Pathological Interplay between Inflammation and Mitochondria Aggravates Glutamate Toxicity.

Mitochondrial dysfunction and glutamate toxicity are associated with neural disorders, including brain trauma. A review of the literature suggests that toxic and transmission actions of neuronal glutamate are spatially and functionally separated. The transmission pathway utilizes synaptic GluN2A receptors, rapidly released pool of glutamate, evoked release of glutamate mediated by Synaptotagmin 1 and the amount of extracellular glutamate regulated by astrocytes. The toxic pathway utilizes extrasynaptic GluN2B receptors and a cytoplasmic pool of glutamate, which results from the spontaneous release of glutamate mediated by Synaptotagmin 7 and the neuronal 2-oxoglutarate dehydrogenase complex (OGDHC), a tricarboxylic acid (TCA) cycle enzyme. Additionally, the inhibition of OGDHC observed upon neuro-inflammation is due to an excessive release of reactive oxygen/nitrogen species by immune cells. The loss of OGDHC inhibits uptake of glutamate by mitochondria, thus facilitating its extracellular accumulation and stimulating toxic glutamate pathway without affecting transmission. High levels of extracellular glutamate lead to dysregulation of intracellular redox homeostasis and cause ferroptosis, excitotoxicity, and mitochondrial dysfunction. The latter affects the transmission pathway demanding high-energy supply and leading to cell death. Mitochondria aggravate glutamate toxicity due to impairments in the TCA cycle and become a victim of glutamate toxicity, which disrupts oxidative phosphorylation. Thus, therapies targeting the TCA cycle in neurological disorders may be more efficient than attempting to preserve mitochondrial oxidative phosphorylation.

Cervical and Vaginal Microbiomes in Early Miscarriages and Ongoing Pregnancy with and without Dydrogesterone Usage.

Emerging evidence suggests that the reproductive tract microbiota is a key modulator of local inflammatory and immune pathways throughout pregnancy and may subsequently impact pregnancy outcomes. In this study, our objective was to analyze the cervical and vaginal microbiomes during early pregnancy among three groups: women with healthy ongoing pregnancies, women undergoing dydrogesterone treatment, and those who experienced miscarriages. The experiment involved 51 women at 8-11 weeks of gestation. The microbiome was examined using 16S rRNA sequencing on the Ion Torrent PGM platform. Across all groups, Lactobacillus iners was predominant, suggesting that the vaginal community type CST III is common among the majority of participants. Notably, our data highlighted the significant roles of Gardnerella vaginalis and Mycoplasma girerdii in the pathogenesis of early miscarriage. Conversely, L. iners and Bifidobacterium longum have a protective effect in early pregnancy. Moreover, dydrogesterone intake appeared to influence notable differences between the cervical and vaginal microbiomes. Overall, our study enhanced our understanding of the cervical and vaginal microbiome composition in the eastern European population during early pregnancy.

Characteristics of the Fecal Microbiome of Piglets with Diarrhea Identified Using Shotgun Metagenomics Sequencing.

Diarrhea in piglets is one of the most common diseases leading to high mortality and, as a result, to economic losses. Shotgun metagenomic sequencing was performed on the DNBSEQ-G50, MGI system to study the role of the fecal microbiome in the development of diarrhea in newborn piglets. Analysis of the study data showed that the composition of the fecal microbiome at the level of bacteria and fungi did not differ in piglets with diarrhea from the healthy group. Bacteria belonging to the phyla Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, and Fusobacteria were the most abundant. However, a higher level of bacterial alpha diversity was observed in the group of piglets with diarrhea, which may be due to dysbacteriosis and inflammation. The study of the virome showed the difference between the two types of phages: Bacteroides B40-8 prevailed in diseased piglets, while Escherichia virus BP4 was found in greater numbers in healthy piglets. The results of our study suggest that the association between the fecal microbiome and susceptibility to diarrhea in suckling piglets may have been previously overestimated.

Changes in the Human Gut Microbiome Caused by the Short-Term Impact of Lactic Acid Bacteria Consumption in Healthy People.

The gut microbiome is one of the main factors affecting human health. It has been proven that probiotics can regulate the metabolism in the host body. A large number of people use probiotics not as medicines, but as a prophylactic supplement. The aim of our study was to evaluate the effect of lactic acid bacteria on the gut microbiome of healthy people using the V3 region of the 16S rRNA gene. Our study showed changes in the generic composition in the gut of healthy people when taking the supplement. There was an increase in the members responsible for the production of short-chain fatty acids in the gut of the host (Blautia, Fusicatenibacter, Eubacterium hallii group, Ruminococcus), as well as bacteria that improve intestinal homeostasis (Dorea and Barnesiella). There was also a decrease in the abundance of bacteria in the genera Catenibacterium, Hungatella, Escherichia-Shigella, and Pseudomonas, associated with an unhealthy profile of the human gut microbiome. An increase in members of the phylum Actinobacteriota was also observed, which has a positive effect on the host organism. Our results indicate that short-term prophylactic use of lactic acid bacteria-based supplements can be effective, as it contributes to a beneficial effect on the gut microbiome of healthy people.

The Ketogenic Diet but not Hydroxycitric Acid Keeps Brain Mitochondria Quality Control and mtDNA Integrity Under Focal Stroke.

Mitochondrial dysfunction in the ischemic brain is one of the hallmarks of stroke. Dietary interventions such as the ketogenic diet and hydroxycitric acid supplementation (a caloric restriction mimetic) may potentially protect neurons from mitochondrial damage induced by focal stroke in mice. We showed that in control mice, the ketogenic diet and the hydroxycitric acid did not impact significantly on the mtDNA integrity and expression of genes involved in the maintenance of mitochondrial quality control in the brain, liver, and kidney. The ketogenic diet changed the bacterial composition of the gut microbiome, which via the gut-brain axis may affect the increase in anxiety behavior and reduce mice mobility. The hydroxycitric acid causes mortality and suppresses mitochondrial biogenesis in the liver. Focal stroke modelling caused a significant decrease in the mtDNA copy number in both ipsilateral and contralateral brain cortex and increased the levels of mtDNA damage in the ipsilateral hemisphere. These alterations were accompanied by a decrease in the expression of some of the genes involved in maintaining mitochondrial quality control. The ketogenic diet consumption before stroke protects mtDNA in the ipsilateral cortex, probably via activation of the Nrf2 signaling. The hydroxycitric acid, on the contrary, increased stroke-induced injury. Thus, the ketogenic diet is the most preferred variant of dietetic intervention for stroke protection compared with the hydroxycitric acid supplementation. Our data confirm some reports about hydroxycitric acid toxicity, not only for the liver but also for the brain under stroke condition.

Association of milk microbiome with bovine mastitis before and after antibiotic therapy.

Background and Aim: Mastitis is recognized as the most common disease in cattle and causes economic losses in the dairy industry. A number of opportunistic bacterial taxa have been identified as causative agents for this disease. Conventionally, antibiotics are used to treat mastitis; however, most bacteria are resistant to the majority of antibiotics. This study aimed to use molecular methods to identify milk microbiome patterns characteristic of mastitis that can help in the early diagnosis of this disease and in the development of new treatment strategies. Materials and Methods: To evaluate the microbiome composition, we performed NGS sequencing of the 16S rRNA gene of the V3 region. Results: An increase in the abundance of the bacterial genera Hymenobacter and Lachnospiraceae NK4A136 group is associated with the development of subclinical and clinical mastitis in dairy cows. These bacteria can be added to the list of markers used to detect mastitis in cows. Furthermore, a decrease in the abundance of Ralstonia, Lachnospiraceae NK3A20 group, Acetitomaculum, Massilia, and Atopostipes in cows with mastitis may indicate their role in maintaining a healthy milk microbiome. Antibiotics reduced the levels of Streptococcus in milk compared to those in the healthy group and cows before antibiotic treatment. Antibiotic therapy also contributed to an increase in the abundance of beneficial bacteria of the genus Asticcacaulis. Conclusion: This study expands our understanding of the association between milk microbiota and mastitis.

Lower Genital Tract Microbiome in Early Pregnancy in the Eastern European Population.

BACKGROUND: It is known that the features of the cervicovaginal microbiome can depend on ethnicity, which might be caused by genetic factors, as well as differences in diet and lifestyle. There is no research on the cervicovaginal microbiome of Eastern European women during early pregnancy. METHODS: We evaluated the cervical and cervicovaginal microbiome of women with first-trimester pregnancy (n = 22), further delivered at term, using the 16S rRNA sequencing method. RESULTS: The predominant bacterial species in both groups was Lactobacillus iners, followed by Prevotella copri, Ileibacterium valens, Gardnerella vaginalis and Muribaculum intestinale in the cervical samples, and Gardnerella vaginalis, Prevotella copri, Bifidobacterium longum, Ileibacterium valens and Muribaculum intestinale in the cervicovaginal samples. The cervical microbiome had higher alpha diversity; a higher abundance of Muribaculum intestinale, Aquabacterium parvum and Methyloversatilis universalis; and a lower abundance of Psychrobacillus psychrodurans. CONCLUSIONS: The Lactobacillus iners-dominated microbiome (CST III) was the predominant type of cervical and cervicovaginal microbiome in early pregnancy in the majority of the women. The presence of soil and animal bacteria in the cervicovaginal microbiome can be explained by the rural origin of patients.

Characteristics of the Gut Bacterial Composition in People of Different Nationalities and Religions.

High-throughput sequencing has made it possible to extensively study the human gut microbiota. The links between the human gut microbiome and ethnicity, religion, and race remain rather poorly understood. In this review, data on the relationship between gut microbiota composition and the nationality of people and their religion were generalized. The unique gut microbiome of a healthy European (including Slavic nationality) is characterized by the dominance of the phyla Firmicutes, Bacteroidota, Actinobacteria, Proteobacteria, Fusobacteria, and Verrucomicrobia. Among the African population, the typical members of the microbiota are Bacteroides and Prevotella. The gut microbiome of Asians is very diverse and rich in members of the genera Prevotella, Bacteroides Lactobacillus, Faecalibacterium, Ruminococcus, Subdoligranulum, Coprococcus, Collinsella, Megasphaera, Bifidobacterium, and Phascolarctobacterium. Among Buddhists and Muslims, the Prevotella enterotype is characteristic of the gut microbiome, while other representatives of religions, including Christians, have the Bacteroides enterotype. Most likely, the gut microbiota of people of different nationalities and religions are influenced by food preferences. The review also considers the influences of pathologies such as obesity, Crohn's disease, cancer, diabetes, etc., on the bacterial composition of the guts of people of different nationalities.

The Effect of Short-Term Consumption of Lactic Acid Bacteria on the Gut Microbiota in Obese People.

Obesity is a problem of modern health care that causes the occurrence of many concomitant diseases: arterial hypertension, diabetes mellitus, non-alcoholic fatty liver disease, and cardiovascular diseases. New strategies for the treatment and prevention of obesity are being developed that are based on using probiotics for modulation of the gut microbiota. Our study aimed to evaluate the bacterial composition of the gut of obese patients before and after two weeks of lactic acid bacteria (Lactobacillus acidophilus, Lactiplantibacillus plantarum, Limosilactobacillus fermentum, and Lactobacillus delbrueckii) intake. The results obtained showed an increase in the number of members of the phylum Actinobacteriota in the group taking nutritional supplements, while the number of phylum Bacteroidota decreased in comparison with the control group. There has also been an increase in potentially beneficial groups: Bifidobacterium, Blautia, Eubacterium, Anaerostipes, Lactococcus, Lachnospiraceae ND3007, Streptococcus, Escherichia-Shigella, and Lachnoclostridium. Along with this, a decrease in the genera was demonstrated: Faecalibacterium, Pseudobutyrivibrio, Subdoligranulum, Faecalibacterium, Clostridium sensu stricto 1 and 2, Catenibacterium, Megasphaera, Phascolarctobacterium, and the Oscillospiraceae NK4A214 group, which contribute to the development of various metabolic disorders. Modulation of the gut microbiota by lactic acid bacteria may be one of the ways to treat obesity.

The effect of polydispersity, shape fluctuations and curvature on small unilamellar vesicle small-angle X-ray scattering curves.

Small unilamellar vesicles (20-100 nm diameter) are model systems for strongly curved lipid membranes, in particular for cell organelles. Routinely, small-angle X-ray scattering (SAXS) is employed to study their size and electron-density profile (EDP). Current SAXS analysis of small unilamellar vesicles (SUVs) often employs a factorization into the structure factor (vesicle shape) and the form factor (lipid bilayer electron-density profile) and invokes additional idealizations: (i) an effective polydispersity distribution of vesicle radii, (ii) a spherical vesicle shape and (iii) an approximate account of membrane asymmetry, a feature particularly relevant for strongly curved membranes. These idealizations do not account for thermal shape fluctuations and also break down for strong salt- or protein-induced deformations, as well as vesicle adhesion and fusion, which complicate the analysis of the lipid bilayer structure. Presented here are simulations of SAXS curves of SUVs with experimentally relevant size, shape and EDPs of the curved bilayer, inferred from coarse-grained simulations and elasticity considerations, to quantify the effects of size polydispersity, thermal fluctuations of the SUV shape and membrane asymmetry. It is observed that the factorization approximation of the scattering intensity holds even for small vesicle radii (∼30 nm). However, the simulations show that, for very small vesicles, a curvature-induced asymmetry arises in the EDP, with sizeable effects on the SAXS curve. It is also demonstrated that thermal fluctuations in shape and the size polydispersity have distinguishable signatures in the SAXS intensity. Polydispersity gives rise to low-q features, whereas thermal fluctuations predominantly affect the scattering at larger q, related to membrane bending rigidity. Finally, it is shown that simulation of fluctuating vesicle ensembles can be used for analysis of experimental SAXS curves.

Density Field Thermodynamic Integration (DFTI): A "Soft" Approach to Calculate the Free Energy of Surfactant Self-Assemblies.

Thermodynamic integration is one of the most established methods to quantify excess free energies between different metastable states. Excess intermolecular interactions in surfactant assemblies are on the scale of the energy of thermal fluctuations. Therefore, these materials can be deformed and topologically altered via relatively small mechanical stresses. It is thus intuitive to design reaction paths and associated order parameters that exploit the "soft" nature of these materials to mechanically rather than alchemically morph surfactant assemblies from state to state. Here, we propose a novel method coined "density field thermodynamic integration" (DFTI) that adopts the universality and transferability of alchemical methods while simultaneously exploiting the soft excess interactions between surfactant molecules. DFTI was designed for a rapid quantification of the free energy differences between different metastable structures in soft fluid materials. The DFTI method uses an external field coupled to the local density to mechanically morph the system between metastable states of interest. Here, we explored the capability of the DFTI method to swiftly and accurately calculate free energy differences between states. To this aim, we studied two different coarse-grained lipidic surfactant systems: (i) a fusion stalk and (ii) a worm-like micelle. Our results illustrate that DFTI can provide an efficient, versatile, and rather reliable method to calculate the free energy differences between surfactant assemblies.

Thermodynamically reversible paths of the first fusion intermediate reveal an important role for membrane anchors of fusion proteins.

Biological membrane fusion proceeds via an essential topological transition of the two membranes involved. Known players such as certain lipid species and fusion proteins are generally believed to alter the free energy and thus the rate of the fusion reaction. Quantifying these effects by theory poses a major challenge since the essential reaction intermediates are collective, diffusive and of a molecular length scale. We conducted molecular dynamics simulations in conjunction with a state-of-the-art string method to resolve the minimum free-energy path of the first fusion intermediate state, the so-called stalk. We demonstrate that the isolated transmembrane domains (TMDs) of fusion proteins such as SNARE molecules drastically lower the free energy of both the stalk barrier and metastable stalk, which is not trivially explained by molecular shape arguments. We relate this effect to the local thinning of the membrane (negative hydrophobic mismatch) imposed by the TMDs which favors the nearby presence of the highly bent stalk structure or prestalk dimple. The distance between the membranes is the most crucial determinant of the free energy of the stalk, whereas the free-energy barrier changes only slightly. Surprisingly, fusion enhancing lipids, i.e., lipids with a negative spontaneous curvature, such as PE lipids have little effect on the free energy of the stalk barrier, likely because of its single molecular nature. In contrast, the lipid shape plays a crucial role in overcoming the hydration repulsion between two membranes and thus rather lowers the total work required to form a stalk.

Mechanics of membrane fusion/pore formation.

Lipid bilayers play a fundamental role in many biological processes, and a considerable effort has been invested in understanding their behavior and the mechanism of topological changes like fusion and pore formation. Due to the time- and length-scale on which these processes occur, computational methods have proven to be an especially useful tool in their study. With their help, a number of interesting findings about the shape of fusion intermediates could be obtained, and novel hypotheses about the mechanism of topological changes and the involvement of peptides therein were suggested. In this work, we try to present a summary of these developments together with some hitherto unpublished results, featuring, among others, the shape of stalks and fusion pores, possible modes of action of the influenza HA fusion peptide and the SNARE protein complex, the mechanism of supported lipid bilayer formation by vesicle spreading, and the free energy and transition pathway of the fusion process.

Free energy landscape of rim-pore expansion in membrane fusion.

The productive fusion pore in membrane fusion is generally thought to be toroidally shaped. Theoretical studies and recent experiments suggest that its formation, in some scenarios, may be preceded by an initial pore formed near the rim of the extended hemifusion diaphragm (HD), a rim-pore. This rim-pore is characterized by a nontoroidal shape that changes with size. To determine this shape as well as the free energy along the pathway of rim-pore expansion, we derived a simple analytical free energy model. We argue that dilation of HD material via expansion of a rim-pore is favored over a regular, circular pore. Further, the expanding rim-pore faces a free energy barrier that linearly increases with HD size. In contrast, the tension required to expand the rim-pore decreases with HD size. Pore flickering, followed by sudden opening, occurs when the tension in the HD competes with the line energy of the rim-pore, and the rim-pore reaches its equilibrium size before reaching the critical pore size. The experimental observation of flickering and closing fusion pores (kiss-and-run) is very well explained by the observed behavior of rim-pores. Finally, the free energy landscape of rim-pore expansion/HD dilation may very well explain why some cellular fusion reactions, in their attempt to minimize energetic costs, progress via alternative formation and dilation of microscopic hemifusion intermediates.

Transition path from two apposed membranes to a stalk obtained by a combination of particle simulations and string method.

The formation of an hourglass-shaped passage (stalk) connecting two apposed membranes is an essential initial step in membrane fusion. The most probable transition path from two separate membranes to a stalk, i.e., the minimum free-energy path (MFEP), is constructed using a combination of particle simulations and string method. For the reversible transition path in the coarse-grained membrane model, a collective order parameter, m, can be identified as the local difference of hydrophilic and hydrophobic densities. In particle simulations, the free energy F[m] as a functional of m is not readily available. This difficulty is overcome by an equation-free approach, where the morphology and the excess free energy along the MFEP are obtained by an on-the-fly string method. The transition state is confirmed by diagonalization of order-parameter fluctuations and by the probability of reaching either stalk or bilayer morphology from different positions along the MFEP.

Line-tension controlled mechanism for influenza fusion.

Our molecular simulations reveal that wild-type influenza fusion peptides are able to stabilize a highly fusogenic pre-fusion structure, i.e. a peptide bundle formed by four or more trans-membrane arranged fusion peptides. We rationalize that the lipid rim around such bundle has a non-vanishing rim energy (line-tension), which is essential to (i) stabilize the initial contact point between the fusing bilayers, i.e. the stalk, and (ii) drive its subsequent evolution. Such line-tension controlled fusion event does not proceed along the hypothesized standard stalk-hemifusion pathway. In modeled influenza fusion, single point mutations in the influenza fusion peptide either completely inhibit fusion (mutants G1V and W14A) or, intriguingly, specifically arrest fusion at a hemifusion state (mutant G1S). Our simulations demonstrate that, within a line-tension controlled fusion mechanism, these known point mutations either completely inhibit fusion by impairing the peptide's ability to stabilize the required peptide bundle (G1V and W14A) or stabilize a persistent bundle that leads to a kinetically trapped hemifusion state (G1S). In addition, our results further suggest that the recently discovered leaky fusion mutant G13A, which is known to facilitate a pronounced leakage of the target membrane prior to lipid mixing, reduces the membrane integrity by forming a 'super' bundle. Our simulations offer a new interpretation for a number of experimentally observed features of the fusion reaction mediated by the prototypical fusion protein, influenza hemagglutinin, and might bring new insights into mechanisms of other viral fusion reactions.

Solvent-exposed tails as prestalk transition states for membrane fusion at low hydration.

Membrane fusion is a key step in intracellular trafficking and viral infection. The underlying molecular mechanism is poorly understood. We have used molecular dynamics simulations in conjunction with a coarse grained model to study early metastable and transition states during the fusion of two planar palmitoyl-oleoyl-phosphatidylcholine (POPC) bilayers separated by five waters per lipid in the cis leaflets at zero tension. This system mimics the contact area between two vesicles with large diameters compared to the membrane thickness at conditions where fusion may start in the core of the contact area. At elevated temperatures, the two proximal leaflets become connected via multiple lipid molecules and form a stalklike structure. At room temperature, this structure has a free energy of 3k(B)T and is separated from the unconnected state by a significant free energy barrier of 20k(B)T. Stalk formation is initiated by the establishment of a localized hydrophobic contact between the bilayers. This contact is either formed by two partially splayed lipids or a single fully splayed one leading to the formation of a (metastable) splayed lipid bond intermediate. These findings indicate that, for low hydration, early membrane fusion kinetics is not determined by the stalk energy but by the energy of prestalk transition states involving solvent-exposed lipid tails.

Nonmonotonic incommensurability effects in lamellar-in-lamellar self-assembled multiblock copolymers.

Using the self-consistent-field theory numerical procedure we find that the period D of the lamellar-in-lamellar morphology formed in symmetric multiblock copolymer melts A(mN/2)(B(N/2)A(N/2))(n)B(mN/2) at intermediate segregations changes nonmonotonically with an increase in the relative tail length m. Therewith D reveals, as a function of the Flory chi-parameter, a drastic change in the vicinity of the internal structure formation, which can be both a drop and a rise, depending on the value of m. It is argued that the unusual behavior found is a particular case of a rather general effect of the incommensurability between the two length scales that characterize the system under consideration.

Nonconventional morphologies in two-length scale block copolymer systems beyond the weak segregation theory.

The order-disorder and order-order transitions (ODT and OOT) in the linear multiblock copolymers with two-length scale architecture A(fmN)(B(N2)A(N2))(n)B((1-f)mN) are studied under intermediate cooling below the ODT critical point where a nonconventional sequence of the OOTs was predicted previously [Smirnova et al., J. Chem. Phys. 124, 054907 (2006)] within the weak segregation theory (WST). To describe the ordered morphologies appearing in block copolymers (BCs) under cooling, we use the pseudospectral version of the self-consistent field theory (SCFT) with some modifications providing a good convergence speed and a high precision of the solution due to using the Ng iterations [J. Chem. Phys. 61, 2680 (1974)] and a reasonable choice of the predefined symmetries of the computation cell as well as initial guess for the iterations. The WST predicted sequence of the phase transitions is found to hold if the tails of the BCs under consideration are symmetric enough (mid R:0.5-fmid R:0.05, a large region of the face-centered cubic phase stability is found (up to our knowledge, first within the SCFT framework) inside of the body-centered cubic phase stability region. Occurrence of the two-dimensional and three-dimensional phases with the micelles formed, unlike the conventional diblock copolymers, by the longer (rather than shorter) tails, and its relationship to the BC architecture is first described in detail. The calculated spectra of the ordered phases show that nonmonotonous temperature dependence of the secondary peak scattering intensities accompanied by their vanishing and reappearance is rather a rule than exception.

Differential expression of duplicated LDH-A genes during temperature acclimation of weatherfish Misgurnus fossilis. Functional consequences for the enzyme.

Temperature acclimation in poikilotherms entails metabolic rearrangements provided by variations in enzyme properties. However, in most cases the underlying molecular mechanisms that result in structural changes in the enzymes are obscure. This study reports that acclimation to low (5 degrees C) and high (18 degrees C) temperatures leads to differential expression of alternative forms of the LDH-A gene in white skeletal muscle of weatherfish, Misgurnus fossilis. Two isoforms of LDH-A mRNA were isolated and characterized: a short isoform (= 1332 bp) and a long isoform ( = 1550 bp), which both have 5'-UTRs and ORFs of the same length (333 amino acid residues), but differ in the length of the 3'-UTR. In addition, these two mRNAs have 44 nucleotide point mismatches of an irregular pattern along the complete sequence, resulting in three amino acid mismatches (Gly214Val; Val304Ile and Asp312Glu) between protein products from the short and long mRNA forms, correspondingly LDH-A(alpha) and LDH-A(beta) subunits. It is expected that the beta-subunit is more aliphatic due to the properties of the mismatched amino acids and therefore sterically more restricted. According to molecular modelling of M. fossilis LDH-A, the Val304Ile mismatch is located in the subunit contact area of the tetramer, whereas the remaining two mismatches surround the contact area; this is expected to manifest in the kinetic and thermodynamic properties of the assembled tetramer. In warm-acclimated fish the relative expression between alpha and beta isoforms of the LDH-A mRNA is around 5 : 1, whereas in cold-acclimated fish expression of is reduced almost to zero. This indicates that at low temperature the pool of total tetrameric LDH-A is more homogeneous in terms of alpha/beta-subunit composition. The temperature acclimation pattern of proportional pooling of subunits with different kinetic and thermodynamic properties of the tetrameric enzyme may result in fine-tuning of the properties of skeletal LDH-A, which is in line with previously observed kinetic and thermodynamic differences between 'cold' and 'warm' LDH-A purified from weatherfish. Also, an irregular pattern of nucleotide mismatches indicates that these mRNAs are the products of two independently evolving genes, i.e. paralogues. Karyotype analysis has confirmed that the experimental population of M. fossilis is tetraploid (2n = 100), therefore gene duplication, possibly through tetraploidy, may contribute to the adaptability towards temperature variation.