An improved and extended dual-index multiplexed 16S rRNA sequencing for the Illumina HiSeq and MiSeq platform.

BACKGROUND: Recent advancements in next-generation sequencing (NGS) technology have ushered in significant improvements in sequencing speed and data throughput, thereby enabling the simultaneous analysis of a greater number of samples within a single sequencing run. This technology has proven particularly valuable in the context of microbial community profiling, offering a powerful tool for characterizing the microbial composition at the species level within a given sample. This profiling process typically involves the sequencing of 16S ribosomal RNA (rRNA) gene fragments. By scaling up the analysis to accommodate a substantial number of samples, sometimes as many as 2,000, it becomes possible to achieve cost-efficiency and minimize the introduction of potential batch effects. Our study was designed with the primary objective of devising an approach capable of facilitating the comprehensive analysis of 1,711 samples sourced from diverse origins, including oropharyngeal swabs, mouth cavity swabs, dental swabs, and human fecal samples. This analysis was based on data obtained from 16S rRNA metagenomic sequencing conducted on the Illumina MiSeq and HiSeq sequencing platforms. RESULTS: We have designed a custom set of 10-base pair indices specifically tailored for the preparation of libraries from amplicons derived from the V3-V4 region of the 16S rRNA gene. These indices are instrumental in the analysis of the microbial composition in clinical samples through sequencing on the Illumina MiSeq and HiSeq platforms. The utilization of our custom index set enables the consolidation of a significant number of libraries, enabling the efficient sequencing of these libraries in a single run. CONCLUSIONS: The unique array of 10-base pair indices that we have developed, in conjunction with our sequencing methodology, will prove highly valuable to laboratories engaged in sequencing on Illumina platforms or utilizing Illumina-compatible kits.

Lactobacilli: Legal Regulation and Prospects for New Generation Drugs.

The global probiotics industry has been undergoing major changes in recent years. Approaches to finding and creating new probiotics, as well as a paradigm of their use in food, medicine, and pharmacology are changing. The catalyst proved to be the increasing popularity and availability of omics technologies, in particular, metagenomic studies of human and animal microbiomes. However, the efficiency and safety of drugs based on probiotic strains, as well as their marketing rates, largely depend on the levels of legal and technical regulation in the field. The present review discusses the aspects of legal regulation in Russia, the European Union and the United States, along with the advantages and disadvantages of probiotics and postbiotics. A consensus is emerging that postbiotics have a number of advantages over classical live probiotic cultures. The review also focuses on the lactobacilli family, which includes the largest number of probiotic strains studied so far and still holds a leading position among probiotics. On the legislative front, Russia is often ahead of its time with adopting such laws as the Federal Law No. 492-FZ on biosecurity, which defined the concept of human and animal microbiota and set forth legislative guidelines for its preservation. The new field of research referred to as microbiome nutrigenomics aims to achieve this goal.

[Effects of diet on the gut microbiome in patients with depression]. / Vliyanie diety na mikrobiom kishechnika u patsientov s depressiei.

OBJECTIVE: To investigate the effects of diet on the gut microbiota and to assess the relationship of these factors with depression. MATERIAL AND METHODS: Microorganisms that predominate in depressed patients were identified and associations of the identified organisms with the patients' diet were performed. Fourteen depressed patients and 14 healthy volunteers with the same socio-demographic parameters were included in the study. The Hamilton Depression Scale, Generalized Anxiety Disorder Questionnaire, and the Center for Epidemiologic Studies Questionnaire were used. RESULTS: Erysipelatoclostridium and Clostridium innocuum species were 11.3 and 14.4 times higher in depressed patients compared with healthy controls. Fusicatenibacter saccharivorans, Faecalibacterium prausnitzii and Roseburia faecis species, as well as members of the genus Roseburia were statistically significantly more abundant in the healthy volunteers group (6.5, 2.14, 8.75 and 5.2 times more frequently compared to patients). The presence of these microorganisms was correlated with dietary components. CONCLUSION: Our study revealed groups of microorganisms that differ in healthy volunteers and depressed patients. The association of these microorganisms with the diet was shown, which partially confirmed the influence of a «healthy diet¼ on the development of depressive disorders.

A Multi-strain Potential Probiotic Formulation of GABA-Producing Lactobacillus plantarum 90sk and Bifidobacterium adolescentis 150 with Antidepressant Effects.

Today, a number of studies conclusively show that certain bacterial strains, mainly from the genera Lactobacillus and Bifidobacterium, influence the functioning of the central nervous system, leading to changes in beahvior, nociception and the cognitive abilities of humans and animals. Such strains serve as the basis for developing probiotics with a curative potential for the central nervous system - psychobioitcs. However, the question of how to find such strains and which criteria to use for their selection remains unanswered. Some compounds produced by bacteria, such as gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter of the central nervous system, are potential mediators between bacterial cells and the host. Previously, we established that some species of Lactobacillus and Bifidobacterium are capable of producing GABA. We presumed that GABA-producing Lactobacillus and Bifidobacterium strains are great candidates to use as psychobiotics. Therefore, we selected the strains Lactobacillus plantarum 90sk and Bifidobacterium adolescentis 150 as efficient GABA producers. The goal of this work was to assess the probiotic properties of the selected strains as well as their antidepressive effects in mice. We established that the ingestion of the probiotic composition based on the selected strains by BALB/c mice for 2 weeks reduced depressive-like behavior in the forced swimming test; the effect was similar to that of fluoxetine.

Microscopic diagnosis of the leaf and stem of Piper solmsianum C.DC.

Piper solmsianum C.DC., which is popularly known as pariparoba, is a shrub that measures 1-3 m in height and it inhabits areas with wet tropical soils. The objective of this study was to analyze the leaf and stem anatomy using light microscopy, scanning electron micrographs, and energy-dispersive X-ray spectroscopy in order to provide information for species identification. The anatomical profile showed the following main microscopic markers: hypostomatic leaf; hypodermis layer on both sides; pearl glands; biconvex midrib shape; five collateral vascular bundles in open arc with the central bundle larger than the others; circular stem shape; collateral vascular bundles arranged in two rings; sinuous sclerenchymatic sheath in the pith; secretory idioblasts; and starch grains in the mesophyll, in the ground parenchyma of the midrib, petiole, and in the stem; and six morphotypes of calcium oxalate crystals (styloids, cuneiform, tabular crystal rosettes, cuneiform crystal rosettes, elongated square dipyramids, as well as very elongated square dipyramids).

The Lactobacillus rhamnosus and Lactobacillus fermentum strains from human biotopes characterized with MLST and toxin-antitoxin gene polymorphism.

The diversity of Lb. rhamnosus and Lb. fermentum strains isolated from feces, saliva, and the vaginal cavity of 18-22-year-old healthy women residing in central regions of the Russian Federation has been characterized. The results obtained using multilocus sequence typing were identical to those obtained with the analysis of genetic and genomic polymorphism in TA systems. Different as well as identical Lb. rhamnosus and Lb. fermentum sequence types (ST) were isolated from various parts of the body of the same person. Identical ST were also isolated from different women, suggesting that such strains belong to a common pool of strains circulating among the population members. Our results demonstrate that TAs are suitable for characterizing intra-specific diversity of Lb. rhamnosus and Lb. fermentum strains. The advantage of using polymorphisms in TA systems for genotyping is based on the weak number of genes used, and consequently, less time is required for the analysis.

GABA production and structure of gadB/gadC genes in Lactobacillus and Bifidobacterium strains from human microbiota.

Gamma-amino butyric acid (GABA) is an active biogenic substance synthesized in plants, fungi, vertebrate animals and bacteria. Lactic acid bacteria are considered the main producers of GABA among bacteria. GABA-producing lactobacilli are isolated from food products such as cheese, yogurt, sourdough, etc. and are the source of bioactive properties assigned to those foods. The ability of human-derived lactobacilli and bifidobacteria to synthesize GABA remains poorly characterized. In this paper, we screened our collection of 135 human-derived Lactobacillus and Bifidobacterium strains for their ability to produce GABA from its precursor monosodium glutamate. Fifty eight strains were able to produce GABA. The most efficient GABA-producers were Bifidobacterium strains (up to 6 g/L). Time profiles of cell growth and GABA production as well as the influence of pyridoxal phosphate on GABA production were studied for L. plantarum 90sk, L. brevis 15f, B. adolescentis 150 and B. angulatum GT102. DNA of these strains was sequenced; the gadB and gadC genes were identified. The presence of these genes was analyzed in 14 metagenomes of healthy individuals. The genes were found in the following genera of bacteria: Bacteroidetes (Bacteroides, Parabacteroides, Alistipes, Odoribacter, Prevotella), Proteobacterium (Esherichia), Firmicutes (Enterococcus), Actinobacteria (Bifidobacterium). These data indicate that gad genes as well as the ability to produce GABA are widely distributed among lactobacilli and bifidobacteria (mainly in L. plantarum, L. brevis, B. adolescentis, B. angulatum, B. dentium) and other gut-derived bacterial species. Perhaps, GABA is involved in the interaction of gut microbiota with the macroorganism and the ability to synthesize GABA may be an important feature in the selection of bacterial strains - psychobiotics.

Neuroendocrine Regulation of Anxiety: Beyond the Hypothalamic-Pituitary-Adrenal Axis.

The central nervous system regulates and responds to endocrine signals, and this reciprocal relationship determines emotional processing and behavioural anxiety. Although the hypothalamic-pituitary-adrenal (HPA) axis remains the best-characterised system for this relationship, other steroid and peptide hormones are increasingly recognised for their effects on anxiety-like behaviour and reward. The present review examines recent developments related to the role of a number of different hormones in anxiety, including pregnane neurosteroids, gut peptides, neuropeptides and hormonal signals derived from fatty acids. Findings from both basic and clinical studies suggest that these alternative systems may complement or occlude stress-induced changes in anxiety and anxiety-like behaviour. By broadening the scope of mechanisms for depression and anxiety, it may be possible to develop novel strategies to attenuate stress-related psychiatric conditions. The targets for these potential therapies, as discussed in this review, encompass multiple circuits and systems, including those outside of the HPA axis.

Molecular Cytotoxic Mechanisms of 1-(3,4,5-Trihydroxyphenyl)-dodecylbenzoate in Human Leukemia Cell Lines.

Recent studies have shown that gallic acid and its alkylesters induce apoptosis in different cell lines. Since new compounds with biological activity and less cytotoxicity to normal cells are necessary for cancer therapy, the aim of this study was to evaluate the cytotoxic effect of 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate on human acute myeloid leukemia K562 cells and on human acute lymphoblastic leukemia Jurkat cells. The cell viability was determined by MTT method. The apoptosis induction was assessed by bromide and acridine orange staining and by Annexin V-FITC Apoptosis Detection kit. The cell cycle analysis was carried out by flow cytometry using propidium iodide. Cytometric analysis was also performed to evaluate the expression of the following proteins: AIF, p53, Bcl-2 and Bax. The mitochondrial potential was also assessed by flow cytometry using MitoView633 kit. The results showed that the compound significantly reduced the cell viability of K562 and Jurkat cells in a concentration and time dependent manner (IC50 of 30 µM). The compound induced cell cycle arrest in G0/G1phase and significantly increased the proportion of cells in the sub-G0/G1phase. Apoptosis was confirmed by the sight of morphological characteristics of apoptosis and by phosphatidylserine externalization (73.47±5.71% of cells expressing annexin). The results also showed that the compound promotes a modification in Bax:Bcl-2 ratio and increases p53 expression. Thus, it is possible to conclude that 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate induces apoptosis by inhibiting the antiapoptotic protein Bcl-2 and by increasing the release of AIF, Bax and p53. In addition, it blocks the cell cycle at G0/G1, stopping cell proliferation. So far, the results suggest that this compound may have a potential therapeutic effect against leukemia cells.

Allopregnanolone and puberty: modulatory effect on glutamate and GABA release and expression of 3α-hydroxysteroid oxidoreductase in the hypothalamus of female rats.

The hypothalamic release of glutamate and GABA regulates neurosecretory functions that may control the onset of puberty. This release may be influenced by neurosteroids such as allopregnanolone. Using superfusion experiments we examined the role of allopregnanolone on the K(+)-evoked and basal [(3)H]-glutamate and [(3)H]-GABA release from mediobasal hypothalamus and anterior preoptic area in prepubertal, vaginal opening and pubertal (P) rats and evaluated its modulatory effect on GABAA and NMDA (N-methyl-d-aspartic acid) receptors. Also, we examined the hypothalamic activity and mRNA expression of 3α-hydroxysteroid oxidoreductase (3α-HSOR) - enzyme that synthesizes allopregnanolone - using a spectrophotometric method and RT-PCR, respectively. Allopregnanolone increased both the K(+)-evoked [(3)H]-glutamate and [(3)H]-GABA release in P rats, being the former effect mediated by the modulation of NMDA receptors - as was reverted by Mg(2+) and by the NMDA receptor antagonist AP-7 and the latter by the modulation of NMDA and GABAA receptors - as was reverted by Mg(2+) and the GABAA receptor antagonist bicuculline. The neurosteroid also increased the basal release of [(3)H]-glutamate in VO rats in an effect that was dependent on the modulation of NMDA receptors as was reverted by Mg(2+). On the other hand we show that allopregnanolone reduced the basal release of [(3)H]-GABA in P rats although we cannot elucidate the precise mechanism by which the neurosteroid exerted this latter effect. The enzymatic activity and the mRNA expression of 3α-HSOR were both increased in P rats regarding the other two studied stages of sexual development. These results suggest an important physiological function of allopregnanolone in the hypothalamus of the P rat where it might be involved in the 'fine tuning' of neurosecretory functions related to the biology of reproduction of the female rats.

Allopregnanolone alters the luteinizing hormone, prolactin, and progesterone serum levels interfering with the regression and apoptosis in rat corpus luteum.

Steroids synthesized in the central nervous system are termed "neurosteroids". They are synthesized and metabolized in several brain areas. The objective of this work was to determine if 1 intracerebroventricular allopregnanolone injection in rats can interfere in luteal regression in a close relationship with modifications in LH, progesterone, and prolactin serum concentrations. Allopregnanolone was injected during proestrus morning and the animals were sacrificed on oestrous morning. Ovulation test and histological analysis were performed in the oestrus morning with light and electron microscopy. Serum prolactin, LH, and progesterone levels were measured by radioimmunoassay. The allopregnanolone injection significantly decreased luteinizing hormone serum level and the number of oocytes on oestrus. Progesterone and prolactin serum levels were increased after this injection. The inhibition of apoptotic figures due to allopregnanolone administration was detected in the already formed corpora lutea belonging to the previous ovary cycle and it was significantly lower than in vehicle group (control). When the GABA(A) antagonist (bicuculline) was administered alone or previously to allopregnanolone, no effect on the ovulation rate was observed. No changes in the apoptotic cell numbers were observed with respect to those of vehicle group. These results show that the effect of centrally injected allopreganolone over reproductive function could be due to a centrally originated LH mediated effect over ovarian function that affects luteal regression, through the inhibition of apoptosis and stimulation of progesterone and prolactin release.

Filicene obtained from Adiantum cuneatum interacts with the cholinergic, dopaminergic, glutamatergic, GABAergic, and tachykinergic systems to exert antinociceptive effect in mice.

In the present study, we describe the antinociceptive effect of filicene, a triterpene isolated from Adiantum cuneatum (Adiantaceae) leaves, in several models of pain in mice. When evaluated against acetic acid-induced abdominal constrictions, filicene (10, 30 and 60 mg/kg, i.p.) produced dose-related inhibition of the number of constrictions, being several times more potent [ID(50)=9.17 (6.27-13.18) mg/kg] than acetaminophen [ID(50)=18.8 (15.7-22.6) mg/kg], diclofenac [ID(50)=12.1(9.40-15.6) mg/kg] and acetylsalicylic acid [ID(50)=24.0(13.1-43.8) mg/kg] in the same doses as those used for the standard drugs. Filicene also produced dose-related inhibition of the pain caused by capsaicin and glutamate, with mean ID(50) values of 11.7 (8.51-16.0) mg/kg and <10 mg/kg, respectively. Its antinociceptive action was significantly reversed by atropine, haloperidol, GABA(A) and GABA(B) antagonists (bicuculline and phaclofen, respectively), but was not affected by L-arginine-nitric oxide, serotonin, adrenergic and the opioid systems. Together, these results indicate that the mechanisms involved in its action are not completely understood, but seem to involve interaction with the cholinergic, dopaminergic, glutamatergic, GABAergic and tachykinergic systems.

Relation between lipophilicity of alkyl gallates and antifungal activity against yeasts and filamentous fungi.

The antifungal activity of a complete series of 15 n-alkyl gallates and six analogues acting against a representative panel of opportunistic pathogenic fungi was studied in order to analyze their role in: the importance of the fungi tested, the importance of the hydroxyls, the influence of the chain length and the hydrophobicity of the compounds. It was demonstrated that dermatophytes were the most susceptible species and that hydroxyls appear to be necessary but not sufficient for the activity. When the logP of each gallate was calculated and related to the different values of MIC against Microsporum gypseum it was observed that hexyl, heptyl, octyl and nonyl gallates exhibit a significant positive deviation from the curve corresponding to a polynomial equation obtained for the other gallates. This suggests that these compounds have a further mode of action besides their hydrophobicity, possibly the inhibition of some enzyme involved in ergosterol biosynthesis.

Two new 11alpha,12alpha-epoxy-ursan-28,13beta-olides and other triterpenes from Cecropia catharinensis.

Three new triterpenes, 2alpha-acetoxy-3beta,19alpha-dihydroxy-11alpha,12alpha-epoxy-ursan-28,13beta-olide, 3beta-acetoxy-2alpha,19alpha-dihydroxy-11alpha,12alpha-epoxy-ursan-28,13beta-olide and 2-O-acetyl-euscaphic acid together eight known triterpenes were isolated from the roots and stems of Cecropia catharinensis. Their structures were determined by detailed analysis of NMR spectra and the relative configurations established by difference nOe experiments. In addition, four flavonoid glucosides (vitexin, isovitexin, orientin and isoorientin) were found in the leaves.

Allopregnanolone modulates striatal dopamingergic activity of rats under different gonadal hormones conditions.

OBJECTIVES: Progesterone modulates dopamine (DA) release in corpus striatum. Our objective was to evaluate the effect of the i.c.v injection of the neurosteroid allopregnanolone (ALL), a progesterone metabolite on dopaminergic activity in the corpus striatum of rats under different gonadal hormonal conditions. METHODS: We have measured the concentrations of DOPA, DA and DOPAC (main metabolite of DA) in the corpus striatum in estrus and diestrus rats and in ovariectomized rats without hormonal replacement (OVX group) and primed with estrogen and progesterone (OVX(i) group). Additionally, we have used the aromatic acid decarboxylase inhibitor NSD in order to evaluate the function of tyrosine hydroxylase (TH), the rate-limiting enzyme of dopamine synthesis. RESULTS: ALL significantly decreased the striatal concentrations of both DA and DOPAC in the estrus. On the other hand, ALL increased significantly the levels of DA in the OVX(i) group. The DOPA accumulation in OVX(i) after NSD treatment in the ALL-treated groups was greater than in the vehicle group. However, the estrus group did not modify the DOPA accumulation after NSD injection. DISCUSSION: Our results suggest that ALL could modulate the dopaminergic transmission in the corpus striatum by causing changes in the activity of TH and/or in the pre- and post-synaptic dopaminergic terminals in the corpus striatum. This neurosteroidal mechanism could be a new kind of neurotransmitter systems modulation accomplished on TH activity itself and/or on the second messengers not related to ionic channels. Additionally, our results reinforce the idea of a close relationship between the fast non-genomic mechanism of ALL and the genomic actions of estrogen and progesterone.

Analgesic potential of marrubiin derivatives, a bioactive diterpene present in Marrubium vulgare (Lamiaceae).

Marrubiin, a furane labdane diterpene, is the main analgesic compound present in Marrubium vulgare, a medicinal plant used in Brazil and other countries to treat several ailments. Considering its important pharmacological action, as well as its high yield, some structural modifications were performed in order to obtain more active compounds. Success was obtained in reducing the lactonic function, in the formation of marrubiinic acid and two esterified derivatives, which exhibited significant analgesic effect against the writhing test in mice. Marrubiinic acid showed better activity and excellent yield, and its analgesic effect was confirmed in other experimental models of pain in mice, suggesting its possible use as a model to obtain new and potent analgesic agents.

Pharmacological and phytochemical investigations of different parts of Calophyllum brasiliense (Clusiaceae).

Continuing our search for antinociceptive agents from natural sources, this study analyzed the antinociceptive effects of some fractions obtained from different parts (roots, flowers and fruits) of Calophyllum brasiliense, a Brazilian medicinal plant used to treat several diseases, including inflammation and pain. For this purpose, the writhing and formalin induced-pain models in mice were used. We also analyzed the chemical composition of these different parts and tested two pure compounds isolated from chloroform fraction (roots) identified as friedelin (1) and 1,5-dihydroxyxanthone (3), by direct comparison with authentic samples. The results showed that some fractions and both compounds exhibited considerable antinociception properties, particularly against the writhing test, and that these are more potent than acetyl salicylic acid and acetaminophen, two reference drugs used here for comparison.

Antifungal activity of fractions and two pure compounds of flowers from Wedelia paludosa (Acmela brasiliensis) (Asteraceae).

Wedelia paludosa (Acmela brasiliensis) (Asteraceae), a traditionally used native Brazilian medicinal plant, showed antifungal activity against dermatophytes in dilution tests. The hexane, dichloromethane and butanol fractions displayed activity against Epidermophyton floccosum, Trichophyton rubrum and Trichophyton mentagrophytes, with minimal inhibitory concentrations between 250 and 1000 microg/mL. Two pure compounds, identified as kaurenoic acid (1) and luteolin (2), also showed activity against these dermatophytes.

Antifungal activity of sterols and triterpenes isolated from Ganoderma annulare.

Three sterols, 5alpha-ergost-7-en-3beta-ol, 5alpha-ergosta-7,22-dien-3beta-ol and 5,8-epidioxy-5alpha,8alpha-ergosta-6,22-dien-3beta-ol and five triterpenes, applanoxidic acids A, C, F, G and H, have been isolated from Ganoderma annulare. The applanoxidic acids A, C and F were found to inhibit the growth of the fungi Microsporum cannis and Trichophyton mentagrophytes at concentrations of 500 to 1000 microg/ml.

Incidence of sperm-tail tyrosine phosphorylation and hyperactivated motility in normozoospermic and asthenozoospermic human sperm samples

Our objective was to study the incidence of sperm-tail phosphotyrosine immunoreactivity in normozoospermic and asthenozoospermic human sperm samples, its association with sperm motion parameters, particularly hyperactivated motility, and its potential involvement in the pathogenesis of asthenozoospermia. The work was conducted as a prospective experimental study in the Sperm Biology and Andrology laboratories of the Jones Institute, a medical school-based fertility center. The study subjects were healthy fertile male donors (normozoospermic samples) and infertile patients (asthenozoospermic samples) attending the center. Recently ejaculated semen samples were washed twice to eliminate seminal plasma and a swim-up was performed to select the motile population which, in turn, was incubated up to 18 h at 37 degrees C in 3.5% human serum albumin-supplemented Hams F10 to allow for capacitation. For evaluation, sperm aliquots were taken pre-swim-up (T0), immediately post swim-up (T1), at 6 h (T6), and 18 h (T18) of incubation. The main outcome measures were computer-analyzed sperm motion parameters and hyperactivated motility, and immunodetection of phosphotyrosine (PY)-containing proteins. During the capacitating incubation, normozoospermic samples displayed maximum motility, velocity, and hyperactivation at T6, significantly decreasing their values at T18. PY-proteins were located both at the tail and head of spermatozoa. Their expression increased progressively during the incubation, being present in about 70% of the sperm tails at T18. Asthenozoospermic samples showed an inability to respond to capacitation with an increase in motion parameters and PY-phosphorylation. At T6, both hyperactivation and PY-phosphorylation were significantly lower than in normal samples. Our results suggest that PY-phosphorylation of tail proteins is highly conspicuous in human spermatozoa, and increases its incidence in a time-dependent manner, as more sperm become capacitated. Asthenozoospermic samples displaying low percentages of motile sperm and altered motion characteristics showed a decreased incidence of PY-phosphorelated sperm. Tail protein PY-phosphorylation may be related to sperm movement, especially to hyperactivated motility and its deficiency may be associated to asthenozoospermia. (AU)