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.

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.

Effects of isochoric freezing on freezing process and quality attributes of chicken breast meat.

This study investigated the potential of isochoric freezing for chicken breast meat for the first time. Chicken breast samples were immersed in isochoric NaCl solutions with various concentrations of 0 (pure water, PW), 1.5 and 2.5 % at -4 and -8 °C, respectively, and effects of process parameters such as temperature, pressure, and solution concentration on quality characteristics of the sample including colour, water holding capacity, weight loss, texture, microstructure, and water mobility were evaluated. Results showed that increasing NaCl concentrations depressed freezing temperature and pressure and samples treated in PW and 1.5 % NaCl solution at -4 and -8 °C exhibited a significant decrease (P < 0.05) in the quality characteristics, while those treated in 2.5 % solution at -4 and -8 °C showed no significant difference compared with the control. These results indicated the potential for enhancing the quality of meats preserved in isochoric systems.

Novel isochoric impregnation to develop high-quality and nutritionally fortified plant materials (apples and sweet potatoes).

Isochoric impregnation was explored as a novel pressure-assisted infusion technique to fortify plant materials with bioactive compounds. Apple and potato cylinders were impregnated with a sucrose solution containing 4% ascorbic acid (AA) while freezing under isochoric conditions. Isochoric impregnation resulted in greater infusion of AA compared to infusion at atmospheric pressure, which demonstrated the feasibility of this impregnation technology. Processing temperatures (-3°C and -5°C) and processing times (1, 3, and 5 h) significantly affected the AA infusion. The AA content values ranged from 446 to 516 mg/100 g for apples and 322 to 831 mg/100 g for sweet potatoes under isochoric conditions. For both plant materials, isochoric impregnation at -3°C did not cause major changes in texture and microstructure of the biological tissues. These results indicated that isochoric impregnation of solid foods could be a feasible technology for infusion of bioactive compounds without significantly altering their matrix. PRACTICAL APPLICATION: The findings of this study showed that the use of isochoric impregnation as a fortification technique is a promising way to develop fresh-like and value-added products with improved nutrition during preservation at subfreezing temperatures.

Stop Codon Usage as a Window into Genome Evolution: Mutation, Selection, Biased Gene Conversion and the TAG Paradox.

Protein coding genes terminate with one of three stop codons (TAA, TGA, or TAG) that, like synonymous codons, are not employed equally. With TGA and TAG having identical nucleotide content, analysis of their differential usage provides an unusual window into the forces operating on what are ostensibly functionally identical residues. Across genomes and between isochores within the human genome, TGA usage increases with G + C content but, with a common G + C → A + T mutation bias, this cannot be explained by mutation bias-drift equilibrium. Increased usage of TGA in G + C-rich genomes or genomic regions is also unlikely to reflect selection for the optimal stop codon, as TAA appears to be universally optimal, probably because it has the lowest read-through rate. Despite TAA being favored by selection and mutation bias, as with codon usage bias G + C pressure is the prime determinant of between-species TGA usage trends. In species with strong G + C-biased gene conversion (gBGC), such as mammals and birds, the high usage and conservation of TGA is best explained by an A + T → G + C repair bias. How to explain TGA enrichment in other G + C-rich genomes is less clear. Enigmatically, across bacterial and archaeal species and between human isochores TAG usage is mostly unresponsive to G + C pressure. This unresponsiveness we dub the TAG paradox as currently no mutational, selective, or gBGC model provides a well-supported explanation. That TAG does increase with G + C usage across eukaryotes makes the usage elsewhere yet more enigmatic. We suggest resolution of the TAG paradox may provide insights into either an unknown but common selective preference (probably at the DNA/RNA level) or an unrecognized complexity to the action of gBGC.

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.

Thermo-mechanics aspects of isochoric cryopreservation: A new modeling approach and comparison with experimental data.

A new mathematical model is proposed for the analysis of thermo-mechanics effects during isochoric cryopreservation. In that process, some ice crystallization in a fixed-volume container drives pressure elevation, which may be favorable to the preservation of biological material when it resides in the unfrozen portion of the same container. The proposed model is comprehensive, integrating for the first time concepts from the disparate fields of thermodynamics, heat transfer, fluid mechanics, and solid mechanics. The novelty in this study is in treating the cryopreserved material as having a pseudo-viscoelastic behavior over a very narrow temperature range, without affecting the mechanical behavior of the material in the rest of the domain. This unique approach permits treating the domain as a continuum, while avoiding the need to trace freezing fronts and sperate the analysis to liquid and solid subdomains. Consistent with the continuum approach, the heat transfer problem is solved using the enthalpy approach. The presented analysis focusses on isochoric cooling of pure water between standard atmospheric conditions and the triple point of liquid water, ice Ih, and ice III (-22°C and 207.4 MPa). The proposed model is also applicable to isochoric vitrification, by substituting the pseudo-viscoelastic material model with the real viscosity model of the vitrifying material. Results of this study display good agreement with phase-diagram data at steady state, and with experimental data from the literature. Furthermore, this study provides a venue to discussing experimentation aspects of isochoric cryopreservation. The proposed model is further demonstrated on a 3D problem, while discussing scale considerations, crystallization conditions, and transient effects. Notably, the new model can be used to bridge the gap between limited pressure and temperature measurements during cryopreservation and the analysis of the continuum. Arguably, this study presents the most advanced thermo-mechanics model to solve practical problems relating to isochoric cryopreservation.

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.

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.

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.

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.

Preservation of grape tomato by isochoric freezing.

This study investigated the potential of isochoric freezing to preserve tomatoes. Isochoric freezing is an emerging technology that preserves biological matter at subfreezing temperatures without any ice damage. Isochoric freezing was compared with freezing under isobaric conditions and with preservation techniques used in the food industry: cold storage at 10 °C and individual quick freezing (IQF). Physicochemical and nutritional properties were evaluated weekly for four weeks. Preservation under isochoric conditions maintained the mass, color, nutrient content (ascorbic acid, lycopene and phenolics) and antioxidant activity of the fresh tomatoes. Also, isochoric preservation led to minimal texture damage. In comparison, mass loss of tomatoes stored at 10 °C for 3 weeks contributed to changes in overall visual quality and firmness as well as significant losses in nutrient content. The greatest mass, texture, and nutrients losses were obtained for tomatoes subjected to IQF and isobaric freezing. The results show that isochoric freezing has the potential to preserve tomatoes while maintaining physicochemical and nutritional properties similar to those of fresh tomatoes which might find application in the commercial preservation of tomatoes.

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.

IsoXpressor: A Tool to Assess Transcriptional Activity within Isochores.

Genomes are characterized by large regions of homogeneous base compositions known as isochores. The latter are divided into GC-poor and GC-rich classes linked to distinct functional and structural properties. Several studies have addressed how isochores shape function and structure. To aid in this important subject, we present IsoXpressor, a tool designed for the analysis of the functional property of transcription within isochores. IsoXpressor allows users to process RNA-Seq data in relation to the isochores, and it can be employed to investigate any biological question of interest for any species. The results presented herein as proof of concept are focused on the preimplantation process in Homo sapiens (human) and Macaca mulatta (rhesus monkey).

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.

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.

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.

A common genomic code for chromatin architecture and recombination landscape.

Recent findings established a link between DNA sequence composition and interphase chromatin architecture and explained the evolutionary conservation of TADs (Topologically Associated Domains) and LADs (Lamina Associated Domains) in mammals. This prompted us to analyse conformation capture and recombination rate data to study the relationship between chromatin architecture and recombination landscape of human and mouse genomes. The results reveal that: (1) low recombination domains and blocks of elevated linkage disequilibrium tend to coincide with TADs and isochores, indicating co-evolving regulatory elements and genes in insulated neighbourhoods; (2) double strand break (DSB) and recombination frequencies increase in the short loops of GC-rich TADs, whereas recombination cold spots are typical of LADs and (3) the binding and loading of proteins, which are critical for DSB and meiotic recombination (SPO11, DMC1, H3K4me3 and PRMD9) are higher in GC-rich TADs. One explanation for these observations is that the occurrence of DSB and recombination in meiotic cells are associated with compositional and epigenetic features (genomic code) that influence DNA stiffness/flexibility and appear to be similar to those guiding the chromatin architecture in the interphase nucleus of pre-leptotene cells.