Effect of hydrothermal pre-treatment on physical properties and co-digestion from food waste and sewage sludge mixture.

Anaerobic digestion (AD) is generally considered to be an economic and environmentally friendly technology for treating waste activated sludge, but has some limitations, such as the time it takes for the sludge to be digested and also the ineffectiveness of degrading the solids. Various pre-treatment technologies have been suggested to overcome these limitations and to improve the biogas production rate by enhancing the hydrolysis of organic matter. This paper studies the use of hydrothermal pre-treatment (HTP) for a food waste and sewage sludge mixture (FW-SS mixture) as pre-treatment of co-digestion. The results of the capillary suction time, time to filter, and particle size decreased with increasing HTP temperature. These results of the assessment that was conducted in this study confirm that the HTP process indeed modifies the physical properties of the FW-SS mixture to enhance the solubilization of organic solids. A maximum increase in biogas production of 50% is achieved with a HTP temperature of 140oC. These findings show that to achieve high conversion efficiency, an accurately designed pre-treatment step must be included in the overall AD process for wastewater treatment.

Optimizing pre-treatment conditions for anaerobic co-digestion of food waste and sewage sludge.

In this study, the optimum conditions of thermal-alkali pre-treatment and the performance of ammonia stripping were investigated for improving solubilization efficiency and methane yield in the anaerobic co-digestion of food waste (FW) and sewage sludge (SS). The reaction temperature, NaOH concentration and reaction time for the thermal-alkali pre-treatment were investigated to determine optimum pre-treatment conditions. Solubilization rate, volatile suspended solids (VSS) reduction rate and total volatile fatty acid (VFAs) yields were improved with increasing reaction temperature, NaOH concentration and reaction time. In addition, by applying the optimum pre-treatment conditions (140 °C, 60 meq/L and 60 min), the experimental methane yield of thermal-alkali pre-treatment of a mixture of FW and SS was 483.0 ±â€¯15.7 mL CH4/g VSadded, which was about 84% higher than that of the untreated one. However, after thermal-alkali pre-treatment, the NH4+ concentration of the thermal-alkali pre-treatment liquid showed a concentration that could inhibit anaerobic digestion, so ammonia stripping was performed to remove NH4+. As a result, the experimental methane yield was increased by about 7% compared to when ammonia stripping was not performed.

Single-molecule FRET studies of the cooperative and non-cooperative binding kinetics of the bacteriophage T4 single-stranded DNA binding protein (gp32) to ssDNA lattices at replication fork junctions.

Gene 32 protein (gp32) is the single-stranded (ss) DNA binding protein of the bacteriophage T4. It binds transiently and cooperatively to ssDNA sequences exposed during the DNA replication process and regulates the interactions of the other sub-assemblies of the replication complex during the replication cycle. We here use single-molecule FRET techniques to build on previous thermodynamic studies of gp32 binding to initiate studies of the dynamics of the isolated and cooperative binding of gp32 molecules within the replication complex. DNA primer/template (p/t) constructs are used as models to determine the effects of ssDNA lattice length, gp32 concentration, salt concentration, binding cooperativity and binding polarity at p/t junctions. Hidden Markov models (HMMs) and transition density plots (TDPs) are used to characterize the dynamics of the multi-step assembly pathway of gp32 at p/t junctions of differing polarity, and show that isolated gp32 molecules bind to their ssDNA targets weakly and dissociate quickly, while cooperatively bound dimeric or trimeric clusters of gp32 bind much more tightly, can 'slide' on ssDNA sequences, and exhibit binding dynamics that depend on p/t junction polarities. The potential relationships of these binding dynamics to interactions with other components of the T4 DNA replication complex are discussed.

Internally labeled Cy3/Cy5 DNA constructs show greatly enhanced photo-stability in single-molecule FRET experiments.

DNA constructs labeled with cyanine fluorescent dyes are important substrates for single-molecule (sm) studies of the functional activity of protein-DNA complexes. We previously studied the local DNA backbone fluctuations of replication fork and primer-template DNA constructs labeled with Cy3/Cy5 donor-acceptor Förster resonance energy transfer (FRET) chromophore pairs and showed that, contrary to dyes linked 'externally' to the bases with flexible tethers, direct 'internal' (and rigid) insertion of the chromophores into the sugar-phosphate backbones resulted in DNA constructs that could be used to study intrinsic and protein-induced DNA backbone fluctuations by both smFRET and sm Fluorescent Linear Dichroism (smFLD). Here we show that these rigidly inserted Cy3/Cy5 chromophores also exhibit two additional useful properties, showing both high photo-stability and minimal effects on the local thermodynamic stability of the DNA constructs. The increased photo-stability of the internal labels significantly reduces the proportion of false positive smFRET conversion 'background' signals, thereby simplifying interpretations of both smFRET and smFLD experiments, while the decreased effects of the internal probes on local thermodynamic stability also make fluctuations sensed by these probes more representative of the unperturbed DNA structure. We suggest that internal probe labeling may be useful in studies of many DNA-protein interaction systems.

Single-molecule FRET and linear dichroism studies of DNA breathing and helicase binding at replication fork junctions.

DNA "breathing" is a thermally driven process in which base-paired DNA sequences transiently adopt local conformations that depart from their most stable structures. Polymerases and other proteins of genome expression require access to single-stranded DNA coding templates located in the double-stranded DNA "interior," and it is likely that fluctuations of the sugar-phosphate backbones of dsDNA that result in mechanistically useful local base pair opening reactions can be exploited by such DNA regulatory proteins. Such motions are difficult to observe in bulk measurements, both because they are infrequent and because they often occur on microsecond time scales that are not easy to access experimentally. We report single-molecule fluorescence experiments with polarized light, in which tens-of-microseconds rotational motions of internally labeled iCy3/iCy5 donor-acceptor Förster resonance energy transfer fluorophore pairs that have been rigidly inserted into the backbones of replication fork constructs are simultaneously detected using single-molecule Förster resonance energy transfer and single-molecule fluorescence-detected linear dichroism signals. Our results reveal significant local motions in the ∼100-µs range, a reasonable time scale for DNA breathing fluctuations of potential relevance for DNA-protein interactions. Moreover, we show that both the magnitudes and the relaxation times of these backbone breathing fluctuations are significantly perturbed by interactions of the fork construct with a nonprocessive, weakly binding bacteriophage T4-coded helicase hexamer initiation complex, suggesting that these motions may play a fundamental role in the initial binding, assembly, and function of the processive helicase-primase (primosome) component of the bacteriophage T4-coded DNA replication complex.

Quantitative detection of target cells using unghosted cells (UGCs) of DxH 800 (Beckman Coulter).

BACKGROUND: In the Retic channel of DxH 800 (Beckman Coulter), the red blood cells (RBCs) resistant to hemoglobin clearing are counted as unghosted cells (UGCs). The aim of this study was to evaluate that the UGC is a surrogate marker for both the detection and counting of target cells. METHODS: In total, 1181 samples including 22 from iron deficiency anemia (IDA) patients, 95 from jaundice, 2 from sickle cell anemia, 3 from thalassemia, 1 cord blood, and 269 from normal controls were analyzed. Slides were prepared from all samples except normal controls and target cells were counted for correlation analysis of target cell counts to UGCs. RESULTS: The normal control samples showed 0.01% (0%-0.01%) UGCs, and the reference range was set at ≤0.02%. The IDA samples showed 0.015% (0.01%-0.03%) UGC count and 0.05% (0%-0.2%) target cell count. The jaundice samples showed 0.98% (0.1%-5.36%) UGC count, and 1.4% (0.1%-7.0%) target cell count. The two sickle cell anemia samples showed 0.41% and 3.74% UGC counts and 0.4% and 11.5% target cell counts. A cord blood sample showed 0.01% UGCs and 0% target cells. The three thalassemia samples showed 0.01%, 1.99%, and 7.82% UGC counts and 0%, 1.4%, and 15.5% target cell counts. The samples showing poikilocytosis other than target cells showed normal UGC count (≤0.02%). The positive predictive value of UGCs was 58.2% (124/213) and the negative predictive value was 96.8% (674/696). The UGC counts were well correlated to the manual target cell counts (r=0.944, p=0.000). CONCLUSIONS: This study demonstrates for the first time in the literature that a hematological parameter obtained automatically every time a reticulocyte counting is performed can be used to both screen for the presence of target cells and reliably quantify them.

A single-molecule view of the assembly pathway, subunit stoichiometry, and unwinding activity of the bacteriophage T4 primosome (helicase-primase) complex.

Single-molecule fluorescence resonance energy transfer (smFRET) methods were used to study the assembly pathway and DNA unwinding activity of the bacteriophage T4 helicase-primase (primosome) complex. The helicase substrates used were surface-immobilized model DNA replication forks "internally" labeled in the duplex region with opposed donor/acceptor (iCy3/iCy5) chromophore pairs in the lagging and leading strands. The time dependence of the smFRET signals was monitored during the unwinding process, and helicase rates and processivities were measured as a function of GTP concentration. This smFRET approach was also used to investigate the subunit stoichiometry of the primosome and the assembly pathway required to form functional and fully active primosome-DNA complexes. We confirmed that gp41 helicase monomer subunits form stable hexameric helicases in the presence of GTP and that the resulting (gp41)(6) complexes bind only weakly at DNA fork junctions. The addition of a single subunit of gp61 primase stabilized the resulting primosome complex at the fork and resulted in fully active and processive primosome helicases with gp41:gp61 subunit ratios of 6:1, while higher and lower subunit ratios substantially reduced the primosome unwinding activity. The use of alternative assembly pathways resulted in a loss of helicase activity and the formation of metastable DNA-protein aggregates, which were easily detected in our smFRET experiments as intense light-scattering foci. These single-molecule experiments provide a detailed real-time visualization of the assembly pathway and duplex DNA unwinding activity of the T4 primosome and are consistent with more indirect equilibrium and steady state results obtained in bulk solution studies.

Temperature-dependent conformations of a membrane supported zinc porphyrin tweezer by 2D fluorescence spectroscopy.

We studied the equilibrium conformations of a zinc porphyrin tweezer composed of two carboxylphenyl-functionalized zinc tetraphenyl porphyrin subunits connected by a 1,4-butyndiol spacer, which was suspended inside the amphiphilic regions of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) liposomes. By combining phase-modulation two-dimensional fluorescence spectroscopy (2D FS) with linear absorbance and fluorimetry, we determined that the zinc porphyrin tweezer adopts a mixture of folded and extended conformations in the membrane. By fitting an exciton-coupling model to a series of data sets recorded over a range of temperatures (17-85 °C) and at different laser center wavelengths, we determined that the folded form of the tweezer is stabilized by a favorable change in the entropy of the local membrane environment. Our results provide insights toward understanding the balance of thermodynamic factors that govern molecular assembly in membranes.

Single-molecule Taq DNA polymerase dynamics.

Taq DNA polymerase functions at elevated temperatures with fast conformational dynamics-regimes previously inaccessible to mechanistic, single-molecule studies. Here, single-walled carbon nanotube transistors recorded the motions of Taq molecules processing matched or mismatched template-deoxynucleotide triphosphate pairs from 22° to 85°C. By using four enzyme orientations, the whole-enzyme closures of nucleotide incorporations were distinguished from more rapid, 20-µs closures of Taq's fingers domain testing complementarity and orientation. On average, one transient closure was observed for every nucleotide binding event; even complementary substrate pairs averaged five transient closures between each catalytic incorporation at 72°C. The rate and duration of the transient closures and the catalytic events had almost no temperature dependence, leaving all of Taq's temperature sensitivity to its rate-determining open state.

Surface modification of droplet polymeric microfluidic devices for the stable and continuous generation of aqueous droplets.

Droplet microfluidics performed in poly(methyl methacrylate) (PMMA) microfluidic devices resulted in significant wall wetting by water droplets formed in a liquid-liquid segmented flow when using a hydrophobic carrier fluid such as perfluorotripropylamine (FC-3283). This wall wetting led to water droplets with nonuniform sizes that were often trapped on the wall surfaces, leading to unstable and poorly controlled liquid-liquid segmented flow. To circumvent this problem, we developed a two-step procedure to hydrophobically modify the surfaces of PMMA and other thermoplastic materials commonly used to make microfluidic devices. The surface-modification route involved the introduction of hydroxyl groups by oxygen plasma treatment of the polymer surface followed by a solution-phase reaction with heptadecafluoro-1,1,2,2-tetrahydrodecyl trichlorosilane dissolved in fluorocarbon solvent FC-3283. This procedure was found to be useful for the modification of PMMA and other thermoplastic surfaces, including polycyclic olefin copolymer (COC) and polycarbonate (PC). Angle-resolved X-ray photoelectron spectroscopy indicated that the fluorination of these polymers took place with high surface selectivity. This procedure was used to modify the surface of a PMMA droplet microfluidic device (DMFD) and was shown to be useful in reducing the wetting problem during the generation of aqueous droplets in a perfluorotripropylamine (FC-3283) carrier fluid and could generate stable segmented flows for hours of operation. In the case of PMMA DMFD, oxygen plasma treatment was carried out after the PMMA cover plate was thermally fusion bonded to the PMMA microfluidic chip. Because the appended chemistry to the channel wall created a hydrophobic surface, it will accommodate the use of other carrier fluids that are hydrophobic as well, such as hexadecane or mineral oils.

Pseudo-analytical solutions for multi-species biofilm model of aerobic granular sludge.

This paper demonstrates modelling of the aerobic granular sludge (AGS) process with the pseudo-analytical solutions (PAS) of a biofilm model. A MATLAB programmed graphical user interface platform was developed to facilitate the model calculation and access. Model calibration and validation were carried out through using experimental data collected from a granular sludge sequencing batch reactor operation. The experimental and modelling results identified the distribution of heterotrophs and nitrifiers on the AGS and its contribution to the performance of wastewater treatment. The model could describe multi-species biofilms according to the distinguishing features among the three levels of PAS models. The models demonstrated increasing degrees of interaction (no interaction, competition for nitrogen and layering and protection) between heterotrophs and nitrifiers. Modelling the AGS process using PAS increases the accessibility of the simulation of multiple species in both biofilm and suspended biomass.

Cross-talk-free dual-color fluorescence cross-correlation spectroscopy for the study of enzyme activity.

We have developed an instrument for spectral cross-talk-free dual-color fluorescence cross-correlation spectroscopy (FCCS), which provides a readout modality for the study of enzyme activity in application areas such as high-throughput screening. Two spectrally distinct (approximately 250 nm) fluorophores, Cy3 and IRD800, were excited simultaneously using two different excitation sources: one poised at 532 nm and the other at 780 nm. The fluorescence information was processed on two different color channels monitored with single-photon avalanche diodes (SPADs) that could transduce events at the single-molecule level. The system provided no color cross-talk (cross-excitation and/or cross-emission) and/or fluorescence resonance energy transfer (FRET), significantly improving data quality. To provide evidence of cross-talk-free operation, the system was evaluated using bright microspheres (lambda(abs) = 532 nm, lambda(em) = 560 nm) and quantum dots (lambda(abs) = 532 nm, lambda(em) = 810 nm). Experimental results indicated that no color leakage from the microspheres or quantum dots into inappropriate color channels was observed. To demonstrate the utility of the system, the enzymatic activity of APE1, which is responsible for nicking the phosphodiester backbone in DNA on the 5' side of an apurinic/apyrimidinic site, was monitored by FCCS using a double-stranded DNA substrate dual labeled with Cy3 and IRD800. Activity of APE1 was also monitored in the presence of an inhibitor (7-nitroindole-2-carboxylic acid) of the enzyme using this cross-talk-free FCCS platform. In all cases, no spectral leakage from single-molecule events into inappropriate color channels was observed.

Nanostructure-dependent water-droplet adhesiveness change in superhydrophobic anodic aluminum oxide surfaces: from highly adhesive to self-cleanable.

Water-droplet adhesiveness was freely controlled on a single platform of superhydrophobic anodized aluminum oxide (AAO) within the range from highly adhesive to self-cleanable. Changing the structure from nanopore to nanopillar arrays at the surface caused a dramatic increase in the receding angle and a decrease in the hysteresis of water contact angles. The presence of dead-end nanopores but not through nanoholes was recognized as one of the main causes of the adhesiveness of superhydrophobic surfaces. The adhesiveness-controllable superhydrophobic AAO can be an excellent platform on which to elucidate the physical nature of the wetting phenomenon related to the nanostructure and has promising potential in technological applications.

Single-pair fluorescence resonance energy transfer (spFRET) for the high sensitivity analysis of low-abundance proteins using aptamers as molecular recognition elements.

We have developed a strategy for the detection of single protein molecules, which uses single-pair fluorescence resonance energy transfer (spFRET) as the readout modality and provides exquisite analytical sensitivity and reduced assay turn-around-time by eliminating various sample pre-processing steps. The single-protein detection assay uses two independent aptamer recognition events to form an assembly conducive to intramolecular hybridization of oligonucleotide complements that are tethered to the aptamers. This hybridization brings a donor-acceptor pair within the Förster distance to create a fluorescence signature indicative of the presence of the protein-aptamer(s) association complex. As an example of spFRET, we demonstrate the technique for the analysis of serum thrombin. The assay requires co-association of two distinct epitope-binding aptamers, each of which is labeled with a donor or acceptor fluorescent dye (Cy3 or Cy5, respectively) to produce a FRET response. The FRET response between Cy3 and Cy5 was monitored by single-molecule photon-burst detection, which provides high analytical sensitivity when the number of single-molecule events is plotted versus the target concentration. We are able to identify thrombin with high efficiency based on photon burst events transduced in the Cy5 detection channel. We also demonstrate that the technique can discriminate thrombin molecules from its analogue prothrombin. The analytical sensitivity was >200-fold better than an ensemble measurement.

Quantitative comparisons of antibody-binding sites of platelet glycoprotein IIb/IIIa in aplastic anemia and idiopathic thrombocytopenic purpura.

The numbers of antibody-binding sites of platelet glycoprotein (GP) IIb/IIIa on circulating platelets were analyzed using 4 kinds of antibodies in 34 aplastic anemia (AA) patients, 20 idiopathic thrombocytopenic purpura (ITP) patients, and 14 normal controls. The numbers of antibody-binding sites of CD41, CD41a, CD41b, and CD61 on platelets of the AA patients were less than in the normal controls (p <0.001). In the ITP patients, the numbers of sites for CD41 and CD41a were less than in normal controls (p <0.05). There were significant positive correlations between CD41 and CD41a, CD41b, and CD61 in the 3 groups. There were significant negative correlations between CD41 and CD41b and between CD41a and CD41b in the normal controls, but not in the AA or ITP patients. In summary, the numbers of the 4 antibody-binding sites of GPIIb/IIIa on platelets of AA and ITP patients are different from those in normal controls. Measurements of the antibody-binding sites of GPIIb/IIIa are not necessary for the differential diagnosis of AA and ITP. However, the differences in correlations between the numbers of epitopes in AA and ITP patients suggest that the epitopes of GPIIb/IIIa are altered in these diseases.

A case of essential thrombocythemia in an 8-year-old boy.

Thrombocytosis in childhood is not rare but essential thrombocythemia is an extremely rare myeloproliferative disorder in childhood. The authors report a case of essential thrombocythemia in an 8-year-old boy who was diagnosed during further evaluation of an incidental finding of thrombocythemia in a school health examination.

Modified anaerobic digestion elutriated phased treatment for the anaerobic co-digestion of sewage sludge and food wastewater.

A modified anaerobic digestion elutriated phased treatment (MADEPT) process was developed for investigating anaerobic co-digestion of sewage sludge and food wastewater. The anaerobic digestion elutriated phased treatment (ADEPT) process is similar to a two-phase system, however, in which the effluent from a methanogenic reactor recycles into an acidogenic reactor to elutriate mainly dissolved organics. Although ADEPT could reduce reactor volume significantly, the unsolubilized solids should be wasted from the system. The MADEPT process combines thermo-alkali solubilization with ADEPT to improve anaerobic performance and to minimize the sludge disposal. It was determined that the optimal volume mixing ratio of sewage sludge and food wastewater was 4 : 1 for the anaerobic co-digestion. The removal efficiencies of total chemical oxygen demand, volatile solids, and volatile suspended solids in the MADEPT process were 73%, 70%, and 64%, respectively. However, those in the ADEPT process were only 48%, 37%, and 40%, respectively, at the same hydraulic retention time (HRT) of 7 days. The gas production of MADEPT was two times higher than that of ADEPT. The thermo-alkali solubilization increased the concentration of dissolved organics so that they could be effectively degraded in a short HRT, implying that MADEPT could improve the performance of ADEPT in anaerobic co-digestion.

Significance of cerebrospinal fluid sIL-2R level as a marker of CNS involvement in acute lymphoblastic leukemia.

Soluble IL-2 receptor (sIL-2R), total protein, uric acid, glucose, aspartate aminotransferase (AST) and lactate dehydrogenase (LD) levels were analyzed in 153 (19 cytology(+), 134 cytology(-)) pairs of CSF and serum samples and the data were compared with the results of cytologic examination to find new CSF markers of CNS involvement in 77 patients with acute lymphoblastic leukemia (ALL). The CSF leukocyte count of cytology(+) samples averaged 107.6+/-362.4 cells/microl, and was higher than that of cytology(-) samples (1.0+/-3.4 cells/microl, p=0.001). The CSF sIL2-R level of cytology(+) samples averaged 162.1+/-247.7 U/ml, and was higher than that of cytology(-) samples (11.2+/-44.6 U/ml, p <0.001). The CSF total protein, uric acid, glucose, AST, and LD levels were not significantly different in cytology(+) and cytology(-) samples (p >0.05). ROC curves showed that the discrimination power of CSF sIL2-R for the presence of leukemic blasts was better than that of CSF leukocyte counts. With a cut-off value for CSF sIL2-R at 10 U/ml, the sensitivity was 89.5% and the specificity was 89.6%. With a cut-off value for CSF leukocyte count at 4 cells/microl, the sensitivity and specificity were 47.4% and 63.2%, respectively. In conclusion, CSF sIL2-R level is a valuable marker of CNS involvement in ALL patients; a level of >10 U/ml may serve as an objective indicator of CNS involvement in conjunction with conventional cytology and the CSF leukocyte count.

Development of a cervical cancer progress prediction tool for human papillomavirus-positive Koreans: A support vector machine-based approach.

OBJECTIVES: To develop a Web-based tool to draw attention to patients positive for human papillomavirus (HPV) who have a high risk of progression to cervical cancer, in order to increase compliance with follow-up examinations and facilitate good doctor-patient communication. METHODS: Records were retrospectively analysed from women who were positive for HPV on initial testing (before any treatment). Information concerning age, Papanicolaou (PAP) smear result and presence of 15 high-risk HPV genotypes was used in a support vector machine (SVM) model, to identify the patient features that maximally contributed to progression to high-risk cervical lesions. RESULTS: Data from 731 subjects were analysed. The maximum number of correct cancer predictions was seen when four features (PAP, HPV16, HPV52 and HPV35) were used, giving an accuracy of 74.41%. A web-based high-risk cervical lesion prediction application tool was developed using the SVM model results. CONCLUSIONS: Use of the web-based prediction tool may help to increase patient compliance with physician advice, and may heighten awareness of the significance of regular follow-up HPV examinations for the prevention of cervical cancer, in Korean women predicted to have heightened risk of the disease.

Evaluation of early post-transplant leukocyte recovery using the undiluted erythrocyte lysing technique.

The undiluted erythrocyte lysing technique was evaluated to see if it provides more accurate total leukocyte counts and differential leukocyte counts of severely leukopenic blood samples, in order to detect the onset of hematopoietic recovery after stem cell transplantation. Leukocyte counts using the conventional automated cell counting technique were found to be inaccurate, especially in blood samples with total leukocyte counts < 500/microl. In cases where the difference between results by the two methods was >100/microl, a positive correlation was found between the difference value and the blood reticulocyte count (r = 0.39, p = 0.002). Hematopoietic recovery after stem cell transplantation in a group of patients with chronic myelogenous leukemia (CML) was different from that of non-CML groups. In the CML group, the initial leukocyte counts were higher and the number of days until neutrophil recovery was higher than in the non-CML groups. Also, the day on which the absolute neutrophil count (ANC) exceeds 100/microl could serve as an indicator of neutrophil recovery. This study shows that the undiluted erythrocyte lysing technique can be used to count leukocytes accurately, especially in severely leukopenic samples. This new method can detect neutrophil recovery at ANC > 100/microl, as well as at an earlier date than the conventional method.