Cold denaturation of DNA origami nanostructures.

The coupling of structural transitions to heat capacity changes leads to destabilization of macromolecules at both elevated and lowered temperatures. DNA origami not only exhibit this property but also provide a nanoscopic observable of cold denaturation processes by directing intramolecular strain to the most sensitive elements within their hierarchical architecture.

Superstructure-dependent stability of DNA origami nanostructures in the presence of chaotropic denaturants.

The structural stability of DNA origami nanostructures in various chemical environments is an important factor in numerous applications, ranging from biomedicine and biophysics to analytical chemistry and materials synthesis. In this work, the stability of six different 2D and 3D DNA origami nanostructures is assessed in the presence of three different chaotropic salts, i.e., guanidinium sulfate (Gdm2SO4), guanidinium chloride (GdmCl), and tetrapropylammonium chloride (TPACl), which are widely employed denaturants. Using atomic force microscopy (AFM) to quantify nanostructural integrity, Gdm2SO4 is found to be the weakest and TPACl the strongest DNA origami denaturant, respectively. Despite different mechanisms of actions of the selected salts, DNA origami stability in each environment is observed to depend on DNA origami superstructure. This is especially pronounced for 3D DNA origami nanostructures, where mechanically more flexible designs show higher stability in both GdmCl and TPACl than more rigid ones. This is particularly remarkable as this general dependence has previously been observed under Mg2+-free conditions and may provide the possibility to optimize DNA origami design toward maximum stability in diverse chemical environments. Finally, it is demonstrated that melting temperature measurements may overestimate the stability of certain DNA origami nanostructures in certain chemical environments, so that such investigations should always be complemented by microscopic assessments of nanostructure integrity.

Evaluating Viscosity and Tear Breakup Time of Contemporary Commercial Ocular Lubricants on an In Vitro Eye Model.

Purpose: To evaluate the link between the viscosity of ophthalmic formulation and tear film stability using a novel in vitro eye model. Methods: The viscosities and noninvasive tear breakup time (NIKBUT) of 13 commercial ocular lubricants were measured to evaluate the correlation between viscosity and NIKBUT. The complex viscosity of each lubricant was measured three times for each angular frequency (ranging from 0.1 to 100 rad/s) using the Discovery HR-2 hybrid rheometer. The NIKBUT measurements were performed eight times for each lubricant using an advanced eye model mounted on the OCULUS Keratograph 5M. A contact lens (CL; ACUVUE OASYS [etafilcon A]) or a collagen shield (CS) was used as the simulated corneal surface. Phosphate-buffered saline was used as a simulated fluid. Results: The results showed a positive correlation between viscosity and NIKBUT at high shear rates (at 10 rad/s, r = 0.67) but not at low shear. This correlation was even better for viscosities between 0 and 100 mPa*s (r = 0.85). Most of the lubricants tested in this study also had shear-thinning properties. OPTASE INTENSE, I-DROP PUR GEL, I DROP MGD, OASIS TEARS PLUS, and I-DROP PUR had higher viscosity in comparison to other lubricants (P < 0.05). All of the formulations had a higher NIKBUT than the control (2.7 ± 1.2 seconds for CS and 5.4 ± 0.9 seconds for CL) without any lubricant (P < 0.05). I-DROP PUR GEL, OASIS TEARS PLUS, I-DROP MGD, REFRESH OPTIVE ADVANCED, and OPTASE INTENSE had the highest NIKBUT using this eye model. Conclusions: The results show that the viscosity is correlated with NIKBUT, but further work is necessary to determine the underlying mechanisms. Translational Relevance: The viscosity of ocular lubricants can affect NIKBUT and tear film stability, so it is an important property to consider when formulating ocular lubricants.

Special Issue "Advances in Monitoring Metabolic Activities of Microorganisms by Calorimetry".

In recent decades, the calorimetric monitoring of microbial metabolism, i [...].

Laparoscopic adhesiolysis versus open adhesiolysis in acute adhesive small bowel obstruction.

Introduction: Exploratory laparotomy is still the standard therapy for patients who need surgical intervention for adhesive small bowel obstruction (SBO). However, the use of laparoscopy in the management of adhesive SBO is still controversial. We aimed to detect the short-term outcomes between open and laparoscopic adhesiolysis for SBO. Patients and Methods: This is a retrospective study of patients with adhesive SBO who underwent either laparoscopic or open surgery from June 2019 to July 2022 at Ain Shams University Hospitals. Intraoperative and early post-operative outcomes were compared in the two groups. Results: A total of 89 patients with adhesive SBO were included in our study. Fifty-one cases underwent open adhesiolysis and 38 cases underwent laparoscopic adhesiolysis. Laparoscopic adhesiolysis is associated with a remarkable decrease in the operative time (71 min vs. 107 min, P = 0.001) and blood loss (50 ml vs. 120 ml, P = 0.001) in comparison to open adhesiolysis. In addition to that, those who underwent adhesiolysis by laparoscopy had a short hospital stay (2.4 days vs. 3.8 days, P = 0.001), early recovery (time to pass flatus 1.3 days vs. 2.8 days) and less post-operative complications (surgical site infection [SSI] 2.6% vs. 19.6%, P = 0.001). Moreover, open adhesiolysis is associated with a higher rate of early post-operative mortality. In addition to that, the incidence of iatrogenic injury was higher in the open group. Conclusion: Laparoscopic adhesiolysis is a safe and feasible approach for the management of SBO and has better short-term outcomes, especially if done by skilled surgeons in advanced laparoscopic techniques.

Exploring Antibacterial Activity and Bacterial-Mediated Allotropic Transition of Differentially Coated Selenium Nanoparticles.

The use of metal nanoparticles (NPs) as antimicrobial agents has become a promising alternative to the problem of antibiotic-resistant bacteria and other applications. Silver nanoparticles (AgNPs) are well-known as one of the most universal biocide compounds. However, selenium nanoparticles (SeNPs) recently gained more attention as effective antimicrobial agents. This study aims to investigate the antibacterial activity of SeNPs with different surface coatings (BSA-coated, chitosan-coated, and undefined coating) on the Gram-negative Stenotrophomonas bentonitica and the Gram-positive Lysinibacillus sphaericus in comparison to AgNPs. The tested NPs had similar properties, including shape (spheres), structure (amorphous), and size (50-90 nm), but differed in their surface charge. Chitosan SeNPs exhibited a positive surface charge, while the remaining NPs assayed had a negative surface charge. We have found that cell growth and viability of both bacteria were negatively affected in the presence of the NPs, as indicated by microcalorimetry and flow cytometry. Specifically, undefined coating SeNPs displayed the highest percentage values of dead cells for both bacteria (85-91%). An increase in reactive oxygen species (ROS) production was also detected. Chitosan-coated and undefined SeNPs caused the highest amount of ROS (299.7 and 289% over untreated controls) for S. bentonitica and L. sphaericus, respectively. Based on DNA degradation levels, undefined-SeNPs were found to be the most hazardous, causing nearly 80% DNA degradation. Finally, electron microscopy revealed the ability of the cells to transform the different SeNP types (amorphous) to crystalline SeNPs (trigonal/monoclinical Se), which could have environmentally positive implications for bioremediation purposes and provide a novel green method for the formation of crystalline SeNPs. The results obtained herein demonstrate the promising potential of SeNPs for their use in medicine as antimicrobial agents, and we propose S. bentonitica and L. sphaericus as candidates for new bioremediation strategies and NP synthesis with potential applications in many fields.

Interdisciplinary biophysical studies of membrane proteins bacteriorhodopsin and rhodopsin.

The centenary of the birth of H. Gobind Khorana provides an auspicious opportunity to review the origins and evolution of parallel advances in biophysical methodology and molecular genetics technology used to study membrane proteins. Interdisciplinary work in the Khorana laboratory in the late 1970s and for the next three decades led to productive collaborations and fostered three subsequent scientific generations whose biophysical work on membrane proteins has led to detailed elucidation of the molecular mechanisms of energy transduction by the light-driven proton pump bacteriorhodopsin (bR) and signal transduction by the G protein-coupled receptor (GPCR) rhodopsin. This review will highlight the origins and advances of biophysical studies of membrane proteins made possible by the application of molecular genetics approaches to engineer site-specific alterations of membrane protein structures.

Distinct Effects of Chemical Toxicity and Radioactivity on Metabolic Heat of Cultured Cells Revealed by "Isotope-Editing".

Studying the toxicity of chemical compounds using isothermal microcalorimetry (IMC), which monitors the metabolic heat from living microorganisms, is a rapidly expanding field. The unprecedented sensitivity of IMC is particularly attractive for studies at low levels of stressors, where lethality-based data are inadequate. We have revealed via IMC the effect of low dose rates from radioactive ß--decay on bacterial metabolism. The low dose rate regime (<400 µGyh-1) is typical of radioactively contaminated environmental sites, where chemical toxicity and radioactivity-mediated effects coexist without a predominance or specific characteristic of either of them. We found that IMC allows distinguishing the two sources of metabolic interference on the basis of "isotope-editing" and advanced thermogram analyses. The stable and radioactive europium isotopes 153Eu and 152Eu, respectively, were employed in monitoring Lactococcus lactis cultures via IMC. ß--emission (electrons) was found to increase initial culture growth by increased nutrient uptake efficiency, which compensates for a reduced maximal cell division rate. Direct adsorption of the radionuclide to the biomass, revealed by mass spectrometry, is critical for both the initial stress response and the "dilution" of radioactivity-mediated damage at later culture stages, which are dominated by the chemical toxicity of Eu.

Simple Growth-Metabolism Relations Are Revealed by Conserved Patterns of Heat Flow from Cultured Microorganisms.

Quantitative analyses of cell replication address the connection between metabolism and growth. Various growth models approximate time-dependent cell numbers in culture media, but physiological implications of the parametrizations are vague. In contrast, isothermal microcalorimetry (IMC) measures with unprecedented sensitivity the heat (enthalpy) release via chemical turnover in metabolizing cells. Hence, the metabolic activity can be studied independently of modeling the time-dependence of cell numbers. Unexpectedly, IMC traces of various origins exhibit conserved patterns when expressed in the enthalpy domain rather than the time domain, as exemplified by cultures of Lactococcus lactis (prokaryote), Trypanosoma congolese (protozoan) and non-growing Brassica napus (plant) cells. The data comply extraordinarily well with a dynamic Langmuir adsorption reaction model of nutrient uptake and catalytic turnover generalized here to the non-constancy of catalytic capacity. Formal relations to Michaelis-Menten kinetics and common analytical growth models are briefly discussed. The proposed formalism reproduces the "life span" of cultured microorganisms from exponential growth to metabolic decline by a succession of distinct metabolic phases following remarkably simple nutrient-metabolism relations. The analysis enables the development of advanced enzyme network models of unbalanced growth and has fundamental consequences for the derivation of toxicity measures and the transferability of metabolic activity data between laboratories.

Effect of Surface Hydrophobicity on the Adsorption of a Pilus-Derived Adhesin-like Peptide.

Bacterial colonization of abiotic surfaces such as those of medical implants, membrane filters, and everyday household items is a process of tremendous importance for public health. Bacteria use adhesive cell surface structures called adhesins to establish contact with abiotic surfaces. Among them, protein filaments called type IV pili are particularly important and found in many Gram-negative pathogens such as Pseudomonas aeruginosa. Understanding the interaction of such adhesin proteins with different abiotic surfaces at the molecular level thus represents a fundamental prerequisite for impeding bacterial colonization and preventing the spread of infectious diseases. In this work, we investigate the interaction of a synthetic adhesin-like peptide, PAK128-144ox, derived from the type IV pilus of P. aeruginosa with hydrophilic and hydrophobic self-assembled monolayers (SAMs). Using a combination of molecular dynamics (MD) simulations, quartz crystal microbalance with dissipation monitoring (QCM-D), and spectroscopic investigations, we find that PAK128-144ox has a higher affinity for hydrophobic than for hydrophilic surfaces. Additionally, PAK128-144ox adsorption on the hydrophobic SAM is furthermore accompanied by a strong increase in α-helix content. Our results show a clear influence of surface hydrophobicity and further indicate that PAK128-144ox adsorption on the hydrophobic surface is enthalpically favored, while on the hydrophilic surface, entropic contributions are more significant. However, our spectroscopic investigations also suggest aggregation of the peptide under the employed experimental conditions, which is not considered in the MD simulations and should be addressed in more detail in future studies.

Anion-specific structure and stability of guanidinium-bound DNA origami.

While the folding of DNA into rationally designed DNA origami nanostructures has been studied extensively with the aim of increasing structural diversity and introducing functionality, the fundamental physical and chemical properties of these nanostructures remain largely elusive. Here, we investigate the correlation between atomistic, molecular, nanoscopic, and thermodynamic properties of DNA origami triangles. Using guanidinium (Gdm) as a DNA-stabilizing but potentially also denaturing cation, we explore the dependence of DNA origami stability on the identity of the accompanying anions. The statistical analyses of atomic force microscopy (AFM) images and circular dichroism (CD) spectra reveals that sulfate and chloride exert stabilizing and destabilizing effects, respectively, already below the global melting temperature of the DNA origami triangles. We identify structural transitions during thermal denaturation and show that heat capacity changes ΔC p determine the temperature sensitivity of structural damage. The different hydration shells of the anions and their potential to form Gdm+ ion pairs in concentrated salt solutions modulate ΔC p by altered wetting properties of hydrophobic DNA surface regions as shown by molecular dynamics simulations. The underlying structural changes on the molecular scale become amplified by the large number of structurally coupled DNA segments and thereby find nanoscopic correlations in AFM images.

Salting-Out of DNA Origami Nanostructures by Ammonium Sulfate.

DNA origami technology enables the folding of DNA strands into complex nanoscale shapes whose properties and interactions with molecular species often deviate significantly from that of genomic DNA. Here, we investigate the salting-out of different DNA origami shapes by the kosmotropic salt ammonium sulfate that is routinely employed in protein precipitation. We find that centrifugation in the presence of 3 M ammonium sulfate results in notable precipitation of DNA origami nanostructures but not of double-stranded genomic DNA. The precipitated DNA origami nanostructures can be resuspended in ammonium sulfate-free buffer without apparent formation of aggregates or loss of structural integrity. Even though quasi-1D six-helix bundle DNA origami are slightly less susceptible toward salting-out than more compact DNA origami triangles and 24-helix bundles, precipitation and recovery yields appear to be mostly independent of DNA origami shape and superstructure. Exploiting the specificity of ammonium sulfate salting-out for DNA origami nanostructures, we further apply this method to separate DNA origami triangles from genomic DNA fragments in a complex mixture. Our results thus demonstrate the possibility of concentrating and purifying DNA origami nanostructures by ammonium sulfate-induced salting-out.

DNA-Mediated Stack Formation of Nanodiscs.

Membrane-scaffolding proteins (MSPs) derived from apolipoprotein A-1 have become a versatile tool in generating nano-sized discoidal membrane mimetics (nanodiscs) for membrane protein research. Recent efforts have aimed at exploiting their controlled lipid protein ratio and size distribution to arrange membrane proteins in regular supramolecular structures for diffraction studies. Thereby, direct membrane protein crystallization, which has remained the limiting factor in structure determination of membrane proteins, would be circumvented. We describe here the formation of multimers of membrane-scaffolding protein MSP1D1-bounded nanodiscs using the thiol reactivity of engineered cysteines. The mutated positions N42 and K163 in MSP1D1 were chosen to support chemical modification as evidenced by fluorescent labeling with pyrene. Minimal interference with the nanodisc formation and structure was demonstrated by circular dichroism spectroscopy, differential light scattering and size exclusion chromatography. The direct disulphide bond formation of nanodiscs formed by the MSP1D1_N42C variant led to dimers and trimers with low yield. In contrast, transmission electron microscopy revealed that the attachment of oligonucleotides to the engineered cysteines of MSP1D1 allowed the growth of submicron-sized tracts of stacked nanodiscs through the hybridization of nanodisc populations carrying complementary strands and a flexible spacer.

Two Be or Not Two Be: The Nuclear Autoantigen La/SS-B Is Able to Form Dimers and Oligomers in a Redox Dependent Manner.

According to the literature, the autoantigen La is involved in Cap-independent translation. It was proposed that one prerequisite for this function is the formation of a protein dimer. However, structural analyses argue against La protein dimers. Noteworthy to mention, these structural analyses were performed under reducing conditions. Here we describe that La protein can undergo redox-dependent structural changes. The oxidized form of La protein can form dimers, oligomers and even polymers stabilized by disulfide bridges. The primary sequence of La protein contains three cysteine residues. Only after mutation of all three cysteine residues to alanine La protein becomes insensitive to oxidation, indicating that all three cysteines are involved in redox-dependent structural changes. Biophysical analyses of the secondary structure of La protein support the redox-dependent conformational changes. Moreover, we identified monoclonal anti-La antibodies (anti-La mAbs) that react with either the reduced or oxidized form of La protein. Differential reactivities to the reduced and oxidized form of La protein were also found in anti-La sera of autoimmune patients.

C. elegans possess a general program to enter cryptobiosis that allows dauer larvae to survive different kinds of abiotic stress.

All organisms encounter abiotic stress but only certain organisms are able to cope with extreme conditions and enter into cryptobiosis (hidden life). Previously, we have shown that C. elegans dauer larvae can survive severe desiccation (anhydrobiosis), a specific form of cryptobiosis. Entry into anhydrobiosis is preceded by activation of a set of biochemical pathways by exposure to mild desiccation. This process called preconditioning induces elevation of trehalose, intrinsically disordered proteins, polyamines and some other pathways that allow the preservation of cellular functionality in the absence of water. Here, we demonstrate that another stress factor, high osmolarity, activates similar biochemical pathways. The larvae that acquired resistance to high osmotic pressure can also withstand desiccation. In addition, high osmolarity significantly increases the biosynthesis of glycerol making larva tolerant to freezing. Thus, to survive abiotic stress, C. elegans activates a combination of genetic and biochemical pathways that serve as a general survival program.

A metabolic switch regulates the transition between growth and diapause in C. elegans.

BACKGROUND: Metabolic activity alternates between high and low states during different stages of an organism's life cycle. During the transition from growth to quiescence, a major metabolic shift often occurs from oxidative phosphorylation to glycolysis and gluconeogenesis. We use the entry of Caenorhabditis elegans into the dauer larval stage, a developmentally arrested stage formed in response to harsh environmental conditions, as a model to study the global metabolic changes and underlying molecular mechanisms associated with growth to quiescence transition. RESULTS: Here, we show that the metabolic switch involves the concerted activity of several regulatory pathways. Whereas the steroid hormone receptor DAF-12 controls dauer morphogenesis, the insulin pathway maintains low energy expenditure through DAF-16/FoxO, which also requires AAK-2/AMPKα. DAF-12 and AAK-2 separately promote a shift in the molar ratios between competing enzymes at two key branch points within the central carbon metabolic pathway diverting carbon atoms from the TCA cycle and directing them to gluconeogenesis. When both AAK-2 and DAF-12 are suppressed, the TCA cycle is active and the developmental arrest is bypassed. CONCLUSIONS: The metabolic status of each developmental stage is defined by stoichiometric ratios within the constellation of metabolic enzymes driving metabolic flux and controls the transition between growth and quiescence.

Calcium binding to a disordered domain of a type III-secreted protein from a coral pathogen promotes secondary structure formation and catalytic activity.

Strains of the Gram-negative bacterium Vibrio coralliilyticus cause the bleaching of corals due to decomposition of symbiotic microalgae. The V. coralliilyticus strain ATCC BAA-450 (Vc450) encodes a type III secretion system (T3SS). The gene cluster also encodes a protein (locus tag VIC_001052) with sequence homology to the T3SS-secreted nodulation proteins NopE1 and NopE2 of Bradyrhizobium japonicum (USDA110). VIC_001052 has been shown to undergo auto-cleavage in the presence of Ca2+ similar to the NopE proteins. We have studied the hitherto unknown secondary structure, Ca2+-binding affinity and stoichiometry of the "metal ion-inducible autocleavage" (MIIA) domain of VIC_001052 which does not possess a classical Ca2+-binding motif. CD and fluorescence spectroscopy revealed that the MIIA domain is largely intrinsically disordered. Binding of Ca2+ and other di- and trivalent cations induced secondary structure and hydrophobic packing after partial neutralization of the highly negatively charged MIIA domain. Mass spectrometry and isothermal titration calorimetry showed two Ca2+-binding sites which promote structure formation with a total binding enthalpy of -110 kJ mol-1 at a low micromolar Kd. Putative binding motifs were identified by sequence similarity to EF-hand domains and their structure analyzed by molecular dynamics simulations. The stoichiometric Ca2+-dependent induction of structure correlated with catalytic activity and may provide a "host-sensing" mechanism that is shared among pathogens that use a T3SS for efficient secretion of disordered proteins.

Body size-dependent energy storage causes Kleiber's law scaling of the metabolic rate in planarians.

Kleiber's law, or the 3/4 -power law scaling of the metabolic rate with body mass, is considered one of the few quantitative laws in biology, yet its physiological basis remains unknown. Here, we report Kleiber's law scaling in the planarian Schmidtea mediterranea. Its reversible and life history-independent changes in adult body mass over 3 orders of magnitude reveal that Kleiber's law does not emerge from the size-dependent decrease in cellular metabolic rate, but from a size-dependent increase in mass per cell. Through a combination of experiment and theoretical analysis of the organismal energy balance, we further show that the mass allometry is caused by body size dependent energy storage. Our results reveal the physiological origins of Kleiber's law in planarians and have general implications for understanding a fundamental scaling law in biology.

DNA-encircled lipid bilayers.

Lipid bilayers and lipid-associated proteins play crucial roles in biology. As in vivo studies and manipulation are inherently difficult, membrane-mimetic systems are useful for the investigation of lipidic phases, lipid-protein interactions, membrane protein function and membrane structure in vitro. In this work, we describe a route to leverage the programmability of DNA nanotechnology and create DNA-encircled bilayers (DEBs). DEBs are made of multiple copies of an alkylated oligonucleotide hybridized to a single-stranded minicircle, in which up to two alkyl chains per helical turn point to the inside of the toroidal DNA ring. When phospholipids are added, a bilayer is observed to self-assemble within the ring such that the alkyl chains of the oligonucleotides stabilize the hydrophobic rim of the bilayer to prevent formation of vesicles and support thermotropic lipid phase transitions. The DEBs are completely free of protein and can be synthesized from commercially available components using routine equipment. The diameter of DEBs can be varied in a predictable manner. The well-established toolbox from structural DNA nanotechnology, will ultimately enable the rational design of DEBs so that their size, shape or functionalization can be adapted to the specific needs of biophysical investigations of lipidic phases and the properties of membrane proteins embedded into DEB nanoparticle bilayers.