Straw from Different Crop Species Recruits Different Communities of Lignocellulose-Degrading Microorganisms in Black Soil.

The biological degradation of plant residues in the soil or on the soil surface is an integral part of the natural life cycle of annual plants and does not have adverse effects on the environment. Crop straw is characterized by a complex structure and exhibits stability and resistance to rapid microbial decomposition. In this study, we conducted a microcosm experiment to investigate the dynamic succession of the soil microbial community and the functional characteristics associated with lignocellulose-degrading pathways. Additionally, we aimed to identify lignocellulose-degrading microorganisms from the straw of three crop species prevalent in Northeast China: soybean (Glycine max Merr.), rice (Oryza sativa L.), and maize (Zea mays L.). Our findings revealed that both the type of straw and the degradation time influenced the bacterial and fungal community structure and composition. Metagenome sequencing results demonstrated that during degradation, different straw types assembled carbohydrate-active enzymes (CAZymes) and KEGG pathways in distinct manners, contributing to lignocellulose and hemicellulose degradation. Furthermore, isolation of lignocellulose-degrading microbes yielded 59 bacterial and 14 fungal strains contributing to straw degradation, with fungi generally exhibiting superior lignocellulose-degrading enzyme production compared to bacteria. Experiments were conducted to assess the potential synergistic effects of synthetic microbial communities (SynComs) comprising both fungi and bacteria. These SynComs resulted in a straw weight loss of 42% at 15 days post-inoculation, representing a 22% increase compared to conditions without any SynComs. In summary, our study provides novel ecological insights into crop straw degradation by microbes.

Soil microbes mediate the effects of resource variability on plant invasion.

A fundamental question in ecology is which species will prevail over others amid changes in both environmental mean conditions and their variability. Although the widely accepted fluctuating resource hypothesis predicts that increases in mean resource availability and variability therein will promote nonnative plant invasion, it remains unclear to what extent these effects might be mediated by soil microbes. We grew eight invasive nonnative plant species as target plants in pot-mesocosms planted with five different synthetic native communities as competitors, and assigned them to eight combinations of two nutrient-fluctuation (constant vs. pulsed), two nutrient-availability (low vs. high) and two soil-microbe (living vs. sterilized) treatments. We found that when plants grew in sterilized soil, nutrient fluctuation promoted the dominance of nonnative plants under overall low nutrient availability, whereas the nutrient fluctuation had minimal effect under high nutrient availability. In contrast, when plants grew in living soil, nutrient fluctuation promoted the dominance of nonnative plants under high nutrient availability rather than under low nutrient availability. Analysis of the soil microbial community suggests that this might reflect that nutrient fluctuation strongly increased the relative abundance of the most dominant pathogenic fungal family or genus under high nutrient availability, while decreasing it under low nutrient availability. Our findings are the first to indicate that besides its direct effect, environmental variability could also indirectly affect plant invasion via changes in soil microbial communities.

Suppression of alias and replica noises in phase holograms using fractal topologies.

Two-dimensional fractal topologies featuring (scaling) self-similarity, dense set of Bragg (diffraction) peaks, and inherent rotation symmetry, which are not achievable with regular grid-matrix geometries, exhibit optical robustness against structural damage and noise immunity of optical transmission paths. In this work, we numerically and experimentally demonstrate phase holograms using fractal plane-divisions. By taking advantage of the symmetries of the fractal topology, we propose numerical algorithms to design the fractal holograms. This algorithm solves the inapplicability of the conventional iterative Fourier transform algorithm (IFTA) method and enables efficient optimizations of millions of adjustable parameters in the optical element. Experimental samples show that the alias and replica noises in the image plane of fractal holograms are clearly suppressed, facilitating applications for high-accuracy and compact requirements.

H∞ Control for Stochastic Singular Systems With Time-Varying Delays via Sampled-Data Controller.

In this article, H∞ control for stochastic singular time-varying delay systems under arbitrarily variable samplings is addressed via designing a sampled-data controller. The first and foremost, a novel time-dependent discontinuous Lyapunov-Krasovskii (L-K) functional is built, which takes good advantage of the factual sampling pattern's available properties. Then, based on the refined input delay method by utilizing the constructed time-dependent L-K functional, the free-weighting matrix method, and the auxiliary vector function approach are adopted to develop conditions ensuring the stochastic admissibility for the studied stochastic singular systems with time-varying delays. On the basis of the derived conditions, the sampled-data H∞ control issue is tackled, and an unambiguous expression for the sampled-data controller design method is obtained. Finally, simulation examples manifest that our proposed results are correct and effective.

Impacts of replanting American ginseng on fungal assembly and abundance in response to disease outbreaks.

Soil physicochemical properties and fungal communities are pivotal factors for continuous cropping of American ginseng (Panax quinquefolium L.). However, the response of soil physicochemical properties and fungal communities to replant disease of American ginseng has not yet been studied. High-throughput sequencing and soil physicochemical analyses were undertaken to investigate the difference of soil fungal communities and environmental driver factors in new and old ginseng fields; the extent of replant disease in old ginseng fields closely related to changes in soil properties and fungal communities was also determined. Results indicated that fungal communities in an old ginseng field were more sensitive to the soil environment than those in a new ginseng field, and fungal communities were mainly driven by soil organic matter (SOM), soil available phosphorus (AP), and available potassium (AK). Notably, healthy ginseng plants in new and old ginseng fields may influence fungal communities by actively recruiting potential disease suppressive fungal agents such as Amphinema, Cladophialophora, Cadophora, Mortierella, and Wilcoxina. When these key groups and members were depleted, suppressive agents in the soil possibly declined, increasing the abundance of pathogens. Soil used to grow American ginseng in the old ginseng field contained a variety of fungal pathogens, including Alternaria, Armillaria, Aphanoascus, Aspergillus, Setophoma, and Rhexocercosporidium. Additionally, micro-ecological factors affecting disease outbreaks in the old ginseng field included a strengthening in competition relationships, a weakening in cooperation relationships, and a change of trophic strategies among fungal communities.

Self-Crossing Leads to Weak Co-Variation of the Bacterial and Fungal Communities in the Rice Rhizosphere.

The rhizomicrobial community is influenced by plant genotype. However, the potential differences in the co-assembly of bacterial and fungal communities between parental lines and different generations of rice progenies have not been examined. Here we compared the bacterial and fungal communities in the rhizomicrobiomes of female parent Oryza rufipogon wild rice; male parent Oryza sativa cultivated rice; their F1 progeny; and the F2, F3 and F4 self-crossing generations. Our results showed that the bacterial and fungal α-diversities of the hybrid F1 and self-crossing generations (F2, F3, F4) were closer to one of the two parental lines, which may indicate a role of the parental line in the diversity of the rhizosphere microbial community assembly. Self-crossing from F1 to F4 led to weak co-variation of the bacterial and fungal communities and distinct rhizosphere microbiomes. In the parental and self-crossing progenies, the reduction of community dissimilarity was higher for the fungal community than for the bacterial community.

Connection the Rhizomicrobiome and Plant MAPK Gene Expression Response to Pathogenic Fusarium oxysporum in Wild and Cultivated Soybean.

Little known the connections between soybeans mitogen-activated protein kinase (MAPK) gene expression and the rhizomicrobiome upon invasion of the root pathogen Fusarium oxysporum. To address this lack of knowledge, we assessed the rhizomicrobiome and root transcriptome sequencing of wild and cultivated soybean during the invasion of F. oxysporum. Results indicated F. oxysporum infection enriched Bradyrhizobium spp. and Glomus spp. and induced the expression of more MAPKs in the wild soybean than cultivated soybean. MAPK gene expression was positively correlated with Pseudomonadaceae but negatively correlated with Sphingomonadaceae and Glomeraceae in both cultivated and wild soybean. Specifically, correlation profiles revealed that Pseudomonadaceae was especially correlated with the induced expression of GmMAKKK13-2 (Glyma.14G195300) and GmMAPK3-2 (Glyma.12G073000) in wild and cultivated soybean during F. oxysporum invasion. Main fungal group Glomeraceae was positively correlated with GmMAPKKK14-1 (Glyma.18G060900) and negatively correlated with GmRaf6-4 (Glyma.02G215300) in the wild soybean response to pathogen infection; while there were positive correlations between Hypocreaceae and GmMAPK3-2 (Glyma.12G073000) and between Glomeraceae and GmRaf49-3 (Glyma.06G055300) in the wild soybean response, these correlations were strongly negative in the response of cultivated soybean to F. oxysporum. Taken together, MAPKs correlated with different rhizomicrobiomes indicating the host plant modulated by the host self-immune systems in response to F. oxysporum.

Strigolactones shape the rhizomicrobiome in rice (Oryza sativa).

The rhizomicrobiome helps the host plant to better adapt to environmental stresses. In contrast, plant-derived metabolic substances, including phytohormones, play an active role in structuring rhizomicrobiome. Although strigolactones (SLs), a group of phytohormones, serve as potential rhizosphere signaling molecules, their contributions in shaping the rice (Oryza sativa) rhizomicrobiome remain elusive. To address this issue, we compared the rhizomicrobiome of rice mutants defective in either SL biosynthesis or signaling and wild-type (WT) plants. To understand whether SL-regulated metabolic pathways shape the rhizomicrobiome, a correlation network analysis was conducted among the metabolic pathway-related genes and the rhizomicrobiome of rice. Compared to WT, higher bacterial richness (evidenced by the operational taxonomic unit richness) and lower fungal diversity (evidenced by the Shannon index) were observed in both SL deficient dwarf17 (d17) and signaling (d14) mutants. Additionally, remarkable differences were observed in the composition of a large number of bacterial communities than the fungal communities in the d17 and d14 mutants with respect to the WT. The abundance of certain beneficial bacterial taxa, including Nitrosomonadaceae and Rhodanobacter, were significantly decreased in both mutants relative to the WT. Correlation network analysis between SL-regulated metabolic pathway-associated genes and rhizomicrobiome proposed a role for SL-dependent metabolic pathways in shaping rhizomicrobiome composition. Taken together, our study suggests that SL biosynthesis and signaling play a key role in determining the rice rhizomicrobiome, directly or indirectly, through the mediation of distinct metabolic pathways. Based on our findings, the genetic modulation of rice SL biosynthesis and/or signaling pathways may help to recruit/increase the abundance of the desired rhizomicrobiome, which may assist in the stress resilience of rice.

Strigolactones positively regulate defense against Magnaporthe oryzae in rice (Oryza sativa).

This study presents evidence that strigolactones (SLs) promote defense against devastating rice blast fungal pathogen Magnaporthe oryzae. Impairment in either SL-biosynthetic dwarf17 (d17) or -signaling (d14) led to increased susceptibility towards M. oryzae. Comparative transcriptome profiling of the SL-signaling d14 mutant and WT plants revealed that a large number of defense-associated genes including hydrogen peroxide (H2O2)-, ethylene- and cell wall-synthesis-related genes were remarkably suppressed in d14 with respect to that of WT plants, during M. oryzae infection. In addition, various KEGG metabolic pathways related to sugar metabolism were significantly suppressed in the d14 plants compared to WT, during M. oryzae infection. Accordingly, WT plants accumulated increased levels of H2O2 and soluble sugar content compared to that of d17 and d14 in response to M. oryzae infection. Altogether, these results propose that SLs positively regulated rice defense against M. oryzae through involvement in the induction of various defense associated genes/pathways.

Plants exhibit significant effects on the rhizospheric microbiome across contrasting soils in tropical and subtropical China.

The rhizospheric microbiome appears to be one of the key determinants of plant health and productivity. In this study, to understand the assembly process of the rhizospheric microbiome, the effects of different sites, soils and plants on the rhizospheric microbiome were compared and examined using high-throughput sequencing. A series of comparisons of rhizospheric microbiomes were conducted using two plants (wild rice (Oryza rufipogon Griff.) and L. hexandra (Leersia hexandra Swartz)), two soils (high nutrient and low nutrient) and two sites (Guangdong and Hainan provinces in China). The results of the redundancy analysis, between-class analysis and coinertia analysis indicated that the factors shaping the rhizospheric microbiome (in decreasing order of strength), were the site, soil and plant. The effects of plants on the rhizospheric microbiome were slight and unobvious, with relatively low-explained variations and few core groups and indicator groups; however, the effects were significant across different sites and soils, especially for specific microbial groups that are closely associated with plants, such as pathogens, symbionts, and saprotrophs. Furthermore, rhizospheric fungi were more strongly influenced by plants than rhizospheric bacteria. Our results provide insights into the relationships among multiple factors that shape the rhizospheric microbiome in natural ecosystems and highlight the effects of plants across regional environmental shifts.

Comparative study of the mycorrhizal root transcriptomes of wild and cultivated rice in response to the pathogen Magnaporthe oryzae.

BACKGROUND: Rice, which serves as a staple food for more than half of the world's population, is very susceptible to the pathogenic fungus, Magnaporthe oryzae. However, common wild rice (Oryza rufipogon), which is the ancestor of Asian cultivated rice (O. sativa), has significant potential as a genetic source of resistance to M. oryzae. Recent studies have shown that the domestication of rice has altered its relationship to symbiotic arbuscular mycorrhizae. A comparative response of wild and domestic rice inhabited by mycorrhizae to infection by M. oryzae has not been documented. RESULTS: In the current study, roots of wild and cultivated rice colonized with the arbuscular mycorrhizal (AM) fungus (AMF) Rhizoglomus intraradices were used to compare the transcriptomic responses of the two species to infection by M. oryzae. Phenotypic analysis indicated that the colonization of wild and cultivated rice with R. intraradices improved the resistance of both genotypes to M. oryzae. Wild AM rice, however, was more resistant to M. oryzae than the cultivated AM rice, as well as nonmycorrhizal roots of wild rice. Transcriptome analysis indicated that the mechanisms regulating the responses of wild and cultivated AM rice to M. oryzae invasion were significantly different. The expression of a greater number of genes was changed in wild AM rice than in cultivated AM rice in response to the pathogen. Both wild and cultivated AM rice exhibited a shared response to M. oryzae which included genes related to the auxin and salicylic acid pathways; all of these play important roles in pathogenesis-related protein synthesis. In wild AM rice, secondary metabolic and biotic stress-related analyses indicated that the jasmonic acid synthesis-related α-linolenic acid pathway, the phenolic and terpenoid pathways, as well as the phenolic and terpenoid syntheses-related mevalonate (MVA) pathway were more affected by the pathogen. Genes related to these pathways were more significantly enriched in wild AM rice than in cultivated AM rice in response to M. oryzae. On the other hand, genes associated with the 'brassinosteroid biosynthesis' were more enriched in cultivated AM rice. CONCLUSIONS: The AMF R. intraradices-colonized rice plants exhibited greater resistance to M. oryzae than non-AMF-colonized plants. The findings of the current study demonstrate the potential effects of crop domestication on the benefits received by the host via root colonization with AMF(s), and provide new information on the underlying molecular mechanisms. In addition, results of this study can also help develop guidelines for the applications of AMF(s) when planting rice.

Preparation and in vivo investigation of oligomeric proanthocyanidins cross-linked collagen serving as synthesized tissue regeneration membrane.

In this study, type I collagen membranes were prepared using oligomeric proanthocyanidins (OPCs) as the cross-linking agent. The fabricated materials were evaluated to be applied as guided tissue regeneration membranes for periodontal defects. The mechanical strength of the cross-linked collagen membranes, namely OPCs-Col films, using different concentrations of OPCs ranged from 30 to 60 kPa. The cross-linked collagen membranes had better thermal stability than non-cross-linked one and could effectively resist the decomposition in collagenase solution as long as fifty days. The results of material characterization showed that 10% OPCs-Col film was ideal for our purpose. In vitro study using L929 and MG-63 cells revealed that 10% OPCs-Col film had great biocompatibility while OPC was demonstrated to be not cytotoxic as glutaraldehyde and genipin but even promote L929 cells. The material was further studied for in vivo studies with two models, subcutaneous and cranium defects in rat. The subcutaneous test showed that the regeneration membrane degraded till one month and the inflammatory response also reduced with implantation time. When implanted into the cranium defect, no lesions of the brain were caused and new bone tissue was observed inside the material. The results of in vivo studies showed that the synthesized membrane was helpful for tissue regeneration with long degradation time. The tissue regeneration membranes can barrier the rapid growing soft tissue, in order to save the capacity for the growth of neo bone.

Performance comparison of III-V//Si and III-V//InGaAs multi-junction solar cells fabricated by the combination of mechanical stacking and wire bonding.

The integration of III-V and Si multi-junction solar cells as photovoltaic devices has been studied in order to achieve high photovoltaic conversion efficiency. However, large differences in the coefficients of thermal expansion and the lattice parameters of GaAs, Si, and InGaAs have made it difficult to obtain high-efficiency solar cells grown as epilayers on Si and InP substrates. In this paper, two types of devices, including GaInP/GaAs stacked on Si (GaInP/GaAs//Si) and GaInP/GaAs stacked on InGaAs (GaInP/GaAs//InGaAs), are fabricated via mechanical stacking and wire bonding technologies. Mechanically stacked GaInP/GaAs//Si and GaInP/GaAs//InGaAs triple-junction solar cells are prepared via glue bonding. Current-voltage measurements of the two samples are made at room temperature. The short-circuit current densities of the GaInP/GaAs//Si and GaInP/GaAs//InGaAs solar cells are 13.37 and 13.66 mA/cm2, while the open-circuit voltages of these two samples are measured to be 2.71 and 2.52 V, respectively. After bonding the GaInP/GaAs dual-junction with the Si and InGaAs solar cells, the conversion efficiency is relatively improved by 32.6% and 30.9%, respectively, compared to the efficiency of the GaInP/GaAs dual-junction solar cell alone. This study demonstrates the high potential of combining mechanical stacked with wire bonding and ITO films to achieve high conversion efficiency in solar cells with three or more junctions.

Differential responses of molecular mechanisms and physiochemical characters in wild and cultivated soybeans against invasion by the pathogenic Fusarium oxysporum Schltdl.

Cultivated soybean (Glycine max) was derived from the wild soybean (Glycine soja), which has genetic resources that can be critically important for improving plant stress resistance. However, little information is available pertaining to the molecular and physiochemical comparison between the cultivated and wild soybeans in response to the pathogenic Fusarium oxysporum Schltdl. In this study, we first used comparative phenotypic and paraffin section analyses to indicate that wild soybean is indeed more resistant to F. oxysporum than cultivated soybean. Genome-wide RNA-sequencing approach was then used to elucidate the genetic mechanisms underlying the differential physiological and biochemical responses of the cultivated soybean, and its relative, to F. oxysporum. A greater number of genes related to cell wall synthesis and hormone metabolism were significantly altered in wild soybean than in cultivated soybean under F. oxysporum infection. Accordingly, a higher accumulation of lignins was observed in wild soybean than cultivated soybean under F. oxysporum infection. Collectively, these results indicated that secondary metabolites and plant hormones may play a vital role in differentiating the response between cultivated and wild soybeans against the pathogen. These important findings may provide future direction to breeding programs to improve resistance to F. oxysporum in the elite soybean cultivars by taking advantage of the genetic resources within wild soybean germplasm.

Comparative analysis of the root transcriptomes of cultivated and wild rice varieties in response to Magnaporthe oryzae infection revealed both common and species-specific pathogen responses.

BACKGROUND: Magnaporthe oryzae, the causal fungus of rice blast disease, negatively impacts global rice production. Wild rice (Oryza rufipogon), a relative of cultivated rice (O. sativa), possesses unique attributes that enable it to resist pathogen invasion. Although wild rice represents a major resource for disease resistance, relative to current cultivated rice varieties, no prior studies have compared the immune and transcriptional responses in the roots of wild and cultivated rice to M. oryzae. RESULTS: In this study, we showed that M. oryzae could act as a typical root-infecting pathogen in rice, in addition to its common infection of leaves, and wild rice roots were more resistant to M. oryzae than cultivated rice roots. Next, we compared the differential responses of wild and cultivated rice roots to M. oryzae using RNA-sequencing (RNA-seq) to unravel the molecular mechanisms underlying the enhanced resistance of the wild rice roots. Results indicated that both common and genotype-specific mechanisms exist in both wild and cultivated rice that are associated with resistance to M. oryzae. In wild rice, resistance mechanisms were associated with lipid metabolism, WRKY transcription factors, chitinase activities, jasmonic acid, ethylene, lignin, and phenylpropanoid and diterpenoid metabolism; while the pathogen responses in cultivated rice were mainly associated with phenylpropanoid, flavone and wax metabolism. Although modulations in primary metabolism and phenylpropanoid synthesis were common to both cultivated and wild rice, the modulation of secondary metabolism related to phenylpropanoid synthesis was associated with lignin synthesis in wild rice and flavone synthesis in cultivated rice. Interestingly, while the expression of fatty acid and starch metabolism-related genes was altered in both wild and cultivated rice in response to the pathogen, changes in lipid acid synthesis and lipid acid degradation were dominant in cultivated and wild rice, respectively. CONCLUSIONS: The response mechanisms to M. oryzae were more complex in wild rice than what was observed in cultivated rice. Therefore, this study may have practical implications for controlling M. oryzae in rice plantings and will provide useful information for incorporating and assessing disease resistance to M. oryzae in rice breeding programs.

Current understanding of pattern-triggered immunity and hormone-mediated defense in rice (Oryza sativa) in response to Magnaporthe oryzae infection.

Plant pathogens represent a huge threat to world food security, affecting both crop production and quality. Although significant progress has been made in improving plant immunity by expressing key, defense-related genes and proteins from different species in transgenic crops, a challenge remains for molecular breeders and biotechnologists to successfully engineer elite, transgenic crop varieties with improved resistance against critical plant pathogens. Upon pathogen attack, including infection of rice (Oryza sativa) by Magnaporthe oryzae, host plants initiate a complex defense response at molecular, biochemical and physiological levels. Plants perceive the presence of pathogens by detecting microbe-associated molecular patterns via pattern recognition receptors, and initiate a first line of innate immunity, the so-called pattern-triggered immunity (PTI). This results in a series of downstream defense responses, including the production of hormones, which collectively function to fend off pathogen attacks. A variety of studies have demonstrated that many genes are involved in the defense response of rice to M. oryzae. In this review, the current understanding of mechanisms that improve rice defense response to M. oryzae will be discussed, with special focus on PTI and the phytohormones ethylene, jasmonic acid, salicylic acid, and abscisic acid; as well as on the mediation of defense signaling mechanisms by PTI and these hormones. Potential target genes that may serve as promising candidates for improving rice immunity against M. oryzae will also be discussed.

Relationship between calorimetric profiles and differential melting curves for natural DNAs.

Many experiments demonstrate that regions with higher GC-content in natural DNAs unwind at higher temperatures adsorbing more heat than equivalently sized regions with lower GC-content. This simple observation implies that normalized calorimetric melting profiles (calorimetric cDMCs) will not be equivalent differential melting curves (DMCs). We propose simple expressions for long natural and random DNA sequences to reciprocally convert DMCs and corresponding calorimetric cDMCs. The expressions are confirmed by the Poland-Fixman-Freire method and an approach based upon mixtures of homopolymeric duplexes. Using these expressions and experimental calorimetric data, we demonstrate that the average relative deviation between DMC and cDMC is proportional to the temperature melting range of the helix-coil transition ΔT. Corresponding difference between melting temperatures is proportional to ΔT2. In general, sequence and ionic conditions influence the deviation through their effect on ΔT. On the basis of the developed approach, we propose a method to determine the thermodynamic melting temperature (ratio of calorimetric enthalpy and entropy of the helix-coil transition) for natural DNAs from optical DMCs without calorimetric experiments.

Estimation of the diversity between DNA calorimetric profiles, differential melting curves and corresponding melting temperatures.

The Poland-Fixman-Freire formalism was adapted for modeling of calorimetric DNA melting profiles, and applied to plasmid pBR 322 and long random sequences. We studied the influence of the difference (HGC -HAT ) between the helix-coil transition enthalpies of AT and GC base pairs on the calorimetric melting profile and on normalized calorimetric melting profile. A strong alteration of DNA calorimetrical profile with HGC -HAT was demonstrated. In contrast, there is a relatively slight change in the normalized profiles and in corresponding ordinary (optical) normalized differential melting curves (DMCs). For fixed HGC -HAT , the average relative deviation (S) between DMC and normalized calorimetric profile, and the difference between their melting temperatures (Tcal -Tm ) are weakly dependent on peculiarities of the multipeak fine structure of DMCs. At the same time, both the deviation S and difference (Tcal -Tm ) enlarge with the temperature melting range of the helix-coil transition. It is shown that the local deviation between DMC and normalized calorimetric profile increases in regions of narrow peaks distant from the melting temperature.

Determination of melting temperature and temperature melting range for DNA with multi-peak differential melting curves.

Many factors that change the temperature position and interval of the DNA helix-coil transition often also alter the shape of multi-peak differential melting curves (DMCs). For DNAs with a multi-peak DMC, there is no agreement on the most useful definition for the melting temperature, Tm, and temperature melting width, ΔT, of the entire DNA transition. Changes in Tm and ΔT can reflect unstable variation of the shape of the DMC as well as alterations in DNA thermal stability and heterogeneity. Here, experiments and computer modeling for DNA multi-peak DMCs varying under different factors allowed testing of several methods of defining Tm and ΔT. Indeed, some of the methods give unreasonable "jagged" Tm and ΔT dependences on varying relative concentration of DNA chemical modifications (rb), [Na(+)], and GC content. At the same time, Tm determined as the helix-coil transition average temperature, and ΔT, which is proportional to the average absolute temperature deviation from this temperature, are suitable to characterize multi-peak DMCs. They give smoothly varying theoretical and experimental dependences of Tm and ΔT on rb, [Na(+)], and GC content. For multi-peak DMCs, Tm value determined in this way is the closest to the thermodynamic melting temperature (the helix-coil transition enthalpy/entropy ratio).

DNA combing on low-pressure oxygen plasma modified polysilsesquioxane substrates for single-molecule studies.

Molecular combing and flow-induced stretching are the most commonly used methods to immobilize and stretch DNA molecules. While both approaches require functionalization steps for the substrate surface and the molecules, conventionally the former does not take advantage of, as the latter, the versatility of microfluidics regarding robustness, buffer exchange capability, and molecule manipulation using external forces for single molecule studies. Here, we demonstrate a simple one-step combing process involving only low-pressure oxygen (O2) plasma modified polysilsesquioxane (PSQ) polymer layer to facilitate both room temperature microfluidic device bonding and immobilization of stretched single DNA molecules without molecular functionalization step. Atomic force microscopy and Kelvin probe force microscopy experiments revealed a significant increase in surface roughness and surface potential on low-pressure O2 plasma treated PSQ, in contrast to that with high-pressure O2 plasma treatment, which are proposed to be responsible for enabling effective DNA immobilization. We further demonstrate the use of our platform to observe DNA-RNA polymerase complexes and cancer drug cisplatin induced DNA condensation using wide-field fluorescence imaging.