Identification of the initial molecular changes in response to circulating angiogenic cells-mediated therapy in critical limb ischemia

BackgroundCritical limb ischemia (CLI) constitutes the most aggressive form of peripheral arterial occlusive disease, characterized by the blockade of arteries supplying blood to the lower extremities, significantly diminishing oxygen and nutrient supply. CLI patients usually undergo amputation of fingers, feet, or extremities, with a high risk of mortality due to associated comorbidities.Circulating angiogenic cells (CACs), also known as early endothelial progenitor cells, constitute promising candidates for cell therapy in CLI due to their assigned vascular regenerative properties. Preclinical and clinical assays with CACs have shown promising results. A better understanding of how these cells participate in vascular regeneration would significantly help to potentiate their role in revascularization.Herein, we analyzed the initial molecular mechanisms triggered by human CACs after being administered to a murine model of CLI, in order to understand how these cells promote angiogenesis within the ischemic tissues.MethodsBalb-c nude mice (n:24) were distributed in four different groups: healthy controls (C, n:4), shams (SH, n:4), and ischemic mice (after femoral ligation) that received either 50 mu l physiological serum (SC, n:8) or 5x10(5) human CACs (SE, n:8). Ischemic mice were sacrificed on days 2 and 4 (n:4/group/day), and immunohistochemistry assays and qPCR amplification of Alu-human-specific sequences were carried out for cell detection and vascular density measurements. Additionally, a label-free MS-based quantitative approach was performed to identify protein changes related.ResultsAdministration of CACs induced in the ischemic tissues an increase in the number of blood vessels as well as the diameter size compared to ischemic, non-treated mice, although the number of CACs decreased within time. The initial protein changes taking place in response to ischemia and more importantly, right after administration of CACs to CLI mice, are shown.ConclusionsOur results indicate that CACs migrate to the injured area; moreover, they trigger protein changes correlated with cell migration, cell death, angiogenesis, and arteriogenesis in the host. These changes indicate that CACs promote from the beginning an increase in the number of vessels as well as the development of an appropriate vascular network.Institute of Health Carlos III, ISCIII; Junta de Andaluci

Appropriate waist circumference cut points for identifying insulin resistance in black youth: a cross sectional analysis of the 1986 Jamaica birth cohort

Background While the International Diabetes Federation (IDF) has ethnic specific waist circumference (WC) cut-points for the metabolic syndrome for Asian populations it is not known whether the cut-points for black populations should differ from those for European populations. We examined the validity of IDF WC cut points for identifying insulin resistance (IR), the underlying cause of the metabolic syndrome, in predominantly black, young Jamaican adults. Methods Participants from a 1986 birth cohort were evaluated between 2005 and 2007 when they were 18-20 years old. Trained observers took anthropometric measurements and collected a fasting blood sample. IR was assessed using the homeostasis model assessment computer programme (HOMA-IR). Sex specific quartiles for IR were generated using HOMA-IR values and participants in the highest quartile were classified as "insulin resistant". Receiver operator characteristic (ROC) curves were used to estimate the best WC to identify insulin resistance. The sensitivity and specificity of these values were compared with the IDF recommended WC cut-points. Results Data from 707 participants (315 males; 392females) were analysed. In both sexes those with IR were more obese, had higher mean systolic blood pressure, glucose and triglycerides and lower mean HDL cholesterol. The WC was a good predictor of IR with an ROC area under the curve (95% CI) of 0.71(0.64,0.79) for men and 0.72(0.65,0.79) for women. Using the Youden Index (J) the best WC cut point for identifying IR in male participants was 82 cm (sensitivity 45%, specificity 93%, J 0.38) while the standard cut point of 94 cm had a sensitivity of 14% and specificity of 98% (J 0.12). In the female participants 82 cm was also a good cut point for identifying IR (sensitivity 52%, specificity 87%, J 0.39) and was similar to the standard IDF 80 cm cut point (sensitivity 53%, specificity 82%, J 0.35). Conclusions The WC that identified IR in young black men is lower than the IDF recommended WC cut point. Sex differences in WC cut points for identifying IR were less marked in this population than in other ethnic groups

Receptor-Mediated Enhancement of Beta Adrenergic Drug Activity by Ascorbate In Vitro and In Vivo

RATIONALE: Previous in vitro research demonstrated that ascorbate enhances potency and duration of activity of agonists binding to alpha 1 adrenergic and histamine receptors. OBJECTIVES: Extending this work to beta 2 adrenergic systems in vitro and in vivo. METHODS: Ultraviolet spectroscopy was used to study ascorbate binding to adrenergic receptor preparations and peptides. Force transduction studies on acetylcholine-contracted trachealis preparations from pigs and guinea pigs measured the effect of ascorbate on relaxation due to submaximal doses of beta adrenergic agonists. The effect of inhaled albuterol with and without ascorbate was tested on horses with heaves and sheep with carbachol-induced bronchoconstriction. MEASUREMENTS: Binding constants for ascorbate binding to beta adrenergic receptor were derived from concentration-dependent spectral shifts. Dose- dependence curves were obtained for the relaxation of pre-contracted trachealis preparations due to beta agonists in the presence and absence of varied ascorbate. Tachyphylaxis and fade were also measured. Dose response curves were determined for the effect of albuterol plus-and-minus ascorbate on airway resistance in horses and sheep. MAIN RESULTS: Ascorbate binds to the beta 2 adrenergic receptor at physiological concentrations. The receptor recycles dehydroascorbate. Physiological and supra-physiological concentrations of ascorbate enhance submaximal epinephrine and isoproterenol relaxation of trachealis, producing a 3-10-fold increase in sensitivity, preventing tachyphylaxis, and reversing fade. In vivo, ascorbate improves albuterol's effect on heaves and produces a 10-fold enhancement of albuterol activity in "asthmatic" sheep. CONCLUSIONS: Ascorbate enhances beta-adrenergic activity via a novel receptor-mediated mechanism; increases potency and duration of beta adrenergic agonists effective in asthma and COPD; prevents tachyphylaxis; and reverses fade. These novel effects are probably caused by a novel mechanism involving phosphorylation of aminergic receptors and have clinical and drug-development applications

Keeping participants on board: increasing uptake by automated respondent reminders in an Internet-based Chlamydia Screening in the Netherlands

<p>Abstract</p> <p>Background</p> <p>Effectiveness of Chlamydia screening programs is determined by an adequate level of participation and the capturing of high-risk groups. This study aimed to evaluate the contribution of automated reminders by letter, email and short message service (SMS) on package request and sample return in an Internet-based Chlamydia screening among people aged 16 to 29 years in the Netherlands.</p> <p>Methods</p> <p>Individuals not responding to the invitation letter received a reminder letter after 1 month. Email- and SMS-reminders were sent to persons who did not return their sample. It was examined to what extent reminders enhanced the response rate (% of package requests) and participation rate (% of sample return). Sociodemographic and behavioural correlates of providing a cell phone number and participation after the reminder(s) were studied by logistic regression models.</p> <p>Results</p> <p>Of all respondents (screening round 1: 52,628, round 2: 41,729), 99% provided an email address and 72% a cell phone number. Forty-two percent of all package requests were made after the reminder letter. The proportion of invitees returning a sample increased significantly from 10% to 14% after email/SMS reminders (round 2: from 7% to 10%). Determinants of providing a cell-phone number were younger age (OR in 25-29 year olds versus 16-19 year olds = 0.8, 95%CI 0.8-0.9), non-Dutch (OR in Surinam/Antillean versus Dutch = 1.3, 95%CI 1.2-1.4, Turkish/Moroccan: 1.1, 95%CI 1.0-1.2, Sub Sahara African: 1.5, 95%CI 1.3-1.8, non-Western other 1.1, 95%CI 1.1-1.2), lower educational level (OR in high educational level versus low level = 0.8, 95%CI 0.7-0.9), no condom use during the last contact with a casual partner (OR no condom use versus condom use 1.2, 95%CI 1.1-1.3), younger age at first sexual contact (OR 19 years or older versus younger than 16: 0.7, 95%CI 0.6-0.8). Determinants for requesting a test-package after the reminder letter were male gender (OR female versus male 0.9 95%CI 0.8-0.9), non-Dutch (OR in Surinam/Antillean versus Dutch 1.3, 95%CI 1.2-1.4, Turkish/Moroccan: 1.4, 95%CI 1.3-1.5, Sub Sahara African: 1.4, 95%CI 1.2-1.5, non-Western other: 1.2, 95%CI 1.1-1.2), having a long-term steady partnership (long-term versus short-term.1.2 95%CI 1.1-1.3). Email/SMS reminders seem to have resulted in more men and people aged 25-29 years returning a sample.</p> <p>Conclusions</p> <p>Nearly all respondents (99.5%) were reachable by modern communication media. Response and participation rates increased significantly after the reminders. The reminder letters also seemed to result in reaching more people at risk. Incorporation of automated reminders in Internet-based (<b>C</b>hlamydia) screening programs is strongly recommended.</p

Transoral laser microsurgery for laryngeal cancer: A primer and review of laser dosimetry

Transoral laser microsurgery (TLM) is an emerging technique for the management of laryngeal and other head and neck malignancies. It is increasingly being used in place of traditional open surgery because of lower morbidity and improved organ preservation. Since the surgery is performed from the inside working outward as opposed to working from the outside in, there is less damage to the supporting structures that lie external to the tumor. Coupling the laser to a micromanipulator and a microscope allows precise tissue cutting and hemostasis; thereby improving visualization and precise ablation. The basic approach and principles of performing TLM, the devices currently in use, and the associated dosimetry parameters will be discussed. The benefits of using TLM over conventional surgery, common complications and the different settings used depending on the location of the tumor will also be discussed. Although the CO2 laser is the most versatile and the best-suited laser for TLM applications, a variety of lasers and different parameters are used in the treatment of laryngeal cancer. Improved instrumentation has lead to an increased utilization of TLM by head and neck cancer surgeons and has resulted in improved outcomes. Laser energy levels and spot size are adjusted to vary the precision of cutting and amount of hemostasis obtained

Identification of Attractive Drug Targets in Neglected-Disease Pathogens Using an In Silico Approach

In cell-based drug development, researchers attempt to create drugs that kill a pathogen without necessarily understanding the details of how the drugs work. In contrast, target-based drug development entails the search for compounds that act on a specific intracellular target—often a protein known or suspected to be required for survival of the pathogen. The latter approach to drug development has been facilitated greatly by the sequencing of many pathogen genomes and the incorporation of genome data into user-friendly databases. The present paper shows how the database TDRtargets.org can identify proteins that might be considered good drug targets for diseases such as African sleeping sickness, Chagas disease, parasitic worm infections, tuberculosis, and malaria. These proteins may score highly in searches of the database because they are dissimilar to human proteins, are structurally similar to other “druggable” proteins, have functions that are easy to measure, and/or fulfill other criteria. Researchers can use the lists of high-scoring proteins as a basis for deciding which potential drug targets to pursue experimentally

A Novel Role of RASSF9 in Maintaining Epidermal Homeostasis

The physiological role of RASSF9, a member of the Ras-association domain family (RASSF), is currently unclear. Here, we report a mouse line in which an Epstein-Barr virus Latent Membrane Protein 1 (LMP1) transgene insertion has created a 7.2-kb chromosomal deletion, which abolished RASSF9 gene expression. The RASSF9-null mice exhibited interesting phenotypes that resembled human ageing, including growth retardation, short lifespan, less subcutaneous adipose layer and alopecia. In the wild-type mice, RASSF9 is predominantly expressed in the epidermal keratinocytes of skin, as determined by quantitative reverse-transcription PCR, immunofluorescence and in situ hybridization. In contrast, RASSF9−/− mice presented a dramatic change in epithelial organization of skin with increased proliferation and aberrant differentiation as detected by bromodeoxyuridine incorporation assays and immunofluorescence analyses. Furthermore, characteristic functions of RASSF9−/− versus wild type (WT) mouse primary keratinocytes showed significant proliferation linked to a reduction of p21Cip1 expression under growth or early differentiation conditions. Additionally, in RASSF9−/− keratinocytes there was a drastic down-modulation of terminal differentiation markers, which could be rescued by infection with a recombinant adenovirus, Adv/HA-RASSF9. Our results indicate a novel and significant role of RASSF9 in epidermal homeostasis

Differential responses to woodland character and landscape context by cryptic bats in urban environments

© 2015 Lintott et al. Urbanisation is one of the most dramatic forms of land use change which relatively few species can adapt to. Determining how and why species respond differently to urban habitats is important in predicting future biodiversity loss as urban areas rapidly expand. Understanding how morphological or behavioural traits can influence species adaptability to the built environment may enable us to improve the effectiveness of conservation efforts. Although many bat species are able to exploit human resources, bat species richness generally declines with increasing urbanisation and there is considerable variation in the responses of different bat species to urbanisation. Here, we use acoustic recordings from two cryptic, and largely sympatric European bat species to assess differential responses in their use of fragmented urban woodland and the surrounding urban matrix. There was a high probability of P. pygmaeus activity relative to P. pipistrellus in woodlands with low clutter and understory cover which were surrounded by low levels of built environment. Additionally, the probability of recording P. pygmaeus relative to P. pipistrellus was considerably higher in urban woodland interior or edge habitat in contrast to urban grey or non-wooded green space. These results show differential habitat use occurring between two morphologically similar species; whilst the underlying mechanism for this partitioning is unknown it may be driven by competition avoidance over foraging resources. Their differing response to urbanisation indicates the difficulties involved when attempting to assess how adaptable a species is to urbanisation for conservation purposes

Transcription Factor NF-κB Is Transported to the Nucleus via Cytoplasmic Dynein/Dynactin Motor Complex in Hippocampal Neurons

Mikenberg I, Widera D, Kaus A, Kaltschmidt B, Kaltschmidt C. Transcription Factor NF-kappa B Is Transported to the Nucleus via Cytoplasmic Dynein/Dynactin Motor Complex in Hippocampal Neurons. PLOS ONE. 2007;2(7):e589.Background. Long-term changes in synaptic plasticity require gene transcription, indicating that signals generated at the synapse must be transported to the nucleus. Synaptic activation of hippocampal neurons is known to trigger retrograde transport of transcription factor NF-kappa B. Transcription factors of the NF-kappa B family are widely expressed in the nervous system and regulate expression of several genes involved in neuroplasticity, cell survival, learning and memory. Principal Findings. In this study, we examine the role of the dynein/dynactin motor complex in the cellular mechanism targeting and transporting activated NF-kappa B to the nucleus in response to synaptic stimulation. We demonstrate that overexpression of dynamitin, which is known to dissociate dynein from microtubules, and treatment with microtubule-disrupting drugs inhibits nuclear accumulation of NF-kappa B p65 and reduces NF-kappa B-dependent transcription activity. In this line, we show that p65 is associated with components of the dynein/dynactin complex in vivo and in vitro and that the nuclear localization sequence (NLS) within NF-kappa B p65 is essential for this binding. Conclusion. This study shows the molecular mechanism for the retrograde transport of activated NF-kappa B from distant synaptic sites towards the nucleus