Multifaceted regulation of the sumoylation of the Sgs1 DNA helicase.

Homologous recombination repairs DNA breaks and sequence gaps via the production of joint DNA intermediates such as Holliday junctions. Dissolving Holliday junctions into linear DNA repair products requires the activity of the Sgs1 helicase in yeast and of its homologs in other organisms. Recent studies suggest that the functions of these conserved helicases are regulated by sumoylation; however, the mechanisms that promote their sumoylation are not well understood. Here, we employed in vitro sumoylation systems and cellular assays to determine the roles of DNA and the scaffold protein Esc2 in Sgs1 sumoylation. We show that DNA binding enhances Sgs1 sumoylation in vitro. In addition, we demonstrate the Esc2's midregion (MR) with DNA-binding activity is required for Sgs1 sumoylation. Unexpectedly, we found that the sumoylation-promoting effect of Esc2-MR is DNA independent, suggesting a second function for this domain. In agreement with our biochemical data, we found the Esc2-MR domain, like its SUMO E2-binding C-terminal domain characterized in previous studies, is required for proficient sumoylation of Sgs1 and its cofactors, Top3 and Rmi1, in cells. Taken together, these findings provide evidence that while DNA binding enhances Sgs1 sumoylation, Esc2-based stimulation of this modification is mediated by two distinct domains.

Using an Automated Speech Recognition Approach to Differentiate Between Normal and Aspirating Swallowing Sounds Recorded from Digital Cervical Auscultation in Children.

Use of machine learning to accurately detect aspirating swallowing sounds in children is an evolving field. Previously reported classifiers for the detection of aspirating swallowing sounds in children have reported sensitivities between 79 and 89%. This study aimed to investigate the accuracy of using an automatic speaker recognition approach to differentiate between normal and aspirating swallowing sounds recorded from digital cervical auscultation in children. We analysed 106 normal swallows from 23 healthy children (median 13 months; 52.1% male) and 18 aspirating swallows from 18 children (median 10.5 months; 61.1% male) who underwent concurrent videofluoroscopic swallow studies with digital cervical auscultation. All swallowing sounds were on thin fluids. A support vector machine classifier with a polynomial kernel was trained on feature vectors that comprised the mean and standard deviation of spectral subband centroids extracted from each swallowing sound in the training set. The trained support vector machine was then used to classify swallowing sounds in the test set. We found high accuracy in the differentiation of aspirating and normal swallowing sounds with 98% overall accuracy. Sensitivity for the detection of aspiration and normal swallowing sounds were 89% and 100%, respectively. There were consistent differences in time, power spectral density and spectral subband centroid features between aspirating and normal swallowing sounds in children. This study provides preliminary research evidence that aspirating and normal swallowing sounds in children can be differentiated accurately using machine learning techniques.

Esc2 orchestrates substrate-specific sumoylation by acting as a SUMO E2 cofactor in genome maintenance.

SUMO modification regulates diverse cellular processes by targeting hundreds of proteins. However, the limited number of sumoylation enzymes raises the question of how such a large number of substrates are efficiently modified. Specifically, how genome maintenance factors are dynamically sumoylated at DNA replication and repair sites to modulate their functions is poorly understood. Here, we demonstrate a role for the conserved yeast Esc2 protein in this process by acting as a SUMO E2 cofactor. Esc2 is required for genome stability and binds to Holliday junctions and replication fork structures. Our targeted screen found that Esc2 promotes the sumoylation of a Holliday junction dissolution complex and specific replisome proteins. Esc2 does not elicit these effects via stable interactions with substrates or their common SUMO E3. Rather, we show that a SUMO-like domain of Esc2 stimulates sumoylation by exploiting a noncovalent SUMO binding site on the E2 enzyme. This role of Esc2 in sumoylation is required for Holliday junction clearance and genome stability. Our findings thus suggest that Esc2 acts as a SUMO E2 cofactor at distinct DNA structures to promote the sumoylation of specific substrates and genome maintenance.

RR-APET - Heart rate variability analysis software.

BACKGROUND AND OBJECTIVES: Heart rate variability (HRV) has increasingly been linked to medical phenomena and several HRV metrics have been found to be good indicators of patient health. This has enabled generalised treatment plans to be developed in order to respond to subtle personal differences that are reflected in HRV metrics. There are several established HRV analysis platforms and methods available within the literature; some of which provide command line operation across databases but do not offer extensive graphical user interface (GUI) and editing functionality, while others offer extensive ECG editing but are not feasible over large datasets without considerable manual effort. The aim of this work is to provide a comprehensive open-source package, in a well known and multi-platform language, that offers considerable graphical signal editing features, flexibility within the algorithms used for R-peak detection and HRV quantification, and includes graphical functionality for batch processing. Thereby, providing a platform suited to either physician or researcher. METHODS: RR-APET's software was developed in the Python language and is modular in format, providing a range of different modules for established R-peak detection algorithms, as well as an embedded template for alternate algorithms. These modules also include several easily adjustable features, allowing the user to optimise any of the algorithms for different ECG signals or databases. Additionally, the software's user-friendly GUI platform can be operated by both researchers or medical professionals to accomplish different tasks, such as: the in-depth visual analysis of a single ECG, or the analysis multiple signals in a single iteration using batch processing. RR-APET also supports several popular data formats, including text, HDF5, Matlab, and Waveform Database (WFDB) files. RESULTS: The RR-APET platform presents multiple metrics that quantify the heart rate variability features of an R-to-R interval series, including time-domain, frequency-domain, and nonlinear metrics. When known R-peak annotations are available, positive predictability, sensitivity, detection error rate, and accuracy measures are also provided to assess the validity of the implemented R-peak detection algorithm. RR-APET scored an overall usability rating of 4.16 out of a possible 5, when released on a trial basis for user evaluation. CONCLUSIONS: With its unique ability to both create and operate on large databases, this software provides a strong platform from which to conduct further research in the field of HRV analytics and its correlation to patient healthcare outcomes. This software is available free of charge at https://gitlab.com/MegMcC/rr-apet-hrv-analysis-software and can be operated as an executable file within Windows, Mac and Linux systems.

Impact of Heat on Coil Hydrodynamic Size Yields the Energetics of Denatured State Conformational Bias.

Conformational equilibria in the protein denatured state have key roles regulating folding, stability, and function. The extent of conformational bias in the protein denatured state under folding conditions, however, has thus far proven elusive to quantify, particularly with regard to its sequence dependence and energetic character. To better understand the structural preferences of the denatured state, we analyzed both the sequence dependence to the mean hydrodynamic size of disordered proteins in water and the impact of heat on the coil dimensions, showing that the sequence dependence and thermodynamic energies associated with intrinsic biases for the α and polyproline II (PPII) backbone conformations can be obtained. Experiments that evaluate how the hydrodynamic size changes with compositional changes in the protein reveal amino acid specific preferences for PPII that are in good quantitative agreement with calorimetry-measured values from unfolded peptides and those inferred by survey of the protein coil library. At temperatures above 25 °C, the denatured state follows the predictions of a PPII-dominant ensemble. Heat effects on coil hydrodynamic size indicate the α bias is comparable to the PPII bias at cold temperatures. Though historically thought to give poor resolution to structural details, the hydrodynamic size of the unfolded state is found to be an effective reporter on the extent of the biases for the α and PPII backbone conformations.

Ppb-level formaldehyde detection using a CW room-temperature interband cascade laser and a miniature dense pattern multipass gas cell.

A ppb-level formaldehyde (H2CO) sensor was developed using a thermoelectrically cooled (TEC), continuous-wave (CW) room temperature interband cascade laser (ICL) emitting at 3.59 µm and a miniature dense pattern multipass gas cell with >50 m optical path length. Performance of the sensor was investigated with two measurement schemes: direct absorption (DAS) and wavelength modulation spectroscopy (WMS). With an integration time of less than 1.5 second, a detection limit of ~3 ppbv for H2CO measurement with precision of 1.25 ppbv for DAS and 0.58 ppbv for WMS, respectively, was achieved without zero air based background subtraction. An Allan-Werle variance analysis indicated that the precisions can be further improved to 0.26 ppbv @ 300s for DAS and 69 pptv @ 90 s for WMS, respectively. A side-by-side comparison between two measurement schemes is also discussed in detail.

Differential expression of galectin-1 and its interactions with cells and laminins in the intervertebral disc.

Galectin-1 (Gal-1), an endogenous ß-galactoside-binding protein, binds to laminins, which are highly expressed in the nucleus pulposus (NP) of the intervertebral disc (IVD). The objective of this study is to evaluate the expression of Gal-1 protein in IVD tissues during aging and the effect of Gal-1 on IVD cell adhesion to laminins. Tissues from rat, porcine, and human (scoliosis or disc degeneration) IVDs were used to evaluate Gal-1 expression via immunostaining, RT-PCR, and Western blot analysis. Attachment of isolated IVD cells (porcine and human) on select laminin isoforms (LM-111 and LM-511) was compared with/without pre-incubation with exogenous Gal-1. A biotinylated Gal-1(B-Gal-1) was used to evaluate for binding to IVD cells and to select for IVD cells by magnetic activated cell sorting (MACS). NP cells expressed high levels of Gal-1 protein as compared to anulus fibrosus (AF) cells in immature tissues, while exogenous Gal-1 increased both NP and AF cell attachment to laminins and exhibited a similar binding to both cell types in vitro. With aging, Gal-1 levels in NP tissue appeared to decrease. In addition, incubation with B-Gal-1 was able to promote the retention of more than 50% of IVD cells via MACS. Our results provide new findings for the presence and functional role of Gal-1 within IVDs. Similar staining patterns for Gal-1 and LM-511 in IVD tissue suggest that Gal-1 may serve as an adhesion molecule to interact with both cells and laminins. This MACS protocol may be useful for selecting pure IVD cells from mixed cells of pathological tissue.

Metered-dose inhaler technique: the effect of two educational interventions delivered in community pharmacy over time.

Instruction is critical in order to ensure correct technique with pressurized metered-dose inhalers (pMDIs) by patients. The aim of this study was to compare the effects over time of two educational interventions delivered in community pharmacy to pMDI users. In this randomized controlled parallel-group study, pMDI technique was assessed before and after written and verbal instruction, alone or with physical demonstration, at baseline and 4, 8, and 16 weeks. The study recruited 52 subjects with asthma or chronic obstructive pulmonary disease (COPD). Initially only 1/52 (6%) subject had correct pMDI technique (= checklist score 8/8), with mean baseline score 5 (SD 1) for both groups. Written and verbal information improved pMDI technique at 16 weeks (7 +/- 1, p < .05). Addition of physical demonstration resulted in significant improvement at weeks 4, 8, and 16 (7 +/- 1, 7 +/- 1, 7 +/- 1 respectively; p < .05 for each). Subjects receiving written and verbal information alone were less likely to return for follow-up than those receiving physical demonstration (8 weeks: 6/25 versus 19/27; p < .001). By the 8-week visit, 80% subjects in the physical demonstration group had correct technique prior to education, compared with 10% of subjects receiving written and verbal information alone (p < .05). There was some decline in inhaler technique by 16 weeks. The results demonstrate that adding a physical demonstration is more effective in improving pMDI technique than written and verbal instructions alone.

Proinflammatory cytokine expression profile in degenerated and herniated human intervertebral disc tissues.

OBJECTIVE: Prior reports document macrophage and lymphocyte infiltration with proinflammatory cytokine expression in pathologic intervertebral disc (IVD) tissues. Nevertheless, the role of the Th17 lymphocyte lineage in mediating disc disease remains uninvestigated. We undertook this study to evaluate the immunophenotype of pathologic IVD specimens, including interleukin-17 (IL-17) expression, from surgically obtained IVD tissue and from nondegenerated autopsy control tissue. METHODS: Surgical IVD tissues were procured from patients with degenerative disc disease (n = 25) or herniated IVDs (n = 12); nondegenerated autopsy control tissue was also obtained (n = 8) from the anulus fibrosus and nucleus pulposus regions. Immunohistochemistry was performed for cell surface antigens (CD68 for macrophages, CD4 for lymphocytes) and various cytokines, with differences in cellularity and target immunoreactivity scores analyzed between surgical tissue groups and between autopsy control tissue regions. RESULTS: Immunoreactivity for IL-4, IL-6, IL-12, and interferon-gamma (IFNgamma) was modest in surgical IVD tissue, although expression was higher in herniated IVD samples and virtually nonexistent in control samples. The Th17 lymphocyte product IL-17 was present in >70% of surgical tissue fields, and among control samples was detected rarely in anulus fibrosus regions and modestly in nucleus pulposus regions. Macrophages were prevalent in surgical tissues, particularly herniated IVD samples, and lymphocytes were expectedly scarce. Control tissue revealed lesser infiltration by macrophages and a near absence of lymphocytes. CONCLUSION: Greater IFNgamma positivity, macrophage presence, and cellularity in herniated IVDs suggests a pattern of Th1 lymphocyte activation in this pathology. Remarkable pathologic IVD tissue expression of IL-17 is a novel finding that contrasts markedly with low levels of IL-17 in autopsy control tissue. These findings suggest involvement of Th17 lymphocytes in the pathomechanism of disc degeneration.

Gait abnormalities and inflammatory cytokines in an autologous nucleus pulposus model of radiculopathy.

STUDY DESIGN: The authors investigated gait abnormalities and mechanical hypersensitivity associated with invertebral disc herniation in a rat model of radiculopathy. Further evaluation involved assessing how nucleus pulposus (NP) injury affected systemic cytokine expression and molecular changes at the dorsal root ganglion (DRG). OBJECTIVE: The objective of this work was to describe the gait and behavioral changes in an animal model of disc-herniation induced radiculopathy. A second objective included examining how these functional changes correlated with neuroinflammation and autoreactive lymphocyte immune activation. SUMMARY OF BACKGROUND DATA: Animal models of radiculopathy describe demyelination, slowed nerve conduction, and heightened pain sensitivity after application of autologous NP to the DRG. The quantitative impact of disc herniation on animal locomotion has not been investigated. Further, while local inflammation occurs at the injury site, the role of autoimmune cytokines reactive against previously immune-sequestered NP requires investigation. METHODS: NP-treated animals (n = 16) received autologous tail NP placed onto the L5 DRG exposed by unilateral facetectomy, and control animals (n = 16) underwent exposure only. At weekly time points, animals were evaluated for mechanical allodynia, thermal hyperalgesia, and gait characteristics through digitized video analysis. Serum cytokine content was measured after animal sacrifice, and immunohistochemistry tested DRG tissue for mediators of inflammation and immune activation. RESULTS: Sensory testing revealed mechanical allodynia in the affected limb of NP-treated rats compared with sham animals (P < 0.01) at all time points. Gait analysis reflected functional locomotive consequences of marked asymmetry (P = 0.048) and preference to bear weight on the contralateral limb (duty factor imbalance, P < 0.01) at early time points. Equivalent serum cytokine expression occurred in both groups, confirming the local inflammatory nature of this disease model. Immunohistochemistry of the sectioned DRGs revealed equivalent postsurgical inflammatory activation (interleukin 23, P = 0.47) but substantial early immune activation in the NP-treated group (interleukin 17, P = 0.01). CONCLUSION: This model of radiculopathy provides evidence of altered gait in a model of noncompressive disc herniation. Systemic inflammation was absent, but mechanical allodynia, local inflammation, and autoreactive immune activation were observed. Future work will involve therapeutic interventions to rescue animals from the phenotype of inflammatory radiculopathy.

Collagen Vitrigel membranes for the in vitro reconstruction of separate corneal epithelial, stromal, and endothelial cell layers.

The goal of this study was to evaluate the potential suitability of collagen Vitrigel (CV) membrane as a substrate for the separate reconstruction of the three main cellular layers of the cornea. Limbal explants, keratocytes, and endothelial cells were cultured on transparent membranes made of type I collagen. The resulting cell sheets were evaluated using RT-PCR, in addition to light and electron microscopy. Tensile testing was also performed to examine the mechanical properties of CV. Limbal explant cultures resulted in partially stratified epithelial sheets with upregulation of the putative stem cell marker p63. Keratocytes cultured in serum on CV exhibited stellate morphology along with a marked increase in expression of corneal crystallin ALDH and keratocan, (a keratan sulphate proteoglycan: KSPG), compared to identical cultures on tissue culture plastic. Endothelial cells formed dense monolayers with uniform cell size, tight intercellular junctions, and expression of voltage-dependent anion channels VDAC2 and VDAC3, chloride channel protein CLCN2, and sodium bicarbonate transporter NBC1. Epithelial and endothelial cells exhibited adhesive structures (desmosomes and hemidesmosomes) and evidence of apical specialization (microplicae), while endothelial cells also produced a Descemet's membrane-like basal lamina. CV was found to possess ultimate tensile strengths of 6.8 +/- 1.5 MPa when hydrated and 28.6 +/- 7.0 MPa when dry. Taken together, these results indicate that CV holds promise as a substrate for corneal reconstruction.

Dual interband cascade laser based trace-gas sensor for environmental monitoring.

The development of an interband cascade laser (ICL) based spectroscopic trace-gas sensor for the simultaneous detection of two atmospheric trace gases is reported. The sensor performance was evaluated using two ICLs capable of targeting formaldehyde (H2CO) and ethane (C2H6). Minimum detection limits of 3.5 ppbV for H2CO and 150 pptV for C2H6 was demonstrated with a 1 s integration time. The sensor was deployed for field measurements of H2CO, and laboratory quantification of both formaldehyde and ethane are reported. A cross comparison of the atmospheric concentration data for H2CO with data collected by a collocated commercial H2CO sensor employing Hantzsch reaction based fluorometric detection was performed. These results show excellent agreement between these two different approaches for trace-gas quantification. In addition, laboratory experiments for dual gas quantification show accurate, fast response with no crosstalk between the two gas channels.

Pulsed quantum-cascade laser-based sensor for trace-gas detection of carbonyl sulfide.

Simultaneous exhaled carbonyl sulfide (OCS) and carbon dioxide concentration measurements in human breath are demonstrated with a compact pulsed quantum-cascade laser-based gas sensor. We achieved a noise-equivalent sensitivity (1sigma) of 1.2 parts per billion by measuring a well-isolated OCS P(11) absorption line in the v3 band at 2057.6 cm(-1) using an astigmatic Herriott cell of 36-m optical path length and 0.4-s acquisition time.

Atmospheric formaldehyde monitoring in the Greater Houston area in 2002.

A laser spectrometer based on difference frequency generation (DFG) was deployed for real-time long-term monitoring of HCHO concentrations at an environmental monitoring site located at Deer Park, Texas, in the Greater Houston area. Three HCHO concentration measurements were made during the periods of July 20-31 (period I), August 2-14 (period II), and August 24-September 25 (period III), 2002. In periods I and II, differences in HCHO concentrations are apparent between day and night measurements, with elevated concentrations during daylight hours. Most of the HCHO peak values are less than 20 ppbV except for two intense peaks on August 02 (approximately 25 ppbV) and August 04 (approximately 30 ppbV). The formaldehyde concentration levels in ambient air at the measurement site are produced mainly by the photochemical oxidation of volatile organic compounds (VOCs) caused by intense sunlight during periods I and II. This observation was made based on a comparison with the ozone concentration, solar radiation, temperature, relative humidity, and wind speed data obtained from the Texas Commission on Environmental Quality (TCEQ). During period III, data collected by a time-integrating wet-chemical technique are compared to the data collected by the spectroscopic instrument.