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The archaea that can be readily cultivated in the laboratory are only a small fraction of the total diversity that exists in nature. Although molecular ecology methods, such as metagenomic sequencing, can provide valuable information independent of cell cultivation, it is only through cultivation-based experiments that they may be fully characterized, both for their physiological and ecological properties. Here, we report our efforts towards enriching and isolation of uncultivated archaea from marine sediments using a refined combination of conventional microbial cultivation methods. Initially, cells were retrieved from the sediment samples through a cell extraction procedure and the sediment-free mixed cells were then divided into different size-range fractions by successive filtration through 0.8 µm, 0.6 µm and 0.2 µm membranes. Archaeal 16S rRNA gene analyses indicated noticeable retention of different archaeal groups in different fractions. For each fraction, supplementation with a variety of defined substrates (e.g., methane, sulfate, and lignin) and stepwise dilutions led to highly active enrichment cultures of several archaeal groups with Bathyarchaeota most prominently enriched. Finally, using a roll-bottle technique, three co-cultures consisting of Bathyarchaeota (subgroup-8) and a bacterial species affiliated with either Pseudomonas or Glutamicibacter were obtained. Our results demonstrate that a combination of cell extraction, size fractionation, and roll-bottle isolation methods could be a useful protocol for the successful enrichment and isolation of numerous slow-growing archaeal groups from marine sediments.

Anaerobic oxidation of methane greatly contributes to global carbon cycling, yet the anaerobic oxidation of non-methane alkanes by archaea was only recently detected in lab enrichments. The distribution and activity of these archaea in natural environments are not yet reported and understood. Here, a combination of metagenomic and metatranscriptomic approaches was utilized to understand the ecological roles and metabolic potentials of methyl-coenzyme M reductase (MCR)-based alkane oxidizing (MAO) archaea in Guaymas Basin sediments. Diverse MAO archaea, including multi-carbon alkane oxidizer Ca. Syntrophoarchaeum spp., anaerobic methane oxidizing archaea ANME-1 and ANME-2c as well as sulfate-reducing bacteria HotSeep-1 and Seep-SRB2 that potentially involved in MAO processes, coexisted and showed activity in Guaymas Basin sediments. High-quality genomic bins of Ca. Syntrophoarchaeum spp., ANME-1 and ANME-2c were retrieved. They all contain and expressed mcr genes and genes in Wood-Ljungdahl pathway for the complete oxidation from alkane to CO2 in local environment, while Ca. Syntrophoarchaeum spp. also possess beta-oxidation genes for multi-carbon alkane degradation. A global survey of potential multi-carbon alkane metabolism archaea shows that they are usually present in organic rich environments but are not limit to hydrothermal or marine ecosystems. Our study provided new insights into ecological and metabolic potentials of MAO archaea in natural environments.

Nutraceutical have emerged as beneficial health products obtained from food, herbal, and pharmaceutical manufacturing industries. Nutraceuticals have gained worldwide popularity and defined as purified or concentrated food and food products with health-promoting and/or disease-preventing properties beyond their basic nutritional function (Gunther et al. 2004). The significance of nutraceuticals has been associated with the treatment of many disorders such as obesity, cancer, metabolic problems, cold and cough, depression, coronary heart disease, osteoporosis, diabetes, cardiovascular problems, delayed gastrointestinal emptying, and other degenerative diseases (Kaur et al. 2015). Nutraceutical has led to the new era of medicine and health, in which the food industry has become a research-oriented sector. Although the use of nutraceuticals by people has a long history, recently it has been scientifically supported by nutritional and medical evidence that has allowed nutraceuticals to emerge as being potentially effective (Dillard and German 2000).

\(\bf Background:\) Previous studies noted that intravitreal injection of S100B triggered a glaucoma-like degeneration of retina and optic nerve as well as microglia activation after 14 days. The precise role of microglia in our intravitreal S100B model is still unclear. Hence, microglia were inhibited through minocycline. The aim is to investigate whether microglia have a significant influence on the degeneration process or whether they are only a side effect in the model studied here. \(\bf Methods:\) Minocycline was applied daily in rats by intraperitoneal injection using two different concentrations (13.5 mg/kg body weight, 25 mg/kg body weight). One day after treatment start, S100B or PBS was intravitreally injected in one eye per rat. The naïve groups received no injections. This resulted in a total of five groups (naïve \(\it n\) = 14, PBS \(\it n\) = 14, S100B \(\it n\) = 13, 13.5 mg/kg mino \(\it n\) = 15, 25 mg/kg mino \(\it n\) = 15). At day 14, electroretinogram measurements were performed, followed by immunofluorescence and label-free quantitative proteomics analysis. The focus of these investigations was on the survival of RGCs as well as their axons, the response of the microglia, and the identification of further pathological modes of action of S100B. \(\bf Results:\) The best signal transmission was detected via ERG in the 13.5 mg/kg mino group. The inhibition of the microglia protected optic nerve neurofilaments and decreased the negative impact of S100B on RGCs. However, the minocycline treatment could not trigger complete protection of RGCs. Furthermore, in retina and optic nerve, the minocycline treatment reduced the number and activity of S100B-triggered microglia in a concentration-dependent manner. Proteomics analysis showed that S100B application led to numerous metabolic functions and cellular stress, mainly an increased inflammatory response, glycolysis, and mitochondrial dysfunction, which caused oxidative stress in the retina. Importantly, the protective capability of lower dose of minocycline was unraveled by suppressing the apoptotic, inflammatory, and the altered metabolic processes caused by S100B insult in the retina. \(\bf Conclusion:\) Intravitreally injected S100B not only led to a pro-inflammatory microglial reaction, but also a mitochondrial and metabolic dysfunction. Also, these results suggest that an excessive microglial response may be a significant degenerative factor, but not the only trigger for increased cell death.

BackgroundDeep-sea hydrothermal vents are highly productive biodiversity hotspots in the deep ocean supported by chemosynthetic microorganisms. Prominent features of these systems are sulfide chimneys emanating high-temperature hydrothermal fluids. While several studies have investigated the microbial diversity in both active and inactive sulfide chimneys that have been extinct for up to thousands of years, little is known about chimneys that have ceased activity more recently, as well as the microbial succession occurring during the transition from active to inactive chimneys.ResultsGenome-resolved metagenomics was applied to an active and a recently extinct (~ 7 years) sulfide chimney from the 9–10° N hydrothermal vent field on the East Pacific Rise. Full-length 16S rRNA gene and a total of 173 high-quality metagenome assembled genomes (MAGs) were retrieved for comparative analysis. In the active chimney (L-vent), sulfide- and/or hydrogen-oxidizingCampylobacteriaandAquificaewith the potential for denitrification were identified as the dominant community members and primary producers, fixing carbon through the reductive tricarboxylic acid (rTCA) cycle. In contrast, the microbiome of the recently extinct chimney (M-vent) was largely composed of heterotrophs from various bacterial phyla, includingDelta-/Beta-/AlphaproteobacteriaandBacteroidetes.Gammaproteobacteriawere identified as the main primary producers, using the oxidation of metal sulfides and/or iron oxidation coupled to nitrate reduction to fix carbon through the Calvin-Benson-Bassham (CBB) cycle. Further analysis revealed a phylogenetically distinctNitrospiraecluster that has the potential to oxidize sulfide minerals coupled to oxygen and/or nitrite reduction, as well as for sulfate reduction, and that might serve as an indicator for the early stages of chimneys after venting has ceased.ConclusionsThis study sheds light on the composition, metabolic functions, and succession of microbial communities inhabiting deep-sea hydrothermal vent sulfide chimneys. Collectively, microbial succession during the life span of a chimney could be described to proceed from a “fluid-shaped” microbial community in newly formed and actively venting chimneys supported by the oxidation of reductants in the hydrothermal fluid to a “mineral-shaped” community supported by the oxidation of minerals after hydrothermal activity has ceased. Remarkably, the transition appears to occur within the first few years, after which the communities stay stable for thousands of years.

Additional file 1: Figure S1. Taxonomic classification of key functional genes retrieved from the L- and M-vent chimney. (a) The key genes enriched in the active L-vent chimney. (b) The key genes enriched in the recently inactive M-vent chimney. Figure S2 Maximum-likelihood phylogeny of dsrA genes retrieved from L-vent and M-vent chimney. The red branches represent the dsrA genes recovered from active the L-vent chimney, while the blue ones are those from recently inactive M-vent chimney. Numbers of dsrA gene for each sample are displayed in the parenthesis after the clade name. Figure S3 Maximum-likelihood phylogeny of soxB genes retrieved from L-vent and M-vent chimney. The red nodes represent the soxB genes recovered from the active L-vent chimney, while the blue ones are those from the recently inactive M-vent chimney. Four soxB genes from M-vent chimney form a distinct clade (marked with red star) that different with other soxB. Figure S4 Distribution and quantity of top 50 RpS3 genes retrieved in the MAGs for two chimney samples. (a) in the active L-vent chimney. (b) in the recently inactive M-vent chimney. The x-axis represents each RpS3 gene identified in two samples, the y-axis is the sequencing depth for each RpS3 gene. The red bars indicate the RpS3 genes identified in the MAGs in corresponding samples, while the blue ones are those are not included in the MAGs. Figure S5 Maximum-likelihood phylogeny of coxL genes retrieved from L- and M-vent chimney. The red branches represent the coxL genes recovered from active the L-vent chimney, while the blue ones are those from recently inactive M-vent chimney. Support for internal nodes was constructed from 1000 bootstrap replicates, black dots represent those nodes with bootstrap value > 75%, which is direct proportional to their diameter. Figure S6 Phylogeny of 6 Nitrospirae MAGs recovered from M-vent chimney. The phylogenetic tree is based on 37 concatenated ribosomal proteins and collapsed at the genus level. The red nodes represent those MAGs recovered from M-vent chimney, the green ones are those from subseafloor massive sulfide deposits [71] and the organe ones are those retrieved from inactive chimneys [37]. The green-shaded branches are proposed as “Sulfide mineral” clade of Nitrospirae in this study. Figure S7 Phylogeny of 20 Gammaproteobacteria MAGs recovered from M- and L-vent chimney. The phylogenetic tree is based on 37 concatenated ribosomal proteins and collapsed at the genus level. The red nodes represent those MAGs recovered from L-vent chimney, and blue for M-vent chimney. The organe ones are those retrieved from inactive chimneys [37]. Figure S8 Phylogeny of 8 Crenarchaeota MAGs recovered from L-vent chimney. The phylogenetic tree is based on 37 concatenated ribosomal proteins and collapsed at the family level. The red nodes represent those MAGs recovered from L-vent chimney. Figure S9 Maximum-likelihood phylogeny of cyc2 genes retrieved from L- and M-vent chimney. The red branches represent the cyc2 genes recovered from the active L-vent chimney, while the blue ones are those from recently inactive M-vent chimney. the green ones are those from subseafloor massive sulfide deposits [71] and the organe ones are those retrieved from inactive chimneys [37]. The other reference sequences are come from Kato et al., 2015 [71]. Support for internal nodes was constructed from 1000 bootstrap replicates, black dots represent those nodes with bootstrap value > 70%, which is direct proportional to their diameter.

The house swine (Sus scrofa domestica Linnaeus 1758) is an important model organism regarding the study of neurodegenerative diseases, especially ocular neuropathies such as glaucoma. This is due to the high comparability of the porcine and human eye regarding anatomy and molecular features. In the pathogenesis of glaucoma, the trabecular meshwork (TM) forms a key ocular component in terms of intraocular pressure (IOP) elevation. Thereby, functional TM abnormalities are correlated with distinct proteomic alterations. However, a detailed analysis of the TM proteome has not been realized so far. Since the porcine eye has high potential as a model system to study ocular diseases such as glaucoma, the present study focuses on the in-depth analysis of the porcine TM proteome. By use of a bottom-up (BU) mass spectrometric (MS) platform utilizing electrospray ionization liquid chromatography tandem MS (LC-ESI-MS/MS) considering database-dependent and peptide de novo sequencing, more than 3000 TM proteins were documented with high confidence (FDR < 1%). A distinct number of proteins with neuronal association were revealed. To the best to our knowledge, many of these protein species have not been reported for TM tissue before such as reelin, centlein and high abundant neuroblast differentiation-associated protein AHNAK (AHNAK). Thereby, AHNAK might play a superordinate role in the TM regarding proposed tissue involvement in barrier function. Also, a high number of secretory proteins could be identified. The generated TM proteomic landscape underlines a multifunctional character of the TM beyond representing a simple drainage system. Finally, the protein catalogue of the porcine TM provides an in-depth view of the TM molecular landscape and will serve as an important reference map in terms of glaucoma research utilizing porcine animal models, porcine TM tissues and/or cultured TM cells.

Aqueous humour (AH) is an important biologic fluid that maintains normal intraocular pressure and contains proteins that regulate the homeostasis of ocular tissues. Any alterations in the protein compositions are correlated to the pathogenesis of various ocular disorders. In recent years, gender-based medicine has emerged as an important research focus considering the prevalence of certain diseases, which are higher in a particular sex. Nevertheless, the inter-gender variations in the AH proteome are unknown. Therefore, this study endeavoured to characterize the AH proteome to assess the differences between genders. Thirty AH samples of patients who underwent cataract surgery were categorized according to their gender. Label-free quantitative discovery mass spectrometry-based proteomics strategy was employed to characterize the AH proteome. A total of 147 proteins were identified with a false discovery rate of less than 1% and only the top 10 major AH proteins make up almost 90% of the total identified proteins. A large number of proteins identified were correlated to defence, immune and inflammatory mechanisms, and response to wounding. Four proteins were found to be differentially abundant between the genders, comprising SERPINF1, SERPINA3, SERPING1 and PTGDS. The findings emerging from our study provide the first insight into the gender-based proteome differences in the AH and also highlight the importance in considering potential sex-dependent changes in the proteome of ocular pathologies in future studies employing the AH.

BACKGROUND: (−)-Hydroxycitric acid (HCA) is the major acid present in the fruit rinds of certain species of Garcinia. HCA has been reported to have several health benefits. As HCA is highly hygroscopic in nature and thermally sensitive, it is difficult to incorporate in foodstuffs. Hence, Garcinia cowa fruit extract was microencapsulated using three different wall materials such as whey protein isolate (WPI), maltodextrin (MD) and a combination of whey protein isolate and maltodextrin (WPI+MD) by spray drying. Further, these microencapsulated powders were evaluated for their impact on bread quality and HCA retention. RESULTS: Maltodextrin (MD) encapsulates had higher free (86%) and net HCA (90%) recovery. Microencapsulates incorporated breads had enhanced qualitative characteristics and higher HCA content thanwater extract incorporated bread due to efficient encapsulation during bread baking. Comparatively, bread with MD encapsulates showed softer crumb texture, desirable sensory attributes with considerable volume and higher HCA content. CONCLUSION: The higher HCA contents of encapsulate incorporated breads were sufficient to claim for functionality of HCA in bread. Comparatively, MD had efficiently encapsulated Garcinia fruit extract during spray drying and bread baking. Spray drying proved to be an excellent encapsulation technique for incorporation into the food system.

Glaucoma related proteomic changes have been documented in cell and animal models. However, proteomic studies investigating on human retina samples are still rare. In the present work, retina samples of glaucoma and non-glaucoma control donors have been examined by a state-of-the-art mass spectrometry (MS) workflow to uncover glaucoma related proteomic changes. More than 600 proteins could be identified with high confidence (FDR