Yeast Diversity in Honey and Pollen Samples from Stingless Bees in the State of Bahia, Brazil: Use of the MALDI-TOF MS/Genbank Proteomic Technique.

(1) Background: The identification of microorganisms includes traditional biochemical methods, molecular biology methods evaluating the conserved regions of rRNA, and the molecular biology of proteins (proteomics), such as MALDI-TOF MS mass spectrometry. This work aimed to identify the biodiversity of yeasts associated with stingless bee species' honey and pollen, Melipona scutellaris, Nannotrigona testaceicornes, and Tetragonisca angustula, from the region of São Gonçalo dos Campos-Bahia (BA) state, Brazil. (2) Methods: Cellular proteins were extracted from 2837 microbial isolates (pollen and honey) and identified via MALDI-TOF MS. The identified yeast species were also compared to the mass spectra of taxonomically well-characterized reference strains, available from the National Center of Biotechnology Information (NCBI) database. (3) Results: Nine yeast species were identified: Candida maltosa, Candida norvegica, Kazachstania telluris, Schizosaccharomyces pombe, Scheffersomyces insectosus, Meyerozyma guilliermondii, Brettanomyces bruxellensis, Kazachstania exigua, and Starmerella lactis-condensi. Nannotrigona testaceicornes pollen had the highest number of yeast colonies. The yeasts Brettanomyces bruxellensis and Kazachstania telluris showed high populations in the samples of Nannotrigona testaceicornes and Melipona scutellaris, respectively. This work shows that there is some sharing of the same species of yeast between honey and pollen from the same beehive. (4) Conclusions: A total of 71.84% of the identified species present a high level of confidence at the species level. Eight yeast species (Candida maltosa, Candida norvegica, Kazachstania telluris, Schizosaccharomyces pombe, Scheffersomyces insectosus, Meyerozyma guilliermondii, Kazachstania exigua, and Starmerella lactis-condensi) were found for the first time in the samples that the authors inspected. This contributes to the construction of new knowledge about the diversity of yeasts associated with stingless bee products, as well as to the possibility of the biotechnological application of some yeast species.

Photodynamic control of citrus crop diseases.

Citrus are economically important fruit crops to which infectious diseases like citrus canker caused by Xanthomonas citri subs. citri, citrus variegated chlorosis caused by Xylella fastidiosa, "huanglongbing" associated with the presence of Candidatus liberibacter species, anthracnose caused by Colletotrichum gloeosporioides and citrus black spot caused by Phyllosticta citricarpa, impose significant losses. Control measures involve chemical treatment of orchards but often, eradication of infected plants is unavoidable. To circumvent the environmental impacts of pesticides and the socio-economic impacts of eradication, innovative antimicrobial approaches like photodynamic inactivation are being tested. There is evidence of the susceptibility of Xanthomonas citri subs. citri and C. gloeosporioides to photodynamic damage. However, the realistic assessment of perspectives for widespread application of photodynamic inactivation in the control of citrus diseases, necessarily implies that other microorganisms are also considered. This review intends to provide a critical summary of the current state of research on photodynamic inactivation of citrus pathogens and to identify some of the current limitations to the widespread use of photodynamic treatments in citrus crops.

Phthalocyanine-sulfonamide conjugates: Synthesis and photodynamic inactivation of Gram-negative and Gram-positive bacteria.

Phthalocyanines bearing four or eight sulfonamide units were synthesized and their efficiency in the photodynamic inactivation of Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria was evaluated. Conjugates with simpler sulfonamide units (N,N-diethylbenzenesulfonamide, N-isopropylbenzenesulfonamide and N-(4-methoxyphenyl)benzenesulfonamide) caused stronger inactivation than those with heterocyclic groups (N-(thiazol-2-yl)benzenesulfonamide) or long alkyl chains (N-dodecylbenzenesulfonamide) in both bacteria. Furthermore, the encapsulation of the phthalocyanine-sulfonamide conjugates within polyvinylpyrrolidone micelles, used as drug delivery vehicles, in general showed to enhance the inactivation efficiency. The results show that encapsulated phthalocyanine-sulfonamide conjugates are a promising class of photosensitizers to be used in photodynamic antimicrobial therapy.

Liquid chromatography-tandem mass spectrometry of phosphatidylserine advanced glycated end products.

Phosphatidylserine (PS) is an aminophospholipid that is prone to glycation. In oxidative conditions, glycated PS may lead to the formation of Amadori compounds and advanced glycated end products (AGEs), which are known to accumulate in diabetic patients. Nevertheless, there have been no studies that identified products from the oxidative reaction of glycated PS. In this study, glycated 1-palmitoyl-2-oleoyl-PS was synthesized and further oxidized by Fenton reagent. The AGES formed were structurally characterized by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in negative mode. The oxidation products from glycated PS that we have found include products arising from the oxidation of the fatty acyl chains (hydroperoxides, hydroxides and keto derivatives), and arising from oxidative cleavage of serine polar head and lyso-glycated PS. Oxidation in C6 of glucose lead to the formation of glucuronyl-PS. In addition, new products arising from oxidative cleavage of glucose moiety (between C1C2, C2C3 and C3C4 bonds) were identified as PS-AGES. The current findings add substantially to the best of our knowledge of PS glycoxidation products, opening new perspectives for the detection of these products in complex biological matrices.

Evaluation of the capacity of oxidized phosphatidylserines to induce the expression of cytokines in monocytes and dendritic cells.

Oxidized phospholipids are known to be key signaling molecules in the onset of several diseases involving inflammation. The aim of this study was to evaluate the effect of oxidized phosphatidylserines (oxPS) in modulating the immune system, through cytokine production. Flow cytometry analysis was used to evaluate the oxPS capacity to induce the expression of different cytokines by monocytes, myeloid dendritic cells (mDCs) and DCs CD14(-/low)CD16(+). oxPS were formed during oxidation induced by the hydroxyl radical. Among the four families of oxPS studied, only oxPS modified in the polar head with formation of a terminal hydroperoxyacetaldehyde upregulated the production of cytokines IL-8 and TNF-α by monocytes and DCs subsets (mDCs and CD14(-/low)CD16(+) DCs). This family of oxPS showed the capacity to upregulate the production of IL-1ß, IL-6, and MIP-1ß from the same type of cells. A significant raise in the percentage of monocytes and dendritic cells producing the studied cytokines was observed, when compared with basal control. Oxidation products modified in the fatty acyl chain did not upregulate TNF and IL-8. oxPS with terminal hydroperoxyacetaldehyde has pro-inflammatory properties. This outcome may help to understand the biological role of phosphatidylserine oxidation products in inflammatory processes and in dysfunctions of immune system.

Structural characterization of oxidized glycerophosphatidylserine: evidence of polar head oxidation.

Non-oxidized phosphatidylserine (PS) is known to play a key role in apoptosis but there is considerable research evidence suggesting that oxidized PS also plays a role in this event, leading to the increasing interest in studying PS oxidative modifications. In this work, different PS (1-palmitoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine (PLPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), and 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS) were oxidized in vitro by hydroxyl radical, generated under Fenton reaction conditions, and the reactions were monitored by ESI-MS in negative mode. Oxidation products were then fractionated by thin layer chromatography (TLC) and characterized by tandem mass spectrometry (MS/MS). This approach allowed the identification of hydroxyl, peroxy, and keto derivatives due to oxidation of unsaturated fatty acyl chains. Oxidation products due to oxidation of serine polar head were also identified. These products, with lower molecular weight than the non-modified PS, were identified as [M - 29 - H](-) (terminal acetic acid), [M - 30 - H](-) (terminal acetamide), [M - 13 - H](-) (terminal hydroperoxyacetaldehyde), and [M - 13 - H](-) (terminal hydroxyacetaldehyde plus hydroxy fatty acyl chain). Phosphatidic acid was also formed in these conditions. These findings confirm the oxidation of the serine polar head induced by the hydroxyl radical. The identification of these modifications may be a valuable tool to evaluate phosphatidylserine alteration under physiopathologic conditions and also to help understand the biological role of phosphatidylserine oxidation in the apoptotic process and other biological functions.