmunoreactive cells surrounded by immunostained cells. We also observed some 5HT-imniunoreactive nerve fibres in the rnedullar tissue. NPY-like itlitnunoreactivity was detected in both chromaffin and ganglion cells in adrenal medulla. NPY-like imrnunoreactivity was also detected in nerve fibres at cortical level. In a few cases, we observed medullar SHT- and NPYinimunoreactive tissue in the adrenal cortex (monotremas)

Clinical cases of VIM-producing Pseudomonas mendocina from two burned patients

Since its first description in 1970 by Palleroni et al. from water and soil samples collected in the province of Mendoza in Argentina [1], few cases of human infection by Pseudomonas mendocina have been reported. The majority of reported cases are associated with endocarditis, lumbar spondylodiscitis and sepsis [2,3]. VIM enzymes are one of the most widespread metallo-β-lactamases (MBLs), which are commonly associated with class 1 integrons or even plasmids, contributing to the global spread of this resistance mechanism.Fil: Almuzara, Marisa. Provincia de Buenos Aires. Ministerio de Salud. Hospital Interzonal de Agudos "Eva Perón"; ArgentinaFil: Montaña, Sabrina Daiana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Carulla, Mariana Estela. Provincia de Buenos Aires. Ministerio de Salud. Hospital Interzonal de Agudos "Eva Perón"; ArgentinaFil: Sly, Gabriela. Provincia de Buenos Aires. Ministerio de Salud. Hospital Interzonal de Agudos "Eva Perón"; ArgentinaFil: Fernandez, Jennifer. California State University; Estados UnidosFil: Hernandez, Marlene. California State University; Estados UnidosFil: Moriano, Alessandro. California State University; Estados UnidosFil: Traglia, German Matias. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Bakai, Romina Vanesa. Provincia de Buenos Aires. Ministerio de Salud. Hospital Interzonal de Agudos "Eva Perón"; ArgentinaFil: Ramirez, Maria Soledad. California State University; Estados Unido

Genomic Analysis of two NDM-1 Providencia stuartii Strains Recovered from a Single Patient

In the last years, an increasing number of untreatable infections caused by drug-resistant microbes have impacted the health care system. Worldwide, infections caused by carbapenem-resistant (CR) Gram-negative bacilli have dramatically increased. Among the CR-Gram-negative bacilli, those producing carbapenemases, such as NDM-1, are the main concern. Different Enterobacterales harboring NDM-1 have been reported lately. Providencia stuartii, a member of the Morganellaceae family, is ubiquitous in the environment, but is also known to cause nosocomial infections. Here we describe the genomic analysis of two NDM-1- producing P. stuartii strains recovered from the same patient as well as other carbapenem resistant strains recovered from the same hospital. As a result of the genomic analysis thirteen resistance genes, including three to β-lactams (blaOXA-1, blaTEM-1, blaNDM-1), four to aminoglycosides (aphA6, aac(3)-IId, aac(2′)-Ia, aac(6′)-Ib-cr5), one to sulfonamides (sul1), two to chloramphenicol (catB3, catA3), one to rifampicin, one to bleomycin (ble), and one to tetracycline (tet(B)) were found. Moreover, a variety of mobile genetic elements, such as insertion sequences, plasmids and phage- related sequences, were found within P. stuartii genomes. The spread of carbapenem-resistant isolates remains a significant clinical and public health concern. Therefore, we considered that the detection of CR isolates is an essential step in addressing this problem.Fil: Hoard, Amparo. California State University; Estados UnidosFil: Montaña, Sabrina Daiana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Moriano, Alessandro. California State University; Estados UnidosFil: Fernandez, Jennifer S.. California State University; Estados UnidosFil: Traglia, German Matias. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Quiroga, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Franchi, Agustina. Gobierno de la Provincia de Buenos Aires. Hospital Interzonal de Agudos Evita.; ArgentinaFil: Cohen, Emilia. Gobierno de la Provincia de Buenos Aires. Hospital Interzonal de Agudos Evita.; ArgentinaFil: Corigliano, Cecilia. Gobierno de la Provincia de Buenos Aires. Hospital Interzonal de Agudos Evita.; ArgentinaFil: Almuzara, Marisa. Gobierno de la Provincia de Buenos Aires. Hospital Interzonal de Agudos Evita.; ArgentinaFil: Ramirez, Maria Soledad. California State University; Estados Unido