2-Desaza-annomontine (C81) impedes angiogenesis through reduced VEGFR2 expression derived from inhibition of CDC2-like kinases.

Angiogenesis is a crucial process in the progression of various pathologies, like solid tumors, wet age-related macular degeneration, and chronic inflammation. Current anti-angiogenic treatments still have major drawbacks like limited efficacy in diseases that also rely on inflammation. Therefore, new anti-angiogenic approaches are sorely needed, and simultaneous inhibition of angiogenesis and inflammation is desirable. Here, we show that 2-desaza-annomontine (C81), a derivative of the plant alkaloid annomontine previously shown to inhibit endothelial inflammation, impedes angiogenesis by inhibiting CDC2-like kinases (CLKs) and WNT/ß-catenin signaling. C81 reduced choroidal neovascularization in a laser-induced murine in vivo model, inhibited sprouting from vascular endothelial growth factor A (VEGF-A)-activated murine aortic rings ex vivo, and reduced angiogenesis-related activities of endothelial cells in multiple functional assays. This was largely phenocopied by CLK inhibitors and knockdowns, but not by inhibitors of the other known targets of C81. Mechanistically, CLK inhibition reduced VEGF receptor 2 (VEGFR2) mRNA and protein expression as well as downstream signaling. This was partly caused by a reduction of WNT/ß-catenin pathway activity, as activating the pathway induced, while ß-catenin knockdown impeded VEGFR2 expression. Surprisingly, alternative splicing of VEGFR2 was not detected. In summary, C81 and other CLK inhibitors could be promising compounds in the treatment of diseases that depend on angiogenesis and inflammation due to their impairment of both processes.

Diffusion and diversion of suboxone: an exploration of illicit street opioid selling.

Interviews with fourteen opioid retail pill sellers provides an exploration into the diversion and diffusion of Suboxone to recreational ("week-end warriors") drug users. The use of social media and electronic devices enables the diffusion of Suboxone to dependent and non-dependent opiate/opioid drug abusers. Overprescribing by physicians and prescribing in drug treatment settings fuels the diversion of Suboxone. The diversion and the diffusion of Suboxone have the potential to delay entrance into drug treatment and promote the misuse of the drug by both dependent opiate/opioid drug abusers and recreational users. The dilemma posed by Suboxone maintenance treatment will not be easily addressed or mitigated in the near future.

Excellence needs training "Certified programme in endoscopic surgery".

The complexity of modern surgery has increased the demands and challenges to surgical education and quality control. Today the endoscopic approach is preferred because it increases the surgical possibilities and decreases patient discomfort. Implementing endoscopic surgery without specific training leads to decrease in surgical performance and increase in patient morbidity and mortality. Research of the European Academy for Gynaecological Surgery (+he Academy) has resulted in the establishment of a structured certification and diploma programme with three levels of expertise. One level should be passed to obtain access to the next level. Furthermore one should first pass +he Academy skill exam before entering the clinical surgical competence programme. The European Society for Gynaecological Endoscopy (ESGE) has defined the different diplomas, whereas the Bachelor diploma is seen as a prerequisite to start the in OR one to one clinical training aiming to provide endoscopic skilled individuals to the clinical one to one training. Further diplomas are the Minimal invasive Gynaecological Surgeon (MIGS), master in hysteroscopy and the laparoscopic pelvic surgeon. This programme is based on the best available scientific evidence. It counteracts the problem of the traditional surgical apprentice tutor model and increases patient safety and surgical performance. It is seen as a major step toward standardization of endoscopic surgical training in general.

Impact of using a fast-freezing technique and different thawing protocols on viability and fertility of frozen equine spermatozoa.

The effects of freezing technique and thawing protocol on thawed semen viability and fertility were studied. Ejaculates from 5 stallions (n = 25) were frozen by conventional or a fast-freezing technique. Frozen semen was thawed by two thawing protocols (37 °C 30 s(-1) or 75 °C 7 s(-1) ). Thawed semen was evaluated by progressive motility, vigour, morphology and plasma membrane integrity. Mares (n = 25) were inseminated with 300 (n = 11) or 150 (n = 14) million spermatozoa. A greater (P < 0.05) vigour and progressively motile spermatozoa were detected, respectively, at thawing and after 20 min post-thawing in the fast-freezing technique than in the conventional one. Plasma membrane integrity was also greater (P < 0.05) in semen frozen with the fast-freezing technique. Semen viability was not affected by thawing protocol. Pregnancy rate using the fast-freezing technique was 76% (19/25), and did not differ (P > 0.05) between insemination doses. We concluded that the 150 million progressively motile spermatozoa per dose using a deep-horn insemination maximises the use of equine semen. The fast-freezing technique, as compared to the conventional one, efficiently preserves the viability and fertilising capacity of spermatozoa, indicating a new method to improve the fertility of frozen equine semen.

A new perspective on old drugs: non-mitotic actions of tubulin-binding drugs play a major role in cancer treatment.

Microtubule-targeting agents (MTAs) are the most frequently used anti-cancer drugs. They can be divided into tubulin stabilizing and destabilizing agents. Their mode of action has been ascribed to their ability to interfere with the spindle apparatus and, thus, to block mitosis leading to tumor cell death. However, this view has been challenged in the last years and it became increasingly evident that non-mitotic actions of MTAs, i.e. their ability to affect the dynamics of interphase microtubules, are the most relevant mechanism underlying their efficacy. In this review we are presenting a distinct selection of examples of studies describing biological effects of MTAs in three areas: (i) mitosis-independent cell death and metastasis, (ii) tumor angiogenesis, and (iii) vascular-disrupting activity.

Suboxone misuse along the opiate maintenance treatment pathway.

This study explores strategies that Suboxone misusers utilize while in drug treatment. Ethnographic interviews were conducted with 14 patients who had cycled in and out of Suboxone treatment. The objective of the study is to identify strategies implemented by patients who intermittently use opiates/opioids while in Suboxone treatment. Findings indicate that some patients serially stop and start treatment in a Harm Reduction setting in New York City. Many patients suggest that they manage their opiate/opioid dependency through a sequential use of Suboxone and heroin to avoid withdrawal and to continue their misuse of opiates/opioids. Results are discussed in conjunction with the difficulties inherent to substance abuse treatment and suggestions for improvement are offered.

Spirostanol saponins and esculin from Rusci rhizoma reduce the thrombin-induced hyperpermeability of endothelial cells.

Rusci rhizoma extracts are traditionally used against chronic venous disorders (CVD). To determine the effect of its secondary plant metabolites on the endothelium, phenolic compounds and saponins from Butcher's broom were isolated from a methanolic extract, and their activity on the thrombin-induced hyperpermeability of human microvascular endothelial cells (HMEC-1) was investigated in vitro. In addition to the six known spirostanol saponins deglucoruscin (5), 22-O-methyl-deglucoruscoside (6), deglucoruscoside (7), ruscin (8), ruscogenin-1-O-(α-l-rhamnopyranosyl-(1→2)-ß-d-galactopyranoside (9) and 1-O-sulpho-ruscogenin (10), three new spirostanol derivatives were isolated and identified: 3'-O-acetyl-4'-O-sulphodeglucoruscin (1), 4'-O-(2-hydroxy-3-methylpentanoyl)-deglucoruscin (2) and 4'-O-acetyl-deglucoruscin (3). Furthermore, the coumarin esculin (4), which is also prominently present in other medicinal plants used in the treatment of CVD, was isolated for the first time from Rusci rhizoma. Five of the isolated steroid derivatives (2, 5, 8, 9 and 10) and esculin (4) were tested for their ability to reduce the thrombin-induced hyperpermeability of endothelial cells in vitro, and the results were compared to those of the aglycone neoruscogenin (11). The latter compound showed a slight but concentration-dependent reduction in hyperpermeability to 71.8% at 100µM. The highest activities were observed for the spirostanol saponins 5 and 8 and for esculin (4) at 10µM, and these compounds resulted in a reduction of the thrombin-induced hyperpermeability to 41.9%, 42.6% and 53.3%, respectively. For 2, 5 and 8, the highest concentration tested (100µM) resulted in a drastic increase of the thrombin effect. The effect of esculin observed at a concentration of 10µM was diminished at 100µM. These in vitro data provide insight into the pharmacological mechanism by which the genuine spirostanol saponins and esculin can contribute to the efficacy of Butcher's broom against chronic venous disorders.

Anti-angiogenic effects of the tubulysin precursor pretubulysin and of simplified pretubulysin derivatives.

BACKGROUND AND PURPOSE: The use of tubulin-binding compounds, which act in part by inhibiting tumour angiogenesis, has become an integral strategy of tumour therapy. Recently, tubulysins were identified as a novel class of natural compounds of myxobacterial origin, which inhibit tubulin polymerization. As these compounds are structurally highly complex, the search for simplified precursors [e.g. pretubulysin (Prt)] and their derivatives is mandatory to overcome supply problems hampering clinical development. We tested the anti-angiogenic efficacy of Prt and seven of its derivatives in comparison to tubulysin A (TubA). EXPERIMENTAL APPROACH: The compounds were tested in cellular angiogenesis assays (proliferation, cytotoxicity, cell cycle, migration, chemotaxis, tube formation) and in vitro (tubulin polymerization). The efficacy of Prt was also tested in vivo in a murine subcutaneous tumour model induced with HUH7 cells; tumour size and vascularization were measured. KEY RESULTS: The anti-angiogenic potency of all the compounds tested ran parallel to their inhibition of tubulin polymerization in vitro. Prt showed nearly the same efficacy as TubA (EC(50) in low nanomolar range in all cellular assays). Some modifications in the Prt molecule caused only a moderate drop in potency, while others resulted in a dramatic loss of action, providing initial insight into structure-activity relations. In vivo, Prt completely prevented tumour growth and reduced vascular density to 30%. CONCLUSIONS AND IMPLICATIONS: Prt, a chemically accessible precursor of some tubulysins is a highly attractive anti-angiogenic compound both in vitro and in vivo. Even more simplified derivatives of this compound still retain high anti-angiogenic efficacy.

Effects of prostaglandin administration 10 days apart on reproductive parameters of cyclic dairy nulliparous goats / Efeitos da aplicação de prostaglandinas intervaladas de 10 dias sobre características reprodutivas de cabras leiteiras nulíparas cíclicas

This study reported the effects of prostaglandin (PGF2a) administration 10 days apart on reproductive parameters of cyclic artificial inseminated (AI) nulliparous Alpine (n=9) and Saanen (n=9) goats. Animals received two doses of 22.5mg PGF2a 10 days apart. After 1st and 2nd PGF2a administrations, estrus was monitored at 12 h intervals, with a buck teaser. Plasma progesterone concentration (ng/mL) was determined from blood sampled on day 0 (1st PGF2a) and the following 5, 10 (2nd PGF2a), 15, 20, 25 and 30 days. After the onset of the second estrus, females were transrectally (5 MHz probe) scanned at 4 hour intervals until at least 8h after ovulation. Pregnancy was checked through transrectal ultrasound on days 20, 25, 30, 35 and 90 after insemination. All parameters studied did not differ between breeds (P>0.05). Estrous response and interval to estrus, respectively, after 1st (78.9% and 50.6±17.2h) and 2nd PGF2a (88.9% and 50.0±14.8h) administration did not differ (P>0.05). Overall animals ovulating (100.0%), interval to ovulation after 2nd PGF2a (64.5±19.5h) and after estrous onset (18.0±9.1h), ovulation rate (1.3±0.5), diameter of ovulatory follicle (8.1±1.1mm) were recorded. Embryo loss occurred before day 30 of pregnancy. Estrus can be efficiently synchronized in nulliparous Alpine and Saanen goats with two doses of prostaglandin 10 days apart.

Relataram-se os efeitos da aplicação de prostaglandina sobre características reprodutivas de cabras leiteiras nulíparas cíclicas. Cabras Alpinas (n=9) e Saanen (n=9) receberam duas doses de 22,5mg PGF2a com 10 dias de intervalo. A progesterona plasmática (ng/mL) foi determinada a partir de amostras de sangue coletadas nos dias 0 (primeira dose), 5, 10 (segunda dose), 15, 20, 25 e 30. Após início do segundo estro, as fêmeas foram monitoradas por ultrassonografia transretal a cada quatro horas até oito horas após a ovulação. A gestação foi verificada por ultrassonografia transretal nos dias 20, 25, 30, 35 e 90 após a segunda dose. As características estudadas foram semelhantes entre as raças (P>0,05). Animais em estro e o intervalo parto-estro de, respectivamente, 78,9% e 50,6±17,2h e 88,9% e 50,0±14,8h após a primeira e segunda administrações de prostaglandina, não diferiram (P>0,05). Todas as cabras ovularam e registraram-se valores do intervalo parto-ovulação após a segunda aplicação de prostaglandina de 64,5±19,5h e após início do estro de 18,0±9,1h, a taxa de ovulação de 1,3±0,5 e diâmetro do folículo ovulatório de 8,1±1,1mm. Perda embrionária ocorreu antes de 30 dias de gestação. O estro pode ser eficientemente sincronizado em cabras leiteiras núliparas com duas doses de prostaglandina intervaladas de 10 dias.

Residual transglutaminase in collagen - effects, detection, quantification, and removal.

In the present study, we developed an enzyme-linked immunosorbent assay (ELISA) for microbial transglutaminase (mTG) from Streptomyces mobaraensis to overcome the lack of a quantification method for mTG. We further performed a detailed follow-on-analysis of insoluble porcine collagen type I enzymatically modified with mTG primarily focusing on residuals of mTG. Repeated washing (4 ×) reduced mTG-levels in the washing fluids but did not quantitatively remove mTG from the material (p < 0.000001). Substantial amounts of up to 40% of the enzyme utilized in the crosslinking mixture remained associated with the modified collagen. Binding was non-covalent as could be demonstrated by Western blot analysis. Acidic and alkaline dialysis of mTG treated collagen material enabled complete removal the enzyme. Treatment with guanidinium chloride, urea, or sodium chloride was less effective in reducing the mTG content.

The transformation of drug markets and its impact on HIV outreach to injection drug users in New York City, 1987-2008.

This oral history describes three periods of street outreach to injection drug users at risk for HIV in New York City: outreach in an era of public drug markets (1987-1993), outreach in an era of private markets (1993-2006), and network-driven outreach (2006-present). Individual interviews with administrators and supervisors of outreach workers are combined with field notes from the ethnographic research experiences of the first two authors to contextualize, compare, and contrast these distinct periods. The combination and triangulation of these sources of data allow for an analysis of both the specific and the wider social and cultural contexts in which outreach intervention efforts were situated. Through these lenses, the article examines some of the reasons why they were or were not successful and discusses prospects for the future.

Ginkgo biloba extract EGb 761 increases endothelial nitric oxide production in vitro and in vivo.

Beneficial effects of Ginkgo biloba on peripheral arterial occlusive disease have been repeatedly shown in clinical trials, especially after use of EGb 761, a standardized special extract. Since the underlying mechanisms are widely unknown, we aimed to elucidate the molecular basis on which EGb 761 protects against endothelial dysfunction in vitro and in vivo. Application of therapeutically feasible doses of EGb 761 for 48 h caused endothelial nitric oxide (NO) production by increasing endothelial nitric oxide synthase (eNOS) promoter activity and eNOS expression in vitro. Phosphorylation of eNOS at a site typical for Akt (Ser 1177) was acutely enhanced by treatment with EGb 761, as was Akt phosphorylation at Ser 478. Furthermore, the extract caused acute relaxation of isolated aortic rings and NO-dependent reduction of blood pressure in vivo in rats. These influences on eNOS represent a putative molecular basis for the protective cardiovascular properties of EGb 761.

A select annotated bibliography: illegal drug research in rural and suburban areas.

As the diffusion of illegal drugs continues to spread to rural and suburban areas, there is a greater need among illegal drug researchers, law enforcement, and policy makers to gain knowledge from previous work done on what is loosely termed as rural substance abuse research. To help serve that need an annotated bibliography is proffered based on three categories and one method of illegal substance abuse research. These are: drug consumption, drug distribution, drug prices, and ethnography. An exhaustive review of the literature was not undertaken, but rather a focus on research that addresses drug consumption and distribution is put forward.

Blood flow study of arteriovenous grafts with homologous and autologous veins in canine femoral vessels.

PURPOSE: The objective of this study was to assess the blood flow in arteriovenous (AV) communications comparing autologous and homologous veins, in the femoral vessels of dogs. METHODS: Ten mongrel dogs were used for the blood flow analysis, and two AV grafts (AVG) were placed in each of them. The grafts were made with an autologous vein in one side, and a omologous vein, kept in a 0.25% glutaraldehyde solution, in the other side. The volumetric flow was measured before and after AVG placement. Fifteen minutes after surgery, the volumetric flow was measured in the cranial artery, in the caudal artery, in the graft and in the vein, and the same procedure was repeated 15 days after surgery. Measurements were done using an eletromagnetic flowmeter calibrated previously. For data analysis, the Wilcoxon test was used (to compare the difference in the results between the times and the techniques used) alfa

Efeito do resfriamento do sêmen eqüino sobre sua congelabilidade / Effect of freezing and thawing protocols on post-thaw quality of equine semen

Utilizaram-se 25 ejaculados de cinco garanhões da raca Mangalarga Marchador, para avaliar dois protocolos de congelamento. No primeiro tratamento, resfriou-se o sêmen até 5ºC (curva de resfriamento - CR) antes do congelamento, no segundo, congelou-se o sêmen sem resfriamento (SC). Compararam-se duas formas de descongelamento, a 37ºC e a 75ºC/sete segundos. Os protocolos foram avaliados pelo teste de termo resistência (TTR - motilidade total e vigor) e pela funcionalidade da membrana plasmática (teste hiposmótico e eosina nigrosina). A motilidade total no tempo zero do TTR foi melhor (P<0,05) para o sêmen do tratamento CR, quando comparado com o do SC, em ambas as temperaturas de descongelamento, respectivamente, 46,7 por cento e 21,0 por cento (descongelamento a 37ºC), e 44,2 por cento e 24,6 por cento (descongelamento a 75ºC). Na mesma ordem, o número de espermatozóides vivos foi maior (P<0,05) no tratamento CR, 71,0 por cento e 54,6 por cento (descongelamento a 37ºC), e 77,4 por cento e 54,1 por cento (descongelamento a 75ºC). O sêmen do tratamento CR apresentou maior reacão (P<0,05) ao teste hiposmótico e maior vigor (P<0,05) que o do SC. O sêmen descongelado a 75ºC apresentou melhor vigor (P<0,05) que o descongelado a 37ºC, independentemente do protocolo de congelamento. Os resultados mostraram, in vitro, o efeito benéfico do resfriamento do sêmen antes do congelamento.

Heroin diffusion in the mid-Hudson region of New York State.

AIMS: Prompted by the history of heroin diffusion in the United States, press reports and building on previous research into retail heroin distribution, ethnographic research was undertaken identifying and describing retail distribution and diffusion of heroin in and into medium- and small-sized towns in the mid-Hudson region of New York State. METHODS: In conjunction with fieldwork, in-depth tape-recorded interviews were conducted with recent admissions (30 days) at 28 different drug treatment facilities located in the region. Interviews were also conducted with drug counselors, narcotic officers, drug treatment administrators and the county commissioners of mental hygiene. FINDINGS: Heroin-dependent individuals who have access to cheaper heroin in urban areas, such as New York City, Newark, and Patterson, New Jersey, drive retail heroin distribution in the mid-Hudson region. They travel to these cities, purchase heroin in quantity (costing $8-10 per bag), return to the region and sell premium-priced heroin ($20-$25 per bag) mostly to irregular users who do not have access to retail drug sellers in urban areas. CONCLUSION: Price disparity contributes to a recurrent process whereby irregular users who are able to gain access to cheaper heroin in urban areas, return to the mid-Hudson and sell premium-priced heroin to other users who do not have access to cheaper heroin. This process contributes to the diffusion of heroin abuse.

Association of genetic traits to estimated haplotypes from SNP genotypes using EM algorithm and Markov Chain Monte Carlo techniques.

In order to find association of genetic traits to some haplotypes from closely spaced multilocus phase-unknown genotypes we use a three-stage approach. Haplotype frequencies and the most likely haplotype pair for each individual are estimated from random samples of individual or small (nuclear) family genotypes via an EM algorithm. If the most likely haplotype pair configuration of the whole sample outweighs the less likely ones, we may consider the estimated haplotypes as alleles of a multi-allelic marker and carry out the conventional statistics, TDT or ANOVA for quantitative traits. If the most likely haplotype pair configuration and the less likely ones do not differ much in their weight, we sample the TDT or ANOVA statistic over all haplotype pair configurations using the Metropolis-Hastings algorithm. Applications of our method to simulated data as well as real data are given.

Entropy as a measure for linkage disequilibrium over multilocus haplotype blocks.

OBJECTIVE: The presence of linkage disequilibrium (LD) forms the basis for a range of uses, including the fine-mapping of diseases and studies on human genealogy. Recent findings indicate that single nucleotide polymorphisms (SNP) can occur in blocks of limited haplotypic diversity with high degrees of LD. Commonly used measures for LD, such as r(2) and D', consider only two loci and might miss information to appropriately describe LD in larger haplotypic structures. METHODS: We introduce the Normalized Entropy Difference, epsilon, as a new multilocus measure for LD. A related quantity, deltaS, provides an approximate chi(2) test for the significance of LD. The ability of the measure to detect haplotype blocks is investigated using simulated data sets as well as a real data set previously analyzed by Daly et al. (2001). RESULTS: epsilon allows for arbitrary numbers of loci, describes LD with regard to the loci sequence, and can be interpreted as a multilocus extension of r(2). The application of epsilon to the data sets demonstrated the measure's ability to appropriately describe simultaneous multilocus LD and to detect haplotype blocks. CONCLUSIONS: epsilon is a reasonable multilocus LD measure and might be of potential use in the construction of the human haplotype map.

Male partners of substance-abusing women in treatment: an exploratory study.

Despite evidence for the importance of significant others in the recovery process, little is known about male partners of female substance abusers. This paper presents a qualitative exploratory study conducted to learn more about male partners of crack/cocaine-addicted women and specifically examines the men's influence on the treatment and recovery process of their female partners. Subjects were found to have an extensive history of substance abuse and some experience with treatment; three-quarters were active crack/cocaine users. Although their attitude toward women's substance abuse was negative and their opinion of treatment for women positive, men's behavioral support for their partners was usually passive and inconsistent. Reasons are discussed why male partners of female drug users in treatment generally were resistant to active participation in family-oriented treatment: the men's active drug use, their desire to maintain the status quo in the relationship, possible focus on their own recovery, divergent views about treatment goals, and the desire to minimize stigma by association.

Drug scene roles and HIV risk.

AIMS: Drug scenes (social and spatial drug-using and drug-selling environments) have complex role structures. Many drug injectors earn money or drugs as drug or syringe sellers, hit doctors (people who help others to inject) commercial sex workers, or in other roles. This paper aims to measure "role behaviors" of drug injectors; describe which drug injectors are more likely to engage in such role behaviors; and to determine whether roles are related to elements of HIV risk. DESIGN: Cross-sectional study of drug injectors. SETTING: Bushwick, a section of Brooklyn, New York, a major location for injection drug use and drug sales. PARTICIPANTS: Seven hundred and sixty-seven street-recruited drug injectors. MEASUREMENTS: Participants were interviewed about their roles, behaviors, socio-demographics and risk networks; sera were collected and assayed for HIV and hepatitis B core antibody. FINDINGS: Socio-demographic variables are related to role-holding in complex ways. Economic need is generally associated with engaging in drug-scene role behaviors. Holders of these roles are at greater behavioral and network risk for HIV and other blood-borne infections than are other drug injectors. They also engage in extensive communication with other drug users, including discussion of HIV risk reduction. CONCLUSION: Role behaviors can be measured in quantitative studies, and seem to be related to HIV risk. Role-holders may be strategic targets for risk-reduction campaigns. It seems feasible and advisable to measure drug scene role-holding in research on drug users.