The clock in growing hyphae and their synchronization in Neurospora crassa.

Utilizing a microfluidic chip with serpentine channels, we inoculated the chip with an agar plug with Neurospora crassa mycelium and successfully captured individual hyphae in channels. For the first time, we report the presence of an autonomous clock in hyphae. Fluorescence of a mCherry reporter gene driven by a clock-controlled gene-2 promoter (ccg-2p) was measured simultaneously along hyphae every half an hour for at least 6 days. We entrained single hyphae to light over a wide range of day lengths, including 6,12, 24, and 36 h days. Hyphae tracked in individual serpentine channels were highly synchronized (K = 0.60-0.78). Furthermore, hyphae also displayed temperature compensation properties, where the oscillation period was stable over a physiological range of temperatures from 24 °C to 30 °C (Q10 = 1.00-1.10). A Clock Tube Model developed could mimic hyphal growth observed in the serpentine chip and provides a mechanism for the stable banding patterns seen in race tubes at the macroscopic scale and synchronization through molecules riding the growth wave in the device.

The macroscopic limit to synchronization of cellular clocks in single cells of Neurospora crassa.

We determined the macroscopic limit for phase synchronization of cellular clocks in an artificial tissue created by a "big chamber" microfluidic device to be about 150,000 cells or less. The dimensions of the microfluidic chamber allowed us to calculate an upper limit on the radius of a hypothesized quorum sensing signal molecule of 13.05 nm using a diffusion approximation for signal travel within the device. The use of a second microwell microfluidic device allowed the refinement of the macroscopic limit to a cell density of 2166 cells per fixed area of the device for phase synchronization. The measurement of averages over single cell trajectories in the microwell device supported a deterministic quorum sensing model identified by ensemble methods for clock phase synchronization. A strong inference framework was used to test the communication mechanism in phase synchronization of quorum sensing versus cell-to-cell contact, suggesting support for quorum sensing. Further evidence came from showing phase synchronization was density-dependent.

Einstein@Home discovers a radio-quiet gamma-ray millisecond pulsar.

Millisecond pulsars (MSPs) are old neutron stars that spin hundreds of times per second and appear to pulsate as their emission beams cross our line of sight. To date, radio pulsations have been detected from all rotation-powered MSPs. In an attempt to discover radio-quiet gamma-ray MSPs, we used the aggregated power from the computers of tens of thousands of volunteers participating in the Einstein@Home distributed computing project to search for pulsations from unidentified gamma-ray sources in Fermi Large Area Telescope data. This survey discovered two isolated MSPs, one of which is the only known rotation-powered MSP to remain undetected in radio observations. These gamma-ray MSPs were discovered in completely blind searches without prior constraints from other observations, raising hopes for detecting MSPs from a predicted Galactic bulge population.

Competition between DNA Methylation, Nucleotide Synthesis, and Antioxidation in Cancer versus Normal Tissues.

Global DNA hypomethylation occurs in many cancer types, but there is no explanation for its differential occurrence or possible impact on cancer cell physiology. Here we address these issues with a computational study of genome-scale DNA methylation in 16 cancer types. Specifically, we identified (i) a possible determinant for global DNA methylation in cancer cells and (ii) a relationship between levels of DNA methylation, nucleotide synthesis, and intracellular oxidative stress in cells. We developed a system of kinetic equations to capture the metabolic relations among DNA methylation, nucleotide synthesis, and antioxidative stress response, including their competitions for methyl and sulfur groups, based on known information about one-carbon metabolism and trans-sulfuration pathways. We observed a kinetic-based regulatory mechanism that controls reaction rates of the three competing processes when their shared resources are limited, particularly when the nucleotide synthesis rates or oxidative states are high. The combination of this regulatory mechanism and the need for rapid nucleotide synthesis, as well as high production of glutathione dictated by cancer-driving forces, led to the nearly universal observations of reduced global DNA methylation in cancer. Our model provides a natural explanation for differential global DNA methylation levels across cancer types and supports the observation that more malignant cancers tend to exhibit reduced DNA methylation levels. Insights obtained from this work provide useful information about the complexities of cancer due to interplays among competing, dynamic biological processes. Cancer Res; 77(15); 4185-95. ©2017 AACR.

Data-driven modeling reveals cell behaviors controlling self-organization during Myxococcus xanthus development.

Collective cell movement is critical to the emergent properties of many multicellular systems, including microbial self-organization in biofilms, embryogenesis, wound healing, and cancer metastasis. However, even the best-studied systems lack a complete picture of how diverse physical and chemical cues act upon individual cells to ensure coordinated multicellular behavior. Known for its social developmental cycle, the bacterium Myxococcus xanthus uses coordinated movement to generate three-dimensional aggregates called fruiting bodies. Despite extensive progress in identifying genes controlling fruiting body development, cell behaviors and cell-cell communication mechanisms that mediate aggregation are largely unknown. We developed an approach to examine emergent behaviors that couples fluorescent cell tracking with data-driven models. A unique feature of this approach is the ability to identify cell behaviors affecting the observed aggregation dynamics without full knowledge of the underlying biological mechanisms. The fluorescent cell tracking revealed large deviations in the behavior of individual cells. Our modeling method indicated that decreased cell motility inside the aggregates, a biased walk toward aggregate centroids, and alignment among neighboring cells in a radial direction to the nearest aggregate are behaviors that enhance aggregation dynamics. Our modeling method also revealed that aggregation is generally robust to perturbations in these behaviors and identified possible compensatory mechanisms. The resulting approach of directly combining behavior quantification with data-driven simulations can be applied to more complex systems of collective cell movement without prior knowledge of the cellular machinery and behavioral cues.

Synchronizing stochastic circadian oscillators in single cells of Neurospora crassa.

The synchronization of stochastic coupled oscillators is a central problem in physics and an emerging problem in biology, particularly in the context of circadian rhythms. Most measurements on the biological clock are made at the macroscopic level of millions of cells. Here measurements are made on the oscillators in single cells of the model fungal system, Neurospora crassa, with droplet microfluidics and the use of a fluorescent recorder hooked up to a promoter on a clock controlled gene-2 (ccg-2). The oscillators of individual cells are stochastic with a period near 21 hours (h), and using a stochastic clock network ensemble fitted by Markov Chain Monte Carlo implemented on general-purpose graphical processing units (or GPGPUs) we estimated that >94% of the variation in ccg-2 expression was stochastic (as opposed to experimental error). To overcome this stochasticity at the macroscopic level, cells must synchronize their oscillators. Using a classic measure of similarity in cell trajectories within droplets, the intraclass correlation (ICC), the synchronization surface ICC is measured on >25,000 cells as a function of the number of neighboring cells within a droplet and of time. The synchronization surface provides evidence that cells communicate, and synchronization varies with genotype.

Patient-reported outcomes and aesthetic evaluation of root coverage procedures: a 12-month follow-up of a randomized controlled clinical trial.

AIM: To assess patient-reported outcome measures (PROMs), aesthetics and stability of root coverage procedures from a previous 6-month RCT after 1 year. MATERIAL & METHODS: Forty-five patients (90 recessions) had received a coronally advanced flap (CAF = control) only or a xenogeneic collagen matrix in addition (CAF + CMX = test). Visual analogue scales (VAS) and questionnaires were used for PROMs and the root coverage aesthetic score (RES) for professional aesthetic evaluations. RESULTS: VAS scores (patient satisfaction) amounted to 8.58 ± 1.86 (test) versus 8.38 ± 2.46 (control). Six patients preferred CAF + CMX concerning surgical procedure and aesthetics, six preferred CAF and 29 were equally satisfied. RES was 7.85 ± 2.42 for the test group versus 7.34 ± 2.90 for the controls. Root coverage (RC) was 76.28% for test and 75.05% for control defects. The mean increase in keratinized tissue width was higher in test (from 1.97 to 3.02 mm) than in controls (from 2.00 to 2.64 mm) (p = 0.0413). Likewise, test sites showed more gain in gingival thickness (0.52 mm) than control sites (0.27 mm) (p = 0.0023). Compared to 6 months, clinical outcomes were stable. CONCLUSIONS: Results for PROMs, RES and RC did not significantly differ between treatment groups. Thickness and width of keratinized tissue were enhanced following CAF + CMX compared to CAF alone.

Treatment of gingival recession defects with a coronally advanced flap and a xenogeneic collagen matrix: a multicenter randomized clinical trial.

AIM: To evaluate the clinical outcomes of the use of a xenogeneic collagen matrix (CM) in combination with the coronally advanced flap (CAF) in the treatment of localized recession defects. MATERIAL & METHODS: In a multicentre single-blinded, randomized, controlled, split-mouth trial, 90 recessions (Miller I, II) in 45 patients received either CAF + CM or CAF alone. RESULTS: At 6 months, root coverage (primary outcome) was 75.29% for test and 72.66% for control defects (p = 0.169), with 36% of test and 31% of control defects exhibiting complete coverage. The increase in mean width of keratinized tissue (KT) was higher in test (from 1.97 to 2.90 mm) than in control defects (from 2.00 to 2.57 mm) (p = 0.036). Likewise, test sites had more gain in gingival thickness (GT) (0.59 mm) than control sites (0.34 mm) (p = 0.003). Larger (≥3 mm) recessions (n = 35 patients) treated with CM showed higher root coverage (72.03% versus 66.16%, p = 0.043), as well as more gain in KT and GT. CONCLUSIONS: CAF + CM was not superior with regard to root coverage, but enhanced gingival thickness and width of keratinized tissue when compared with CAF alone. For the coverage of larger defects, CAF + CM was more effective.

A multi-centre randomized controlled clinical trial on the treatment of intra-bony defects with enamel matrix derivatives/synthetic bone graft or enamel matrix derivatives alone: results after 12 months.

OBJECTIVES: Comparison of the clinical and radiographic outcomes of a combination of enamel matrix derivatives (EMD) and a synthetic bone graft (EMD/SBG) with EMD alone in wide (≥2 mm) and deep (≥4 mm) one- and two- wall intra-bony defects 12 months after treatment. MATERIALS AND METHODS: Seventy-three patients with chronic periodontitis and one wide (≥2 mm) and deep (≥4 mm) intra-bony defect were recruited in five centres in Germany. During surgery, defects were randomly assigned to EMD/SBG (test) or EMD (control). Assessments at baseline, after 6 and 12 months included bone sounding, attachment levels, probing pocket depths, bleeding on probing, and recessions. Changes in defect fill were recorded radiographically. RESULTS: Both treatment modalities led to significant clinical improvements. In the EMD/SBG group a mean defect fill of 2.7 ± 1.9 mm was calculated, in the EMD group the defect fill was 2.8 ± 1.6 mm. A mean gain in clinical attachment of 1.7 ± 2.1 mm in the test group and 1.9 ± 1.7 mm in the control group after 1 year was observed. Radiographic analysis confirmed for both groups that deeper defects were associated with greater defect fill. CONCLUSION: The results show comparable clinical and radiographic outcomes following both treatment modalities 12 months after treatment.

Time-resolved NMR: extracting the topology of complex enzyme networks.

The use of nondestructive NMR spectroscopy for enzymatic studies offers unique opportunities to identify nearly all enzymatic byproducts and detect unstable short-lived products or intermediates at the molecular level; however, numerous challenges must be overcome before it can become a widely used tool. The biosynthesis of acetyl-coenzyme A (acetyl-CoA) by acetyl-CoA synthetase is used here as a case study for the development of an analytical NMR-based time-course assay platform. We describe an algorithm to deconvolve superimposed spectra into spectra for individual molecules, and further develop a model to simulate the acetyl-CoA synthetase enzyme reaction network using the data derived from time-course NMR. Simulation shows indirectly that synthesis of acetyl-CoA is mediated via an enzyme-bound intermediate (possibly acetyl-AMP) and is accompanied by a nonproductive loss from an intermediate. The ability to predict enzyme function based on partial knowledge of the enzymatic pathway topology is also discussed.

Cellulose hydrolysis in evolving substrate morphologies III: time-scale analysis.

We present a time-scale analysis for the enzymatic hydrolysis of solid cellulosic substrates, based on our recently developed kinetic model (Zhou et al., 2009a, Biotechnol Bioeng 104:261-274; Zhou et al., 2009b, Biotechnol Bioeng 104:275-289) which incorporates both enzymatic chain fragmentation and hydrolytic time evolution of the solid substrate morphology. Analytical order-of-magnitude estimates of the relevant single-layer chain depolymerization times are first discussed. These time-scale estimates for pure and mixed enzyme systems can be employed to calculate the degree of synergy between endo- and exo-acting enzymes in a mixed enzyme system. By the way of a quasi-steady-state approximation which allows for a greatly simplified analytical solution of the model, we also explain the origin and give order-of-magnitude estimates of the two characteristic hydrolysis time scales which arise in this model when the solid substrate morphology is taken into account. These analytically derived time-scale relations explain how the embedding of cellulose chains in a solid substrate acts as a crucial rate-limiting factor and results in a substantial slowing down of the hydrolytic conversion process, compared to a hypothetical substrate of immediately enzyme-accessible, isolated chains. The analytical time-scale results are verified by numerical simulations and compared to experimental observations.

Clinical effects of nanocrystalline hydroxyapatite paste in the treatment of intrabony periodontal defects: a randomized controlled clinical study.

The purpose of the present randomized controlled clinical study was to compare the clinical outcomes of papilla preservation flap surgery with or without the application of a novel nanocrystalline hydroxyapatite (nano-HA) bone graft substitute. Fourteen patients with paired intrabony periodontal defects of ≥ 4 mm participated in this split-mouth design study. The defects in each subject were randomly selected to receive nano-HA paste in conjunction with papilla preservation flaps or papilla preservation flaps alone. Probing bone levels (PBL) from a customized acrylic stent and probing pocket depths (PPD) were measured at baseline and again 6 months following surgery. No differences in any of the investigated parameters were observed at baseline between the two groups. Healing was uneventful in all patients. Both treatments resulted in significant improvements between baseline and 6 months (p < 0.05). At 6 months after therapy, the sites treated with nano-HA paste showed a reduction in mean PPD from 8.3 ± 1.2 to 4.0 ± 1.1 mm and a gain in PBL of 4.3 ± 1.4 mm, whereas in the control group, the mean PPD changed from 7.9 ± 1.2 mm to 5.0 ± 1.2 mm and PBL gain was 2.6 ± 1.4 mm. Results demonstrated statistically greater PPD reduction and PBL gain (p < 0.05) in the test group as compared with the control group. In conclusion, after 6 months, the treatment of intrabony periodontal defects with a nano-HA paste leads to significantly improved clinical outcomes when compared with papilla preservation flap surgery alone.

Cellulose hydrolysis in evolving substrate morphologies I: A general modeling formalism.

We develop a general framework for a realistic rate equation modeling of cellulose hydrolysis using non-complexed cellulase. Our proposed formalism, for the first time, takes into account explicitly the time evolution of the random substrate morphology resulting from the hydrolytic cellulose chain fragmentation and solubilization. This is achieved by integrating novel geometrical concepts to quantitatively capture the time-dependent random morphology, together with the enzymatic chain fragmentation, into a coupled morphology-plus-kinetics rate equation approach. In addition, an innovative site number representation, based on tracking available numbers of beta(1,4) glucosidic bonds, of different "site" types, exposed to attacks by different enzyme types, is presented. This site number representation results in an ordinary differential equation (ODE) system, with a substantially reduced ODE system size, compared to earlier chain fragmentation kinetics approaches. This formalism enables us to quantitatively simulate both the hydrolytically evolving random substrate morphology and the profound, and heretofore neglected, morphology effects on the hydrolysis kinetics. By incorporating the evolving morphology on an equal footing with the hydrolytic chain fragmentation, our formalism provides a framework for the realistic modeling of the entire solubilization process, beyond the short-time limit and through near-complete hydrolytic conversion. As part I of two companion papers, the present paper focuses on the development of the general modelling formalism. Results and testable experimental predictions from detailed numerical simulations are presented in part II.

Cellulose hydrolysis in evolving substrate morphologies II: Numerical results and analysis.

Numerical simulation results are presented for a cellulose hydrolysis model which incorporates both the enzymatic glucan chain fragmentation kinetics and the hydrolytic substrate morphology evolution within the general framework of our companion article I. To test the local Poisson (LP) approximation employed in the site number formalism of I, we numerically compare it to the corresponding exact chain number formalism of I. The LP results agree to very high accuracy with the exact chain number kinetics, assuming realistic parameters. From simulations of different types of random and non-random model morphologies, we then show that the details of the random substrate morphology distribution, and its hydrolytic time evolution, profoundly affect the hydrolysis kinetics. Essential, likely very general, experimentally testable features of such morphology-based hydrolysis models are (i) the existence of two distinct time scales, associated with the hydrolysis of the outermost surface-exposed cellulose chains and, respectively, of the entire substrate; (ii) a strongly morphology-dependent hydrolysis slow-down effect, which has also been observed in previous experimental work. Our results also suggest that previously proposed non-morphologic chain fragmentation models can only be applied to describe the hydrolytic short-time behavior in the low enzyme limit. Further experiments to test our modeling framework and its potential applications to the optimization of the hydrolytic conversion process are discussed.

Genome-wide expression analysis of genetic networks in Neurospora crassa.

The products of five structural genes and two regulatory genes of the qa gene cluster of Neurospora crassa control the metabolism of quinic acid (QA) as a carbon source. A detailed genetic network model of this metabolic process has been reported. This investigation is designed to expand the current model of the QA reaction network. The ensemble method of network identification was used to model RNA profiling data on the qa gene cluster. Through microarray and cluster analysis, genome-wide identification of RNA transcripts associated with quinic acid metabolism in N. crassa is described and suggests a connection to other metabolic circuits. More than 100 genes whose products include carbon metabolism, protein degradation and modification, amino acid metabolism and ribosome synthesis appear to be connected to quinic acid metabolism. The core of the qa gene cluster network is validated with respect to RNA profiling data obtained from microarrays.

A genetic network for the clock of Neurospora crassa.

A diverse array of organisms from bacteria to humans may have evolved the ability to tell time in the presence or absence of external environmental cues. In the lowly bread mould, Neurospora crassa, biomolecular reactions involving the white-collar-1 (wc-1), white-collar-2 (wc-2), and frequency (frq) genes and their products constitute building blocks of a biological clock. Here we use genetic network models to explain quantitatively, from a systems perspective, how these building blocks interact, and how a complex trait like clock oscillation emerges from these interactions. We use a recently developed method of genetic network identification to find an ensemble of oscillating network models quantitatively consistent with available RNA and protein profiling data on the N. crassa clock. Predicted key features of the N. crassa clock system are a dynamically frustrated closed feedback loop, cooperativity in frq gene activation, and/or WC-1/WC-2 protein complex deactivation and substantial posttranscriptional enhancement of wc-1 RNA lifetime. Measuring the wc-1 mRNA lifetime provides a critical test of the genetic networks.

A randomized clinical multicentre trial comparing enamel matrix derivative and membrane treatment of buccal class II furcation involvement in mandibular molars. Part III: patient factors and treatment outcome.

OBJECTIVE: Evaluation of effects of patient factors on the outcome of regenerative treatment of buccal mandibular class II furcation defects. MATERIAL AND METHODS: Fifty-one patients were recruited. In the intention-to-treat population 21 patients were allocated into the sequence left treatment with enamel matrix protein derivative (EMD) and right guided tissue regeneration (GTR) and 27 in the sequence left GTR and right EMD. Evaluated patient factors were: smoking, age, gender, hypertension and oral hygiene status. Outcome parameters included change of: (a) horizontal depth of the defect at the deepest point (b) distance from the fornix of the furcation to bone crest of the defect, (c) distance from stent to the bottom of the defect, (d) pocket depth and (e) attachment level at the middle of the furcation. RESULTS: In patients 54 years of age and older, in males, in non-smokers and in patients with "poor" hygiene EMD-treated sites showed a significant higher mean reduction of the parameters d (age), b (gender, hygiene) a (smoking, hygiene) when compared with sites treated with GTR. CONCLUSIONS: These data provided an indication of a possible effect of patient factors on the outcome of regenerative treatment of buccal mandibular class II furcation defects.

A randomized clinical trial comparing enamel matrix derivative and membrane treatment of buccal class II furcation involvement in mandibular molars. Part II: secondary outcomes.

BACKGROUND: This multicenter, randomized trial compared enamel matrix derivative (EMD) with barrier membranes for the treatment of Class II mandibular furcations with regard to secondary outcomes. The influence of furcation morphology on the effectiveness of either treatment was also evaluated. METHODS: Forty-eight patients (age range 28 to 73 years; 22 females, 26 males) with buccal Class II furcation involvements in both contralateral lower first or second molars were included. After initial periodontal treatment, defects were randomized to either EMD or bioabsorbable guided tissue regeneration (GTR) barrier. Study design and the results for the primary parameter were previously described. Results of the following secondary outcome variables are reported here: changes of the hard tissue boundaries describing the anatomical situation of the furcation defect and changes in the following clinical parameters between baseline and 14 months: plaque, level of gingival margin, probing depth, bleeding on probing, attachment level, and bone sounding at five sites/tooth at the buccal side. Descriptive statistics were applied for changes in clinical parameters and measurements of hard tissue boundaries. The differences observed under treatment with EMD or membrane were analyzed by means of the Wilcoxon two-sample test. The difference between the effect of the EMD and membrane treatment was estimated by means of the Hodges-Lehmann estimator. RESULTS: Overall, similar healing results were observed for both treatments. However, there was slightly more recession in the mid-furcation site following membrane treatment (P = 0.04). Additionally, different treatment effects could be detected for the distances from the stent or cemento-enamel junction (CEJ) to the buccal bone crest, mid-distal root (Pstent = 0.01; PCEJ = 0.07) and for the distance from the stent or CEJ to the buccal bone crest, mid-mesial root (Pstent = 0.01; PCEJ = 0.01). There was no measurable bone resorption in EMD sites, whereas a slight resorption occurred with membrane treatment. Furcation morphology at the time of surgery was not associated with clinical outcome, irrespective of the treatment. CONCLUSION: With regard to secondary outcome parameters, enamel matrix derivative treatment led to a similar regenerative result as the membrane procedure.

A randomized clinical trial comparing enamel matrix derivative and membrane treatment of buccal Class II furcation involvement in mandibular molars. Part I: Study design and results for primary outcomes.

BACKGROUND: The objective of this multicenter, randomized trial was to compare enamel matrix derivative (EMD; test) with barrier membranes (control) for the treatment of mandibular buccal Class II furcation defects. METHODS: Forty-five patients with 90 comparable defects on contralateral molars were included. Defects were randomly assigned to EMD or bioabsorbable barrier membrane; the contralateral defect received the alternative treatment. Assessments at baseline and 8 and 14 months included gingival margin levels, probing depths, bleeding on probing, vertical attachment levels, and vertical bone sounding from a stent at five buccal sites/ tooth. Defect dimensions were recorded at surgery and during reentry at 14 months. Change of open horizontal furcation depth was the primary outcome variable. Adverse reactions and patient perceptions were also noted. RESULTS: Both treatment modalities led to significant clinical improvements. The median reduction of open horizontal furcation depth was 2.8 mm with the corresponding interquartile interval (1.5 mm, 3.5 mm) at test sites compared with 1.8 mm (1.0 mm, 2.8 mm) at control sites. The Hodges-Lehmann estimator of the advantage (reduction test versus control) was 0.75 mm (95% confidence interval [CI]: 0.125 mm, 1.375 mm, P = 0.033, Wilcoxon). The frequency of complete furcation closure was 8/45 (test) and 3/45 (control); partial closure, 27/45 in both groups; no change, 9/45 and 11/45, respectively; and deterioration, 1/45 and 4/45, respectively. The frequency of no pain or no swelling at 1 week post-surgery was 62% and 44%, respectively, at the test sites and 12% and 6% at the control sites. CONCLUSION: There was a significantly greater reduction in horizontal furcation depth and a comparatively lower incidence of postoperative pain/swelling following enamel matrix derivative compared to membrane therapy.