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Abstract A clear understanding of real-world uptake of nirmatrelvir–ritonavir for treatment of SARS-CoV-2 can inform treatment allocation strategies and improve interpretation of effectiveness studies. We used data from a large US healthcare system to describe nirmatrelvir–ritonavir dispenses among all SARS-CoV-2 positive patients aged ≥ 12 years meeting recommended National Institutes of Health treatment eligibility criteria for the study period between 1 January and 31 December, 2022. Overall, 10.9% (N = 34,791/319,900) of treatment eligible patients with SARS-CoV-2 infections received nirmatrelvir–ritonavir over the study period. Although uptake of nirmatrelvir–ritonavir increased over time, by the end of 2022, less than a quarter of treatment eligible patients with SARS-CoV-2 infections had received nirmatrelvir–ritonavir. Across patient demographics, treatment was generally consistent with tiered treatment guidelines, with dispenses concentrated among patients aged ≥ 65 years (14,706/63,921; 23.0%), and with multiple comorbidities (10,989/54,431; 20.1%). However, neighborhoods of lower socioeconomic status (upper third of neighborhood deprivation index [NDI]) had between 12% (95% CI: 7–18%) and 28% (25–32%) lower odds of treatment dispense over the time periods studied compared to the lower third of NDI distribution, even after accounting for demographic and clinical characteristics. A limited chart review (N = 40) confirmed that in some cases a decision not to treat was appropriate and aligned with national guidelines to use clinical judgement on a case-by-case basis. There is a need to enhance patient and provider awareness on the availability and benefits of nirmatrelvir–ritonavir for the treatment of COVID-19 illness.

Abstract Burn wounds are a common challenge for medical professionals. Current burn wound models hold several limitations, including a lack of comparability due to the heterogeneity of wounds and differences in individual wound healing. Hence, there is a need for reproducible in vivo models. In this study, we established a new burn wound model using the chorioallantoic membrane assay (CAM) as a surrogate model for animal experiments. The new experimental setup was tested by investigating the effects of the auspicious biophysical therapy, photobiomodulation (PBM), on the wound healing of an induced CAM burn wound with a metal stamp. PBM has been shown to positively influence wound healing through vascular proliferative effects and the increased secretion of chemotactic substances. The easily accessible burn wounds can be treated with various therapies. The model enables the analysis of ingrowing blood vessels (angiogenesis) and diameter and area of the wounds. The established model was used to test the effects of PBM on burn wound healing. PBM promoted angiogenesis in burn wounds on day 4 (p = 0.005). Furthermore, there was a not significant trend toward a higher number of vessels for day 6 (p = 0.065) in the irradiated group. Changes in diameter (p = 0.129) and the burn area (p = 0.131) were not significant. Our results suggest that CAM can be a suitable model for studying burn wounds. The novel experimental design enables reproducible and comparable studies on burn wound treatment.

Thrombosis can cause life-threatening disorders. Unfortunately, current therapeutic methods for thrombosis using injecting thrombolytic medicines systemically resulted in unexpected bleeding complications. Moreover, the absence of practical imaging tools for thrombi raised dangers of undertreatment and overtreatment. This study develops a theranostic drug carrier, Pkr(IR-Ca/Pda-uPA)-cRGD, that enables real-time monitoring of the targeted thrombolytic process of deep vein thrombosis (DVT). Pkr(IR-Ca/Pda-uPA)-cRGD, which is prepared from a Pickering-emulsion-like system, encapsulates both near-infrared-II (NIR-II) contrast agent (IR-1048 dye, loading capacity: 28%) and urokinase plasminogen activators (uPAs, encapsulation efficiency: 89%), pioneering the loading of multiple drugs with contrasting hydrophilicity into one single-drug carrier. Upon intravenous injection, Pkr(IR-Ca/Pda-uPA)-cRGD considerably targets to thrombi selectively (targeting rate: 91%) and disintegrates in response to acidic thrombi to release IR-1048 dye and uPA for imaging and thrombolysis, respectively. Investigations indicate that Pkr(IR-Ca/Pda-uPA)-cRGD enabled real-time visualization of targeted thrombolysis using NIR-II imaging in DVT models, in which thrombi were eliminated (120 min after drug injection) without bleeding complications. This may be the first study using convenient NIR-II imaging for real-time visualization of targeted thrombolysis. It represents the precision medicine that enables rapid response to acquire instantaneous medical images and make necessary real-time adjustments to diagnostic and therapeutic protocols during treatment.

Background Research infrastructures are facilities or resources that have proven fundamental for supporting scientific research and innovation. However, they are also known to be very expensive in their establishment, operation and maintenance. As by far the biggest share of these costs is always borne by public funders, there is a strong interest and indeed a necessity to develop alternative business models for such infrastructures that allow them to function in a more sustainable manner that is less dependent on public financing. Methods In this article, we describe a feasibility study we have undertaken to develop a potentially sustainable business model for a vaccine research and development (R&D) infrastructure. The model we have developed integrates two different types of business models that would provide the infrastructure with two different types of revenue streams which would facilitate its establishment and would be a measure of risk reduction. For the business model we are proposing, we have undertaken an ex ante impact assessment that estimates the expected impact for a vaccine R&D infrastructure based on the proposed models along three different dimensions: health, society and economy. Results Our impact assessment demonstrates that such a vaccine R&D infrastructure could achieve a very significant socio-economic impact, and so its establishment is therefore considered worthwhile pursuing. Conclusions The business model we have developed, the impact assessment and the overall process we have followed might also be of interest to other research infrastructure initiatives in the biomedical field.

Abstract Dehydration of the oceanic subducting slab promotes the formation of magmatic arcs, intra-slab intermediate-depth seismicity, and hydration of the overlying mantle wedge. However, the complex permeability structure of the overriding plate controls the magma and fluid migration and their accumulation at shallower depths. In this regard, mapping the inner structure of the overriding crust and mantle is crucial to understand the magmatic and hydrological processes in subduction zones. We integrate 3-D P-wave, $$V_p/V_s$$ V p / V s , and electrical resistivity tomographic models of the northern Chilean subduction zone to map the magmatic and fluids derived from the subducting oceanic Nazca plate. Results show a continental crust relatively thick (50–65 km) characterized by a lower zone of high $$V_p$$ V p values (7.2–7.6 km/s), which is interpreted as the presence of plutonic rocks. The mantle lithospheric wedge is weakly hydrated ( $$V_p/V_s$$ V p / V s = 1.75–1.8) while the forearc continental crust is traversed by regions of reduced electrical resistivity values ( $$< 10^2$$ < 10 2 $$\Omega m$$ Ω m ) interpreted as zones of relatively high permeability/fracturing and fluid content. These regions spatially correlate with upper plate trans-lithospheric deformation zones. Ascending melts accumulate preferentially in the back-arc, whereas hydrothermal systems form trenchward of the volcanic arc. The results highlight the complex permeability structure of the upper South American plate.

To be able to use infectious disease next generation sequencing as a diagnostic tool, appropriate reference datasets are required. Here, Sichtig et al. describe FDA-ARGOS, a reference database for high-quality microbial reference genomes, and demonstrate its utility on the example of two use cases.

BackgroundOccupational health hazards are ubiquitously found in the operating room, guaranteeing an inevitable risk of exposure to the surgeon. Although provisions on occupational health and safety in healthcare exist, they do not address non-traditional hazards found in the operating room. In order to determine whether surgeons or trainees receive any form of occupational health training, we examine the associations between occupational health training and exposure rate.Study designA cross-sectional survey was distributed. Respondent characteristics included academic level, race/ethnicity, and gender. The survey evaluated seven surgical disciplines and 13 occupational hazards. Multivariable logistic regression was used to examine the association between academic level, surgical specialty, and exposure rate.ResultsOur cohort of 183 respondents (33.1% response rate) consisted of attendings (n = 72, 39.3%) and trainees (n = 111, 60.7%). Surgical trainees were less likely to have been trained in cytotoxic drugs (OR 0.22, pConclusionAlthough surgeon safety is not a domain in residency training, standardized efforts to educate and change the culture of safety in residency programs is warranted. Our study demonstrates a disparity between trainees and attendings with a recommendation to provide formal training to trainees independent of their anticipated risk of exposure.

Pulse is the base timing to which western music is commonly notated, generally expressed by a listener by performing periodic taps with their hand or foot. This cognitive construction helps organize the perception of timed events in music and is the most basic expectation in rhythms. The analysis of expectations, and more specifically the strength with which the beat is felt-the pulse clarity-has been used to analyze affect in music. Most computational models of pulse clarity, and rhythmic expectation in general, analyze the input as a whole, without exhibiting changes through a rhythmic passage. We present Tactus Hypothesis Tracker (THT), a model of pulse clarity over time intended for symbolic rhythmic stimuli. The model was developed based on ideas of beat tracking models that extract beat times from musical stimuli. Our model also produces possible beat interpretations for the rhythm, a fitness score for each interpretation and how these evolve in time. We evaluated the model's pulse clarity by contrasting against tapping variability of human annotators achieving results comparable to a state-of-the-art pulse clarity model. We also analyzed the clarity metric dynamics on synthetic data that introduced changes in the beat, showing that our model presented doubt in the pulse estimation process and adapted accordingly to beat changes. Finally, we assessed if the beat tracking generated by the model was correct regarding listeners tapping data. We compared our beat tracking results with previous beat tracking models. The THT model beat tracking output showed generally correct estimations in phase but exhibits a bias towards a musically correct subdivision of the beat.

IntroductionDifferences in academic qualifications are cited as the reason behind the documented gender gap in industry sponsorship to academic plastic surgeons. Gendered imbalances in academic metrics narrow among senior academic plastic surgeons. However, it is unknown whether this gender parity translates to industry payments.MethodsWe conducted a cross-sectional analysis of industry payments disbursed to plastic surgeons in 2018. Inclusion criteria encompassed (i) faculty with the rank of professor or a departmental leadership position. Exclusion criteria included faculty (i) who belonged to a speciality besides plastic surgery; (ii) whose gender could not be determined; or (iii) whose name could not be located on the Open Payment Database. Faculty and title were identified using departmental listings of ACGME plastic surgery residency programs. We extracted industry payment data through the Open Payment Database. We also collected details on H-index and time in practice. Statistical analysis included odds ratios (OR) and Pearson's correlation coefficient (R).ResultsWe identified 316 senior academic plastic surgeons. The cohort was predominately male (88%) and 91% held a leadership role. Among departmental leaders, women were more likely to be an assistant professor (OR 3.9, p = 0.0003) and heads of subdivision (OR 2.1, p = 0.0382) than men. Industry payments were distributed equally to male and female senior plastic surgeons except for speakerships where women received smaller amounts compared to their male counterparts (median payments of $3,675 vs $7,134 for women and men respectively, pConclusionDisparity in industry funding narrows at senior levels in academic plastic surgery. At higher academic levels, industry sponsorship may preferentially fund individuals based on academic productivity and career length. Increased transparency in selection criteria for speakerships is warranted.

Pulse is the base timing to which western music is commonly notated, generally expressed by a listener by performing periodic taps with their hand or foot. This cognitive construction helps organize the perception of timed events in music and is the most basic expectation in rhythms. The analysis of expectations, and more specifically the strength with which the beat is felt—the pulse clarity—has been used to analyze affect in music. Most computational models of pulse clarity, and rhythmic expectation in general, analyze the input as a whole, without exhibiting changes through a rhythmic passage. We present Tactus Hypothesis Tracker (THT), a model of pulse clarity over time intended for symbolic rhythmic stimuli. The model was developed based on ideas of beat tracking models that extract beat times from musical stimuli. Our model also produces possible beat interpretations for the rhythm, a fitness score for each interpretation and how these evolve in time. We evaluated the model’s pulse clarity by contrasting against tapping variability of human annotators achieving results comparable to a state-of-the-art pulse clarity model. We also analyzed the clarity metric dynamics on synthetic data that introduced changes in the beat, showing that our model presented doubt in the pulse estimation process and adapted accordingly to beat changes. Finally, we assessed if the beat tracking generated by the model was correct regarding listeners tapping data. We compared our beat tracking results with previous beat tracking models. The THT model beat tracking output showed generally correct estimations in phase but exhibits a bias towards a musically correct subdivision of the beat.

Abstract About 70 genetic studies have already addressed the need of biomarkers to predict the response of patients with rheumatoid arthritis (RA) to methotrexate (MTX) treatment. However, no genetic biomarker has yet been sufficiently validated. Here, we aimed to replicate a selection of 25 SNPs in the largest collection of patients up to date, which consisted of 915 patients treated with MTX. The change in disease activity (measured as ΔDAS28) from baseline was considered the primary outcome. In addition, response according to widely used criteria (EULAR) was taken as secondary outcome. We considered consistency between outcomes, P values accounting for the number of SNPs, and independence from potential confounders for interpretation of the results. Only the rs1801394 SNP in MTRR fulfilled the high association standards. Its minor allele was associated with less improvement than the major allele according to ΔDAS28 (p = 0.0016), and EULAR response (p = 0.004), with independence of sex, age, baseline DAS28, smoking, seropositivity, concomitant corticosteroid use or previous treatments. In addition, previous evidence suggests the association of this SNP with response to MTX in another autoimmune disease, juvenile idiopathic arthritis, and with high intracellular folate levels, which could contribute to poor response.

Abstract The concentrations of testosterone deposited in hair during hair growth may provide a retrospective reflection of the concentrations of bioactive testosterone in plasma. The objective of this study was to develop a radioimmunoassay with a sufficiently low limit of detection to measure the testosterone-like immunoreactivity in smaller hair samples (5 mg) than used in earlier studies, and to compare three different extraction procedures. The competitive radioimmunoassay consisted of a polyclonal antiserum (immunogen testosterone-7α-BSA) and a radioligand synthesised from testosterone-3-CMO-histamine. The within-assay and total coefficients of variation in the working range was 3% and 4.5%, respectively. The limit of detection was 0.87 pg/mL, which is equivalent to 0.12 pg/mg testosterone in 5 mg of hair. The concentration of testosterone-like immunoreactivity in hair samples was 1.23 (SD 0.47) pg/mg in women and 2.67 (SD 0.58) pg/mg in men (pulverised hair). Significantly improved precision was found when pulverised hair was used compared to non-pulverised hair. Our data indicate that pulverisation of the hair prior to hormone extraction is crucial. Detection limits fit for the intended purpose are achievable with 5 mg samples of hair.

Abstract The discovery of processes for the synthesis of new materials involves many decisions about process design, operation, and material properties. Experimentation is crucial but as complexity increases, exploration of variables can become impractical using traditional combinatorial approaches. We describe an iterative method which uses machine learning to optimise process development, incorporating multiple qualitative and quantitative objectives. We demonstrate the method with a novel fluid processing platform for synthesis of short polymer fibers, and show how the synthesis process can be efficiently directed to achieve material and process objectives.

The problem of skill acquisition is ubiquitous and fundamental to life. Most tasks in modern society involve the cooperation with other subjects. Notwithstanding its fundamental importance, teammate selection is commonly overlooked when studying learning. We exploit the virtually infinite repository of human behavior available in Internet to study a relevant topic in anthropological science: how grouping strategies may affect learning. We analyze the impact of team play strategies in skill acquisition using a turn-based game where players can participate individually or in teams. We unveil a subtle but strong effect in skill acquisition based on the way teams are formed and maintained during time. "Faithfulness-boost effect" provides a skill boost during the first games that would only be acquired after thousands of games. The tendency to play games in teams is associated with a long-run skill improvement while playing loyally with the same teammate significantly accelerates short-run skill acquisition.

Microarrays have proven to be useful in rapid detection of many viruses and bacteria. Pathogen detection microarrays have been used to diagnose viral and bacterial infections in clinical samples and to evaluate the safety of biological drug materials. In this study, the Axiom Microbiome Array was evaluated to determine its sensitivity, specificity and utility in microbiome analysis of veterinary clinical samples. The array contains probes designed to detect more than 12,000 species of viruses, bacteria, fungi, protozoa and archaea, yielding the most comprehensive microbial detection platform built to date. The array was able to detect Shigella and Aspergillus at 100 genome copies, and vaccinia virus DNA at 1,000 genome copies. The Axiom Microbiome Array made correct species-level calls in mock microbial community samples. When tested against serum, tissue, and fecal samples from pigs experimentally co-infected with porcine reproductive and respiratory syndrome virus and porcine circovirus type 2, the microarray correctly detected these two viruses and other common viral and bacterial microbiome species. This cost-effective and high-throughput microarray is an efficient tool to rapidly analyze large numbers of clinical and environmental samples for the presence of multiple viral and bacterial pathogens.

Kawasaki disease (KD), first identified in 1967, is a pediatric vasculitis of unknown etiology that has an increasing incidence in Japan and many other countries. KD can cause coronary artery aneurysms. Its epidemiological characteristics, such as seasonality and clinical picture of acute systemic inflammation with prodromal intestinal/respiratory symptoms, suggest an infectious etiology for KD. Interestingly, multiple host genotypes have been identified as predisposing factors for KD. To explore experimental methodology for identifying etiological agent(s) for KD and to optimize epidemiological study design (particularly the sample size) for future studies, we conducted a pilot study. For a 1-year period, we prospectively enrolled 11 patients with KD. To each KD patient, we assigned two control individuals (one with diarrhea and the other with respiratory infections), matched for age, sex, and season of diagnosis. During the acute phase of disease, we collected peripheral blood, nasopharyngeal aspirate, and feces. We also determined genotypes, to identify those that confer susceptibility to KD. There was no statistically significant difference in the frequency of the risk genotypes between KD patients and control subjects. We also used unbiased metagenomic sequencing to analyze these samples. Metagenomic sequencing and PCR detected torque teno virus 7 (TTV7) in two patients with KD (18%), but not in control subjects (P = 0.111). Sanger sequencing revealed that the TTV7 found in the two KD patients contained almost identical variants in nucleotide and identical changes in resulting amino acid, relative to the reference sequence. Additionally, we estimated the sample size that would be required to demonstrate a statistical correlation between TTV7 and KD. Future larger scale studies with carefully optimized metagenomic sequencing experiments and adequate sample size are warranted to further examine the association between KD and potential pathogens, including TTV7.

Symptomatic intracerebral hemorrhage (sICH) after bridging thrombolysis for acute ischemic stroke is a devastating complication. We aimed to assess whether the additional administration of aspirin during endovascular intervention increases bleeding rates.We retrospectively compared bleeding complications and outcome in stroke patients who received bridging thrombolysis with (tPA+ASA) and without (tPA-ASA) aspirin during endovascular intervention between November 2008 and March 2014. Furthermore, we analyzed bleeding complications and outcome in antiplatelet naïve patients with those with prior or acute antiplatelet therapy.Baseline characteristics, previous medication, and dosage of rtPA did not differ between 50 tPA+ASA (39 aspirin naïve, 11 preloaded) and 181 tPA-ASA patients (p>0.05). tPA+ASA patients had more often internal carotid artery (ICA) occlusion (p0.1). There were no differences in bleeding complications and mortality among 112 bridging patients with antiplatelet therapy (62 preloaded, 39 acute administration, 11 both) and 117 antiplatelet naïve patients. In a logistic regression analysis, aspirin administration during endovascular procedure was not a predictor of sICH.Antiplatelet therapy before or during bridging thrombolysis in patients with acute ischemic stroke did not increase the risk of bleeding complications and had no impact on outcome. This finding has to be confirmed in larger studies.

The paper aims at detecting the existing safety risks on the minor railway-tracks being operated in accordance with the provision CD-D3 of the Czech Railways, and at providing the solutions and technologies suitable for the mentioned minor railway-tracks. These tracks are currently equipped with out of date safety related systems monitoring the railway, whose operation depends purely on human factor. The latest railway accidents were reported just on these low traffic frequency tracks.

Levitated optomechanical systems, and particularly particles trapped in vacuum, provide unique platforms for studying the mechanical behavior of objects well-isolated from their environment. Ultimately, such systems may enable the study of fundamental questions in quantum mechanics, gravity, and other weak forces. While the optical trapping of nanoparticles has emerged as the prototypical levitated optomechanical system, it is not without problems due to the heating from the high optical intensity required, particularly when combined with a high vacuum environment. Here we investigate a magneto-gravitational trap in ultra-high vacuum. In contrast to optical trapping, we create an entirely passive trap for diamagnetic particles by utilizing the magnetic field generated by permanent magnets and the gravitational interaction. We demonstrate cooling the center of mass motion of a trapped silica microsphere from ambient temperature to an effective temperature near or below one milliKelvin in two degrees of freedom by optical feedback damping.

There is a prevailing belief that interruptions using cellular phones during face to face interactions may affect severely how people relate and perceive each other. We set out to determine this cost quantitatively through an experiment performed in dyads, in a large audience in a TEDx event. One of the two participants (the speaker) narrates a story vividly. The listener is asked to deliberately ignore the speaker during part of the story (for instance, attending to their cell-phone). The speaker is not aware of this treatment. We show that total amount of attention is the major factor driving subjective beliefs about the story and the conversational partner. The effects are mostly independent on how attention is distributed in time. All social parameters of human communication are affected by attention time with a sole exception: the perceived emotion of the story. Interruptions during day-to-day communication between peers are extremely frequent. Our data should provide a note of caution, by indicating that they have a major effect on the perception people have about what they say (whether it is interesting or not . . .) and about the virtues of the people around them.

Inner concepts are much richer than the words that describe them. Our general objective is to inquire what are the best procedures to communicate conceptual knowledge. We construct a simplified and controlled setup emulating important variables of pedagogy amenable to quantitative analysis. To this aim, we designed a game inspired in Chinese Whispers, to investigate which attributes of a description affect its capacity to faithfully convey an image. This is a two player game: an emitter and a receiver. The emitter was shown a simple geometric figure and was asked to describe it in words. He was informed that this description would be passed to the receiver who had to replicate the drawing from this description. We capitalized on vast data obtained from an android app to quantify the effect of different aspects of a description on communication precision. We show that descriptions more effectively communicate an image when they are coherent and when they are procedural. Instead, the creativity, the use of metaphors and the use of mathematical concepts do not affect its fidelity.

Immune cell-type specific miRNA expression patterns have been described but the detailed role of single miRNAs in the function of T-cells remains largely unknown. We investigated the role of miR-21 in the function of primary human CD4+ T-cells. MiR-21 is substantially expressed in T-cells with a memory phenotype, and is robustly upregulated upon αCD3/CD28 activation of both naive and memory T-cells. By inhibiting the endogenous miR-21 function in activated naive and memory T-cells, we showed that miR-21 regulates fundamentally different aspects of T-cell biology, depending on the differentiation status of the T-cell. Stable inhibition of miR-21 function in activated memory T-cells led to growth disadvantage and apoptosis, indicating that the survival of memory T-cells depends on miR-21 function. In contrast, stable inhibition of miR-21 function in activated naive T-cells did not result in growth disadvantage, but led to a significant induction of CCR7 protein expression. Direct interaction between CCR7 and miR-21 was confirmed in a dual luciferase reporter assay. Our data provide evidence for a dual role of miR-21 in CD4+ T cells; Regulation of T-cell survival is confined to activated memory T-cells, while modulation of potential homing properties, through downregulation of CCR7 protein expression, is observed in activated naive T-cells.

Processing of miRNAs occurs simultaneous with the transcription and splicing of their primary transcripts. For the small subset of exonic miRNAs it is unclear if the unspliced and/or spliced transcripts are used for miRNA biogenesis. We assessed endogenous levels and cellular location of primary transcripts of three exonic miRNAs. The ratio between unspliced and spliced transcripts varied markedly, i.e. >1 for BIC,

The high mutation rate of RNA viruses enables a diverse genetic population of viral genotypes to exist within a single infected host. In-host genetic diversity could better position the virus population to respond and adapt to a diverse array of selective pressures such as host-switching events. Multiple new coronaviruses, including SARS, have been identified in human samples just within the last ten years, demonstrating the potential of coronaviruses as emergent human pathogens. Deep sequencing was used to characterize genomic changes in coronavirus quasispecies during simulated host-switching. Three bovine nasal samples infected with bovine coronavirus were used to infect human and bovine macrophage and lung cell lines. The virus reproduced relatively well in macrophages, but the lung cell lines were not infected efficiently enough to allow passage of non lab-adapted samples. Approximately 12 kb of the genome was amplified before and after passage and sequenced at average coverages of nearly 950×(454 sequencing) and 38,000×(Illumina). The consensus sequence of many of the passaged samples had a 12 nucleotide insert in the consensus sequence of the spike gene, and multiple point mutations were associated with the presence of the insert. Deep sequencing revealed that the insert was present but very rare in the unpassaged samples and could quickly shift to dominate the population when placed in a different environment. The insert coded for three arginine residues, occurred in a region associated with fusion entry into host cells, and may allow infection of new cell types via heparin sulfate binding. Analysis of the deep sequencing data indicated that two distinct genotypes circulated at different frequency levels in each sample, and support the hypothesis that the mutations present in passaged strains were "selected" from a pre-existing pool rather than through de novo mutation and subsequent population fixation.

Neuropathic pain elevates spinal anandamide (AEA) levels in a way further increased when URB597, an inhibitor of AEA hydrolysis by fatty acid amide hydrolase (FAAH), is injected intrathecally. Spinal AEA reduces neuropathic pain by acting at both cannabinoid CB1 receptors and transient receptor potential vanilloid-1 (TRPV1) channels. Yet, intrathecal URB597 is only partially effective at counteracting neuropathic pain. We investigated the effect of high doses of intrathecal URB597 on allodynia and hyperalgesia in rats with chronic constriction injury (CCI) of the sciatic nerve. Among those tested, the 200 µg/rat dose of URB597 was the only one that elevated the levels of the FAAH non-endocannabinoid and anti-inflammatory substrates, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), and of the endocannabinoid FAAH substrate, 2-arachidonoylglycerol, and fully inhibited thermal and tactile nociception, although in a manner blocked almost uniquely by TRPV1 antagonism. Surprisingly, this dose of URB597 decreased spinal AEA levels. RT-qPCR and western blot analyses demonstrated altered spinal expression of lipoxygenases (LOX), and baicalein, an inhibitor of 12/15-LOX, significantly reduced URB597 analgesic effects, suggesting the occurrence of alternative pathways of AEA metabolism. Using immunofluorescence techniques, FAAH, 15-LOX and TRPV1 were found to co-localize in dorsal spinal horn neurons of CCI rats. Finally, 15-hydroxy-AEA, a 15-LOX derivative of AEA, potently and efficaciously activated the rat recombinant TRPV1 channel. We suggest that intrathecally injected URB597 at full analgesic efficacy unmasks a secondary route of AEA metabolism via 15-LOX with possible formation of 15-hydroxy-AEA, which, together with OEA and PEA, may contribute at producing TRPV1-mediated analgesia in CCI rats.

While it is often thought that the geometric phase is less sensitive to fluctuations in the control fields, a very general feature of adiabatic Hamiltonians is the unavoidable dynamic phase that accompanies the geometric phase. The effect of control field noise during adiabatic geometric quantum gate operations has not been probed experimentally, especially in the canonical spin qubit system that is of interest for quantum information. We present measurement of the Berry phase and carry out adiabatic geometric phase gate in a single solid-state spin qubit associated with the nitrogen-vacancy center in diamond. We manipulate the spin qubit geometrically by careful application of microwave radiation that creates an effective rotating magnetic field, and observe the resulting Berry phase signal via spin echo interferometry. Our results show that control field noise at frequencies higher than the spin echo clock frequency causes decay of the quantum phase, and degrades the fidelity of the geometric phase gate to the classical threshold after a few (∼10) operations. This occurs inspite of the geometric nature of the state preparation, due to unavoidable dynamic contributions. We have carried out systematic analysis and numerical simulations to study the effects of the control field noise and imperfect driving waveforms on the quantum phase gate.

MicroRNA (miRNA) sponges are transcripts with repeated miRNA antisense sequences that can sequester miRNAs from endogenous targets. MiRNA sponges are valuable tools for miRNA loss-of-function studies both in vitro and in vivo. We developed a fast and flexible method to generate miRNA sponges and tested their efficiency in various assays. Using a single directional ligation reaction we generated sponges with 10 or more miRNA binding sites. Luciferase and AGO2-immuno precipitation (IP) assays confirmed effective binding of the miRNAs to the sponges. Using a GFP competition assay we showed that miR-19 sponges with central mismatches in the miRNA binding sites are efficient miRNA inhibitors while sponges with perfect antisense binding sites are not. Quantification of miRNA sponge levels suggests that this is at least in part due to degradation of the perfect antisense sponge transcripts. Finally, we provide evidence that combined inhibition of miRNAs of the miR-17∼92 cluster results in a more effective growth inhibition as compared to inhibition of individual miRNAs. In conclusion, we describe and validate a method to rapidly generate miRNA sponges for miRNA loss-of-function studies.

BACKGROUND: The vast computational resources that became available during the past decade enabled the development and simulation of increasingly complex mathematical models of cancer growth. These models typically involve many free parameters whose determination is a substantial obstacle to model development. Direct measurement of biochemical parameters in vivo is often difficult and sometimes impracticable, while fitting them under data-poor conditions may result in biologically implausible values. RESULTS: We discuss different methodological approaches to estimate parameters in complex biological models. We make use of the high computational power of the Blue Gene technology to perform an extensive study of the parameter space in a model of avascular tumor growth. We explicitly show that the landscape of the cost function used to optimize the model to the data has a very rugged surface in parameter space. This cost function has many local minima with unrealistic solutions, including the global minimum corresponding to the best fit. CONCLUSIONS: The case studied in this paper shows one example in which model parameters that optimally fit the data are not necessarily the best ones from a biological point of view. To avoid force-fitting a model to a dataset, we propose that the best model parameters should be found by choosing, among suboptimal parameters, those that match criteria other than the ones used to fit the model. We also conclude that the model, data and optimization approach form a new complex system and point to the need of a theory that addresses this problem more generally.

Emerging known and unknown pathogens create profound threats to public health. Platforms for rapid detection and characterization of microbial agents are critically needed to prevent and respond to disease outbreaks. Available detection technologies cannot provide broad functional information about known or novel organisms. As a step toward developing such a system, we have produced and tested a series of high-density functional gene arrays to detect elements of virulence and antibiotic resistance mechanisms. Our first generation array targets genes from Escherichia coli strains K12 and CFT073, Enterococcus faecalis and Staphylococcus aureus. We determined optimal probe design parameters for gene family detection and discrimination. When tested with organisms at varying phylogenetic distances from the four target strains, the array detected orthologs for the majority of targeted gene families present in bacteria belonging to the same taxonomic family. In combination with whole-genome amplification, the array detects femtogram concentrations of purified DNA, either spiked in to an aerosol sample background, or in combinations from one or more of the four target organisms. This is the first report of a high density NimbleGen microarray system targeting microbial antibiotic resistance and virulence mechanisms. By targeting virulence gene families as well as genes unique to specific biothreat agents, these arrays will provide important data about the pathogenic potential and drug resistance profiles of unknown organisms in environmental samples.