Trimers Conjugated to Fibrin Hydrogels Promote Salivary Gland Function.

New strategies for tissue engineering have great potential for restoring and revitalizing impaired tissues and organs, including the use of smart hydrogels that can be modified to enhance organization and functionality of the salivary glands. For instance, monomers of laminin-111 peptides chemically conjugated to fibrin hydrogel (L1pM-FH) promote cell cluster formation in vitro and salivary gland regeneration in vivo when compared with fibrin hydrogel (FH) alone; however, L1pM-FH produce only weak expression of acinar differentiation markers in vivo (e.g., aquaporin-5 and transmembrane protein 16). Since previous studies demonstrated that a greater impact can be achieved when trimeric forms were used as compared with monomeric or dimeric forms, we investigated the extent to which trimers of laminin-111 chemically conjugated to FH (L1pT-FH) can increase the expression of acinar differentiation markers and elevate saliva secretion. In vitro studies using Par-C10 acinar cells demonstrated that when compared with L1pM-FH, L1pT-FH induced similar levels of acinar-like cell clustering, polarization, lumen formation, and calcium signaling. To assess the performance of the trimeric complex in vivo, we compared the ability of L1pM-FH and L1pT-FH to increase acinar differentiation markers and restore saliva flow rate in a salivary gland wound model of C57BL/6 mice. Our results show that L1pT-FH applied to wounded mice significantly improved the expression of the acinar differentiation markers and saliva secretion when compared with the monomeric form. Together, these positive effects of L1pT-FH warrant its future testing in additional models of hyposalivation with the ultimate goal of applying this technology in humans.

Linking imaging to omics utilizing image-guided tissue extraction.

Phenotypic heterogeneity is commonly observed in diseased tissue, specifically in tumors. Multimodal imaging technologies can reveal tissue heterogeneity noninvasively in vivo, enabling imaging-based profiling of receptors, metabolism, morphology, or function on a macroscopic scale. In contrast, in vitro multiomics, immunohistochemistry, or histology techniques accurately characterize these heterogeneities in the cellular and subcellular scales in a more comprehensive but ex vivo manner. The complementary in vivo and ex vivo information would provide an enormous potential to better characterize a disease. However, this requires spatially accurate coregistration of these data by image-driven sampling as well as fast sample-preparation methods. Here, a unique image-guided milling machine and workflow for precise extraction of tissue samples from small laboratory animals or excised organs has been developed and evaluated. The samples can be delineated on tomographic images as volumes of interest and can be extracted with a spatial accuracy better than 0.25 mm. The samples remain cooled throughout the procedure to ensure metabolic stability, a precondition for accurate in vitro analysis.

SINCERITIES: inferring gene regulatory networks from time-stamped single cell transcriptional expression profiles.

MOTIVATION: Single cell transcriptional profiling opens up a new avenue in studying the functional role of cell-to-cell variability in physiological processes. The analysis of single cell expression profiles creates new challenges due to the distributive nature of the data and the stochastic dynamics of gene transcription process. The reconstruction of gene regulatory networks (GRNs) using single cell transcriptional profiles is particularly challenging, especially when directed gene-gene relationships are desired. RESULTS: We developed SINCERITIES (SINgle CEll Regularized Inference using TIme-stamped Expression profileS) for the inference of GRNs from single cell transcriptional profiles. We focused on time-stamped cross-sectional expression data, commonly generated from transcriptional profiling of single cells collected at multiple time points after cell stimulation. SINCERITIES recovers directed regulatory relationships among genes by employing regularized linear regression (ridge regression), using temporal changes in the distributions of gene expressions. Meanwhile, the modes of the gene regulations (activation and repression) come from partial correlation analyses between pairs of genes. We demonstrated the efficacy of SINCERITIES in inferring GRNs using in silico time-stamped single cell expression data and single cell transcriptional profiles of THP-1 monocytic human leukemia cells. The case studies showed that SINCERITIES could provide accurate GRN predictions, significantly better than other GRN inference algorithms such as TSNI, GENIE3 and JUMP3. Moreover, SINCERITIES has a low computational complexity and is amenable to problems of extremely large dimensionality. Finally, an application of SINCERITIES to single cell expression data of T2EC chicken erythrocytes pointed to BATF as a candidate novel regulator of erythroid development. AVAILABILITY AND IMPLEMENTATION: MATLAB and R version of SINCERITIES are freely available from the following websites: http://www.cabsel.ethz.ch/tools/sincerities.html and https://github.com/CABSEL/SINCERITIES. The single cell THP-1 and T2EC transcriptional profiles are available from the original publications (Kouno et al., 2013; Richard et al., 2016). The in silico single cell data are available on SINCERITIES websites. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

TRaCE+: Ensemble inference of gene regulatory networks from transcriptional expression profiles of gene knock-out experiments.

BACKGROUND: The inference of gene regulatory networks (GRNs) from transcriptional expression profiles is challenging, predominantly due to its underdetermined nature. One important consequence of underdetermination is the existence of many possible solutions to this inference. Our previously proposed ensemble inference algorithm TRaCE addressed this issue by inferring an ensemble of network directed graphs (digraphs) using differential gene expressions from gene knock-out (KO) experiments. However, TRaCE could not deal with the mode of the transcriptional regulations (activation or repression), an important feature of GRNs. RESULTS: In this work, we developed a new algorithm called TRaCE+ for the inference of an ensemble of signed GRN digraphs from transcriptional expression data of gene KO experiments. The sign of the edges indicates whether the regulation is an activation (positive) or a repression (negative). TRaCE+ generates the upper and lower bounds of the ensemble, which define uncertain regulatory interactions that could not be verified by the data. As demonstrated in the case studies using Escherichia coli GRN and 100-gene gold-standard GRNs from DREAM 4 network inference challenge, by accounting for regulatory signs, TRaCE+ could extract more information from the KO data than TRaCE, leading to fewer uncertain edges. Importantly, iterating TRaCE+ with an optimal design of gene KOs could resolve the underdetermined issue of GRN inference in much fewer KO experiments than using TRaCE. CONCLUSIONS: TRaCE+ expands the applications of ensemble GRN inference strategy by accounting for the mode of the gene regulatory interactions. In comparison to TRaCE, TRaCE+ enables a better utilization of gene KO data, thereby reducing the cost of tackling underdetermined GRN inference. TRaCE+ subroutines for MATLAB are freely available at the following website: http://www.cabsel.ethz.ch/tools/trace.html .

Optimal design of gene knockout experiments for gene regulatory network inference.

MOTIVATION: We addressed the problem of inferring gene regulatory network (GRN) from gene expression data of knockout (KO) experiments. This inference is known to be underdetermined and the GRN is not identifiable from data. Past studies have shown that suboptimal design of experiments (DOE) contributes significantly to the identifiability issue of biological networks, including GRNs. However, optimizing DOE has received much less attention than developing methods for GRN inference. RESULTS: We developed REDuction of UnCertain Edges (REDUCE) algorithm for finding the optimal gene KO experiment for inferring directed graphs (digraphs) of GRNs. REDUCE employed ensemble inference to define uncertain gene interactions that could not be verified by prior data. The optimal experiment corresponds to the maximum number of uncertain interactions that could be verified by the resulting data. For this purpose, we introduced the concept of edge separatoid which gave a list of nodes (genes) that upon their removal would allow the verification of a particular gene interaction. Finally, we proposed a procedure that iterates over performing KO experiments, ensemble update and optimal DOE. The case studies including the inference of Escherichia coli GRN and DREAM 4 100-gene GRNs, demonstrated the efficacy of the iterative GRN inference. In comparison to systematic KOs, REDUCE could provide much higher information return per gene KO experiment and consequently more accurate GRN estimates. CONCLUSIONS: REDUCE represents an enabling tool for tackling the underdetermined GRN inference. Along with advances in gene deletion and automation technology, the iterative procedure brings an efficient and fully automated GRN inference closer to reality. AVAILABILITY AND IMPLEMENTATION: MATLAB and Python scripts of REDUCE are available on www.cabsel.ethz.ch/tools/REDUCE CONTACT: rudi.gunawan@chem.ethz.ch SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Ensemble inference and inferability of gene regulatory networks.

The inference of gene regulatory network (GRN) from gene expression data is an unsolved problem of great importance. This inference has been stated, though not proven, to be underdetermined implying that there could be many equivalent (indistinguishable) solutions. Motivated by this fundamental limitation, we have developed new framework and algorithm, called TRaCE, for the ensemble inference of GRNs. The ensemble corresponds to the inherent uncertainty associated with discriminating direct and indirect gene regulations from steady-state data of gene knock-out (KO) experiments. We applied TRaCE to analyze the inferability of random GRNs and the GRNs of E. coli and yeast from single- and double-gene KO experiments. The results showed that, with the exception of networks with very few edges, GRNs are typically not inferable even when the data are ideal (unbiased and noise-free). Finally, we compared the performance of TRaCE with top performing methods of DREAM4 in silico network inference challenge.

Storm-induced sea-ice breakup and the implications for ice extent.

The propagation of large, storm-generated waves through sea ice has so far not been measured, limiting our understanding of how ocean waves break sea ice. Without improved knowledge of ice breakup, we are unable to understand recent changes, or predict future changes, in Arctic and Antarctic sea ice. Here we show that storm-generated ocean waves propagating through Antarctic sea ice are able to transport enough energy to break sea ice hundreds of kilometres from the ice edge. Our results, which are based on concurrent observations at multiple locations, establish that large waves break sea ice much farther from the ice edge than would be predicted by the commonly assumed exponential decay. We observed the wave height decay to be almost linear for large waves--those with a significant wave height greater than three metres--and to be exponential only for small waves. This implies a more prominent role for large ocean waves in sea-ice breakup and retreat than previously thought. We examine the wider relevance of this by comparing observed Antarctic sea-ice edge positions with changes in modelled significant wave heights for the Southern Ocean between 1997 and 2009, and find that the retreat and expansion of the sea-ice edge correlate with mean significant wave height increases and decreases, respectively. This includes capturing the spatial variability in sea-ice trends found in the Ross and Amundsen-Bellingshausen seas. Climate models fail to capture recent changes in sea ice in both polar regions. Our results suggest that the incorporation of explicit or parameterized interactions between ocean waves and sea ice may resolve this problem.

Kinetics of optical control of enzyme activity with photoswitchable inhibitors.

This paper presents theoretical and simulation studies on controlling enzymatic reactions with photoswitchable inhibitors. It is found that the maximum attainable switching ratio (ratio of the steady state rates of product formation in the "on" and the "off" state) of a photoswitchable inhibitor is dependent on its photoswitching factor (ratio of the equilibrium constants of the photostationary states under the "off" and the "on" illuminations). Attachment of multiple photoswitchable groups to an inhibitor molecule increases the theoretically attainable switching ratio. The affinity of the enzyme for the substrate and the inhibitor is the rate-limiting factor of the switching between active and inactive states. Use of inhibitors with high enzyme affinity and photoswitchable groups with high photoswitching factor would provide high switching ratio. These results may help to design better systems for optical control of biochemical processes.

Structural explanation for the effect of humidity on persistence of airborne virus: seasonality of influenza.

Seasonal variation of infectious diseases is one of the oldest observations in epidemiology, most particularly for Influenza and other respiratory viral infections. The reason for this seasonality is poorly understood, despite the profound importance of these infections as communicable diseases capable of causing global epidemics. Environmental factors including relative humidity, vapor pressure and temperature are known to affect seasonal virus survival and transmission. Immunological status of the host and evolution of the virus have also been proposed to be the reason behind the cyclic recurrence. The molecular basis of these effects or their interplay with biological factors has not been reported before. Here a theoretical analysis shows that the structure of the viral envelope determines its persistence and transmission in various environmental conditions. Physico-chemical properties of the virus particles and their interaction with atmospheric processes along with the availability and susceptibility of hosts generates the conspicuous seasonality prevalent in the temperate zones and the apparent lack of it in the tropics. Additionally this model can estimate virus transmission in different weather conditions. This model may help to determine the right actions effective in preventing outbreaks of the flu-like respiratory viruses.

Nanoreactors for pH controlled sequential activity switching in multistep enzymatic processes.

This theoretical model predicts that the activity of multiple enzymes may be controlled simultaneously with superior efficiency in nanosized reactors by adjusting pH. Multistep enzymatic processes employed for various purposes including organic biotransformation may require application of multiple reactions and isolation of intermediates. Sequential activity switching would offer substantial advantages. Nanoreactors would provide better option to fully appreciate the pH switching approach.

A theoretical model for template-free synthesis of long DNA sequence.

This theoretical scheme is intended to formulate a potential method for high fidelity synthesis of Nucleic Acid molecules towards a few thousand bases using an enzyme system. Terminal Deoxyribonucleotidyl Transferase, which adds a nucleotide to the 3'OH end of a Nucleic Acid molecule, may be used in combination with a controlled method for nucleotide addition and degradation, to synthesize a predefined Nucleic Acid sequence. A pH control system is suggested to regulate the sequential activity switching of different enzymes in the synthetic scheme. Current practice of synthetic biology is cumbersome, expensive and often error prone owing to the dependence on the ligation of short oligonucleotides to fabricate functional genetic parts. The projected scheme is likely to render synthetic genomics appreciably convenient and economic by providing longer DNA molecules to start with.

Phenanthrene degradation in soils co-inoculated with phenanthrene-degrading and biosurfactant-producing bacteria.

Contaminant sorption within the soil matrix frequently limits biodegradation. However, contaminant bioavailability can be species-specific. This study investigated bioavailability of phenanthrene (PHE) to two PHE-degrading bacteria (Pseudomonas strain R and isolate P5-2) in the presence of rhamnolipid biosurfactant and/or a biosurfactant-producing bacterium, Pseudomonas aeruginosa ATCC 9027. Pseudomonas strain R mineralized more soil-sorbed PHE than strain P5-2, but in aqueous cultures the rate and extent of PHE mineralization by P5-2 exceeded that by P. strain R. In Fallsington sandy loam (fine-loamy, mixed, active, mesic Typic Endoaquult) (high PHE-sorption capacity) the addition of rhamnolipid increased PHE mineralization by P. strain R. Phenanthrene mineralization in soils inoculated with P5-2 was minimal and no enhancement in PHE degradation was observed when biosurfactant was added. Co-inoculation of Fallsington sandy loam with the biosurfactant producer did not affect PHE mineralization by isolate P5-2, but significantly enhanced PHE mineralization by P. strain R. The enhancement of PHE mineralization could not be explained by P. aeruginosa-mediated PHE degradation. The addition of rhamnolipid at concentrations above the critical micelle concentration (CMC) resulted in enhanced PHE release from test soils. These results suggest that the PHE-degrading strains were able to access different pools of PHE and that the biosurfactant-enhanced release of PHE from soils did not result in enhanced biodegradation. The results also demonstrated that bacteria with the catabolic potential to degrade sorbed hydrophobic contaminants could interact commensally with surfactant-producing strains by an unknown mechanism to hasten the biodegradation of aromatic hydrocarbons. Thus, understanding interactions among microbes may provide opportunities to further enhance biodegradation of soil-bound organic contaminants.

Three cases of digital ischemia successfully treated with cilostazol.

Three patients were evaluated for refractory digital ischemia. The first patient presented with chronic, post-traumatic, unremitting, cold, painful, right fourth and fifth fingers. The symptoms had failed to improve despite topical nitroglycerin and a calcium channel blocker. Baseline digital plethysmography documented impaired perfusion within the affected digits. Cilostazol (Pletal) was added to the medical regimen and at the 8-week follow-up the fourth and fifth fingers were warm with repeat plethysmography displaying normal perfusion. A second patient had CREST syndrome-associated painful bilateral index finger ulcerations that had evolved despite taking a calcium channel blocker. Consequently the patient was started on cilostazol and within 4 weeks the digital ulcerations and pain had resolved. The third patient with traumatic right fifth digital arterial thrombosis was seen for persistent pain and cyanosis in spite of undergoing thrombolysis and subsequent anticoagulation with vasodilator therapy. Digital plethysmography established fixed ischemia within the fifth finger; subsequently, cilostazol was prescribed. Four weeks later the digital pain and cyanosis had essentially resolved. A follow-up plethysmographic waveform documented restored perfusion. Although approved for the treatment of intermittent claudication, cilostazol was successfully utilized in the setting of severe digital ischemia.

Calciphylaxis: a favorable outcome with hyperbaric oxygen.

A 66-year-old female with diabetes mellitus and end-stage renal disease presented with painful bilateral lower extremity livedo reticularis and necrotic ulcerations. Her distal lower extremity pulses were intact and plethysmographic studies confirmed relatively normal large vessel arterial perfusion. Extensive laboratory analysis was remarkable for an elevated calcium x phosphorous product and parathyroid hormone level. An ulcer biopsy revealed small vessel medial calcinosis, and calciphylaxis was subsequently diagnosed. Despite aggressive wound debridements, antibiotics and subtotal parathyroidectomy, her ulcers failed to improve significantly prompting a trial of hyperbaric oxygen therapy. After 7 weeks of hyperbaric treatments, her ulcers had essentially healed.

Ultrasound-guided compression closure of postcatheterization pseudoaneurysms during concurrent anticoagulation: a review of seventy-seven patients.

PURPOSE: Data from our institution and elsewhere have demonstrated that ultrasound-guided compression closure (UGCC) is an effective method of treating postcatheterization pseudoaneurysms. Whereas patients receiving anticoagulation do not have as high a success rate as those not receiving anticoagulants, there have been no large series evaluating the factors associated with success or failure in patients receiving anticoagulation. The purpose of this study is to determine whether uninterrupted anticoagulation interferes with successful UGCC of pseudoaneurysms and to identify factors associated with success or failure. METHODS: From May 1991 to September 1994, 238 cases of attempted UGCC of pseudoaneurysms were performed in our vascular laboratory. Only patients who received uninterrupted heparin, warfarin, or both at the time of pseudoaneurysm compression were eligible for inclusion into the study. Seventy-seven patients were identified who met the study criteria. RESULTS: Successful pseudoaneurysm compression was obtained in 56 (73%) patients, whereas 21 (27%) patients had a failed UGCC. In the successfully treated group, seven (12.5%) required between two to three compression attempts to induce sustained thrombosis. There was no statistical difference in age, sex, sheath size, days after procedure, location of pseudoaneurysm, or number of chambers in the pseudoaneurysm between those patients who had a successful repair and those who did not. If the pseudoaneurysm was less than 4 cm in diameter, 51 of 65 patients (78%) had a successful repair compared with 5 of 12 patients (42%) with a pseudoaneurysm of 4 cm or greater (p = 0.013). There was no statistical difference between success and failure in patients receiving warfarin alone (3.73 mean international normalized ratio, 72% success rate), heparin alone (mean activated partial thromboplastin time of 63 seconds, 92% success rate), or heparin and warfarin (mean activated partial thromboplastin time of 70 seconds, mean international normalized ratio of 4, success rate of 67%). No arterial or venous thrombosis occurred during pseudoaneurysm compression. CONCLUSION: Successful UGCC of pseudoaneurysms occurred in a large percentage of patients receiving full-dose, uninterrupted anticoagulation. The only factor influencing success was the size of the pseudoaneurysm.

Evaluation of lumbar spine fusion. Plain radiographs versus direct surgical exploration and observation.

STUDY DESIGN: In a retrospective study, the incidence of false positive and false negative interpretation of x-rays for solid spinal arthrodesis with spinal instrumentation was evaluated in 75 patients. OBJECTIVE: To evaluate the accuracy of the interpretation of x-rays for diagnosing solid spinal arthrodesis in patients with spinal instrumentation. SUMMARY OF BACKGROUND DATA: This retrospective study compared spinal fusion, as determined by direct observation and radiographic evaluation, in 75 patients with instrumented lumbar fusions using multiple devices. The fusions included posterolateral fusions or posterolateral with interbody fusions. Autograft, allograft, and a combination of these also were used. METHODS: A single blinded examiner reviewed all x-rays immediately before the spinal hardware was removed and the fusion mass was explored by the surgeon. RESULTS: There was a positive correlation between x-rays and the observations at the time of surgery in only 68% of the patients. CONCLUSION: This study indicates that the accuracy of x-ray interpretation for spinal arthrodesis is only 68%. The L4-L5 level was the most difficult level to fuse and the most difficult to interpret using x-rays. Patients with persistent back pain, when nonmechanical causes have been ruled out, should be considered for surgical exploration of the fusion mass even if x-rays appear to indicate a solid fusion.

Flashlamp-excited dye laser therapy of idiopathic vulvodynia is safe and efficacious.

OBJECTIVE: The management of chronic vulvovaginal pain, not explicable on specific histologic grounds, presents a major problem in referral centers for lower genital tract diseases. STUDY DESIGN: This article reports on a two-step protocol in a sample of 175 medical nonresponders, drawn from a 2-year cohort of 725 women with vulvovaginal pain. The first maneuver was the use of a flashlamp-excited dye laser to selectively photocoagulate symptomatic subepithelial blood vessels in 168 women; the second was the microsurgical removal of chronically painful Bartholin's glands in 52 women not responsive or not suited to flashlamp-excited dye laser photothermolysis. RESULTS: Dye laser response rates were independent of whether patients manifested macroscopic foci of painful erythema ("vestibular adenitis") or just colposcopically apparent hyperemia-ectasia of the individual blood vessels ("pruritic papillomatosis") (56% vs 45% after a single surgical procedure; 76% vs 65% after serial retreatment; p not significant). Conversely, response rates were much lower among women in whom pressure on the Bartholin's glands produced sharp, lancinating pain (15% vs 66% after a single surgical procedure; 22% vs 93% after serial retreatment; p < 0.001). Forty-two (85%) of 50 patients with flashlamp-excited dye laser failure had deep pain; however, the impasse to progress was broken by gland removal. Final response rates were 92.5% (complete response 62%; partial response 30%) in the "surface-only" group and 80.3% in the "surface-plus-deep" group (chi 2 = 14.9; p < 0.001). The major complication was acute bacterial cellulitis, occurring in the first postoperative week. Modification of the treatment protocol to include topical antibiotics with an occlusive dressing reduced the cellulitis rate from 17.2% to 2.5%. In four women (1.8%) Koebner-like exophytic condylomas also developed within 1 month of flashlamp-excited dye laser surgery. CONCLUSION: The availability of a safe, efficacious, and relatively noninvasive treatment should reduce the need for resective surgery in most patients with idiopathic vulvodynia.

Silencer region of a chalcone synthase promoter contains multiple binding sites for a factor, SBF-1, closely related to GT-1.

Bean nuclear extracts were used in gel retardation assays and DNase I footprinting experiments to identify a protein factor, designated SBF-1, that specifically interacts with regulatory sequences in the promoter of the bean defense gene CHS15, which encodes the flavonoid biosynthetic enzyme chalcone synthase. SBF-1 binds to three short sequences designated boxes 1, 2 and 3 in the region -326 to - 173. This cis-element, which is involved in organ-specific expression in plant development, functions as a transcriptional silencer in electroporated protoplasts derived from undifferentiated suspension-cultured soybean cells. The silencer element activates in trans a co-electroporated CHS15-chloramphenicol acetyl-transferase gene fusion, indicating that the factor acts as a repressor in these cells. SBF-1 binding in vitro is rapid, reversible and sensitive to prior heat or protease treatment. Competitive binding assays show that boxes 1, 2 and 3 interact cooperatively, but that each box can bind the factor independently, with box 3 showing the strongest binding and box 2 the weakest binding. GGTTAA(A/T)(A/T)(A/T), which forms a consensus sequence common to all three boxes, resembles the binding site for the GT-1 factor in light-responsive elements of the pea rbcS-3A gene, which encodes the small subunit of ribulose bisphosphate carboxylase. Binding to the CHS15 -326 to -173 element, and to boxes 1, 2 or 3 individually, is competed by the GT-1 binding sequence of rbcS-3A, but not by a functionally inactive form, and likewise the CHS sequences can compete with authentic GT-1 sites from the rbcS-3A promoter for binding.(ABSTRACT TRUNCATED AT 250 WORDS)