Oligodeoxynucleotides containing conformationally constrained abasic sites: a UV and fluorescence spectroscopic investigation on duplex stability and structure

The synthesis and incorporation into oligodeoxy­nucleotides of two novel, conformationally restricted abasic (AB) site analogs are described. The stability of oligonucleotide 18mer duplexes containing one such AB site opposite any of the four natural DNA bases was investigated by UV melting curve analysis and compared to that of duplexes containing a conformationally flexible propanediol unit 1 or a tetrahydrofuran unit 2 as an AB site analog. No major differences in the melting temperatures (ΔT_m 0–3°C) between the different abasic duplexes were observed. All AB duplexes were found to have T_ms that were lower by 9–15°C relative to a fully matched 18mer control duplex, and by 4–10°C relative to the corresponding 19mer duplexes in which the AB site is replaced by a mismatched nucleobase. Thus we conclude that the loss of stability of a duplex that is encountered by removal of a nucleobase from the stack cannot be compensated with conformational restriction of the AB site. From the van’t Hoff transition enthalpies obtained from the melting curves, it appears that melting cooperativity is higher for the duplexes containing the conformationally rigid AB sites. Fluorescence quenching experiments with duplexes containing the fluorescent base 2-amino­purine (2AP) opposite the AB sites showed a weak tendency towards more efficient stacking of this base in duplexes containing the conformationally constrained AB sites. Thus, such AB sites may structurally stabilize the cavity formed by the removal of a base. Potential applications emerging from the properties of such conformationally constrained AB sites in DNA diagnostics are discussed.     

Thermodynamic and hydration effects for the incorporation of a cationic 3-aminopropyl chain into DNA

The introduction of cationic 5-(ω-aminoalkyl)-2′-deoxypyrimidines into duplex DNA has been shown to induce DNA bending. In order to understand the energetic and hydration contributions for the incorporation of a cationic side chain in DNA a combination of spectroscopy, calorimetry and density techniques were used. Specifically, the temperature unfolding and isothermal formation was studied for a pair of duplexes with sequence d(CGTAGUCG TGC)/d(GCACGACTACG), where U represents 2′-deoxyuridine (‘control’) or 5-(3-aminopropyl)-2′-deoxyuridine (‘modified’). Continuous variation experiments confirmed 1:1 stoichiometries for each duplex and the circular dichroism spectra show that both duplexes adopted the B conformation. UV and differential scanning calorimetry melting experiments reveal that each duplex unfolds in two-state transitions. In low salt buffer, the ‘modified’ duplex is more stable and unfolds with a lower endothermic heat and lower release of counterion and water. This electrostatic stabilization is entropy driven and disappears at higher salt concentrations. Complete thermodynamic profiles at 15°C show that the favorable formation of each duplex results from the compensation of a favorable exothermic heat with an unfavorable entropy contribution. However, the isothermal profiles yielded a differential enthalpy of 8.8 kcal/mol, which is 4.3 kcal/mol higher than the differential enthalpy observed in the unfolding profiles. This indicates that the presence of the aminopropyl chain induces an increase in base stacking interactions in the modified single strand and a decrease in base stacking interactions in the modified duplex. Furthermore, the formation of the ‘control’ duplex releases water while the ‘modified’ duplex takes up water. Relative to the control duplex, formation of the modified duplex at 15°C yielded a marginal differential ΔG° term, positive ΔΔH_ITC–Δ(TΔS) compensation, negative ΔΔV and a net release of counterions. The opposite signs of the differential enthalpy–entropy compensation and differential volume change terms show a net uptake of structural water around polar and non-polar groups. This indicates that incorporation of the aminopropyl chain induces a higher exposure of aromatic bases to the solvent, which may be consistent with a small and local bend in the ‘modified’ duplex.     

Energetic signatures of single base bulges: thermodynamic consequences and biological implications

DNA bulges are biologically consequential defects that can arise from template-primer misalignments during replication and pose challenges to the cellular DNA repair machinery. Calorimetric and spectroscopic characterizations of defect-containing duplexes reveal systematic patterns of sequence-context dependent bulge-induced destabilizations. These distinguishing energetic signatures are manifest in three coupled characteristics, namely: the magnitude of the bulge-induced duplex destabilization (ΔΔG_Bulge); the thermodynamic origins of ΔΔG_Bulge (i.e. enthalpic versus entropic); and, the cooperativity of the duplex melting transition (i.e. two-state versus non-two state). We find moderately destabilized duplexes undergo two-state dissociation and exhibit ΔΔG_Bulge values consistent with localized, nearest neighbor perturbations arising from unfavorable entropic contributions. Conversely, strongly destabilized duplexes melt in a non-two-state manner and exhibit ΔΔG_Bulge values consistent with perturbations exceeding nearest-neighbor expectations that are enthalpic in origin. Significantly, our data reveal an intriguing correlation in which the energetic impact of a single bulge base centered in one strand portends the impact of the corresponding complementary bulge base embedded in the opposite strand. We discuss potential correlations between these bulge-specific differential energetic profiles and their overall biological implications in terms of DNA recognition, repair and replication.     

DNA multiplex hybridization on microarrays and thermodynamic stability in solution: a direct comparison

Hybridization intensities of 30 distinct short duplex DNAs measured on spotted microarrays, were directly compared with thermodynamic stabilities measured in solution. DNA sequences were designed to promote formation of perfect match, or hybrid duplexes containing tandem mismatches. Thermodynamic parameters ΔH°, ΔS° and ΔG° of melting transitions in solution were evaluated directly using differential scanning calorimetry. Quantitative comparison with results from 63 multiplex microarray hybridization experiments provided a linear relationship for perfect match and most mismatch duplexes. Examination of outliers suggests that both duplex length and relative position of tandem mismatches could be important factors contributing to observed deviations from linearity. A detailed comparison of measured thermodynamic parameters with those calculated using the nearest-neighbor model was performed. Analysis revealed the nearest-neighbor model generally predicts mismatch duplexes to be less stable than experimentally observed. Results also show the relative stability of a tandem mismatch is highly dependent on the identity of the flanking Watson–Crick (w/c) base pairs. Thus, specifying the stability contribution of a tandem mismatch requires consideration of the sequence identity of at least four base pair units (tandem mismatch and flanking w/c base pairs). These observations underscore the need for rigorous evaluation of thermodynamic parameters describing tandem mismatch stability.     

The First Identification of Carbohydrate Binding Modules Specific to Chitosan

Two carbohydrate binding modules (DD1 and DD2) belonging to CBM32 are located at the C terminus of a chitosanase from Paenibacillus sp. IK-5. We produced three proteins, DD1, DD2, and tandem DD1/DD2 (DD1+DD2), and characterized their binding ability. Transition temperature of thermal unfolding (T_m) of each protein was elevated by the addition of cello-, laminari-, chitin-, or chitosan-hexamer (GlcN)₆. The T_m elevation (ΔT_m) in DD1 was the highest (10.3 °C) upon the addition of (GlcN)₆ and was markedly higher than that in DD2 (1.0 °C). A synergistic effect was observed (ΔT_m = 13.6 °C), when (GlcN)₆ was added to DD1+DD2. From isothermal titration calorimetry experiments, affinities to DD1 were not clearly dependent upon chain length of (GlcN)_n; ΔG_r° values were −7.8 (n = 6), −7.6 (n = 5), −7.6 (n = 4), −7.6 (n = 3), and −7.1 (n = 2) kcal/mol, and the value was not obtained for GlcN due to the lowest affinity. DD2 bound (GlcN)_n with the lower affinities (ΔG_r° = −5.0 (n = 3) ∼ −5.2 (n = 6) kcal/mol). Isothermal titration calorimetry profiles obtained for DD1+DD2 exhibited a better fit when the two-site model was used for analysis and provided greater affinities to (GlcN)₆ for individual DD1 and DD2 sites (ΔG_r° = −8.6 and −6.4 kcal/mol, respectively). From NMR titration experiments, (GlcN)_n (n = 2∼6) were found to bind to loops extruded from the core β-sandwich of individual DD1 and DD2, and the interaction sites were similar to each other. Taken together, DD1+DD2 is specific to chitosan, and individual modules synergistically interact with at least two GlcN units, facilitating chitosan hydrolysis.     

Detection of guanine-adenine mismatches by surface plasmon resonance sensor carrying naphthyridine-azaquinolone hybrid on the surface

We have discovered a new molecule naphthyridine–azaquinolone hybrid (Npt–Azq) that strongly stabilized the guanine-adenine (G-A) mismatch in duplex DNA. In the presence of Npt–Azq, the melting temperature (T_m) of 5′-d(CTA ACG GAA TG)-3′/3′-d(GAT TGA CTT AC)-5′ containing a single G-A mismatch increased by 15.4°C, whereas fully matched duplex increased its T_m only by 2.2°C. Npt–Azq was immobilized on the sensor surface for the surface plasmon resonance (SPR) assay to examine SPR detection of duplexes containing a G-A mismatch. Distinct SPR signals were observed when 27mer DNA containing a G-A mismatch was analyzed by the Npt–Azq immobilized sensor surfaces, whereas the signal of the fully matched duplex was ~6-fold weaker in intensity. The SPR signals for the G-A mismatch were proportional to the concentration of DNA in a range up to 1 µM, confirming that the SPR signal is in fact due to the binding of the G-A mismatch to Npt–Azq immobilized on the surface. Examination of all 16 G-A mismatches regarding the flanking sequence revealed that the sensor surface reported here is applicable to eight flanking sequences, covering 50% of all possible G-A mismatches.     

Determination of base and backbone contributions to the thermodynamics of premelting and melting transitions in B DNA

In previous papers of this series the temperature-dependent Raman spectra of poly(dA)·poly(dT) and poly(dA–dT)·poly(dA–dT) were used to characterize structurally the melting and premelting transitions in DNAs containing consecutive A·T and alternating A·T/T·A base pairs. Here, we describe procedures for obtaining thermodynamic parameters from the Raman data. The method exploits base-specific and backbone-specific Raman markers to determine separate thermodynamic contributions of A, T and deoxyribosyl-phosphate moieties to premelting and melting transitions. Key findings include the following: (i) Both poly(dA)·poly(dT) and poly(dA–dT)· poly(dA–dT) exhibit robust premelting transitions, due predominantly to backbone conformational changes. (ii) The significant van’t Hoff premelting enthalpies of poly(dA)·poly(dT) [ΔH_vH^pm = 18.0 ± 1.6 kcal·mol^–1 (kilocalories per mole cooperative unit)] and poly(dA–dT)·poly(dA–dT) (ΔH_vH^pm = 13.4 ± 2.5 kcal·mol^–1) differ by an amount (~4.6 kcal·mol^–1) estimated as the contribution from three-centered inter-base hydrogen bonding in (dA)_n·(dT)_n tracts. (iii) The overall stacking free energy of poly(dA)· poly(dT) [–6.88 kcal·mol_bp^–1 (kilocalories per mole base pair)] is greater than that of poly(dA–dT)· poly(dA–dT) (–6.31 kcal·mol_bp^–1). (iv) The difference between stacking free energies of A and T is significant in poly(dA)·poly(dT) (ΔΔG_st = 0.8 ± 0.3 kcal· mol_bp^–1), but marginal in poly(dA–dT)·poly(dA–dT) (ΔΔG_st = 0.3 ± 0.3 kcal·mol_bp^–1). (v) In poly(dA)· poly(dT), the van’t Hoff parameters for melting of A (ΔH_vH^A = 407 ± 23 kcal·mol^–1, ΔS_vH^A = 1166 ± 67 cal·°K^–1·mol^–1, ΔG_vH(25°C)^A = 60.0 ± 3.2 kcal·mol^–1) are clearly distinguished from those of T (ΔH_vH^T = 185 ± 38 kcal·mol^–1, ΔS_vH^T = 516 ± 109 cal·°K^–1·mol^–1, ΔG_vH(25°C)^T = 27.1 ± 5.5 kcal·mol^–1). (vi) Similar relative differences are observed in poly(dA–dT)· poly(dA–dT) (ΔH_vH^A = 333 ± 54 kcal·mol^–1, ΔS_vH^A = 961 ± 157 cal·°K^–1·mol^–1, ΔG_vH(25°C)^A = 45.0 ± 7.6 kcal· mol^–1; ΔH_vH^T = 213 ± 30 kcal·mol^–1, ΔS_vH^T = 617 ± 86 cal·°K^–1·mol^–1, ΔG_vH(25°C)^T = 29.3 ± 4.9 kcal·mol^–1). The methodology employed here distinguishes thermodynamic contributions of base stacking, base pairing and backbone conformational ordering in the molecular mechanism of double-helical B DNA formation.     

Conformational constraints of cyclopentane peptide nucleic acids facilitate tunable binding to DNA

We report a series of synthetic, nucleic acid mimics with highly customizable thermodynamic binding to DNA. Incorporation of helix-promoting cyclopentanes into peptide nucleic acids (PNAs) increases the melting temperatures (T_m) of PNA+DNA duplexes by approximately +5°C per cyclopentane. Sequential addition of cyclopentanes allows the T_m of PNA + DNA duplexes to be systematically fine-tuned from +5 to +50°C compared with the unmodified PNA. Containing only nine nucleobases and an equal number of cyclopentanes, cpPNA-9 binds to complementary DNA with a T_m around 90°C. Additional experiments reveal that the cpPNA-9 sequence specifically binds to DNA duplexes containing its complementary sequence and functions as a PCR clamp. An X-ray crystal structure of the cpPNA-9–DNA duplex revealed that cyclopentanes likely induce a right-handed helix in the PNA with conformations that promote DNA binding.     

Optimal design of parallel triplex forming oligonucleotides containing Twisted Intercalating Nucleic Acids—TINA

Twisted intercalating nucleic acid (TINA) is a novel intercalator and stabilizer of Hoogsteen type parallel triplex formations (PT). Specific design rules for position of TINA in triplex forming oligonucleotides (TFOs) have not previously been presented. We describe a complete collection of easy and robust design rules based upon more than 2500 melting points (T_m) determined by FRET. To increase the sensitivity of PT, multiple TINAs should be placed with at least 3 nt in-between or preferable one TINA for each half helixturn and/or whole helixturn. We find that ΔT_m of base mismatches on PT is remarkably high (between 7.4 and 15.2°C) compared to antiparallel duplexes (between 3.8 and 9.4°C). The specificity of PT by ΔT_m increases when shorter TFOs and higher pH are chosen. To increase ΔTms, base mismatches should be placed in the center of the TFO and when feasible, A, C or T to G base mismatches should be avoided. Base mismatches can be neutralized by intercalation of a TINA on each side of the base mismatch and masked by a TINA intercalating direct 3′ (preferable) or 5′ of it. We predict that TINA stabilized PT will improve the sensitivity and specificity of DNA based clinical diagnostic assays.     

Improved nearest-neighbor parameters for the stability of RNA/DNA hybrids under a physiological condition

The stability of Watson–Crick paired RNA/DNA hybrids is important for designing optimal oligonucleotides for ASO (Antisense Oligonucleotide) and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)–Cas9 techniques. Previous nearest-neighbour (NN) parameters for predicting hybrid stability in a 1 M NaCl solution, however, may not be applicable for predicting stability at salt concentrations closer to physiological condition (e.g. ∼100 mM Na⁺ or K⁺ in the presence or absence of Mg²⁺). Herein, we report measured thermodynamic parameters of 38 RNA/DNA hybrids at 100 mM NaCl and derive new NN parameters to predict duplex stability. Predicted ΔG°₃₇ and T_m values based on the established NN parameters agreed well with the measured values with 2.9% and 1.1°C deviations, respectively. The new results can also be used to make precise predictions for duplexes formed in 100 mM KCl or 100 mM NaCl in the presence of 1 mM Mg²⁺, which can mimic an intracellular and extracellular salt condition, respectively. Comparisons of the predicted thermodynamic parameters with published data using ASO and CRISPR–Cas9 may allow designing shorter oligonucleotides for these techniques that will diminish the probability of non-specific binding and also improve the efficiency of target gene regulation.     

3'-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures

DNA probes with conjugated minor groove binder (MGB) groups form extremely stable duplexes with single-stranded DNA targets, allowing shorter probes to be used for hybridization based assays. In this paper, sequence specificity of 3′-MGB probes was explored. In comparison with unmodified DNA, MGB probes had higher melting temperature (T_m) and increased specificity, especially when a mismatch was in the MGB region of the duplex. To exploit these properties, fluorogenic MGB probes were prepared and investigated in the 5′-nuclease PCR assay (real-time PCR assay, TaqMan assay). A 12mer MGB probe had the same T_m (65°C) as a no-MGB 27mer probe. The fluorogenic MGB probes were more specific for single base mismatches and fluorescence quenching was more efficient, giving increased sensitivity. A/T rich duplexes were stabilized more than G/C rich duplexes, thereby leveling probe T_m and simplifying design. In summary, MGB probes were more sequence specific than standard DNA probes, especially for single base mismatches at elevated hybridization temperatures.     

RNase H mediated cleavage of RNA by cyclohexene nucleic acid (CeNA)

Cyclohexene nucleic acid (CeNA) forms a duplex with RNA that is more stable than a DNA–RNA duplex (ΔT_m per modification: +2°C). A cyclohexenyl A nucleotide adopts a 3′-endo conformation when introduced in dsDNA. The neighbouring deoxynucleotide adopts an O4′-endo conformation. The CeNA:RNA duplex is cleaved by RNase H. The V_max and K_m of the cleavage reaction for CeNA:RNA and DNA:RNA is in the same range, although the k_cat value is about 600 times lower in the case of CeNA:RNA.     

Markov state models elucidate the stability of DNA influenced by the chiral 5S-Tg base

The static and dynamic structures of DNA duplexes affected by 5S-Tg (Tg, Thymine glycol) epimers were studied using MD simulations and Markov State Models (MSMs) analysis. The results show that the 5S,6S-Tg base caused little perturbation to the helix, and the base-flipping barrier was determined to be 4.4 kcal mol⁻¹ through the use of enhanced sampling meta-eABF calculations, comparable to 5.4 kcal mol⁻¹ of the corresponding thymine flipping. Two conformations with the different hydrogen bond structures between 5S,6R-Tg and A19 were identified in several independent MD trajectories. The 5S,6R-Tg:O6H_O6•••N1:A19 hydrogen bond is present in the high-energy conformation displaying a clear helical distortion, and near barrier-free Tg base flipping. The low-energy conformation always maintains Watson–Crick base pairing between 5S,6R-Tg and A19, and 5S-Tg base flipping is accompanied by a small barrier of ca. 2.0 K_BT (T = 298 K). The same conformations are observed in the MSMs analysis. Moreover, the transition path and metastable structures of the damaged base flipping are for the first time verified through MSMs analysis. The data clearly show that the epimers have completely different influence on the stability of the DNA duplex, thus implying different enzymatic mechanisms for DNA repair.     

Cold denaturation of monoclonal antibodies

The susceptibility of monoclonal antibodies (mAbs) to undergo cold denaturation remains unexplored. In this study, the phenomenon of cold denaturation was investigated for a mAb, mAb1, through thermodynamic and spectroscopic analyses. tryptophan fluorescence and circular dichroism (CD) spectra were recorded for the guanidine hydrochloride (GuHCl)-induced unfolding of mAb1 at pH 6.3 at temperatures ranging from −5 to 50°C. A three-state unfolding model incorporating the linear extrapolation method was fit to the fluorescence data to obtain an apparent free energy of unfolding, ΔG_u, at each temperature. CD studies revealed that mAb1 exhibited polyproline II helical structure at low temperatures and at high GuHCl concentrations. the Gibbs-Helmholtz expression fit to the ΔG_u versus temperature data from fluorescence gave a ΔC_p of 8.0 kcal mol⁻¹ K⁻¹, a maximum apparent stability of 23.7 kcal mol⁻¹ at 18°C, and an apparent cold denaturation temperature (T_CD) of −23°C. ΔG_u values for another mAb (mAb2) with a similar framework exhibited less stability at low temperatures, suggesting a depressed protein stability curve and a higher relative T_CD. Direct experimental evidence of the susceptibility of mAb1 and mAb2 to undergo cold denaturation in the absence of denaturant was confirmed at pH 2.5. thus, mAbs have a potential to undergo cold denaturation at storage temperatures near −20°C (pH 6.3), and this potential needs to be evaluated independently for individual mAbs.Key words: monoclonal antibodies, thermodynamic stability, cold denaturation, free energy, fluorescence     

Clamping down on weak terminal base pairs: oligonucleotides with molecular caps as fidelity-enhancing elements at the 5'- and 3'-terminal residues

The base-pairing fidelity of oligonucleotides depends on the identity of the nucleobases involved and the position of matched or mismatched base pairs in the duplex. Nucleobases forming weak base pairs, as well as a terminal position favor mispairing. We have searched for 5′-appended acylamido caps that enhance the stability and base-pairing fidelity of oligonucleotides with a 5′-terminal 2′-deoxyadenosine residue using combinatorial synthesis and MALDI-monitored nuclease selections. This provided the residue of 4-(pyren-1-yl)butyric acid as a lead. Lead optimization gave (S)-N-(pyren-1-ylmethyl)pyrrolidine-3-phosphate as a cap that increases duplex stability and base-pairing fidelity. For the duplex of 5′-AGGTTGAC-3′ with its fully complementary target, this cap gives an increase in the UV melting point T_m of +10.9°C. The T_m is 6.3–8.3°C lower when a mismatched nucleobase faces the 5′-terminal dA residue. The optimized cap can be introduced via automated DNA synthesis. It was combined with an anthraquinone carboxylic acid residue as a cap for the 3′-terminal residue. A doubly capped dodecamer thus prepared gives a melting point decrease for double-terminal mismatches that is 5.7–5.9°C greater than that for the unmodified control duplex.     

New insights into DNA triplexes: residual twist and radial difference as measures of base triplet non-isomorphism and their implication to sequence-dependent non-uniform DNA triplex

DNA triplexes are formed by both isomorphic (structurally alike) and non-isomorphic (structurally dissimilar) base triplets. It is espoused here that (i) the base triplet non-isomorphism may be articulated in structural terms by a residual twist (Δt°), the angle formed by line joining the C1′…C1′ atoms of the adjacent Hoogsteen or reverse Hoogsteen (RH) base pairs and the difference in base triplet radius (Δr Å), and (ii) their influence on DNA triplex is largely mechanistic, leading to the prediction of a high (t + Δt)° and low (t − Δt)° twist at the successive steps of Hoogsteen or RH duplex of a parallel or antiparallel triplex. Efficacy of this concept is corroborated by molecular dynamics (MD) simulation of an antiparallel DNA triplex comprising alternating non-isomorphic G*GC and T*AT triplets. Conformational changes necessitated by base triplet non-isomorphism are found to induce an alternating (i) high anti and anti glycosyl and (ii) BII and an unusual BIII conformation resulting in a zigzag backbone for the RH strand. Thus, base triplet non-isomorphism causes DNA triplexes into exhibiting sequence-dependent non-uniform conformation. Such structural variations may be relevant in deciphering the specificity of interaction with DNA triplex binding proteins. Seemingly then, residual twist (Δt°) and radial difference (Δr Å) suffice as indices to define and monitor the effect of base triplet non-isomorphism in nucleic acid triplexes.     

A thermodynamic overview of naturally occurring intramolecular DNA quadruplexes

Loop length and its composition are important for the structural and functional versatility of quadruplexes. To date studies on the loops have mainly concerned model sequences compared with naturally occurring quadruplex sequences which have diverse loop lengths and compositions. Herein, we have characterized 36 quadruplex-forming sequences from the promoter regions of various proto-oncogenes using CD, UV and native gel electrophoresis. We examined folding topologies and determined the thermodynamic profile for quadruplexes varying in total loop length (5–18 bases) and composition. We found that naturally occurring quadruplexes have variable thermodynamic stabilities (ΔG₃₇) ranging from −1.7 to −15.6 kcal/mol. Overall, our results suggest that both loop length and its composition affect quadruplex structure and thermodynamics, thus making it difficult to draw generalized correlations between loop length and thermodynamic stability. Additionally, we compared the thermodynamic stability of quadruplexes and their respective duplexes to understand quadruplex–duplex competition. Our findings invoke a discussion on whether biological function is associated with quadruplexes with lower thermodynamic stability which undergo facile formation and disruption, or by quadruplexes with high thermodynamic stability.     

Pure-Culture Growth of Fermentative Bacteria, Facilitated by H2 Removal: Bioenergetics and H2 Production

We used an H₂-purging culture vessel to replace an H₂-consuming syntrophic partner, allowing the growth of pure cultures of Syntrophothermus lipocalidus on butyrate and Aminobacterium colombiense on alanine. By decoupling the syntrophic association, it was possible to manipulate and monitor the single organism's growth environment and determine the change in Gibbs free energy yield (ΔG) in response to changes in the concentrations of reactants and products, the purging rate, and the temperature. In each of these situations, H₂ production changed such that ΔG remained nearly constant for each organism (−11.1 ± 1.4 kJ mol butyrate⁻¹ for S. lipocalidus and −58.2 ± 1.0 kJ mol alanine⁻¹ for A. colombiense). The cellular maintenance energy, determined from the ΔG value and the hydrogen production rate at the point where the cell number was constant, was 4.6 × 10⁻¹³ kJ cell⁻¹ day⁻¹ for S. lipocalidus at 55°C and 6.2 × 10⁻¹³ kJ cell⁻¹ day⁻¹ for A. colombiense at 37°C. S. lipocalidus, in particular, seems adapted to thrive under conditions of low energy availability.     

Mitochondrial Networks in Cardiac Myocytes Reveal Dynamic Coupling Behavior

Oscillatory behavior of mitochondrial inner membrane potential (ΔΨ_m) is commonly observed in cells subjected to oxidative or metabolic stress. In cardiac myocytes, the activation of inner membrane pores by reactive oxygen species (ROS) is a major factor mediating intermitochondrial coupling, and ROS-induced ROS release has been shown to underlie propagated waves of ΔΨ_m depolarization as well as synchronized limit cycle oscillations of ΔΨ_m in the network. The functional impact of ΔΨ_m instability on cardiac electrophysiology, Ca²⁺ handling, and even cell survival, is strongly affected by the extent of such intermitochondrial coupling. Here, we employ a recently developed wavelet-based analytical approach to examine how different substrates affect mitochondrial coupling in cardiac cells, and we also determine the oscillatory coupling properties of mitochondria in ventricular cells in intact perfused hearts. The results show that the frequency of ΔΨ_m oscillations varies inversely with the size of the oscillating mitochondrial cluster, and depends on the strength of local intermitochondrial coupling. Time-varying coupling constants could be quantitatively determined by applying a stochastic phase model based on extension of the well-known Kuramoto model for networks of coupled oscillators. Cluster size-frequency relationships varied with different substrates, as did mitochondrial coupling constants, which were significantly larger for glucose (7.78 × 10⁻² ± 0.98 × 10⁻² s⁻¹) and pyruvate (7.49 × 10⁻² ± 1.65 × 10⁻² s⁻¹) than lactate (4.83 × 10⁻² ± 1.25 × 10⁻² s⁻¹) or β-hydroxybutyrate (4.11 × 10⁻² ± 0.62 × 10⁻² s⁻¹). The findings indicate that mitochondrial spatiotemporal coupling and oscillatory behavior is influenced by substrate selection, perhaps through differing effects on ROS/redox balance. In particular, glucose-perfusion generates strong intermitochondrial coupling and temporal oscillatory stability. Pathological changes in specific catabolic pathways, which are known to occur during the progression of cardiovascular disease, could therefore contribute to altered sensitivity of the mitochondrial network to oxidative stress and emergent ΔΨ_m instability, ultimately scaling to produce organ level dysfunction.     

Design Maps for the Hyperthermic Treatment of Tumors with Superparamagnetic Nanoparticles

A plethora of magnetic nanoparticles has been developed and investigated under different alternating magnetic fields (AMF) for the hyperthermic treatment of malignant tissues. Yet, clinical applications of magnetic hyperthermia are sporadic, mostly due to the low energy conversion efficiency of the metallic nanoparticles and the high tissue concentrations required. Here, we study the hyperthermic performance of commercially available formulations of superparamagnetic iron oxide nanoparticles (SPIOs), with core diameter of 5, 7 and 14 nm, in terms of absolute temperature increase ΔT and specific absorption rate (SAR). These nanoparticles are operated under a broad range of AMF conditions, with frequency f varying between 0.2 and 30 MHz; field strength H ranging from 4 to 10 kA m⁻¹; and concentration c_MNP varying from 0.02 to 3.5 mg ml⁻¹. At high frequency field (∼30 MHz), non specific heating dominates and ΔT correlates with the electrical conductivity of the medium. At low frequency field (<1 MHz), non specific heating is negligible and the relaxation of the SPIO within the AMF is the sole energy source. We show that the ΔT of the medium grows linearly with c_MNP, whereas the SAR_MNP of the magnetic nanoparticles is independent of c_MNP and varies linearly with f and H². Using a computational model for heat transport in a biological tissue, the minimum requirements for local hyperthermia (T_tissue >42°C) and thermal ablation (T_tissue >50°C) are derived in terms of c_MNP, operating AMF conditions and blood perfusion. The resulting maps can be used to rationally design hyperthermic treatments and identifying the proper route of administration – systemic versus intratumor injection – depending on the magnetic and biodistribution properties of the nanoparticles.