Publisher Correction: Heterochromatin drives compartmentalization of inverted and conventional nuclei.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Heterochromatin drives compartmentalization of inverted and conventional nuclei.

The nucleus of mammalian cells displays a distinct spatial segregation of active euchromatic and inactive heterochromatic regions of the genome1,2. In conventional nuclei, microscopy shows that euchromatin is localized in the nuclear interior and heterochromatin at the nuclear periphery1,2. Genome-wide chromosome conformation capture (Hi-C) analyses show this segregation as a plaid pattern of contact enrichment within euchromatin and heterochromatin compartments3, and depletion between them. Many mechanisms for the formation of compartments have been proposed, such as attraction of heterochromatin to the nuclear lamina2,4, preferential attraction of similar chromatin to each other1,4-12, higher levels of chromatin mobility in active chromatin13-15 and transcription-related clustering of euchromatin16,17. However, these hypotheses have remained inconclusive, owing to the difficulty of disentangling intra-chromatin and chromatin-lamina interactions in conventional nuclei18. The marked reorganization of interphase chromosomes in the inverted nuclei of rods in nocturnal mammals19,20 provides an opportunity to elucidate the mechanisms that underlie spatial compartmentalization. Here we combine Hi-C analysis of inverted rod nuclei with microscopy and polymer simulations. We find that attractions between heterochromatic regions are crucial for establishing both compartmentalization and the concentric shells of pericentromeric heterochromatin, facultative heterochromatin and euchromatin in the inverted nucleus. When interactions between heterochromatin and the lamina are added, the same model recreates the conventional nuclear organization. In addition, our models allow us to rule out mechanisms of compartmentalization that involve strong euchromatin interactions. Together, our experiments and modelling suggest that attractions between heterochromatic regions are essential for the phase separation of the active and inactive genome in inverted and conventional nuclei, whereas interactions of the chromatin with the lamina are necessary to build the conventional architecture from these segregated phases.

Histological Changes in Category 2 Animal Byproducts under the Influence of a Ferment Preparation.

The paper deals with the possibility of processing category 2 animal byproducts with a ferment preparation. We chose collagen-rich cattle lips and ears as the category 2 byproducts for our study. The selected samples were processed in the following sequence: fixation, rinsing with running water, densifying the samples, slicing into sections, dying sections, and enclosing the sections under cover glass. The pathohistomorphologic changes found in the control samples significantly differ both before the processing and after the use of the ferment preparation Protepsin, which caused destructive metabolic and hydrolytic processes in the dense connective tissue of the ear and lip framework and led to softening of the muscle parenchyma of the organs.

Human Gut Microbiome Response to Short-Term Bifidobacterium-Based Probiotic Treatment.

Gut microbiota is believed to play a crucial role in modulating obesity in humans, and probiotics affecting gut microbiota can alleviate some of the obesity-related health complications. The study was aimed to investigate changes in the composition of the gut microbiome in obese humans due to short-term (2 weeks) treatment of obese patients with a probiotic preparation containing Bifidobacterium longum. Faecal microbiome diversity was studied using the 16S amplicon sequencing by Illumina MiSeq. Bioinformatic analysis showed distribution across 14 phyla (with Firmicutes and Bacteroidetes dominating), 21 class, 125 genera and 973 OTUs. The probiotic treatment decreased relative abundance of Bacteroidetes (Prevotellaceae and Bacteroidaceae), while increasing that of Actinobacteria (Bifidobacteriaceae and Coriobacteriaceae), and Firmicutes (Negativicutes: Veillonellaceae and Clostridia: Peptostreptococcaceae). The probiotic treatment decreased total blood sugar and increased patients' assessment of their physical and mental health. Thus even the short-term Bifidobacterium-based probiotic treatment brought significant compositional changes in the 16S rRNA gene diversity in faecal bacterial assemblages by increasing beneficial and decreasing pathogenic or opportunistic bacteria; the related shifts in life quality assessment necessitate further research into the causal relationships involved.

Primary progressive multiple sclerosis in a Russian cohort: relationship with gut bacterial diversity.

BACKGROUND: Gut microbiota has been increasingly acknowledged to shape significantly human health, contributing to various autoimmune diseases, both intestinal and non-intestinal, including multiple sclerosis (MS). Gut microbiota studies in patients with relapsing remitting MS strongly suggested its possible role in immunoregulation; however, the profile and potential of gut microbiota involvement in patients with primary progressive MS (PPMS) patients has received much less attention due to the rarity of this disease form. We compared the composition and structure of faecal bacterial assemblage using Illumina MiSeq sequencing of V3-V4 hypervariable region of 16S rRNA genes amplicons in patients with primary progressive MS and in the healthy controls. RESULTS: Over all samples 12 bacterial phyla were identified, containing 21 classes, 25 orders, 54 families, 174 genera and 1256 operational taxonomic units (OTUs). The Firmicutes phylum was found to be ultimately dominating both in OTUs richness (68% of the total bacterial OTU number) and in abundance (71% of the total number of sequence reads), followed by Bacteroidetes (12 and 16%, resp.) and Actinobacteria (7 and 6%, resp.). Summarily in all samples the number of dominant OTUs, i.e. OTUs with ≥1% relative abundance, was 13, representing much less taxonomic richness (three phyla, three classes, four orders, six families and twelve genera) as compared to the total list of identified OTUs and accounting for 30% of the sequence reads number in the healthy cohort and for 23% in the PPMS cohort. Human faecal bacterial diversity profiles were found to differ between PPMS and healthy cohorts at different taxonomic levels in minor or rare taxa. Marked PPMS-associated increase was found in the relative abundance of two dominant OTUs (Gemmiger sp. and an unclassified Ruminococcaceae). The MS-related differences were also found at the level of minor and rare OTUs (101 OTUs). These changes in OTUs' abundance translated into increased bacterial assemblage diversity in patients. CONCLUSION: The findings are important for constructing a more detailed global picture of the primary progressive MS-associated gut microbiota, contributing to better understanding of the disease pathogenesis.

Crystal structure of the DNA binding domain of the transcription factor T-bet suggests simultaneous recognition of distant genome sites.

The transcription factor T-bet (Tbox protein expressed in T cells) is one of the master regulators of both the innate and adaptive immune responses. It plays a central role in T-cell lineage commitment, where it controls the TH1 response, and in gene regulation in plasma B-cells and dendritic cells. T-bet is a member of the Tbox family of transcription factors; however, T-bet coordinately regulates the expression of many more genes than other Tbox proteins. A central unresolved question is how T-bet is able to simultaneously recognize distant Tbox binding sites, which may be located thousands of base pairs away. We have determined the crystal structure of the Tbox DNA binding domain (DBD) of T-bet in complex with a palindromic DNA. The structure shows a quaternary structure in which the T-bet dimer has its DNA binding regions splayed far apart, making it impossible for a single dimer to bind both sites of the DNA palindrome. In contrast to most other Tbox proteins, a single T-bet DBD dimer binds simultaneously to identical half-sites on two independent DNA. A fluorescence-based assay confirms that T-bet dimers are able to bring two independent DNA molecules into close juxtaposition. Furthermore, chromosome conformation capture assays confirm that T-bet functions in the direct formation of chromatin loops in vitro and in vivo. The data are consistent with a looping/synapsing model for transcriptional regulation by T-bet in which a single dimer of the transcription factor can recognize and coalesce distinct genetic elements, either a promoter plus a distant regulatory element, or promoters on two different genes.

Iterative correction of Hi-C data reveals hallmarks of chromosome organization.

Extracting biologically meaningful information from chromosomal interactions obtained with genome-wide chromosome conformation capture (3C) analyses requires the elimination of systematic biases. We present a computational pipeline that integrates a strategy to map sequencing reads with a data-driven method for iterative correction of biases, yielding genome-wide maps of relative contact probabilities. We validate this ICE (iterative correction and eigenvector decomposition) technique on published data obtained by the high-throughput 3C method Hi-C, and we demonstrate that eigenvector decomposition of the obtained maps provides insights into local chromatin states, global patterns of chromosomal interactions, and the conserved organization of human and mouse chromosomes.

Analysis of long-range chromatin interactions using Chromosome Conformation Capture.

Chromosome Conformation Capture, or 3C, is a pioneering method for investigating the three-dimensional structure of chromatin. 3C is used to analyze long-range looping interactions between any pair of selected genomic loci. Most 3C studies focus on defined genomic regions of interest that can be up to several hundred Kb in size. The method has become widely adopted and has been modified to increase throughput to allow unbiased genome-wide analysis. These large-scale adaptations are presented in other articles in this issue of Methods. Here we describe the 3C procedure in detail, including the appropriate use of the technology, the experimental set-up, an optimized protocol and troubleshooting guide, and considerations for data analysis. The protocol described here contains previously unpublished improvements, which save time and reduce labor. We pay special attention to primer design, appropriate controls and data analysis. We include notes and discussion based on our extensive experience to help researchers understand the principles of 3C-based techniques and to avoid common pitfalls and mistakes. This paper represents a complete resource and detailed guide for anyone who desires to perform 3C.

From cells to chromatin: capturing snapshots of genome organization with 5C technology.

In eukaryotes, genome organization can be observed on many levels and at different scales. This organization is important not only to reduce chromosome length but also for the proper execution of various biological processes. High-resolution mapping of spatial chromatin structure was made possible by the development of the chromosome conformation capture (3C) technique. 3C uses chemical cross-linking followed by proximity-based ligation of fragmented DNA to capture frequently interacting chromatin segments in cell populations. Several 3C-related methods capable of higher chromosome conformation mapping throughput were reported afterwards. These techniques include the 3C-carbon copy (5C) approach, which offers the advantage of being highly quantitative and reproducible. We provide here an updated reference protocol for the production of 5C libraries analyzed by next-generation sequencing or onto microarrays. A procedure used to verify that 3C library templates bear the high quality required to produce superior 5C libraries is also described. We believe that this detailed protocol will help guide researchers in probing spatial genome organization and its role in various biological processes.

Hi-C: a comprehensive technique to capture the conformation of genomes.

We describe a method, Hi-C, to comprehensively detect chromatin interactions in the mammalian nucleus. This method is based on Chromosome Conformation Capture, in which chromatin is crosslinked with formaldehyde, then digested, and re-ligated in such a way that only DNA fragments that are covalently linked together form ligation products. The ligation products contain the information of not only where they originated from in the genomic sequence but also where they reside, physically, in the 3D organization of the genome. In Hi-C, a biotin-labeled nucleotide is incorporated at the ligation junction, enabling selective purification of chimeric DNA ligation junctions followed by deep sequencing. The compatibility of Hi-C with next generation sequencing platforms makes it possible to detect chromatin interactions on an unprecedented scale. This advance gives Hi-C the power to both explore the biophysical properties of chromatin as well as the implications of chromatin structure for the biological functions of the nucleus. A massively parallel survey of chromatin interaction provides the previously missing dimension of spatial context to other genomic studies. This spatial context will provide a new perspective to studies of chromatin and its role in genome regulation in normal conditions and in disease.

West-Siberian Chernozem: How Vegetation and Tillage Shape Its Bacteriobiome.

Managing soil biodiversity using reduced tillage is a popular approach, yet soil bacteriobiomes in the agroecosystems of Siberia has been scarcely studied, especially as they are related to tillage. We studied bacteriobiomes in Chernozem under natural steppe vegetation and cropped for wheat using conventional or no tillage in a long-term field trial in the Novosibirsk region, Russia, by using the sequence diversity of the V3/V4 region of 16S rRNA genes. Actinobacteria, Acidobacteria, and Proteobacteria summarily accounted for 80% of the total number of sequences, with Actinobacteria alone averaging 51%. The vegetation (natural vs. crop) and tillage (ploughed vs. no-till) affected the bacterial relative abundance at all taxonomic levels and many taxa, e.g., hundreds of OTUs. However, such changes did not translate into α-biodiversity changes, i.e., observed and potential OTUs' richness, Shannon, and Simpson, excepting the slightly higher evenness and equitability in the top 0-5 cm of the undisturbed soil. As for the ß-biodiversity, substituting conventional ploughing with no tillage and maintaining the latter for 12 years notably shifted the soil bacteriobiome closer to the one in the undisturbed soil. This study, presenting the first inventory of soil bacteriobiomes under different tillage in the south of West Siberia, underscores the need to investigate the seasonality and longevity aspects of tillage, especially as they are related to crop production.

Association of autoantibodies targeting endothelin type-A receptors with no-reflow in ST-elevation myocardial infarction.

BACKGROUND AND AIMS: No-reflow (NR), where the coronary artery is patent after treatment of ST-elevation myocardial infarction (STEMI) but tissue perfusion is not restored, is associated with worse outcomes. We aimed to investigate the relationship between autoantibodies activating endothelin-1 receptor type A (ETAR-AAs) and NR after primary percutaneous coronary intervention (PPCI) in STEMI. METHODS: We studied 50 patients (age 59 ± 11 years, 40 males) with STEMI who underwent PPCI within 6 h after the onset of symptoms. Blood samples were obtained from all patients within 12 h following PPCI for ETAR-AA level measurement. The seropositive threshold was provided by the manufacturer (>10 U/ml). NR was assessed by cardiac magnetic resonance imaging (MVO, microvascular obstruction). As a control group, 40 healthy subjects matched for age and sex were recruited from the general population. RESULTS: MVO was observed in 24 patients (48%). The prevalence of MVO was higher in patients with ETAR-AAs seropositivity (72% vs. 38%, p = 0.03). ETAR-AAs were higher in patients with MVO (8.9 U/mL (interquartile range [IQR] 6.8-16.2 U/mL) vs. 5.7 U/mL [IQR 4.3-7.7 U/mL], p = 0.003). ETAR-AAs seropositivity was independently associated with MVO (OR 3.2, 95% CI 1.3-7.1; p = 0.03). We identified ≥6.74 U/mL as the best cut-off for prediction of MVO (sensitivity 79%; specificity 65%; NPV 71%; PPV 74%; accuracy 72%). CONCLUSIONS: The ETAR-AAs seropositivity is associated with NR in STEMI patients. These findings may open up new options in the management of myocardial infarction even if confirmation in a larger trial is needed.

Integrating one-dimensional and three-dimensional maps of genomes.

Genomes exist in vivo as complex physical structures, and their functional output (i.e. the gene expression profile of a cell) is related to their spatial organization inside the nucleus as well as to local chromatin status. Chromatin modifications and chromosome conformation are distinct in different tissues and cell types, which corresponds closely with the diversity in gene-expression patterns found in different tissues of the body. The biological processes and mechanisms driving these general correlations are currently the topic of intense study. An emerging theme is that genome compartmentalization - both along the linear length of chromosomes, and in three dimensions by the spatial colocalization of chromatin domains and genomic loci from across the genome - is a crucial parameter in regulating genome expression. In this Commentary, we propose that a full understanding of genome regulation requires integrating three different types of data: first, one-dimensional data regarding the state of local chromatin - such as patterns of protein binding along chromosomes; second, three-dimensional data that describe the population-averaged folding of chromatin inside cells and; third, single-cell observations of three-dimensional spatial colocalization of genetic loci and trans factors that reveal information about their dynamics and frequency of colocalization.

Soil Mycobiome Diversity under Different Tillage Practices in the South of West Siberia.

Managing soil biodiversity by reduced or no tillage is an increasingly popular approach. Soil mycobiome in Siberian agroecosystems has been scarcely studied; little is known about its changes due to tillage. We studied mycobiome in Chernozem under natural steppe vegetation and cropped for wheat by conventional or no tillage in a long-term field trial in West Siberia, Russia, by using ITS2 rDNA gene marker (Illumina MiSeq sequencing). Half of the identified OTUs were Ascomycota with 82% of the total number of sequence reads and showing, like other phyla (Basidiomycota, Zygomycota, Mortierellomycota, Chytridiomycota, Glomeromycota), field-related differential abundance. Several dominant genera (Mortierella, Chaetomium, Clonostachys, Gibberella, Fusarium, and Hypocrea) had increased abundance in both cropped soils as compared with the undisturbed one and therefore can be safely assumed to be associated with wheat residues. Fungal OTUs' richness in cropped soils was less than in the undisturbed one; however, no tillage shifted soil mycobiome composition closer to the latter, albeit, it was still similar to the ploughed soil, despite different organic matter and wheat residue content. The study provided the first inventory of soil mycobiome under different tillage treatments in the south of West Siberia, where wheat production is an important section of the regional economy.

The Core of Gut Life: Firmicutes Profile in Patients with Relapsing-Remitting Multiple Sclerosis.

The multiple sclerosis (MS) incidence rate has been increasing in Russia, but the information about the gut bacteriobiome in the MS-afflicted patients is scarce. Using the Illumina MiSeq sequencing of 16S rRNA gene amplicons, we aimed to analyze the Firmicutes phylum and its taxa in a cohort of Moscow patients with relapsing-remitting MS, assessing the effects of age, BMI, disease modifying therapy (DMT), disability (EDSS), and gender. Among 1252 identified bacterial OTUs, 857 represented Firmicutes. The phylum was the most abundant also in sequence reads, overall averaging 74 ± 13%. The general linear model (GLM) analysis implicated Firmicutes/Clostridia/Clostridiales/Lachospiraceae/Blautia/Blautia wexlerae as increasing with BMI, and only Lachospiraceae/Blautia/Blautia wexlerae as increasing with age. A marked DMT-related decrease in Firmicutes was observed in females at the phylum, class (Clostridia), and order (Clostridiales) levels. The results of our study implicate DMT and gender as factors shaping the fecal Firmicutes assemblages. Together with the gender-dependent differential MS incidence growth rate in the country, the results suggest the likely involvement of gender-specific pathoecological mechanisms underlying the occurrence of the disease, switching between its phenotypes and response to disease-modifying therapies. Overall, the presented profile of Firmicutes can be used as a reference for more detailed research aimed at elucidating the contribution of this core phylum and its lower taxa into the etiology and progression of relapsing-remitting multiple sclerosis.

Gut Microbiome in a Russian Cohort of Pre- and Post-Cholecystectomy Female Patients.

The last decade saw extensive studies of the human gut microbiome and its relationship to specific diseases, including gallstone disease (GSD). The information about the gut microbiome in GSD-afflicted Russian patients is scarce, despite the increasing GSD incidence worldwide. Although the gut microbiota was described in some GSD cohorts, little is known regarding the gut microbiome before and after cholecystectomy (CCE). By using Illumina MiSeq sequencing of 16S rRNA gene amplicons, we inventoried the fecal bacteriobiome composition and structure in GSD-afflicted females, seeking to reveal associations with age, BMI and some blood biochemistry. Overall, 11 bacterial phyla were identified, containing 916 operational taxonomic units (OTUs). The fecal bacteriobiome was dominated by Firmicutes (66% relative abundance), followed by Bacteroidetes (19%), Actinobacteria (8%) and Proteobacteria (4%) phyla. Most (97%) of the OTUs were minor or rare species with ≤1% relative abundance. Prevotella and Enterocossus were linked to blood bilirubin. Some taxa had differential pre- and post-CCE abundance, despite the very short time (1-3 days) elapsed after CCE. The detailed description of the bacteriobiome in pre-CCE female patients suggests bacterial foci for further research to elucidate the gut microbiota and GSD relationship and has potentially important biological and medical implications regarding gut bacteria involvement in the increased GSD incidence rate in females.

The mitochondrial permeability transition pore: an evolving concept critical for cell life and death.

In this review, we summarize current knowledge of perhaps one of the most intriguing phenomena in cell biology: the mitochondrial permeability transition pore (mPTP). This phenomenon, which was initially observed as a sudden loss of inner mitochondrial membrane impermeability caused by excessive calcium, has been studied for almost 50 years, and still no definitive answer has been provided regarding its mechanisms. From its initial consideration as an in vitro artifact to the current notion that the mPTP is a phenomenon with physiological and pathological implications, a long road has been travelled. We here summarize the role of mitochondria in cytosolic calcium control and the evolving concepts regarding the mitochondrial permeability transition (mPT) and the mPTP. We show how the evolving mPTP models and mechanisms, which involve many proposed mitochondrial protein components, have arisen from methodological advances and more complex biological models. We describe how scientific progress and methodological advances have allowed milestone discoveries on mPTP regulation and composition and its recognition as a valid target for drug development and a critical component of mitochondrial biology.

Pair creation in QED-strong pulsed laser fields interacting with electron beams.

QED effects are known to occur in a strong laser pulse interaction with a counterpropagating electron beam, among these effects being electron-positron pair creation. We discuss the range of laser pulse intensities of J≥5×10(22) W/cm2 combined with electron beam energies of tens of GeV. In this regime multiple pairs may be generated from a single beam electron, some of the newborn particles being capable of further pair production. Radiation backreaction prevents avalanche development and limits pair creation. The system of integro-differential kinetic equations for electrons, positrons and γ photons is derived and solved numerically.

Regulation of Cell Death by Mitochondrial Transport Systems of Calcium and Bcl-2 Proteins.

Mitochondria represent the fundamental system for cellular energy metabolism, by not only supplying energy in the form of ATP, but also by affecting physiology and cell death via the regulation of calcium homeostasis and the activity of Bcl-2 proteins. A lot of research has recently been devoted to understanding the interplay between Bcl-2 proteins, the regulation of these interactions within the cell, and how these interactions lead to the changes in calcium homeostasis. However, the role of Bcl-2 proteins in the mediation of mitochondrial calcium homeostasis, and therefore the induction of cell death pathways, remain underestimated and are still not well understood. In this review, we first summarize our knowledge about calcium transport systems in mitochondria, which, when miss-regulated, can induce necrosis. We continue by reviewing and analyzing the functions of Bcl-2 proteins in apoptosis. Finally, we link these two regulatory mechanisms together, exploring the interactions between the mitochondrial Ca2+ transport systems and Bcl-2 proteins, both capable of inducing cell death, with the potential to determine the cell death pathway-either the apoptotic or the necrotic one.

Effect of soil sample preservation, compared to the effect of other environmental variables, on bacterial and eukaryotic diversity.

Archived soil samples are a valuable source for retrospective ecological studies, and their recent analysis using molecular ecological approaches has drawn significant attention within the scientific community. However, the possibility of addressing ecological questions regarding detectable microbiota in dried and extensively stored soils has not yet been fully evaluated. To achieve this, soil samples collected from two long-term grassland experiments in the United Kingdom and The Netherlands were subjected to air-drying at 40-42 degrees C and stored at room temperature. Total bacterial, Bacillus benzoevorans-related and eukaryotic communities associated with these samples were analyzed by DGGE-fingerprinting of PCR-amplified ribosomal RNA gene fragments. Changes in microbial community structure due to drying and storage were evaluated by multivariate analysis in relation to changes caused by other environmental conditions, such as soil pH, type of fertilizer and vegetation. Soil drying and storage affected the detectable community structure, but did not materially impair our capacity to identify the effect of soil parameters studied in long-term grassland experiments. Although, in some cases, the amplitude of the influence of a given parameter changed due to sample preservation, analyses revealed that pH, fertilization and soil type significantly influenced microbial community structure in the analyzed samples.