Emergence and influence of sequence bias in evolutionarily malleable, mammalian tandem arrays.

BACKGROUND: The radiation of mammals at the extinction of the dinosaurs produced a plethora of new forms-as diverse as bats, dolphins, and elephants-in only 10-20 million years. Behind the scenes, adaptation to new niches is accompanied by extensive innovation in large families of genes that allow animals to contact the environment, including chemosensors, xenobiotic enzymes, and immune and barrier proteins. Genes in these "outward-looking" families are allelically diverse among humans and exhibit tissue-specific and sometimes stochastic expression. RESULTS: Here, we show that these tandem arrays of outward-looking genes occupy AT-biased isochores and comprise the "tissue-specific" gene class that lack CpG islands in their promoters. Models of mammalian genome evolution have not incorporated the sharply different functions and transcriptional patterns of genes in AT- versus GC-biased regions. To examine the relationship between gene family expansion, sequence content, and allelic diversity, we use population genetic data and comparative analysis. First, we find that AT bias can emerge during evolutionary expansion of gene families in cis. Second, human genes in AT-biased isochores or with GC-poor promoters experience relatively low rates of de novo point mutation today but are enriched for non-synonymous variants. Finally, we find that isochores containing gene clusters exhibit low rates of recombination. CONCLUSIONS: Our analyses suggest that tolerance of non-synonymous variation and low recombination are two forces that have produced the depletion of GC bases in outward-facing gene arrays. In turn, high AT content exerts a profound effect on their chromatin organization and transcriptional regulation.

Isochores and Heat Capacity of Liquid Water in Terms of the Ion-Molecular Model.

Thermodynamics of liquid water in terms of a non-standard approach-the ion-molecular model-is considered. Water is represented as a dense gas of neutral H2O molecules and single charged H3O+ and OH- ions. The molecules and ions perform thermal collisional motion and interconvert due to ion exchange. The energy-rich process-vibrations of an ion in a hydration shell of molecular dipoles-well known to spectroscopists with its dielectric response at 180 cm-1 (5 THz), is suggested to be key for water dynamics. Taking into account this ion-molecular oscillator, we compose an equation of state of liquid water to obtain analytical expressions for the isochores and heat capacity.

Abandoning the Isochore Theory Can Help Explain Genome Compositional Organization in Fish.

The organization of the genome nucleotide (AT/GC) composition in vertebrates remains poorly understood despite the numerous genome assemblies available. Particularly, the origin of the AT/GC heterogeneity in amniotes, in comparison to the homogeneity in anamniotes, is controversial. Recently, several exceptions to this dichotomy were confirmed in an ancient fish lineage with mammalian AT/GC heterogeneity. Hence, our current knowledge necessitates a reevaluation considering this fact and utilizing newly available data and tools. We analyzed fish genomes in silico with as low user input as possible to compare previous approaches to assessing genome composition. Our results revealed a disparity between previously used plots of GC% and histograms representing the authentic distribution of GC% values in genomes. Previous plots heavily reduced the range of GC% values in fish to comply with the alleged AT/GC homogeneity and AT-richness of their genomes. We illustrate how the selected sequence size influences the clustering of GC% values. Previous approaches that disregarded chromosome and genome sizes, which are about three times smaller in fish than in mammals, distorted their results and contributed to the persisting confusion about fish genome composition. Chromosome size and their transposons may drive the AT/GC heterogeneity apparent on mammalian chromosomes, whereas far less in fishes.

Isochoric supercooling cryomicroscopy.

We introduce an isochoric (constant-volume) supercooling cryomicroscope (ISCM), enabling the ice-free study of biological systems and biochemical reactions at subzero temperatures at atmospheric pressure absent ice. This technology draws from thermodynamic findings on the behavior of water in isochoric systems at subfreezing temperatures. A description of the design of the ISCM and a demonstration of the stability of the supercooled solution in the ISCM is followed by an illustration of the possible use of the ISCM in the preservation of biological matter research. A comparison was made between the survival of HeLa cells in the University of Wisconsin (UW) solution in the ISCM at +4 °C under conventional atmospheric conditions and at -5 °C under isochoric supercooled conditions. Continuous real-time monitoring at cryopreservation temperature via fluorescence microscopy showed that after three days of isochoric supercooling storage, the percentage of compromised cells remained similar to fresh controls, while storage at +4 °C yielded approximately three times the mortality rate of cells preserved at -5 °C.

Methods to stabilize aqueous supercooling identified by use of an isochoric nucleation detection (INDe) device.

Stable aqueous supercooling has shown significant potential as a technique for human tissue preservation, food cold storage, conservation biology, and beyond, but its stochastic nature has made its translation outside the laboratory difficult. In this work, we present an isochoric nucleation detection (INDe) platform for automated, high-throughput characterization of aqueous supercooling at >1 mL volumes, which enables statistically-powerful determination of the temperatures and time periods for which supercooling in a given aqueous system will remain stable. We employ the INDe to investigate the effects of thermodynamic, surface, and chemical parameters on aqueous supercooling, and demonstrate that various simple system modifications can significantly enhance supercooling stability, including isochoric (constant-volume) confinement, hydrophobic container walls, and the addition of even mild concentrations of solute. Finally, in order to enable informed design of stable supercooled biopreservation protocols, we apply a statistical model to estimate stable supercooling durations as a function of temperature and solution chemistry, producing proof-of-concept supercooling stability maps for four common cryoprotective solutes.

Slaying (Yet Again) the Brain-Eating Zombie Called the "Isochore Theory": A Segmentation Algorithm Used to "Confirm" the Existence of Isochores Creates "Isochores" Where None Exist.

The isochore theory, which was proposed more than 40 years ago, depicts the mammalian genome as a mosaic of long, homogeneous regions that are characterized by their guanine and cytosine (GC) content. The human genome, for instance, was claimed to consist of five compositionally distinct isochore families. The isochore theory, in all its reincarnations, has been repeatedly falsified in the literature, yet isochore proponents have persistently resurrected it by either redefining isochores or by proposing alternative means of testing the theory. Here, I deal with the latest attempt to salvage this seemingly immortal zombie-a sequence segmentation method called isoSegmenter, which was claimed to "identify" isochores while at the same time disregarding the main characteristic attribute of isochores-compositional homogeneity. I used a series of controlled, randomly generated simulated sequences as a benchmark to study the performance of isoSegmenter. The main advantage of using simulated sequences is that, unlike real data, the exact start and stop point of any isochore or homogeneous compositional domain is known. Based on three key performance metrics-sensitivity, precision, and Jaccard similarity index-isoSegmenter was found to be vastly inferior to isoPlotter, a segmentation algorithm with no user input. Moreover, isoSegmenter identified isochores where none exist and failed to identify compositionally homogeneous sequences that were shorter than 100-200 kb. Will this zillionth refutation of "isochores" ensure a final and permanent entombment of the isochore theory? This author is not holding his breath.

Nanodosimetric Calculations of Radiation-Induced DNA Damage in a New Nucleus Geometrical Model Based on the Isochore Theory.

Double-strand breaks (DSBs) in nuclear DNA represents radiation-induced damage that has been identified as particularly deleterious. Calculating this damage using Monte Carlo track structure modeling could be a suitable indicator to better assess and anticipate the side-effects of radiation therapy. However, as already demonstrated in previous work, the geometrical description of the nucleus and the DNA content used in the simulation significantly influence damage calculations. Therefore, in order to obtain accurate results, this geometry must be as realistic as possible. In this study, a new geometrical model of an endothelial cell nucleus and DNA distribution according to the isochore theory are presented and used in a Monte Carlo simulation chain based on the Geant4-DNA toolkit. In this theory, heterochromatin and euchromatin compaction are distributed along the genome according to five different families (L1, L2, H1, H2, and H3). Each of these families is associated with a different hetero/euchromatin rate related to its compaction level. In order to compare the results with those obtained using a previous nuclear geometry, simulations were performed for protons with linear energy transfers (LETs) of 4.29 keV/µm, 19.51 keV/µm, and 43.25 keV/µm. The organization of the chromatin fibers at different compaction levels linked to isochore families increased the DSB yield by 6-10%, and it allowed the most affected part of the genome to be identified. These new results indicate that the genome core is more radiosensitive than the genome desert, with a 3-8% increase in damage depending on the LET. This work highlights the importance of using realistic distributions of chromatin compaction levels to calculate radio-induced damage using Monte Carlo simulation methods.

A state of the art review of isochoric cryopreservation and cryoprotectants.

There is a developing enthusiasm for discovering new methods, cryoprotectants, systems and devices for cells, tissues, and organ preservation in medicine, in sub-zero temperature conditions and a growing interest in developing more efficient and economical methods for long-term preservation of food in a frozen state. Most of the preservation protocols currently used in medicine and food preservation involve the use of atmospheric pressure, and temperatures lower than normal body temperature in medicine, or lower than room temperature in the food industry. In this state of the art review, we analyzed the results of a new preservation method that uses an isochoric system. We aimed to offer a clear overview of the potential of this new technology. Firstly, to study the origins of isochoric preservation, we searched using the WoS Database. A search with the world "isochoric" returned 488 results. A more specific search of the term isochoric freezing returned 94 results. From these searches, we selected the 12 most relevant articles and discuss them here in detail. We present an overall characterization and criticism of the current use and potential of this new preservation method that can be used in the medicine and food industry. The main findings indicate encouraging results for the tested biological matter, including for the preservation of food products (e.g. cherries, spinach, potatoes), biological organisms (e.g. Caenorhabditis elegans, Escherichia coli, Listeria, Salmonella typhimurium), organs (e.g. rat hearts), tissues (e.g., tilapia fish filets) or cells (e.g., mammalian cells, pancreatic cells). Accordingly, we conclude that the isochoric system holds huge potential as a new technique in the field of preservation. doi.org/10.54680/fr22410110112.

The Genomic Code: A Pervasive Encoding/Molding of Chromatin Structures and a Solution of the "Non-Coding DNA" Mystery.

Recent investigations have revealed 1) that the isochores of the human genome group into two super-families characterized by two different long-range 3D structures, and 2) that these structures, essentially based on the distribution and topology of short sequences, mold primary chromatin domains (and define nucleosome binding). More specifically, GC-poor, gene-poor isochores are low-heterogeneity sequences with oligo-A spikes that mold the lamina-associated domains (LADs), whereas GC-rich, gene-rich isochores are characterized by single or multiple GC peaks that mold the topologically associating domains (TADs). The formation of these "primary TADs" may be followed by extrusion under the action of cohesin and CTCF. Finally, the genomic code, which is responsible for the pervasive encoding and molding of primary chromatin domains (LADs and primary TADs, namely the "gene spaces"/"spatial compartments") resolves the longstanding problems of "non-coding DNA," "junk DNA," and "selfish DNA" leading to a new vision of the genome as shaped by DNA sequences.

Mass transfer into biological matter using isochoric freezing.

This paper is a theoretical study of a protocol for transport of high concentrations of cryoprotectants into biological matter, using isochoric freezing. Unlike isobaric freezing, where the entire system freezes at temperatures lower than the freezing temperature, in isochoric freezing a substantial portion of the system remains unfrozen at temperatures below freezing. In isochoric freezing cryopreservation, the system is designed in such a way that the biological matter remains unfrozen and surrounded by an unfrozen solution. The protocol in this study involves the freezing of an isochoric systems along the "liquidus line" at which water and ice are in thermodynamic equilibrium. Rejection of solutes by ice increases the concentration of the solutes in the unfrozen solution surrounding the unfrozen biological matter, leading, thereby, to transport of increasingly higher concentrations of cryoprotectants into the biological matter, as the temperature of the system is lowered and the toxicity of the cryoprotectants is reduced.

Evidence of distinct gene functional patterns in GC-poor and GC-rich isochores in Bos taurus.

Vertebrate genomes are mosaics of megabase-size DNA segments with a fairly homogeneous base composition, called isochores. They are divided into five families characterized by different guanine-cytosine (GC) levels and linked to several functional and structural properties. The increased availability of fully sequenced genomes allows the investigation of isochores in several species, assessing their level of conservation across vertebrate genomes. In this work, we characterized the isochores in Bos taurus using the ARS-UCD1.2 genome version. The comparison of our results with the well-studied human isochores and those of other mammals revealed a large conservation in isochore families, in number, average GC levels and gene density. Exceptions to the established increase in gene density with the increase in isochores (GC%) were observed for the following gene biotypes: tRNA, small nuclear RNA, small nucleolar RNA and pseudogenes that have their maximum number in H2 and H1 isochores. Subsequently, we assessed the ontology of all gene biotypes looking for functional classes that are statistically over- or under-represented in each isochore. Receptor activity and sensory perception pathways were significantly over-represented in L1 and L2 (GC-poor) isochores. This was also validated for the horse genome. Our analysis of housekeeping genes confirmed a preferential localization in GC-rich isochores, as reported in other species. Finally, we assessed the SNP distribution of a bovine high-density SNP chip across the isochores, finding a higher density in the GC-rich families, reflecting a potential bias in the chip, widely used for genetic selection and biodiversity studies.

Evidence of low-density and high-density liquid phases and isochore end point for water confined to carbon nanotube.

Possible transition between two phases of supercooled liquid water, namely the low- and high-density liquid water, has been only predicted to occur below 230 K from molecular dynamics (MD) simulation. However, such a phase transition cannot be detected in the laboratory because of the so-called "no-man's land" under deeply supercooled condition, where only crystalline ices have been observed. Here, we show MD simulation evidence that, inside an isolated carbon nanotube (CNT) with a diameter of 1.25 nm, both low- and high-density liquid water states can be detected near ambient temperature and above ambient pressure. In the temperature-pressure phase diagram, the low- and high-density liquid water phases are separated by the hexagonal ice nanotube (hINT) phase, and the melting line terminates at the isochore end point near 292 K because of the retracting melting line from 292 to 278 K. Beyond the isochore end point (292 K), low- and high-density liquid becomes indistinguishable. When the pressure is increased from 10 to 600 MPa along the 280-K isotherm, we observe that water inside the 1.25-nm-diameter CNT can undergo low-density liquid to hINT to high-density liquid reentrant first-order transitions.

Isochoric Containers and Its Frontier between Cryopreservation and Sterilization.

BACKGROUND: The use of isochoric containers below the freezing point was proposed for the reduction of freezing damage. OBJECTIVE: To determine mathematically the dielectric constant (k') of a sample inside an isochoric container depending on a suggested nucleated volume, and to compare the values with the reported k' for an isochoric container. METHODS: Different nucleation arrangements inside a cylindrical capacitor filled with water was considered, and the way that ice Ih changes the capacitance and the expected k' was examined. RESULTS: Dielectric constant for different nucleation arrangements decreases proportionally with the nucleated volume, reaching smaller values when the nucleation is supposed only over the internal electrode. However, the nucleation proposed don't reproduce the experimental behavior. CONCLUSION: When compared with experimental results, k' values suggest the water inside an isochoric container remains in liquid state (-4 ~ 0 degree C), which may explain that there is no biological damage for this temperature range.

Time-dependent Effects of Pressure during Preservation of Rat Hearts in an Isochoric System at Subfreezing Temperatures.

BACKGROUND: Isochoric freezing systems enable ice-free preservation of biological matter at subfreezing temperatures under the increased hydrostatic pressure. OBJECTIVE: To examine the effects of pressure and exposure period on rat hearts preserved in an isochoric chamber. MATERIALS AND METHODS: Rat hearts were preserved in the UW solution in isochoric chambers at temperatures from -2°C to -8°C and pressure from the atmospheric level to 78 MPa for up to eight hours, with and without the addition of glycerol. Hearts were evaluated via Langendorff perfusion and HE histology. RESULTS: Hearts were compromised quickly as pressure increased, suggesting an acute time-pressure sensitivity. With the addition of 1 M glycerol, which reduces the pressure experienced at a given temperature, the survival time at -4°C was doubled. CONCLUSION: The enhanced hydrostatic pressure encountered during isochoric preservation yields time-dependent negative effects on the heart, which can potentially be alleviated by the addition of a cryoprotectant.

The Isochores as a Fundamental Level of Genome Structure and Organization: A General Overview.

The recent availability of a number of fully sequenced genomes (including marine organisms) allowed to map very precisely the isochores, based on DNA sequences, confirming the results obtained before genome sequencing by the ultracentrifugation in CsCl. In fact, the analytical profile of human DNA showed that the vertebrate genome is a mosaic of isochores, typically megabase-size DNA segments that belong to a small number of families characterized by different GC levels. In this review, we will concentrate on some general genome features regarding the compositional organization from different organisms and their evolution, ranging from vertebrates to invertebrates until unicellular organisms. Since isochores are tightly linked to biological properties such as gene density, replication timing, and recombination, the new level of detail provided by the isochore map helped the understanding of genome structure, function, and evolution. All the findings reported here confirm the idea that the isochores can be considered as a "fundamental level of genome structure and organization." We stress that we do not discuss in this review the origin of isochores, which is still a matter of controversy, but we focus on well established structural and physiological aspects.

A genomic view on epilepsy and autism candidate genes.

Epilepsy is a common complex disorder most frequently associated with psychiatric and neurological diseases. Massive parallel sequencing of individual or cohort genomes and exomes led the identification of several disease associated genes. We review here the candidate genes in epilepsy genetics with focus on exome and gene panel data. Together with the examination of brain expressed genes and post synaptic proteome the results show that: (1) Non-metabolic epilepsies and autism candidate genes tend to be AT-rich and (2) large transcript size and local AT-richness are characteristic features of genes involved in developmental brain disorders and synaptic functions. These results point to the preferential location of core epilepsy and autism candidate genes in late replicating, GC-poor chromosomal regions (isochores). These results indicate that the genomic alterations leading to some brain disorders are confined to responsive chromatin areas harboring brain critical genes.

Phase behavior and critical activated dynamics of limited-valence DNA nanostars.

Colloidal particles with directional interactions are key in the realization of new colloidal materials with possibly unconventional phase behaviors. Here we exploit DNA self-assembly to produce bulk quantities of "DNA stars" with three or four sticky terminals, mimicking molecules with controlled limited valence. Solutions of such molecules exhibit a consolution curve with an upper critical point, whose temperature and concentration decrease with the valence. Upon approaching the critical point from high temperature, the intensity of the scattered light diverges with a power law, whereas the intensity time autocorrelation functions show a surprising two-step relaxation, somehow reminiscent of glassy materials. The slow relaxation time exhibits an Arrhenius behavior with no signs of criticality, demonstrating a unique scenario where the critical slowing down of the concentration fluctuations is subordinate to the large lifetime of the DNA bonds, with relevant analogies to critical dynamics in polymer solutions. The combination of equilibrium and dynamic behavior of DNA nanostars demonstrates the potential of DNA molecules in diversifying the pathways toward collective properties and self-assembled materials, beyond the range of phenomena accessible with ordinary molecular fluids.

Effects of DNA methylation on nucleosome stability.

Methylation of DNA at CpG dinucleotides represents one of the most important epigenetic mechanisms involved in the control of gene expression in vertebrate cells. In this report, we conducted nucleosome reconstitution experiments in conjunction with high-throughput sequencing on 572 KB of human DNA and 668 KB of mouse DNA that was unmethylated or methylated in order to investigate the effects of this epigenetic modification on the positioning and stability of nucleosomes. The results demonstrated that a subset of nucleosomes positioned by nucleotide sequence was sensitive to methylation where the modification increased the affinity of these sequences for the histone octamer. The features that distinguished these nucleosomes from the bulk of the methylation-insensitive nucleosomes were an increase in the frequency of CpG dinucleotides and a unique rotational orientation of CpGs such that their minor grooves tended to face toward the histones in the nucleosome rather than away. These methylation-sensitive nucleosomes were preferentially associated with exons as compared to introns while unmethylated CpG islands near transcription start sites became enriched in nucleosomes upon methylation. The results of this study suggest that the effects of DNA methylation on nucleosome stability in vitro can recapitulate what has been observed in the cell and provide a direct link between DNA methylation and the structure and function of chromatin.

Twisted signatures of GC-biased gene conversion embedded in an evolutionary stable karyotype.

The genomes of many vertebrates show a characteristic heterogeneous distribution of GC content, the so-called GC isochore structure. The origin of isochores has been explained via the mechanism of GC-biased gene conversion (gBGC). However, although the isochore structure is declining in many mammalian genomes, the heterogeneity in GC content is being reinforced in the avian genome. Despite this discrepancy, which remains unexplained, examinations of individual substitution frequencies in mammals and birds are both consistent with the gBGC model of isochore evolution. On the other hand, a negative correlation between substitution and recombination rate found in the chicken genome is inconsistent with the gBGC model. It should therefore be important to consider along with gBGC other consequences of recombination on the origin and fate of mutations, as well as to account for relationships between recombination rate and other genomic features. We therefore developed an analytical model to describe the substitution patterns found in the chicken genome, and further investigated the relationships between substitution patterns and several genomic features in a rigorous statistical framework. Our analysis indicates that GC content itself, either directly or indirectly via interrelations to other genomic features, has an impact on the substitution pattern. Further, we suggest that this phenomenon is particularly visible in avian genomes due to their unusually low rate of chromosomal evolution. Because of this, interrelations between GC content and other genomic features are being reinforced, and are as such more pronounced in avian genomes as compared with other vertebrate genomes with a less stable karyotype.

Comparative genomics of the neglected human malaria parasite Plasmodium vivax.

The human malaria parasite Plasmodium vivax is responsible for 25-40% of the approximately 515 million annual cases of malaria worldwide. Although seldom fatal, the parasite elicits severe and incapacitating clinical symptoms and often causes relapses months after a primary infection has cleared. Despite its importance as a major human pathogen, P. vivax is little studied because it cannot be propagated continuously in the laboratory except in non-human primates. We sequenced the genome of P. vivax to shed light on its distinctive biological features, and as a means to drive development of new drugs and vaccines. Here we describe the synteny and isochore structure of P. vivax chromosomes, and show that the parasite resembles other malaria parasites in gene content and metabolic potential, but possesses novel gene families and potential alternative invasion pathways not recognized previously. Completion of the P. vivax genome provides the scientific community with a valuable resource that can be used to advance investigation into this neglected species.