Search

Tissue engineering predominantly relies on trial and error in vitro and ex vivo experiments to develop protocols and bioreactors to generate functional tissues. As an alternative, in silico methods have the potential to significantly reduce the timelines and costs of experimental programs for tissue engineering. In this paper, we propose a methodology to formulate, select, calibrate, and test mathematical models to predict cell population growth as a function of the biochemical environment and to design optimal experimental protocols for model inference of in silico model parameters. We systematically combine methods from the experimental design, mathematical statistics, and optimization literature to develop unique and explainable mathematical models for cell population dynamics. The proposed methodology is applied to the development of this first published model for a population of the airway-relevant bronchio-alveolar epithelial (BEAS-2B) cell line as a function of the concentration of metabolic-related biochemical substrates. The resulting model is a system of ordinary differential equations that predict the temporal dynamics of BEAS-2B cell populations as a function of the initial seeded cell population and the glucose, oxygen, and lactate concentrations in the growth media, using seven parameters rigorously inferred from optimally designed in vitro experiments.

El Gigante rockshelter in western Honduras provides a deeply stratified archaeological record of human-environment interaction spanning the entirety of the Holocene. Botanical materials are remarkably well preserved and include important tree (e.g., ciruela (Spondias), avocado (Persea americana)) and field (maize (Zea mays), beans (Phaseolus), and squash (Cucurbita)) crops. Here we provide a major update to the chronology of tree and field crop use evident in the sequence. We report 375 radiocarbon dates, a majority of which are for short-lived botanical macrofossils (e.g., maize cobs, avocado seeds, or rinds). Radiocarbon dates were used in combination with stratigraphic details to establish a Bayesian chronology for ~9,800 identified botanical samples spanning the last 11,000 years. We estimate that at least 16 discrete intervals of use occurred during this time, separated by gaps of ~100-2,000 years. The longest hiatus in rockshelter occupation was between ~6,400 and 4,400 years ago and the deposition of botanical remains peaked at ~2,000 calendar years before present (cal BP). Tree fruits and squash appeared early in the occupational sequence (~11,000 cal BP) with most other field crops appearing later in time (e.g., maize at ~4,400 cal BP; beans at ~2,200 cal BP). The early focus on tree fruits and squash is consistent with early coevolutionary partnering with humans as seed dispersers in the wake of megafaunal extinction in Mesoamerica. Tree crops predominated through much of the Holocene, and there was an overall shift to field crops after 4,000 cal BP that was largely driven by increased reliance on maize farming.

IntroductionGeriatric co-management is known to improve treatment of older adults in various clinical settings, however, widespread application of the concept is limited due to restricted resources. Digitalization may offer options to overcome these shortages by providing structured, relevant information and decision support tools for medical professionals. We present the SURGE-Ahead project (Supporting SURgery with GEriatric co-management and Artificial Intelligence) addressing this challenge.MethodsA digital application with a dashboard-style user interface will be developed, displaying 1) evidence-based recommendations for geriatric co-management and 2) artificial intelligence-enhanced suggestions for continuity of care (COC) decisions. The development and implementation of the SURGE-Ahead application (SAA) will follow the Medical research council framework for complex medical interventions. In the development phase a minimum geriatric data set (MGDS) will be defined that combines parametrized information from the hospital information system with a concise assessment battery and sensor data. Two literature reviews will be conducted to create an evidence base for co-management and COC suggestions that will be used to display guideline-compliant recommendations. Principles of machine learning will be used for further data processing and COC proposals for the postoperative course. In an observational and AI-development study, data will be collected in three surgical departments of a University Hospital (trauma surgery, general and visceral surgery, urology) for AI-training, feasibility testing of the MGDS and identification of co-management needs. Usability will be tested in a workshop with potential users. During a subsequent project phase, the SAA will be tested and evaluated in clinical routine, allowing its further improvement through an iterative process.DiscussionThe outline offers insights into a novel and comprehensive project that combines geriatric co-management with digital support tools to improve inpatient surgical care and continuity of care of older adults.Trial registrationGerman clinical trials registry (Deutsches Register für klinische Studien, DRKS00030684), registered on 21st November 2022.

BackgroundRetrospective data demonstrates that robotic-assisted thoracoscopic surgery provides many benefits, such as decreased postoperative pain, lower mortality, shorter length of stay, shorter chest tube duration, and reductions in the incidence of common postoperative pulmonary complications, when compared to video-assisted thoracoscopic surgery. Despite the potential benefits of robotic surgery, there are two major barriers against its widespread adoption in thoracic surgery: lack of high-quality prospective data, and the perceived higher cost of it. Therefore, in the face of these barriers, a prospective randomized controlled trial comparing robotic- to video-assisted thoracoscopic surgery is needed. The RAVAL trial is a two-phase, international, multi-centered, blinded, parallel, randomized controlled trial that is comparing robotic- to video-assisted lobectomy for early-stage non-small cell lung cancer that has been enrolling patients since 2016.MethodsThe RAVAL trial will be conducted in two phases: Phase A will enroll 186 early-stage non-small cell lung cancer patients who are candidates for minimally invasive pulmonary lobectomy; while Phase B will continue to recruit until 592 patients are enrolled. After consent, participants will be randomized in a 1:1 ratio to either robotic- or video-assisted lobectomy, and blinded to the type of surgery they are allocated to. Health-related quality of life questionnaires will be administered at baseline, postoperative day 1, weeks 3, 7, 12, months 6, 12, 18, 24, and years 3, 4, 5. The primary objective of the RAVAL trial is to determine the difference in patient-reported health-related quality of life outcomes between the robotic- and video-assisted lobectomy groups at 12 weeks. Secondary objectives include determining the differences in cost-effectiveness, and in the 5-year survival data between the two arms. The results of the primary objective will be reported once Phase A has completed accrual and the 12-month follow-ups are completed. The results of the secondary objectives will be reported once Phase B has completed accrual and the 5-year follow-ups are completed.DiscussionIf successfully completed, the RAVAL Trial will have studied patient-reported outcomes, cost-effectiveness, and survival of robotic- versus video-assisted lobectomy in a prospective, randomized, blinded fashion in an international setting.Trial registrationClinicalTrials.gov, NCT02617186. Registered 22-September-2015. https://clinicaltrials.gov/ct2/show/NCT02617186.

[This corrects the article DOI: 10.1371/journal.pone.0252425.].

Accurate computation of joint angles from optical marker data using inverse kinematics methods requires that the locations of markers on a model match the locations of experimental markers on participants. Marker registration is the process of positioning the model markers so that they match the locations of the experimental markers. Markers are typically registered using a graphical user interface (GUI), but this method is subjective and may introduce errors and uncertainty to the calculated joint angles and moments. In this investigation, we use OpenSim to isolate and quantify marker registration-based error from other sources of error by analyzing the gait of a bipedal humanoid robot for which segment geometry, mass properties, and joint angles are known. We then propose a marker registration method that is informed by the orientation of anatomical reference frames derived from surface-mounted optical markers as an alternative to user registration using a GUI. The proposed orientation registration method reduced the average root-mean-square error in both joint angles and joint moments by 67% compared to the user registration method, and eliminated variability among users. Our results show that a systematic method for marker registration that reduces subjective user input can make marker registration more accurate and repeatable.

Structural variation (SV) is typically defined as variation within the human genome that exceeds 50 base pairs (bp). SV may be copy number neutral or it may involve duplications, deletions, and complex rearrangements. Recent studies have shown SV to be associated with many human diseases. However, studies of SV have been challenging due to technological constraints. With the advent of third generation (long-read) sequencing technology, exploration of longer stretches of DNA not easily examined previously has been made possible. In the present study, we utilized third generation (long-read) sequencing techniques to examine SV in the EGFR landscape of four haplotypes derived from two human samples. We analyzed the EGFR gene and its landscape (+/- 500,000 base pairs) using this approach and were able to identify a region of non-coding DNA with over 90% similarity to the most common activating EGFR mutation in non-small cell lung cancer. Based on previously published Alu-element genome instability algorithms, we propose a molecular mechanism to explain how this non-coding region of DNA may be interacting with and impacting the stability of the EGFR gene and potentially generating this cancer-driver gene. By these techniques, we were also able to identify previously hidden structural variation in the four haplotypes and in the human reference genome (hg38). We applied previously published algorithms to compare the relative stabilities of these five different EGFR gene landscape haplotypes to estimate their relative potentials to generate the EGFR exon 19, 15 bp canonical deletion. To our knowledge, the present study is the first to use the differences in genomic architecture between targeted cancer-linked phased haplotypes to estimate their relative potentials to form a common cancer-linked driver mutation.

Quantifying ecosystem-level processes that drive community structure and function is key to the development of effective environmental restoration and management programs. To assess the effects of large-scale aquatic vegetation loss on fish and invertebrate communities in Florida estuaries, we quantified and compared the food webs of two adjacent spring-fed rivers that flow into the Gulf of Mexico. We constructed a food web model using field-based estimates of community absolute biomass and trophic interactions of a highly productive vegetated river, and modeled long-term simulations of vascular plant decline coupled with seasonal production of filamentous macroalgae. We then compared ecosystem model predictions to observed community structure of the second river that has undergone extensive vegetative habitat loss, including extirpation of several vascular plant species. Alternative models incorporating bottom-up regulation (decreased primary production resulting from plant loss) versus coupled top-down effects (compensatory predator search efficiency) were ranked by total absolute error of model predictions compared to the empirical community observations. Our best model for predicting community responses to vascular plant loss incorporated coupled effects of decreased primary production (bottom-up), increased prey search efficiency of large-bodied fishes at low vascular plant density (top-down), and decreased prey search efficiency of small-bodied fishes with increased biomass of filamentous macroalgae (bottom-up). The results of this study indicate that the loss of vascular plants from the coastal river ecosystem may alter the food web structure and result in a net decline in the biomass of fishes. These results are highly relevant to ongoing landscape-level restoration programs intended to improve aesthetics and ecosystem function of coastal spring-fed rivers by highlighting how the structure of these communities can be regulated both by resource availability and consumption. Restoration programs will need to acknowledge and incorporate both to be successful.

BackgroundMarinobufagenin (MBG) is an endogenous cardiotonic steroid (CTS) that inhibits the Na+/K+-ATPase. Human MBG is significantly increased in end-stage renal disease and immunization against MBG attenuates cardiovascular fibrosis in a rat model of uremic cardiomyopathy. Mineralocorticoid antagonists (MRA) block MBG binding sites and decrease proteinuria in chronic kidney disease (CKD) patients. We therefore aimed to investigate the association of MBG and albuminuria, as a marker of renal damage, as well as MBG and decline of glomerular filtration rate (GFR).MethodsThe Graz endocrine causes of hypertension (GECOH) study is a single center study of adults routinely referred for screening of endocrine hypertension. Plasma MBG was measured by an enzyme-linked immunoassay, and in a post-hoc analysis, follow-up creatinine levels were obtained. Patients with proteinuria >3.5g/day at baseline were excluded from further evaluation.ResultsWe measured MBG concentrations in 40 hypertensive subjects and excluded one patient due to pre-existing proteinuria. Plasma MBG was significantly correlated with albuminuria (Spearman ρ = .357; p = .028) and proteinuria (ρ = .336; p = .039). In linear regression analysis, the association remained significant after adjustment for age, sex, and BMI (β = .306; p = .036), and for mean systolic blood pressure (β = .352; p = .034). In follow-up analyses (N = 30), MBG was significantly associated with decline in GFR after adjustment for time-to-follow-up (β = -.374; p = .042).ConclusionThe findings suggest that MBG plasma concentrations were associated with albuminuria as well as decline in kidney function. Whether MBG predicts hard renal endpoints warrants further investigations.

The fall armyworm (FAW) Spodoptera frugiperda (J. E. Smith) is a species native to the Americas. This polyphagous lepidopteran pest was first reported in Nigeria and the Democratic Republic of São Tomé and Principe in 2016, but its presence in eastern Africa has not been confirmed via molecular characterisation. In this study, FAW specimens from western and central Uganda were identified based on the partial mtDNA COI gene sequences, with mtDNA COI haplotypes matching those identified in Nigeria and São Tomé. In this study, we sequence an additional partial mtDNA Cyt b gene and also the partial mtDNA COIII gene in Ugandan FAW samples. We detected identical mitochondrial DNA haplotypes for both the mtDNA Cyt b and COI partial genes, while combining the mtDNA COI/Cyt b haplotypes and mtDNA COIII haplotypes enabled a new maternal lineage in the Ugandan corn-preferred FAW samples to be identified. Our results suggested that the African incursions of S. frugiperda involved at least three maternal lineages. Recent full genome, phylogenetic and microsatellite analyses provided evidence to support S. frugiperda as likely consisted of two sympatric sister species known as the corn-preferred and rice-preferred strains. In our Ugandan FAW populations, we identified the presence of mtDNA haplotypes representative of both sister species. It is not known if both FAW sister species were originally introduced together or separately, and whether they have since spread as a single population. Further analyses of additional specimens originally collected from São Tomé, Nigeria and throughout Africa would be required to clarify this issue. Importantly, our finding showed that the genetic diversity of the African corn-preferred FAW species is higher than previously reported. This potentially contributed to the success of FAW establishment in Africa. Furthermore, with the additional maternal lineages detected, there is likely an increase in paternal lineages, thereby increasing the diversity of the African FAW population. Knowledge of the FAW genetic diversity will be needed to assess the risks of introducing Bt-resistance traits and to understand the FAW incursion pathways into the Old World and its potential onward spread. The agricultural implications of the presence of two evolutionary divergent FAW lineages (the corn and the rice lineage) in the African continent are further considered and discussed.

The earliest evidence for cheese production in the Mediterranean is revealed by stable carbon isotope analyses of individual fatty acids in pottery residues from the Dalmatian coast of Croatia. Lipid residue data indicate the presence of milk in the earliest pottery, Impressed Ware, by 5700 cal. BCE (7700 BP). In contrast, by 5200 cal BCE (7200 BP), milk was common in refined Figulina pottery, meat was mostly associated with Danilo ware, cheese occurred in Rhyta, and sieves contained fermented dairy, representing strong links between specific function and stylistically distinctive pottery vessels. Genetic data indicate the prevalence of lactose intolerance among early farming populations. However, young children are lactase persistent until after weaning and could consume milk as a relatively pathogen-free and nutrient rich food source, enhancing their chances of survival into adulthood. Fermentation of milk into yogurt and cheese decreases lactose content. The evidence for fermented dairy products by 5200 cal BCE indicates a larger proportion of the population was able to consume dairy products and benefit from their significant nutritional advantages. We suggest that milk and cheese production among Europe's early farmers reduced infant mortality and helped stimulate demographic shifts that propelled farming communities to expand to northern latitudes.

There is worldwide concern about the status of elasmobranchs, primarily as a result of overfishing and bycatch with subsequent ecosystem effects following the removal of top predators. Whilst abundant and wide-ranging, blue sharks (Prionace glauca) are the most heavily exploited shark species having suffered marked declines over the past decades, and there is a call for robust abundance estimates. In this study, we utilized depth data collected from two blue sharks using pop-up satellite archival tags, and modelled the proportion of time the sharks were swimming in the top 1-meter layer and could therefore be detected by observers conducting aerial surveys. The availability models indicated that the tagged sharks preferred surface waters whilst swimming over the continental shelf and during daytime, with a model-predicted average proportion of time spent at the surface of 0.633 (SD = 0.094) for on-shelf, and 0.136 (SD = 0.075) for off-shelf. These predicted values were then used to account for availability bias in abundance estimates for the species over a large area in the Northeast Atlantic, derived through distance sampling using aerial survey data collected in 2015 and 2016 and modelled with density surface models. Further, we compared abundance estimates corrected with model-predicted availability to uncorrected estimates and to estimates that incorporated the average time the sharks were available for detection. The mean abundance (number of individuals) corrected with modelled availability was 15,320 (CV = 0.28) in 2015 and 11,001 (CV = 0.27) in 2016. Depending on the year, these estimates were ~7 times higher compared to estimates without the bias correction, and ~3 times higher compared to the abundances corrected with average availability. When the survey area contains habitat heterogeneity that may affect surfacing patterns of animals, modelling animals' availability provides a robust alternative to correcting for availability bias and highlights the need for caution when applying "average" correction factors.

The transcription factor SOX2 has been identified as a lineage survival oncogene in squamous cell carcinoma and copy number gain is a common event in several human malignancies including head and neck cancer. However, the regulation and function of SOX2 during carcinogenesis as well as its prognostic value appears to be highly context dependent. As an example, high SOX2 expression in lung squamous cell carcinoma (SCC) is related to a favorable prognosis, while it is associated with poor outcome in lung adenocarcinoma. More recently, higher SOX2 levels and improved survival was also reported for head and neck SCC (HNSCC), and silencing of SOX2 expression in HNSCC cell lines revealed a mesenchymal-like phenotype with prominent vimentin expression. So far, SOX2 expression and its clinical relevance for other head and neck cancers, such as adenoid cystic carcinoma (HNACC) have not been sufficiently investigated.SOX2, vimentin and E-cadherin expression was assessed by immunohistochemical staining on serial sections from formalin fixed and paraffin embedded tissue samples of a patient cohort (n = 45) with primary ACC and correlated with patient and tumor characteristics as well as survival.High SOX2 expression was found in 14 (31%) primary tumor specimens and was significantly correlated with a N0 lymph node status (p = 0.04), while low SOX2 expression was correlated with a solid growth pattern (p = 0.031). Of the 45 patients, 27 tumor samples resembled an EMT-like phenotype, as assessed by high vimentin and low E-cadherin levels. However, in HNACC SOX2 levels were neither correlated with vimentin nor with E-cadherin expression, further supporting a context dependent regulation and function of SOX2 in distinct tumor entities.The absence of SOX2 was predominantly found in solid HNACC, which are characterized by a more aggressive phenotype in ACC. However, the underlying molecular mechanisms of SOX2 regulation and function in distinct HNACC subgroups remain to be fully elucidated.

The cotton bollworm, Helicoverpa armigera (Hübner) is one of the most serious insect pest species to evolve resistance against many insecticides from different chemical classes. This species has evolved resistance to the pyrethroid insecticides across its native range and is becoming a truly global pest after establishing in South America and having been recently recorded in North America. A chimeric cytochrome P450 gene, CYP337B3, has been identified as a resistance mechanism for resistance to fenvalerate and cypermethrin. Here we show that this resistance mechanism is common around the world with at least eight different alleles. It is present in South America and has probably introgressed into its closely related native sibling species, Helicoverpa zea. The different alleles of CYP337B3 are likely to have arisen independently in different geographic locations from selection on existing diversity. The alleles found in Brazil are those most commonly found in Asia, suggesting a potential origin for the incursion of H. armigera into the Americas.

Next-generation sequencing can be costly and labour intensive. Usually, the sequencing cost per sample is reduced by pooling amplified DNA = amplicons) derived from different individuals on the same sequencing lane. Barcodes unique to each amplicon permit short-read sequences to be assigned appropriately. However, the cost of the library preparation increases with the number of barcodes used. We propose an alternative to barcoding: by using different known proportions of individually-derived amplicons in a pooled sample, each is characterised a priori by an expected depth of coverage. We have developed a Hidden Markov Model that uses these expected proportions to reconstruct the input sequences. We apply this method to pools of mitochondrial DNA amplicons extracted from kangaroo meat, genus Macropus. Our experiments indicate that the sequence coverage can be efficiently used to index the short-reads and that we can reassemble the input haplotypes when secondary factors impacting the coverage are controlled. We therefore demonstrate that, by combining our approach with standard barcoding, the cost of the library preparation is reduced to a third.

Wearable robotic devices can restore and enhance mobility. There is growing interest in designing devices that reduce the metabolic cost of walking; however, designers lack guidelines for which joints to assist and when to provide the assistance. To help address this problem, we used musculoskeletal simulation to predict how hypothetical devices affect muscle activity and metabolic cost when walking with heavy loads. We explored 7 massless devices, each providing unrestricted torque at one degree of freedom in one direction (hip abduction, hip flexion, hip extension, knee flexion, knee extension, ankle plantarflexion, or ankle dorsiflexion). We used the Computed Muscle Control algorithm in OpenSim to find device torque profiles that minimized the sum of squared muscle activations while tracking measured kinematics of loaded walking without assistance. We then examined the metabolic savings provided by each device, the corresponding device torque profiles, and the resulting changes in muscle activity. We found that the hip flexion, knee flexion, and hip abduction devices provided greater metabolic savings than the ankle plantarflexion device. The hip abduction device had the greatest ratio of metabolic savings to peak instantaneous positive device power, suggesting that frontal-plane hip assistance may be an efficient way to reduce metabolic cost. Overall, the device torque profiles generally differed from the corresponding net joint moment generated by muscles without assistance, and occasionally exceeded the net joint moment to reduce muscle activity at other degrees of freedom. Many devices affected the activity of muscles elsewhere in the limb; for example, the hip flexion device affected muscles that span the ankle joint. Our results may help experimentalists decide which joint motions to target when building devices and can provide intuition for how devices may interact with the musculoskeletal system. The simulations are freely available online, allowing others to reproduce and extend our work.

To compare the efficacy of silver in situ hybridization (SISH) and immunohistochemistry (IHC) in detecting MET and IGF1R alterations and to investigate their prevalence and prognostic significance. A possible correlation between MET receptor expression, MET gene alterations and the two most frequent occurring EGFR gene mutations was also investigated.Stage I to IIIA tumors from 326 patients with NSCLC were immunohistochemically tested for protein expression of MET and IGF-1. Their cytoplasmic expression was compared with the gene copy number of the MET and IGF1Rgenes by SISH in paraffin-embedded, formalin-fixed material. Correlations were made with the immunohistochemical expression of two frequent EGFR mutations and clinicopathological variables. Univariate and multivariate survival analyses was used to evaluate the prognostic efficacy of the tested markers.In univariate analyses, high cytoplasmic MET expression showed a significant negative prognostic effect in adenocarcinoma patients (p = 0.026). MET gene to chromosome 7 ratio was a significant positive prognostic marker (p = 0.005), probably only due to the highly negative prognostic significance of chromosome 7 polysomy (p = 0.002). High IGF1R gene copy number was a negative prognostic marker for all NSCLC patients (p = 0.037). In the multivariate analysis, polysomy of chromosome 7 in tumor cells correlated significantly and independently with a poor prognosis (p = 0.011). In patients with adenocarcinoma, a high cytoplasmic MET expression was an independent negative prognostic marker (p = 0.013). In males a high IGF1R gene copy number to chromosome 15 count ratio was significantly and independently correlated to a poor prognosis (p = 0.018).MET protein expression provides superior prognostic information compared with SISH. Polysomy of chromosome 7 is an independent negative prognostic factor in NSCLC patients. This finding has an important implication while examining genes located on chromosome 7 by means of SISH. High IGF1R gene copy number to chromosome 15 count ratio is an independent predictor of inferior survival in male patients with primary NSCLC.

Muscles attach to bones via tendons that stretch and recoil, affecting muscle force generation and metabolic energy consumption. In this study, we investigated the effect of tendon compliance on the metabolic cost of running using a full-body musculoskeletal model with a detailed model of muscle energetics. We performed muscle-driven simulations of running at 2-5 m/s with tendon force-strain curves that produced between 1 and 10% strain when the muscles were developing maximum isometric force. We computed the average metabolic power consumed by each muscle when running at each speed and with each tendon compliance. Average whole-body metabolic power consumption increased as running speed increased, regardless of tendon compliance, and was lowest at each speed when tendon strain reached 2-3% as muscles were developing maximum isometric force. When running at 2 m/s, the soleus muscle consumed less metabolic power at high tendon compliance because the strain of the tendon allowed the muscle fibers to operate nearly isometrically during stance. In contrast, the medial and lateral gastrocnemii consumed less metabolic power at low tendon compliance because less compliant tendons allowed the muscle fibers to operate closer to their optimal lengths during stance. The software and simulations used in this study are freely available at simtk.org and enable examination of muscle energetics with unprecedented detail.

Toxin/antitoxin (TA) systems reduce metabolism under stress; for example, toxin YafQ of the YafQ/DinJ Escherichia coli TA system reduces growth by cleaving transcripts with in-frame 5'-AAA-G/A-3' sites, and antitoxin DinJ is a global regulator that represses its locus as well as controls levels of the stationary sigma factor RpoS. Here we investigated the influence on cell growth at various temperatures and found that deletion of the antitoxin gene, dinJ, resulted in both reduced metabolism and slower growth at 18°C but not at 37°C. The reduction in growth could be complemented by producing DinJ from a plasmid. Using a transposon screen to reverse the effect of the absence of DinJ, two mutations were found that inactivated the toxin YafQ; hence, the toxin caused the slower growth only at low temperatures rather than DinJ acting as a global regulator. Corroborating this result, a clean deletion of yafQ in the ΔdinJ ΔKmR strain restored both metabolism and growth at 18°C. In addition, production of YafQ was more toxic at 18°C compared to 37°C. Furthermore, by overproducing all the E. coli proteins, the global transcription repressor Mlc was found that counteracts YafQ toxicity only at 18°C. Therefore, YafQ is more effective at reducing metabolism at low temperatures, and Mlc is its putative target.

CDK9 is the catalytic subunit of positive elongation factor b (P-TEFb) that controls the transition of RNA polymerase II (RNAPII) into elongation. CDK9 inhibitors block mRNA synthesis and trigger activation of the stress-sensitive p53 protein. This in turn induces transcription of CDKN1A (p21) and other cell cycle control genes. It is presently unclear if and how p53 circumvents a general P-TEFb-requirement when it activates its target genes. Our investigations using a panel of specific inhibitors reason for a critical role of CDK9 also in the case of direct inhibition of the kinase. At the prototypic p21 gene, the activator p53 initially accumulates at the pre-bound upstream enhancer followed-with significant delay-by de novo binding to a secondary enhancer site within the first intron of p21. This is accompanied by recruitment of the RNAPII initiation machinery to both elements. ChIP and functional analyses reason for a prominent role of CDK9 itself and elongation factor complexes PAF1c and SEC involved in pause and elongation control. It appears that the strong activation potential of p53 facilitates gene activation in the situation of global repression of RNAPII transcription. The data further underline the fundamental importance of CDK9 for class II gene transcription.

OBJECTIVES:Social factors have profound effects on health. Children are especially vulnerable to social influences, particularly in their early years. Adverse social exposures in childhood can lead to chronic disorders later in life. Here, we sought to identify and evaluate the impact of social factors on child health in Ghana. As Ghana is unlikely to achieve the Millennium Development Goals' target of reducing child mortality by two-thirds between 1990 and 2015, we deemed it necessary to identify social determinants that might have contributed to the non-realisation of this goal. METHODS:ScienceDirect, PubMed, MEDLINE via EBSCO and Google Scholar were searched for published articles reporting on the influence of social factors on child health in Ghana. After screening the 98 articles identified, 34 of them that met our inclusion criteria were selected for qualitative review. RESULTS:Major social factors influencing child health in the country include maternal education, rural-urban disparities (place of residence), family income (wealth/poverty) and high dependency (multiparousity). These factors are associated with child mortality, nutritional status of children, completion of immunisation programmes, health-seeking behaviour and hygiene practices. CONCLUSIONS:Several social factors influence child health outcomes in Ghana. Developing more effective responses to these social determinants would require sustainable efforts from all stakeholders including the Government, healthcare providers and families. We recommend the development of interventions that would support families through direct social support initiatives aimed at alleviating poverty and inequality, and indirect approaches targeted at eliminating the dependence of poor health outcomes on social factors. Importantly, the expansion of quality free education interventions to improve would-be-mother's health knowledge is emphasised.

Tools have been used for millions of years to augment the capabilities of the human body, allowing us to accomplish tasks that would otherwise be difficult or impossible. Powered exoskeletons and other assistive devices are sophisticated modern tools that have restored bipedal locomotion in individuals with paraplegia and have endowed unimpaired individuals with superhuman strength. Despite these successes, designing assistive devices that reduce energy consumption during running remains a substantial challenge, in part because these devices disrupt the dynamics of a complex, finely tuned biological system. Furthermore, designers have hitherto relied primarily on experiments, which cannot report muscle-level energy consumption and are fraught with practical challenges. In this study, we use OpenSim to generate muscle-driven simulations of 10 human subjects running at 2 and 5 m/s. We then add ideal, massless assistive devices to our simulations and examine the predicted changes in muscle recruitment patterns and metabolic power consumption. Our simulations suggest that an assistive device should not necessarily apply the net joint moment generated by muscles during unassisted running, and an assistive device can reduce the activity of muscles that do not cross the assisted joint. Our results corroborate and suggest biomechanical explanations for similar effects observed by experimentalists, and can be used to form hypotheses for future experimental studies. The models, simulations, and software used in this study are freely available at simtk.org and can provide insight into assistive device design that complements experimental approaches.

BackgroundCancer Associated Fibroblasts (CAFs) are thought to regulate tumor growth and metastasis. Fibroblast Activating Protein 1 (FAP-1) is a marker for fibroblast activation and by many recognized as the main marker of CAFs. Alpha Smooth Muscle Actin (α-SMA) is a general myofibroblast marker, and can be used to identify CAFs. This study investigates the prognostic impact of FAP-1 and α-SMA in non-small cell lung cancer (NSCLC) patients and correlates their expression to 105 proteins investigated in the same cohort.MethodsTumor specimens from 536 NSCLC patients were obtained and tissue micro-arrays were constructed. Immunohistochemistry was used to evaluate the expression of FAP-1 and α-SMA and explore their impact on survival and association with other tumor molecular markers in NSCLC patients.ResultsHigh expression of FAP-1, but not α-SMA, in squamous cell carcinoma (SCC, P = 0.043, HR = 0.63 95% CI 0.40-0.99) was significantly associated with increased disease-specific survival. FAP-1 and α-SMA were not significantly correlated to each other. Analyses of FAP-1 and α-SMA associated with other tumor-related proteins revealed histotype-specific correlation patterns.ConclusionThe presence of FAP-1 expressing CAFs is an indicator of positive outcome for NSCLC-SCC patients. In addition, correlation analyses suggest FAP-1 and α-SMA to label different subsets of fibroblasts and their associations with other tumor-related proteins diverge according to histological subtype.

The use of near-infrared (NIR) fluorescence imaging with indocyanine green (ICG) for sentinel lymph node (SN) mapping has been investigated in lung cancer; however, this has not been fully adapted for minimally invasive surgery (MIS). The aim of our study was to develop a minimally invasive SN mapping integrating pre-operative electro-magnetic navigational bronchoscopy (ENB)-guided transbronchial ICG injection and intraoperative NIR thoracoscopic imaging.A NIR thoracoscope was used to visualize ICG fluorescence. ICG solutions in a 96-well plate and ex vivo porcine lungs were examined to optimize ICG concentrations and injection volumes. Transbronchial ICG injection (n=4) was assessed in comparison to a traditional transpleural approach (n=3), where after thoracotomy an ICG solution (100 μL at 100 μg/mL) was injected into the porcine right upper lobe for SN identification. For further translation into clinical use, transbronchial ICG injection prior to thoracotomy followed by NIR thoracoscopic imaging was validated (n=3). ENB was used for accurate targeting in two pigs with a pseudo-tumor.The ICG fluorescence at 10 μg/mL was the brightest among various concentrations, unchanged by the distance between the thoracoscope and ICG solutions. Injected ICG of no more than 500μ L showed a localized fluorescence area. All 7 pigs showed a bright paratracheal lymph node within 15 minutes post-injection, with persistent fluorescence for 60 minutes. The antecedent transbronchial ICG injection succeeded in SN identification in all 3 cases at the first thoracoscopic inspection within 20 minutes post-injection. The ENB system allowed accurate ICG injection surrounding the pseudo-tumors.ENB-guided ICG injection followed by NIR thoracoscopy was technically feasible for SN mapping in the porcine lung. This promising platform may be translated into human clinical trials and is suited for MIS.

Hairless (H) is the major antagonist within the Notch signalling pathway of Drosophila melanogaster. By binding to Suppressor of Hairless [Su(H)] and two co-repressors, H induces silencing of Notch target genes in the absence of Notch signals. We have applied genomic engineering to create several new H alleles. To this end the endogenous H locus was replaced with an attP site by homologous recombination, serving as a landing platform for subsequent site directed integration of different H constructs. This way we generated a complete H knock out allele HattP, reintroduced a wild type H genomic and a cDNA-construct (Hgwt, Hcwt) as well as two constructs encoding H proteins defective of Su(H) binding (HLD, HiD). Phenotypes regarding viability, bristle and wing development were recorded, and the expression of Notch target genes wingless and cut was analysed in mutant wing discs or in mutant cell clones. Moreover, genetic interactions with Notch (N5419) and Delta (DlB2) mutants were addressed. Overall, phenotypes were largely as expected: both HLD and HiD were similar to the HattP null allele, indicating that most of H activity requires the binding of Su(H). Both rescue constructs Hgwt and Hcwt were homozygous viable without phenotype. Unexpectedly, the hemizygous condition uncovered that they were not identical to the wild type allele: notably Hcwt showed a markedly reduced activity, suggesting the presence of as yet unidentified regulatory or stabilizing elements in untranslated regions of the H gene. Interestingly, Hgwt homozygous cells expressed higher levels of H protein, perhaps unravelling gene-by-environment interactions.

Aedes aegypti, the principal vector of yellow fever and dengue fever, is responsible for more than 30,000 deaths annually. Compounds such as carbon dioxide, amino acids, fatty acids and other volatile organic compounds (VOCs) have been widely studied for their role in attracting Ae. aegypti to hosts. Many VOCs from humans are produced by associated skin microbiota. Staphyloccocus epidermidis, although not the most abundant bacteria according to surveys of relative 16S ribosomal RNA abundance, commonly occurs on human skin. Bacteria demonstrate population level decision-making through quorum sensing. Many quorum sensing molecules, such as indole, volatilize and become part of the host odor plum. To date, no one has directly demonstrated the link between quorum sensing (i.e., decision-making) by bacteria associated with a host as a factor regulating arthropod vector attraction. This study examined this specific question with regards to S. epidermidis and Ae. aegypti. Pairwise tests were conducted to examine the response of female Ae. aegypti to combinations of tryptic soy broth (TSB) and S. epidermidis wildtype and agr- strains. The agr gene expresses an accessory gene regulator for quorum sensing; therefore, removing this gene inhibits quorum sensing of the bacteria. Differential attractiveness of mosquitoes to the wildtype and agr- strains was observed. Both wildtype and the agr- strain of S. epidermidis with TSB were marginally more attractive to Ae. aegypti than the TSB alone. Most interestingly, the blood-feeder treated with wildtype S. epidermidis/TSB attracted 74% of Ae. aegypti compared to the agr- strain of S. epidermidis/TSB (P ≤ 0.0001). This study is the first to suggest a role for interkingdom communication between host symbiotic bacteria and mosquitoes. This may have implications for mosquito decision-making with regards to host detection, location and acceptance. We speculate that mosquitoes "eavesdrop" on the chemical discussions occurring between host-associated microbes to determine suitability for blood feeding. We believe these data suggest that manipulating quorum sensing by bacteria could serve as a novel approach for reducing mosquito attraction to hosts, or possibly enhancing the trapping of adults at favored oviposition sites.

In non-small cell lung cancer (NSCLC), nodal metastasis is an adverse prognostic factor. Several mediating factors have been implied in the development of nodal metastases and investigated for predictive and prognostic properties in NSCLC. However, study results differ. In this structured review and meta-analysis we explore the published literature on commonly recognized pathways for molecular regulation of lymphatic metastasis in NSCLC.A structured PubMed search was conducted for papers reporting on the expression of known markers of lymhangiogenesis in NSCLC patients. Papers of sufficient quality, presenting survival and/or correlation data were included.High levels of vascular endothelial growth factor C (VEGF-C, HR 1.57 95% CI 1.34-1.84) and high lymphatic vascular density (LVD, HR 1.84 95% CI 1.18-2.87) were significant prognostic markers of poor survival and high expression of VEGF-C, vascular endothelial growth factor receptor 3 (VEGFR3) and LVD was associated with lymph node metastasis in NSCLC.Lymphangiogenic markers are prognosticators of survival and correlate with lymph node metastasis in NSCLC. Their exact role and clinical implications should be further elucidated.

BACKGROUND:Persistent pain after inguinal herniorrhaphy is a disabling condition with a lack of evidence-based pharmacological treatment options. This randomized placebo-controlled trial investigated the efficacy of a capsaicin 8% cutaneous patch in the treatment of severe persistent inguinal postherniorrhaphy pain. METHODS:Forty-six patients with persistent inguinal postherniorrhaphy pain were randomized to receive either a capsaicin 8% patch or a placebo patch. Pain intensity (Numerical Rating Scale [NRS 0-10]) was evaluated under standardized conditions (at rest, during movement, and during pressure) at baseline and at 1, 2 and 3 months after patch application. Skin punch biopsies for intraepidermal nerve fiber density (IENFD) measurements were taken at baseline and 1 month after patch application. Quantitative sensory testing was performed at baseline and at 1, 2, and 3 months after patch application. The primary outcome was comparisons of summed pain intensity differences (SPIDs) between capsaicin and placebo treatments at 1, 2 and 3 months after patch application (significance level P < 0.01). RESULTS:The maximum difference in SPID, between capsaicin and placebo treatments, was observed at 1 month after patch application, but the pain reduction was not significant (NRS, mean difference [95% CI]: 5.0 [0.09 to 9.9]; P = 0.046). No differences in SPID between treatments were observed at 2 and 3 months after patch application. Changes in IENFD on the pain side, from baseline to 1 month after patch application, did not differ between capsaicin and placebo treatment: 1.9 [-0.1 to 3.9] and 0.6 [-1.2 to 2.5] fibers/mm, respectively (P = 0.32). No significant changes in sensory function, sleep quality or psychological factors were associated with capsaicin patch treatment. CONCLUSIONS:The study did not demonstrate significant differences in pain relief between capsaicin and placebo treatment, although a trend toward pain improvement in capsaicin treated patients was observed 1 month after patch application. TRIAL REGISTRATION:Clinicaltrialsregister.eu 2012-001540-22 ClinicalTrials.gov NCT01699854.

The impact of BRAF tumor mutations on the natural course of disease of melanoma patients is controversial.We analyzed the mutational status and overall survival of 215 patients receiving treatment with dacarbazine or temozolomide. All patients who started first-line treatment at our institution between 2000 and 2010 were included to prevent selection and bias due to thereafter arising therapeutic options.No patient received BRAF- or MEK-inhibitors during follow-up. Survival was associated with the pattern of visceral involvement, the presence of brain metastases and the serum lactate dehydrogenase level (all p

The Escherichia coli protein BdcA (previously referred to as YjgI) plays a key role in the dispersal of cells from bacterial biofilms, and its constitutive activation provides an attractive therapeutic target for dismantling these communities. In order to investigate the function of BdcA at a molecular level, we integrated structural and functional studies. Our 2.05 Å structure of BdcA shows that it is a member of the NAD(P)(H)-dependent short-chain dehydrogenase/reductase (SDR) superfamily. Structural comparisons with other members of the SDR family suggested that BdcA binds NADP(H). This was demonstrated experimentally using thermal denaturation studies, which showed that BcdA binds specifically to NADPH. Subsequent ITC experiments further confirmed this result and reported a Kd of 25.9 µM. Thus, BdcA represents the newest member of the limited number of oxidoreductases shown to be involved in quorum sensing and biofilm dispersal.

The prognostic impact of BRAF-V600 tumor mutations in stage I/II melanoma patients has not yet been analyzed in detail. We investigated primary tumors of 437 patients diagnosed between 1989 and 2006 by Sanger sequencing. Mutations were detected in 38.7% of patients and were associated with age, histological subtype as well as mitotic rate. The mutational rate was 36.7% in patients with disease-free course and 51.7% in those with subsequent distant metastasis (p = 0.031). No difference in overall survival (p = 0.119) but a trend for worse distant-metastasis-free survival (p = 0.061) was observed in BRAF mutant compared to BRAF wild-type patients. Independent prognostic factors for overall survival were tumor thickness, mitotic rate and ulceration. An interesting significant prognostic impact was observed in patients with tumor thickness of 1 mm or less, with the mutation present in 6 of 7 patients dying from melanoma. In conclusion, no significant survival differences were found according to BRAF-V600 tumor mutations in patients with primary melanoma but an increasing impact of the mutational status was observed in the subgroup of patients with tumor thickness of 1 mm or less. A potential role of the mutational status as a prognostic factor especially in this subgroup needs to be investigated in larger studies.

D1 and D2 receptor expressing striatal medium spiny neurons (MSNs) are ascribed to striatonigral ("direct") and striatopallidal ("indirect") pathways, respectively, that are believed to function antagonistically in motor control. Glutamatergic synaptic transmission onto the two types is differentially affected by Dopamine (DA), however, less is known about the effects on MSN intrinsic electrical properties. Using patch clamp recordings, we comprehensively characterized the two pathways in rats and mice, and investigated their DA modulation. We identified the direct pathway by retrograde labeling in rats, and in mice we used transgenic animals in which EGFP is expressed in D1 MSNs. MSNs were subjected to a series of current injections to pinpoint differences between the populations, and in mice also following bath application of DA. In both animal models, most electrical properties were similar, however, membrane excitability as measured by step and ramp current injections consistently differed, with direct pathway MSNs being less excitable than their counterparts. DA had opposite effects on excitability of D1 and D2 MSNs, counteracting the initial differences. Pronounced changes in AP shape were seen in D2 MSNs. In direct pathway MSNs, excitability increased across experimental conditions and parameters, and also when applying DA or the D1 agonist SKF-81297 in presence of blockers of cholinergic, GABAergic, and glutamatergic receptors. Thus, DA induced changes in excitability were D1 R mediated and intrinsic to direct pathway MSNs, and not a secondary network effect of altered synaptic transmission. DAergic modulation of intrinsic properties therefore acts in a synergistic manner with previously reported effects of DA on afferent synaptic transmission and dendritic processing, supporting the antagonistic model for direct vs. indirect striatal pathway function.

Coral reefs are damaged by natural disturbances and local and global anthropogenic stresses. As stresses intensify, so do debates about whether reefs will recover after significant damage. True headway in this debate requires documented temporal trajectories for coral assemblages subjected to various combinations of stresses; therefore, we report relevant changes in coral assemblages at Little Cayman Island. Between 1999 and 2012, spatiotemporal patterns in cover, densities of juveniles and size structure of assemblages were documented inside and outside marine protected areas using transects, quadrats and measurements of maximum diameters. Over five years, bleaching and disease caused live cover to decrease from 26% to 14%, with full recovery seven years later. Juvenile densities varied, reaching a maximum in 2010. Both patterns were consistent within and outside protected areas. In addition, dominant coral species persisted within and outside protected areas although their size frequency distributions varied temporally and spatially. The health of the coral assemblage and the similarity of responses across levels of protection suggested that negligible anthropogenic disturbance at the local scale was a key factor underlying the observed resilience.

Understanding how stem cells interact with cardiomyocytes is crucial for cell-based therapies to restore the cardiomyocyte loss that occurs during myocardial infarction and other cardiac diseases. It has been thought that functional myocardial repair and regeneration could be regulated by stem cell-cardiomyocyte contact. However, because various contact modes (junction formation, cell fusion, partial cell fusion, and tunneling nanotube formation) occur randomly in a conventional coculture system, the particular regulation corresponding to a specific contact mode could not be analyzed. In this study, we used laser-patterned biochips to define cell-cell contact modes for systematic study of contact-mediated cellular interactions at the single-cell level. The results showed that the biochip design allows defined stem cell-cardiomyocyte contact-mode formation, which can be used to determine specific cellular interactions, including electrical coupling, mechanical coupling, and mitochondria transfer. The biochips will help us gain knowledge of contact-mediated interactions between stem cells and cardiomyocytes, which are fundamental for formulating a strategy to achieve stem cell-based cardiac tissue regeneration.

Development of secondary hyperalgesia following a cutaneous injury is a centrally mediated, robust phenomenon. The pathophysiological role of endogenous opioid signalling to the development of hyperalgesia is unclear. Recent animal studies, carried out after the resolution of inflammatory pain, have demonstrated reinstatement of tactile hypersensitivity following administration of μ-opioid-receptor-antagonists. In the present study in humans, we analyzed the effect of naloxone when given after the resolution of secondary hyperalgesia following a first-degree burn injury.Twenty-two healthy volunteers were included in this placebo-controlled, randomized, double-blind, cross-over study. Following baseline assessment of thermal and mechanical thresholds, a first-degree burn injury (BI; 47°C, 7 minutes, thermode area 12.5 cm(2)) was induced on the lower leg. Secondary hyperalgesia areas around the BI-area, and separately produced by brief thermal sensitization on the contralateral thigh (BTS; 45°C, 3 minutes, area 12.5 cm(2)), were assessed using a polyamide monofilament at pre-BI and 1, 2, and 3 hours post-BI. At 72 hrs, BI and BTS secondary hyperalgesia areas were assessed prior to start of a 30 minutes intravenous infusion of naloxone (total dose 21 microg/kg) or placebo. Fifteen minutes after start of the infusion, BI and BTS secondary hyperalgesia areas were reassessed, along with mechanical and thermal thresholds.Secondary hyperalgesia areas were demonstrable in all volunteers 1-3 hrs post-BI, but were not demonstrable at 72 hrs post-burn in 73-86% of the subjects. Neither magnitude of secondary hyperalgesia areas nor the mechanical and thermal thresholds were associated with naloxone-treated compared to placebo-treated subjects.Naloxone (21 microg/kg) did not reinstate secondary hyperalgesia when administered 72 hours after a first-degree burn injury and did not increase BTS-generated hyperalgesia. The negative results may be due to the low dose of naloxone or insufficient tissue injury to generate latent sensitization.

Prevention of the initiation of biofilm formation is the most important step for combating biofilm-associated pathogens, as the ability of pathogens to resist antibiotics is enhanced 10 to 1000 times once biofilms are formed. Genes essential to bacterial growth in the planktonic state are potential targets to treat biofilm-associated pathogens. However, the biofilm formation capability of strains with mutations in these essential genes must be evaluated, since the pathogen might form a biofilm before it is eliminated. In order to address this issue, this work proposes a systems-level approach to quantifying the biofilm formation capability of mutants to determine target genes that are essential for bacterial metabolism in the planktonic state but do not induce biofilm formation in their mutants. The changes of fluxes through the reactions associated with the genes positively related to biofilm formation are used as soft sensors in the flux balance analysis to quantify the trend of biofilm formation upon the mutation of an essential gene. The essential genes whose mutants are predicted not to induce biofilm formation are regarded as gene targets. The proposed approach was applied to identify target genes to treat Pseudomonas aeruginosa infections. It is interesting to find that most essential gene mutants exhibit high potential to induce the biofilm formation while most non-essential gene mutants do not. Critically, we identified four essential genes, lysC, cysH, adk, and galU, that constitute gene targets to treat P. aeruginosa. They have been suggested by existing experimental data as potential drug targets for their crucial role in the survival or virulence of P. aeruginosa. It is also interesting to find that P. aeruginosa tends to survive the essential-gene mutation treatment by mainly enhancing fluxes through 8 metabolic reactions that regulate acetate metabolism, arginine metabolism, and glutamate metabolism.

Complex motor skills are more difficult to perform at certain points in the day (for example, shortly after waking), but the daily trajectory of motor-skill error is more difficult to predict. By undertaking a quantitative analysis of the fundamental frequency (FF) and amplitude of hundreds of zebra finch syllables per animal per day, we find that zebra finch song follows a previously undescribed daily oscillation. The FF and amplitude of harmonic syllables rises across the morning, reaching a peak near mid-day, and then falls again in the late afternoon until sleep. This oscillation, although somewhat variable, is consistent across days and across animals and does not require serotonin, as animals with serotonergic lesions maintained daily oscillations. We hypothesize that this oscillation is driven by underlying physiological factors which could be shared with other taxa. Song production in zebra finches is a model system for studying complex learned behavior because of the ease of gathering comprehensive behavioral data and the tractability of the underlying neural circuitry. The daily oscillation that we describe promises to reveal new insights into how time of day affects the ability to accomplish a variety of complex learned motor skills.

The microtubule (MT) "plus end" constitutes the platform for the accumulation of a structurally and functionally diverse group of proteins, collectively called "MT plus-end tracking proteins" (+TIPs). +TIPs control MT dynamics and link MTs to diverse sub-cellular structures. Neurospora crassaMicroTubule Binding protein-3 (MTB-3) is the homolog of yeast EB1, a highly conserved +TIP. To address the function of MTB-3, we examined strains with mtb-3 deletions, and we tagged MTB-3 with GFP to assess its dynamic behavior. MTB-3-GFP was present as comet-like structures distributed more or less homogeneously within the hyphal cytoplasm, and moving mainly towards the apex at speeds up to 4× faster than the normal hyphal elongation rates. MTB-3-GFP comets were present in all developmental stages, but were most abundant in mature hyphae. MTB-3-GFP comets were observed moving in anterograde and retrograde direction along the hypha. Retrograde movement was also observed as originating from the apical dome. The integrity of the microtubular cytoskeleton affects the presence and dynamics of MTB-3-GFP comets, while actin does not seem to play a role. The size of MTB-3-GFP comets is affected by the absence of dynactin and conventional kinesin. We detected no obvious morphological phenotypes in Δmtb-3 mutants but there were fewer MTs in Δmtb-3, MTs were less bundled and less organized. Compared to WT, both MT polymerization and depolymerization rates were significantly decreased in Δmtb-3. In summary, the lack of MTB-3 affects overall growth and morphological phenotypes of N. crassa only slightly, but deletion of mtb-3 has strong effect on MT dynamics.

Emerging evidence suggests that excessive exposure to traffic-derived air pollution during pregnancy may increase the vulnerability to neurodevelopmental alterations that underlie a broad array of neuropsychiatric disorders. We present a mouse model for prenatal exposure to urban freeway nanoparticulate matter (nPM). In prior studies, we developed a model for adult rodent exposure to re-aerosolized urban nPM which caused inflammatory brain responses with altered neuronal glutamatergic functions. nPMs are collected continuously for one month from a local freeway and stored as an aqueous suspension, prior to re-aerosolization for exposure of mice under controlled dose and duration. This paradigm was used for a pilot study of prenatal nPM impact on neonatal neurons and adult behaviors. Adult C57BL/6J female mice were exposed to re-aerosolized nPM (350 µg/m(3)) or control filtered ambient air for 10 weeks (3×5 hour exposures per week), encompassing gestation and oocyte maturation prior to mating. Prenatal nPM did not alter litter size, pup weight, or postnatal growth. Neonatal cerebral cortex neurons at 24 hours in vitro showed impaired differentiation, with 50% reduction of stage 3 neurons with long neurites and correspondingly more undifferentiated neurons at Stages 0 and 1. Neuron number after 24 hours of culture was not altered by prenatal nPM exposure. Addition of exogenous nPM (2 µg/ml) to the cultures impaired pyramidal neuron Stage 3 differentiation by 60%. Adult males showed increased depression-like responses in the tail-suspension test, but not anxiety-related behaviors. These pilot data suggest that prenatal exposure to nPM can alter neuronal differentiation with gender-specific behavioral sequelae that may be relevant to human prenatal exposure to urban vehicular aerosols.

Fibrillar collagen is the primary component of the cardiac interstitial extracellular matrix. This extracellular matrix undergoes dramatic changes from birth to adulthood and then into advanced age. As evidence, fibrillar collagen content was compared in sections from neonates, adult, and old hearts and was found to increase at each respective age. Cardiac fibroblasts are the principle cell type that produce and control fibrillar collagen content. To determine whether fibroblast production, processing, and deposition of collagen differed with age, primary cardiac fibroblasts from neonate, adult, and old mice were isolated and cultured in 3-dimensional (3D) fibrin gels. Fibroblasts from each age aligned in fibrin gels along points of tension and deposited extracellular matrix. By confocal microscopy, wild-type neonate fibroblasts appeared to deposit less collagen into fibrillar structures than fibroblasts from adults. However, by immunoblot analysis, differences in procollagen production and processing of collagen I were not detected in neonate versus adult fibroblasts. In contrast, fibroblasts from old mice demonstrated increased efficiency of procollagen processing coupled with decreased production of total collagen. SPARC is a collagen-binding protein previously shown to affect cardiac collagen deposition. Accordingly, in the absence of SPARC, less collagen appeared to be associated with fibroblasts of each age grown in fibrin gels. In addition, the increased efficiency of procollagen alpha 1(I) processing in old wild-type fibroblasts was not detected in old SPARC-null fibroblasts. Increased levels of fibronectin were detected in wild-type neonate fibroblasts over that of adult and old fibroblasts but not in SPARC-null neonate fibroblasts versus older ages. Immunostaining of SPARC overlapped with that of collagen I but not to that of fibronectin in 3D cultures. Hence, whereas increases in procollagen processing, influenced by SPARC expression, plausibly contribute to increased collagen deposition in old hearts, other cellular mechanisms likely affect differential collagen deposition by neonate fibroblasts.

Integrins constitute a superfamily of transmembrane signaling receptors that play pivotal roles in cutaneous homeostasis by modulating cell growth and differentiation as well as inflammatory responses in the skin. Subrabasal expression of integrins α2 and/or β1 entails hyperproliferation and aberrant differentiation of keratinocytes and leads to dermal and epidermal influx of activated T-cells. The anatomical and physiological similarities between porcine and human skin make the pig a suitable model for human skin diseases. In efforts to generate a porcine model of cutaneous inflammation, we employed the Sleeping Beauty DNA transposon system for production of transgenic cloned Göttingen minipigs expressing human β1 or α2 integrin under the control of a promoter specific for subrabasal keratinocytes. Using pools of transgenic donor fibroblasts, cloning by somatic cell nuclear transfer was utilized to produce reconstructed embryos that were subsequently transferred to surrogate sows. The resulting pigs were all transgenic and harbored from one to six transgene integrants. Molecular analyses on skin biopsies and cultured keratinocytes showed ectopic expression of the human integrins and localization within the keratinocyte plasma membrane. Markers of perturbed skin homeostasis, including activation of the MAPK pathway, increased expression of the pro-inflammatory cytokine IL-1α, and enhanced expression of the transcription factor c-Fos, were identified in keratinocytes from β1 and α2 integrin-transgenic minipigs, suggesting the induction of a chronic inflammatory phenotype in the skin. Notably, cellular dysregulation obtained by overexpression of either β1 or α2 integrin occurred through different cellular signaling pathways. Our findings mark the creation of the first cloned pig models with molecular markers of skin inflammation. Despite the absence of an overt psoriatic phenotype, these animals may possess increased susceptibility to severe skin damage-induced inflammation and should be of great potential in studies aiming at the development and refinement of topical therapies for cutaneous inflammation including psoriasis.

BACKGROUND: This study investigated survival probabilities and prognostic factors in sentinel lymph node biopsy (SLNB) staged patients with cutaneous melanoma (CM) with the aim of defining subgroups of patients who are at higher risk for recurrences and who should be considered for adjuvant clinical trials. METHODS: Patients with primary CM who underwent SLNB in the Department of Dermatology, University of Tuebingen, Germany, between 1996 and 2009 were included into this study. Survival probabilities and prognostic factors were evaluated by Kaplan-Meier and multivariate Cox proportional hazard models. RESULTS: 1909 SLNB staged patients were evaluated. Median follow-up time was 44 months. Median tumor thickness was 1.8 mm, ulceration was present in 31.8% of cases. The 5-year Overall Survival (OS) was 90.3% in SLNB negative patients (IB 96.2%, IIA 87.0%, IIB 78.1%, IIC 72.6%). Patients with micrometastases (stage IIIA/B) had a 5-year OS rate of 70.9% which was clearly less favorable than for stages I-II. Multivariate analysis revealed tumor thickness, ulceration, body site, histopathologic subtype and SLNB status as independent significant prognostic factors. CONCLUSION: Survival rates of patients with primary CM in stages I-II were shown to be much more favorable than previously reported from non sentinel node staged collectives. For future clinical trials, sample size calculations should be adapted using survival probabilities based on sentinel node staging.

Sclerotinia homoeocarpa causes dollar spot disease, the predominate disease on highly-maintained turfgrass. Currently, there are major gaps in our understanding of the molecular interactions between S. homoeocarpa and creeping bentgrass. In this study, 454 sequencing technology was used in the de novo assembly of S. homoeocarpa and creeping bentgrass transcriptomes. Transcript sequence data obtained using Illumina's first generation sequencing-by-synthesis (SBS) were mapped to the transcriptome assemblies to estimate transcript representation in different SBS libraries. SBS libraries included a S. homoeocarpa culture control, a creeping bentgrass uninoculated control, and a library for creeping bentgrass inoculated with S. homoeocarpa and incubated for 96 h. A Fisher's exact test was performed to determine transcripts that were significantly different during creeping bentgrass infection with S. homoeocarpa. Fungal transcripts of interest included glycosyl hydrolases, proteases, and ABC transporters. Of particular interest were the large number of glycosyl hydrolase transcripts that target a wide range of plant cell wall compounds, corroborating the suggested wide host range and saprophytic abilities of S. homoeocarpa. Several of the multidrug resistance ABC transporters may be important for resistance to both fungicides and plant defense compounds. Creeping bentgrass transcripts of interest included germins, ubiquitin transcripts involved in proteasome degradation, and cinnamoyl reductase, which is involved in lignin production. This analysis provides an extensive overview of the S. homoeocarpa-turfgrass pathosystem and provides a starting point for the characterization of potential virulence factors and host defense responses. In particular, determination of important host defense responses may assist in the development of highly resistant creeping bentgrass varieties.

The purpose of this study is to clarify the prognostic significance of expression of Jab1, p16, p21, p62, Ki67 and Skp2 in soft tissue sarcomas (STS). Optimised treatment of STS requires better identification of high risk patients who will benefit from adjuvant therapy. The prognostic significance of Jab1, p16, p21, p62, Ki67 and Skp2 in STS has not been sufficiently investigated.Tissue microarrays from 193 STS patients were constructed from duplicate cores of viable and representative neoplastic tumor areas. Immunohistochemistry was used to evaluate the expression of Jab1, p16, p21, p62, Ki67 and Skp2.In univariate analyses, high tumor expression of Ki67 (P = 0.007) and Skp2 (P = 0.050) correlated with shorter disease-specific survival (DSS). In subgroup analysis, a correlation between Skp2 and DSS was seen in patients with malignancy grade 1 or 2 (P = 0.027), tumor size >5 cm (P = 0.018), no radiotherapy given (P = 0.029) and no chemotherapy given (P = 0.017). No such relationship was apparent for Jab1, p16, p21 and p62; but p62 showed a positive correlation to malignancy grade (P = 0.019). Ki67 was strongly positively correlated to malignancy grade (P = 0.001). In multivariate analyses, Skp2 was an independent negative prognostic factor for DSS in women (P = 0.009) and in patients without administered chemotherapy or radiotherapy (P = 0.026).Increased expression of Skp2 in patients with soft tissue sarcomas is an independent negative prognostic factor for disease-specific survival in women and in patients not administered chemotherapy or radiotherapy. Besides, further studies are warranted to explore if adjuvant chemotherapy or radiotherapy improve the poor prognosis of STS with high Skp2 expression.

Somatic forward genetic screens have the power to interrogate thousands of genes in a single animal. Retroviral and transposon mutagenesis systems in mice have been designed and deployed in somatic tissues for surveying hematopoietic and solid tumor formation. In the context of cancer, the ability to visually mark mutant cells would present tremendous advantages for identifying tumor formation, monitoring tumor growth over time, and tracking tumor infiltrations and metastases into wild-type tissues. Furthermore, locating mutant clones is a prerequisite for screening and analyzing most other somatic phenotypes. For this purpose, we developed a system using the piggyBac (PB) transposon for somatic mutagenesis with an activated reporter and tracker, called PB-SMART. The PB-SMART mouse genetic screening system can simultaneously induce somatic mutations and mark mutated cells using bioluminescence or fluorescence. The marking of mutant cells enable analyses that are not possible with current somatic mutagenesis systems, such as tracking cell proliferation and tumor growth, detecting tumor cell infiltrations, and reporting tissue mutagenesis levels by a simple ex vivo visual readout. We demonstrate that PB-SMART is highly mutagenic, capable of tumor induction with low copy transposons, which facilitates the mapping and identification of causative insertions. We further integrated a conditional transposase with the PB-SMART system, permitting tissue-specific mutagenesis with a single cross to any available Cre line. Targeting the germline, the system could also be used to conduct F1 screens. With these features, PB-SMART provides an integrated platform for individual investigators to harness the power of somatic mutagenesis and phenotypic screens to decipher the genetic basis of mammalian biology and disease.

BACKGROUND: Over recent decades jellyfish have caused fish kill events and recurrent gill problems in marine-farmed salmonids. Common jellyfish (Aurelia spp.) are among the most cosmopolitan jellyfish species in the oceans, with populations increasing in many coastal areas. The negative interaction between jellyfish and fish in aquaculture remains a poorly studied area of science. Thus, a recent fish mortality event in Ireland, involving Aurelia aurita, spurred an investigation into the effects of this jellyfish on marine-farmed salmon. METHODOLOGY/PRINCIPAL FINDINGS: To address the in vivo impact of the common jellyfish (A. aurita) on salmonids, we exposed Atlantic salmon (Salmo salar) smolts to macerated A. aurita for 10 hrs under experimental challenge. Gill tissues of control and experimental treatment groups were scored with a system that rated the damage between 0 and 21 using a range of primary and secondary parameters. Our results revealed that A. aurita rapidly and extensively damaged the gills of S. salar, with the pathogenesis of the disorder progressing even after the jellyfish were removed. After only 2 hrs of exposure, significant multi-focal damage to gill tissues was apparent. The nature and extent of the damage increased up to 48 hrs from the start of the challenge. Although the gills remained extensively damaged at 3 wks from the start of the challenge trial, shortening of the gill lamellae and organisation of the cells indicated an attempt to repair the damage suffered. CONCLUSIONS: Our findings clearly demonstrate that A. aurita can cause severe gill problems in marine-farmed fish. With aquaculture predicted to expand worldwide and evidence suggesting that jellyfish populations are increasing in some areas, this threat to aquaculture is of rising concern as significant losses due to jellyfish could be expected to increase in the future.

AIMS: Transforming growth factor-β (TGF-β), fascin, nuclear factor-kappa B (NF-κB) p105, protein-kinase C-zeta (PKC-ζ), partioning-defective protein-6 (Par-6), E-cadherin and vimentin are tumor promoting molecules through mechanisms involved in cell dedifferentiation. In soft tissue sarcomas, their expression profile is poorly defined and their significance is uncertain. We aimed to investigate the prognostic impact of TGF-β1, NF-κB p105, PKC-ζ, Par-6α, E-cadherin and vimentin in non-gastrointestinal stromal tumor soft tissue sarcomas (non-GIST STSs). PATIENTS AND METHODS: Tumor samples and clinical data from 249 patients with non-GIST STS were obtained, and tissue microarrays (TMAs) were constructed for each specimen. Immunohistochemistry (IHC) was used to evaluate marker expression in tumor cells. RESULTS: In univariate analysis, the expression levels of TGF-β1 (P = 0.016), fascin (P = 0.006), NF-κB p105 (P = 0.022) and PKC-ζ, (P = 0.042) were significant indicators for disease specific survival (DSS). In the multivariate analysis, high TGF-β1 expression was an independent negative prognostic factor for DSS (HR = 1.6, 95% CI = 1.1-2.4, P = 0.019) in addition to tumor depth, malignancy grade, metastasis at diagnosis, surgery and positive resection margins. CONCLUSION: Expression of TGF-β1 was significantly associated with aggressive behavior and shorter DSS in non-GIST STSs.

Initiation, growth, recurrence, and metastasis of head and neck squamous cell carcinomas (HNSCC) have been related to the behavior of cancer stem cells (CSC) that can be identified by their aldehyde-dehydrogenase-isoform-1 (ALDH1) activity. We quantified and enriched ALDH1(+) cells within HNSCC cell lines and subsequently characterized their phenotypical and functional properties like invasion capacity and epithelial-mesenchymal transition (EMT). Spheroid culture enriched CSC from five HNSCC cell lines by up to 5-fold. In spheroid-derived cells (SDC) and the parental monolayer-derived cell line ALDH1, CD44, CD24, E-Cadherin, α-SMA, and Vimentin expression was compared by flow-cytometry and immunofluorescence together with proliferation and cell cycle analysis. Invasion activity was evaluated by Matrigel assay and expression of stemness-related transcription factors (TF) Nanog, Oct3/4, Sox2 and EMT-related genes Snail1 and 2, and Twist by real-time PCR. All cell lines formed spheroids that could self-renew and be serially re-passaged. ALDH1 expression was significantly higher in SDC. ALDH1(+) cells showed increased colony-formation. The proportion of cells with a putative CSC marker constellation of CD44(+)/CD24(-) was highly variable (0.5% to 96%) in monolayer and spheroid cultures and overlapped in 0%-33% with the CD44(+)/CD24(-)/ALDH1(+) cell subset. SDC had significantly higher invading activity. mRNA of the stemness-related genes Sox2, Nanog, and Oct3/4 was significantly increased in SDC of all cell lines. Twist was significantly increased in two while Snail2 showed a significant increase in one and a significant decrease in SDC of two cell lines. SDC had a higher G0 phase proportion, showed high-level expression of α-SMA and Vimentin, but significantly decreased E-Cadherin expression. HNSCC-lines harbor potential CSC, characterized by ALDH1 and stemness marker TF expression as well as properties like invasiveness, quiescence, and EMT. CSC can be enriched by anchorage-independent culture techniques, which may be important for the investigation of their contribution to therapy resistance, tumor recurrence and metastasis.