Optimisation of sample storage and DNA extraction for human gut microbiota studies​.

BACKGROUND: New developments in next-generation sequencing technologies and massive data received from this approach open wide prospects for personalised medicine and nutrition studies. Metagenomic analysis of the gut microbiota is paramount for the characterization of human health and wellbeing. Despite the intensive research, there is a huge gap and inconsistency between different studies due to the non-standardised and biased pipeline. Methodical and systemic understanding of every stage in the process is necessary to overcome all bottlenecks and grey zones of gut microbiota studies, where all details and interactions between processes are important. RESULTS: Here we show that an inexpensive, but reliable iSeq 100 platform is an excellent tool to perform the analysis of the human gut microbiota by amplicon sequencing of the 16 S rRNA gene. Two commercial DNA extraction kits and different starting materials performed similarly regarding the taxonomic distribution of identified bacteria. DNA/RNA Shield reagent proved to be a reliable solution for stool samples collection, preservation, and storage, as the storage of faecal material in DNA/RNA Shield for three weeks at different temperatures and thawing cycles had a low impact on the bacterial distribution. CONCLUSIONS: Altogether, a thoroughly elaborated pipeline with close attention to details ensures high reproducibility with significant biological but not technical variations.

GLI2 cell-specific activity is controlled at the level of transcription and RNA processing: Consequences to cancer metastasis.

High activity of GLI family zinc finger protein 2 (GLI2) promotes tumor progression. Removal of the repressor domain at the N terminus (GLI2∆N) by recombinant methods converts GLI2 into a powerful transcriptional activator. However, molecular mechanisms leading to the formation of GLI2∆N activator proteins have not been established. Herein we report for the first time that the functional activities of GLI2 are parted into different protein isoforms by alternative promoter usage, selection of alternative splicing, transcription initiation and termination sites. Functional studies using melanoma cells revealed that transcriptional regulation of GLI2 is TGFbeta-dependent and supports the predominant production of GLI2∆N and C-terminally truncated GLI2 (GLI2∆C) isoforms in cells with high migratory and invasive phenotype. Taken together, these results highlight the role of transcription and RNA processing as major processes in the regulation of GLI2 activity with severe impacts in cancer development.

Diversity in TAF proteomics: consequences for cellular differentiation and migration.

Development is a highly controlled process of cell proliferation and differentiation driven by mechanisms of dynamic gene regulation. Specific DNA binding factors for establishing cell- and tissue-specific transcriptional programs have been characterised in different cell and animal models. However, much less is known about the role of "core transcription machinery" during cell differentiation, given that general transcription factors and their spatiotemporally patterned activity govern different aspects of cell function. In this review, we focus on the role of TATA-box associated factor 4 (TAF4) and its functional isoforms generated by alternative splicing in controlling lineage-specific differentiation of normal mesenchymal stem cells and cancer stem cells. In the light of our recent findings, induction, control and maintenance of cell differentiation status implies diversification of the transcription initiation apparatus orchestrated by alternative splicing.

Short-term pectin-enriched smoothie consumption has beneficial effects on the gut microbiota of low-fiber consumers.

Adequate consumption of fiber has a positive effect on health. The crossover study examined the effect of a pectin-enriched smoothie on gut microbiota and health parameters. During 3 weeks, 31 adults consumed two smoothies (11.6 or 4.8 g of fiber/day), alternating with washout periods in different order. At the end of each period, weekly food diaries, blood samples, and stool microbiota were collected. Changes in the microbiota during smoothie consumption were associated with baseline fiber intake. A greater proportion of up- (Lachnospira, Colidextribacter, and Bacteroides) or down-shifts (Streptococcus, Holdemanella) was observed in low-fiber (n = 22) compared to high-fiber consumers (n = 9). In both groups, the pectin-enriched smoothie reduced the number of the Ruminococcus torques group bacteria. Our results showed that the short-term approach is effective to estimate relationships between food components and gut bacteria.

Variability and bias in microbiome metagenomic sequencing: an interlaboratory study comparing experimental protocols.

Several studies have documented the significant impact of methodological choices in microbiome analyses. The myriad of methodological options available complicate the replication of results and generally limit the comparability of findings between independent studies that use differing techniques and measurement pipelines. Here we describe the Mosaic Standards Challenge (MSC), an international interlaboratory study designed to assess the impact of methodological variables on the results. The MSC did not prescribe methods but rather asked participating labs to analyze 7 shared reference samples (5 × human stool samples and 2 × mock communities) using their standard laboratory methods. To capture the array of methodological variables, each participating lab completed a metadata reporting sheet that included 100 different questions regarding the details of their protocol. The goal of this study was to survey the methodological landscape for microbiome metagenomic sequencing (MGS) analyses and the impact of methodological decisions on metagenomic sequencing results. A total of 44 labs participated in the MSC by submitting results (16S or WGS) along with accompanying metadata; thirty 16S rRNA gene amplicon datasets and 14 WGS datasets were collected. The inclusion of two types of reference materials (human stool and mock communities) enabled analysis of both MGS measurement variability between different protocols using the biologically-relevant stool samples, and MGS bias with respect to ground truth values using the DNA mixtures. Owing to the compositional nature of MGS measurements, analyses were conducted on the ratio of Firmicutes: Bacteroidetes allowing us to directly apply common statistical methods. The resulting analysis demonstrated that protocol choices have significant effects, including both bias of the MGS measurement associated with a particular methodological choices, as well as effects on measurement robustness as observed through the spread of results between labs making similar methodological choices. In the analysis of the DNA mock communities, MGS measurement bias was observed even when there was general consensus among the participating laboratories. This study was the result of a collaborative effort that included academic, commercial, and government labs. In addition to highlighting the impact of different methodological decisions on MGS result comparability, this work also provides insights for consideration in future microbiome measurement study design.

Absolute quantification of viable bacteria abundances in food by next-generation sequencing: Quantitative NGS of viable microbes.

Next-generation sequencing (NGS) is an important tool for taxonomical bacteria identification. Recent technological developments have led to its improvement and availability. Despite the undeniable advantages of this approach, it has several limitations and shortcomings. The usual outcome of microbiota sequencing is a relative abundance of bacterial taxa. The information about bacteria viability or enumeration is missing. However, this knowledge is crucial for many applications. In the current study, we elaborated the complete workflow for the absolute quantification of living bacteria based on 16S rRNA gene amplicon sequencing. A fluorescent PMAxx reagent penetrating a damaged cell membrane was used to discriminate between the total and viable bacterial population. Bacteria enumeration was estimated by the spike-in technique or qPCR quantification. For method optimization, twenty bacterial species were taken, and the results of the workflow were validated by widely accepted methodologies: flow cytometry, microbiological plating, and viability-qPCR. Despite the minor discrepancy between all methods used, they all showed compatible results. Finally, we tested the workflow with actual food samples and received a good correlation between the methods regarding the estimation of the number of viable bacteria. Overall, the elaborated and integrated NGS approach could be the next step in perceiving a holistic picture of a sample microbiota.

Evaluation of Microbial Dynamics of Kombucha Consortia upon Continuous Backslopping in Coffee and Orange Juice.

The kombucha market is diverse, and competitors constantly test new components and flavours to satisfy customers' expectations. Replacing the original brewing base, adding flavours, or using "backslopping" influence the composition of the symbiotic starter culture of bacteria and yeast (SCOBY). Yet, deep characterisation of microbial and chemical changes in kombucha consortia in coffee and orange juice during backslopping has not been implemented. This study aimed to develop new kombucha beverages in less-conventional matrices and characterise their microbiota. We studied the chemical properties and microbial growth dynamics of lactic-acid-bacteria-tailored (LAB-tailored) kombucha culture by 16S rRNA next-generation sequencing in coffee and orange juice during a backslopping process that spanned five cycles, each lasting two to four days. The backslopping changed the culture composition and accelerated the fermentation. This study gives an overview of the pros and cons of backslopping technology for the production of kombucha-based beverages. Based on research conducted using two different media, this work provides valuable information regarding the aspects to consider when using the backslopping method to produce novel kombucha drinks, as well as identifying the main drawbacks that need to be addressed.

Characterisation of chemical, microbial and sensory profiles of commercial kombuchas.

The kombucha market is a fast-growing segment in the functional beverage category. The selection of kombuchas on the market varies between the traditional and flavoured kombuchas. Our research aimed to characterise the chemical, microbial, and sensory profiles of the commercial kombuchas. We analysed 16 kombuchas from 6 producers. The dominant metabolites were acetate, lactate, and ethanol, the last of which might put some kombuchas into the alcoholic beverage section in some countries. The metagenomic analyses demonstrated that LAB dominates in green tea, and AAB in black tea kombuchas. The main bacterial species were Komagataeibacter rhaeticus and Lactobacillus ssp, and yeast species Dekkera anomala and Dekkera bruxellensis. The sweet and sour balance correlated with acid concentrations. The free sorting task showed that commercial kombuchas clustered into three main categories "fruity and artificial flavour", herbal and tea notes", and "classical notes". Our research results showed the necessity of the definition of kombucha.

Determination of Technological Parameters and Characterization of Microbiota of the Spontaneous Sourdough Fermentation of Hull-Less Barley.

The development of microorganisms of sourdough and biodiversity of microbiota can be influenced by changing the parameters of the technological process such as the ratio of flour and added water, the fermentation temperature and time. The Box-Behnken design methodology was used to determine the optimal parameters for the three-phase spontaneous backslopping fermentation process of hull-less barley sourdough, as well as to characterize the microbiological diversity. The optimized parameters of backslopping fermentation are flour and water ratio 1:1.13, temperature 30 °C, time 24 h in the 1st backslopping; the inoculate, flour and water ratio 1:1:1.3, temperature 31 °C, time 14 h in the 2nd backslopping, and the inoculate, flour and water ratio 1:1:1.5, and temperature 28.5 °C, time 12 h in the 3rd step of backslopping. In the controlled spontaneous fermentation environment in three backslopping steps, the microbiological research of hull-less barley sourdough has confirmed the dominance of species Pediococcus pentosaceus in the 3rd backslopping step of spontaneous fermentation. The developed spontaneous hull-less barley sourdough is consistent with the number of lactic bacteria and yeasts in line with that seen by the active sourdough.

Biomechanical Features of Graphene-Augmented Inorganic Nanofibrous Scaffolds and Their Physical Interaction with Viruses.

Nanofibrous substrates and scaffolds are widely being studied as matrices for 3D cell cultures, and disease models as well as for analytics and diagnostic purposes. These scaffolds usually comprise randomly oriented fibers. Much less common are nanofibrous scaffolds made of stiff inorganic materials such as alumina. Well-aligned matrices are a promising tool for evaluation of behavior of biological objects affected by micro/nano-topologies as well as anisotropy. In this work, for the first time, we report a joint analysis of biomechanical properties of new ultra-anisotropic, self-aligned ceramic nanofibers augmented with two modifications of graphene shells (GAIN scaffolds) and their interaction of three different viral types (influenza virus A, picornavirus (human parechovirus) and potato virus). It was discovered that nano-topology and structure of the graphene layers have a significant implication on mechanical properties of GAIN scaffolds resulting in non-linear behavior. It was demonstrated that the viral adhesion to GAIN scaffolds is likely to be guided by physical cues in dependence on mutual steric factors, as the scaffolds lack common cell membrane proteins and receptors which viruses usually deploy for transfection. The study may have implications for selective viral adsorption, infected cells analysis, and potentially opening new tools for anti-viral drugs development.

Selective laser sintered bio-inspired silicon-wollastonite scaffolds for bone tissue engineering.

The scaffolds, which morphologically and physiologically mimic natural features of the bone, are of high demand for regenerative medicine. To address this challenge, we have developed innovative bioactive porous silicon- wollastonite substrates for bone tissue engineering. Additive manufacturing through selective laser melting approach has been exploited to fabricate scaffolds of different architecture. Unique material combining osteoinductivity, osteoconductivity and bioactive elements allows flexibility in design. As the porous structure is required for the ingrowth of the bone tissue, the CAD designed scaffolds with pore size of 400 µm and hierarchical gradient of pore size from 50 µm to 350 µm have been 3D printed and tested in vitro. The scaffolds have demonstrated not only the enhanced viability and differential patterning of human mesenchymal cells (hMSC) guided by the biomimetic design onto extra and intra scaffold space but also promoted the osteogenic differentiation in vitro. Both homogeneous and gradient scaffolds have shown the differential expression of primary transcription factors (RUNX2, OSX), anti-inflammatory factors and cytokines, which are important for the regulation of ossification. The effective elastic modulus and compressive strength of scaffolds have been calculated as 1.1 ± 0.9 GPa and 37 ± 13.5 MPa with progressive failure for homogeneous structured scaffold; and 1.8 ± 0.9 GPa and 71 ± 9.5 MPa for gradient-structured scaffold with saw-tooth fracture mode and sudden incognito failure zones. The finite element analysis reveals more bulk stress onto the gradient scaffolds when compared to the homogeneous counterpart. The findings demonstrate that as-produced composite ceramic scaffolds can pave the way for treating specific orthopaedic defects by tailoring the design through additive manufacturing.

N-terminally truncated BAF57 isoforms contribute to the diversity of SWI/SNF complexes in neurons.

The SWItch/Sucrose NonFermentable, a nucleosome remodeling complex (SWI/SNF) chromatin-remodelling complexes act upon the nucleosomal structure and regulate transcription, replication, repair of chromatin and splicing. In this study, we present evidence that human, mouse and rat genes encoding one of the SWI/SNF complex subunits, BAF57, undergo neuron-specific splicing of exons II, III and IV. Alternative splicing yields in at least three isoforms of BAF57 protein that have truncated N-termini (N-BAF57s). The transcripts encoding N-BAF57 isoforms are predominantly expressed in the nervous system. The biochemical fractionation data supported by the results of the co-immunoprecipitation analysis show that N-BAF57 isoforms associate into protein complexes together with Brg1, Brm, BAF155 and BAF170. Transient over-expression of N-BAF57 isoforms in non-neural cells affects the level of expression of certain neuron-restrictive silencer element-containing genes. Together these data suggest that neuronal isoforms of BAF57 contribute to functional SWI/SNF complexes regulating neurogenesis.

Oxidative switches in functioning of mammalian copper chaperone Cox17.

Cox17, a copper chaperone for cytochrome-c oxidase, is an essential and highly conserved protein in eukaryotic organisms. Yeast and mammalian Cox17 share six conserved cysteine residues, which are involved in complex redox reactions as well as in metal binding and transfer. Mammalian Cox17 exists in three oxidative states, each characterized by distinct metal-binding properties: fully reduced mammalian Cox17(0S-S) binds co-operatively to four Cu+; Cox17(2S-S), with two disulfide bridges, binds to one of either Cu+ or Zn2+; and Cox17(3S-S), with three disulfide bridges, does not bind to any metal ions. The E(m) (midpoint redox potential) values for two redox couples of Cox17, Cox17(3S-S)<-->Cox17(2S-S) (E(m1)) and Cox17(2S-S)<-->Cox17(0S-S) (E(m2)), were determined to be -197 mV and -340 mV respectively. The data indicate that an equilibrium exists in the cytosol between Cox17(0S-S) and Cox17(2S-S), which is slightly shifted towards Cox17(0S-S). In the IMS (mitochondrial intermembrane space), the equilibrium is shifted towards Cox17(2S-S), enabling retention of Cox17(2S-S) in the IMS and leading to the formation of a biologically competent form of the Cox17 protein, Cox17(2S-S), capable of copper transfer to the copper chaperone Sco1. XAS (X-ray absorption spectroscopy) determined that Cu4Cox17 contains a Cu4S6-type copper-thiolate cluster, which may provide safe storage of an excess of copper ions.

Graphene-Augmented Nanofiber Scaffolds Trigger Gene Expression Switching of Four Cancer Cell Types.

Three-dimensional (3D) customized scaffolds are anticipated to provide new frontiers in cell manipulation and advanced therapy methods. Here, we demonstrate the application of hybrid 3D porous scaffolds, representing networks of highly aligned self-assembled ceramic nanofibers, for culturing four types of cancer cells. Ultrahigh aspect ratio (∼107) of graphene augmented fibers of tailored nanotopology is shown as an alternative tool to substantially affect cancerous gene expression, eventually due to differences in local biomechanical features of the cell-matrix interactions. Here, we report a clear selective up- and down-regulation of groups of markers for breast cancer (MDA-MB231), colorectal cancer (CaCO2), melanoma (WM239A), and neuroblastoma (Kelly) depending on only fiber orientation and morphology without application of any other stimulus. Changes in gene expression are also revealed for Mitomycin C treatment of MDA-MB231, making the scaffold a suitable platform for testing of anticancer agents. This allows an opportunity for selective "clean" guidance to a deep understanding of mechanisms of cancer cells progressive growth and tumor formation without possible side effects by manipulation with the specific markers.

Hybrid graphene-ceramic nanofibre network for spontaneous neural differentiation of stem cells.

A challenge in regenerative medicine is governed by the need to have control over the fate of stem cells that is regulated by the physical and chemical microenvironment in vitro and in vivo. The differentiation of the stem cells into specific lineages is commonly guided by use of specific culture media. For the first time, we demonstrate that human mesenchymal stem cells are capable of turning spontaneously towards neurogenic lineage when seeded on graphene-augmented, highly anisotropic ceramic nanofibres without special differentiation media, contrary to commonly thought requirement of 'soft' substrates for the same purpose. Furthermore, pro-inflammatory gene expression is simultaneously suppressed, and expression of factors promoting focal adhesion and monocytes taxis is upregulated. This opens new possibilities of using local topo-mechanical cues of the 'graphenized' scaffold surfaces to guide stem cell proliferation and differentiation, which can be used in studies of neurological diseases and cell therapy.

Targeted alternative splicing of TAF4: a new strategy for cell reprogramming.

Reprogramming of somatic cells has become a versatile tool for biomedical research and for regenerative medicine. In the current study, we show that manipulating alternative splicing (AS) is a highly potent strategy to produce cells for therapeutic applications. We demonstrate that silencing of hTAF4-TAFH activity of TAF4 converts human facial dermal fibroblasts to melanocyte-like (iMel) cells. iMel cells produce melanin and express microphthalmia-associated transcription factor (MITF) and its target genes at levels comparable to normal melanocytes. Reprogramming of melanoma cells by manipulation with hTAF4-TAFH activity upon TAFH RNAi enforces cell differentiation towards chondrogenic pathway, whereas ectoptic expression of TAF4 results in enhanced multipotency and neural crest-like features in melanoma cells. In both cell states, iMels and cancer cells, hTAF4-TAFH activity controls migration by supporting E- to N-cadherin switches. From our data, we conclude that targeted splicing of hTAF4-TAFH coordinates AS of other TFIID subunits, underscoring the role of TAF4 in synchronised changes of Pol II complex composition essential for efficient cellular reprogramming. Taken together, targeted AS of TAF4 provides a unique strategy for generation of iMels and recapitulating stages of melanoma progression.

Graphene-augmented nanofiber scaffolds demonstrate new features in cells behaviour.

Three-dimensional (3D) customized scaffolds capable to mimic a native extracellular matrix open new frontiers in cells manipulation and advanced therapy. The major challenge is in a proper substrate for in vitro models on engineered scaffolds, capable to modulate cells differentiation. Here for the first time we demonstrate novel design and functionality of the 3D porous scaffolds of aligned, self-assembled ceramic nanofibers of ultra-high anisotropy ratio (~10(7)), augmented into graphene shells. This unique hybrid nano-network allows an exceptional combination of selective guidance stimuli of stem cells differentiation, immune reactions variations, and local immobilization of cancer cells, which was not available before. The scaffolds were shown to be able to direct human mesenchymal stem cells (important for stimulation of neuronal and muscle cells) preferential orientation, to suppress major inflammatory factors, and to localize cancer cells; all without additions of specific culture media. The selective downregulation of specific cytokines is anticipated as a new tool for understanding of human immune system and ways of treatment of associated diseases. The effects observed are self-regulated by cells only, without side effects, usually arising from use of external factors. New scaffolds may open new horizons for stem cells fate control such as towards axons and neurites regeneration (Alzheimer's disease) as well as cancer therapy development.

TAF4 controls differentiation of human neural progenitor cells through hTAF4-TAFH activity.

Expression of general transcription factor and co-activator TAF4 varies during development and in the processes of cell differentiation with suggested connection to neurodegenerative diseases. Here, we show that expression of TAF4 alternative splice variants is different in various regions of the human brain, substantiating the role of alternative splicing of TAF4 in the regulation of neural development and brain function. Most of the described splicing events affect the TAFH homology domain of TAF4 (hTAF4-TAFH). Besides, differentiated towards neural lineages, normal human neural progenitors (NHNPs) lose canonical full-length TAF4 isoform. To study the effects of hTAF4-TAFH splicing on neuronal differentiation, we used RNAi approach to target hTAF4-TAFH-encoding domain in NHNPs. Results show that inactivation of hTAF4-TAFH domain accelerates differentiation of human neural progenitor cells. Conversely, enhanced expression of TAF4 suppresses differentiation and keeps neural progenitor cells in a stem cell-like state. Finally, we provide data on the involvement of TP53 and noncanonical WNT signaling pathways in mediating effects of TAF4 on neuronal differentiation. Overall, our data suggest that specific isoforms of TAF4 may selectively and efficiently control neurogenesis.

Alternative splicing targeting the hTAF4-TAFH domain of TAF4 represses proliferation and accelerates chondrogenic differentiation of human mesenchymal stem cells.

Transcription factor IID (TFIID) activity can be regulated by cellular signals to specifically alter transcription of particular subsets of genes. Alternative splicing of TFIID subunits is often the result of external stimulation of upstream signaling pathways. We studied tissue distribution and cellular expression of different splice variants of TFIID subunit TAF4 mRNA and biochemical properties of its isoforms in human mesenchymal stem cells (hMSCs) to reveal the role of different isoforms of TAF4 in the regulation of proliferation and differentiation. Expression of TAF4 transcripts with exons VI or VII deleted, which results in a structurally modified hTAF4-TAFH domain, increases during early differentiation of hMSCs into osteoblasts, adipocytes and chondrocytes. Functional analysis data reveals that TAF4 isoforms with the deleted hTAF4-TAFH domain repress proliferation of hMSCs and preferentially promote chondrogenic differentiation at the expense of other developmental pathways. This study also provides initial data showing possible cross-talks between TAF4 and TP53 activity and switching between canonical and non-canonical WNT signaling in the processes of proliferation and differentiation of hMSCs. We propose that TAF4 isoforms generated by the alternative splicing participate in the conversion of the cellular transcriptional programs from the maintenance of stem cell state to differentiation, particularly differentiation along the chondrogenic pathway.

Cox17, a copper chaperone for cytochrome c oxidase: expression, purification, and formation of mixed disulphide adducts with thiol reagents.

Copper chaperone for cytochrome c oxidase (Cox17) is a 7 kDa copper-binding protein, which facilitates incorporation of copper ions into Cu(A) site of cytochrome c oxidase. Cox17 contains six conserved Cys residues and occurs in three different oxidative states, which display different metal-binding properties and stability. In the present study, we have elaborated technologies for production of partially oxidized human recombinant Cox17 in a bacterial expression system and purification of fully oxidized Cox17. For this purpose we used Escherichia coli Origami strain, which is deficient in thioredoxin and thioredoxin reductase systems and allows formation of disulfide bonds in cytoplasmic proteins. Fully oxidized Cox17 was purified by a simplified two-step procedure including gel filtration and cation exchange chromatography. By using mass spectrometry we demonstrated that application of 2-mercaptoethanol (2-ME) during purification leads to formation of its mixed disulfide adducts with Cox17. Moreover, partially reduced Cox17 can form mixed disulfide adducts also with the cellular reducing agent glutathione, which abolishes copper-binding ability of partially reduced Cox17.