First Report of Eutypella spp. Associated with Branch Canker of Citrus in California.
Eutypella is one of the few genera in the Diatrypaceae considered plant pathogens (1). In California, E. vitis and other members of the Diatrypaceae cause branch and trunk canker on grapevine (3,4). Eutypella spp. have not previously been documented as pathogens of citrus. In a 2010 survey on citrus branch canker and dieback in six citrus-growing counties of California, four isolates of Eutypella species were detected in Riverside and San Diego counties. Canker symptoms included dieback and bark cracking, and cuts made through symptomatic trees showed that the cankers were expanding through the center of the tree. Branch samples were collected from 10 trees per orchard and 5 to 10 orchards per county (102 trees for two counties). Pieces of symptomatic tissue (1 to 2 mm2) were plated onto potato dextrose agar amended with 0.01% tetracycline (PDA-tet) and incubated at 25°C for 4 days. All isolates were identified by morphological and molecular characteristics. PCR of isolates was performed in a thermal cycler using two primer pairs, ITS4/5 and Bt2a/2b for amplifying the internal transcribed spacer (ITS1), 5.8S, and ITS2 region and the β-tubulin gene, respectively (2,3). PCR products were sequenced at the University of California, Riverside Genomics Core and the sequences compared in a BLAST search. Four isolates identified as Eutypella spp. included two (UCR1088 and UCR1101) from San Diego County and two (UCR1148 and UCR1149) from the Riverside County samples. The sequences were deposited in GenBank (HQ880579, JF758610, HQ880581, and HQ880582 and HQ880583, JF758611, HQ880585, and HQ880586 for the ITS regions and β-tubulin gene, respectively. ITS sequences for UCR1088 and UCR1101 had 98 and 100% match, respectively, to Eutypella spp. ITS sequences in GenBank (GQ293959 to GQ293961), while UCR1148 and UCR1149 matched 99% (GQ293956 to GQ293958). On the basis of morphological characteristics, UCR1088 and UCR1101 were similar to Eutypella spp. group 1, while UCR1148 and UCR1149 were similar to Eutypella spp. group 3 (4). Pathogenicity tests were conducted with all four isolates on detached shoots from healthy citrus trees of the same cultivar/rootstock from which each isolate was obtained. One wound per shoot was made on 1-year-old, green, detached shoots using a 3-mm-diameter cork borer and the wounded surfaces were inoculated with 3-mm-diameter mycelial plugs of 5-day-old cultures of each isolate growing on PDA-tet. Inoculated wounds and shoot ends were covered with petroleum jelly and wrapped with Parafilm (3). Control shoots were inoculated with sterile agar plugs. There were 10 inoculated shoots per isolate and noninoculated control treatment. Shoots were incubated at 25°C in moist chambers for 6 weeks. Lesions similar to those on the original infected shoots were observed on all inoculated shoots except the control treatment. Reisolation and identification of fungi from inoculated and control shoots were done using methods described above. Inoculated isolates were recovered from 100% of inoculated shoots but none was recovered from noninoculated shoots, indicating association of Eutypella spp. with citrus branch canker. To our knowledge, this is the first report of Eutypella spp. associated with cankers on citrus in California. References: (1) B. Piskur et al. Plant Dis. 91:1579, 2007. (2) B. Slippers et al. Mycologia 96:83, 2004. (3) F. P. Trouillas and W. D. Gubler. Plant Dis. 94:867, 2010. (4) F. P. Trouillas et al. Mycologia 102:319, 2010.


The influence of literacy on patient-reported experiences of diabetes self-management support.
Variability in disease-related outcomes may relate to how patients experience self-management support in clinical settings. The purpose of this study was to identify factors associated with experiences of self-management support during primary care encounters. A cross-sectional survey was conducted of 208 patients seen in a multidisciplinary diabetes program in an academic medicine clinic. Multiple regression analysis was used to test associations between patient-rated experiences of self-management support (Patient Assessment of Chronic Illness Care) and race, gender, insurance status, literacy, duration of diabetes, and intensity of care management. The Patient Assessment of Chronic Illness Care ratings decreased with age (r = -.235, p = .001), were higher for women than for men (3.95 vs. 3.65, t = 2.612, p= .010), and were greater for those with more education (F= 3.927, p = .009) and greater literacy skills (t = 3.839, p< .001). The ratings did not vary between racial (t = -1.108, p = .269) or insurance (F = 1.045, p = .374) groups and were unaffected by the duration of diabetes (r= .052, p = .466) and the intensity of care management (F = 1.028, p = .360). In multivariate models, literacy was the only variable contributing significantly to variation in self-management support ratings. Even when considering the objective intensity of health services delivered, literacy was the sole variable contributing to differences in patient ratings of self-management support. Although conclusions are limited by the cross-sectional nature of this study, the results emphasize the need to consider literacy when developing and communicating treatment plans requiring self-management skills.


Influenza Virus NS1 Protein-RNA Interactome Reveals Intron Targeting.
The NS1 protein of influenza A virus is a multifunctional virulence factor that inhibits cellular processes to facilitate viral gene expression. While NS1 is known to interact with RNA and proteins to execute these functions, the cellular RNAs that physically interact with NS1 have not been systematically identified. Here we reveal a NS1 protein-RNA interactome and show that NS1 primarily binds intronic sequences. Among this subset of pre-mRNAs is the RIG-I pre-mRNA, which encodes the main cytoplasmic antiviral sensor of influenza virus infection. This suggested that NS1 interferes with the antiviral response at a posttranscriptional level by virtue of its RNA binding properties. Indeed, we show that NS1 is necessary in the context of viral infection and sufficient upon transfection to decrease the rate of RIG-I intron removal. This NS1 function requires a functional RNA binding domain and is independent of the NS1 interaction with the cleavage and polyadenylation specificity factor CPSF30. NS1 has been previously shown to abrogate RIG-I-mediated antiviral immunity by inhibiting its protein function. Our data further suggest that NS1 also posttranscriptionally alters RIG-I pre-mRNA processing by binding to the RIG-I pre-mRNA.IMPORTANCE A key virulence factor of influenza A virus is the NS1 protein, which inhibits various cellular processes to facilitate viral gene expression. The NS1 protein is localized in the nucleus and in the cytoplasm during infection. In the nucleus, NS1 has functions related to inhibition of gene expression that involve protein-protein and protein-RNA interactions. While several studies have elucidated the protein interactome of NS1, we still lack a clear and systematic understanding of the NS1-RNA interactome. Here we reveal a nuclear NS1-RNA interactome and show that NS1 primarily binds intronic sequences within a subset of pre-mRNAs, including the RIG-I pre-mRNA that encodes the main cytoplasmic antiviral sensor of influenza virus infection. Our data here further suggest that NS1 is necessary and sufficient to impair intron processing of the RIG-I pre-mRNA. These findings support a posttranscriptional role for NS1 in the inhibition of RIG-I expression.


Effect of ammonia on Na+ transport across isolated rumen epithelium of sheep is diet dependent.
The cellular uptake of ammonia affects the intracellular pH (pHi) of polar and non-polar cells. A predominant uptake of NH3 and its intracellular protonation tend to alkalinise the cytoplasm, whereas a predominant uptake of NH4(+) acidifies the cytoplasm by reversing this reaction. Hence, the well-known absorption of ammonia across the rumen epithelium probably causes a change in the pHi. The magnitude and direction of this change in pHi (acid or alkaline) depends on the relative transport rates of NH3 and NH4(+). Consequently, the intracellular availability of protons will influence the activity of the Na(+)-H(+) exchanger, which could affect transepithelial Na(+) transport. The aim of the present study has been to test this possible interaction between ruminal ammonia concentrations and Na(+) transport. The term ammonia is used to designate the sum of the protonated (NH4(+)) and unprotonated (NH3) forms. Isolated ruminal epithelium of sheep was investigated by using the Ussing-chamber technique in vitro. The present results indicate that ammonia inhibits Na(+) transport across the rumen epithelium of hay-fed sheep, probably by binding intracellular protons and thus inhibiting Na(+)-H(+) exchange. By contrast, ammonia stimulates Na(+) transport in concentrate-fed and urea-fed sheep, which develop an adaptation mechanism in the form of an increased metabolism of ammonia in the rumen mucosa and/or an increased permeability of rumen epithelium to the charged ammonium ion (NH4(+)). Intracellular dissociation of NH4(+) increases the availability of protons, which stimulate Na(+)-H(+) exchange. This positive effect of ruminal ammonia on Na(+) absorption may significantly contribute to the regulation of osmotic pressure of the ruminal fluid, because intraruminal ammonia concentrations up to 40 mmol/l have been reported.


Insulin resistance: an additional risk factor in the pathogenesis of cardiovascular disease in type 2 diabetes.
Sedentary life style and high calorie dietary habits are prominent leading cause of metabolic syndrome in modern world. Obesity plays a central role in occurrence of various diseases like hyperinsulinemia, hyperglycemia and hyperlipidemia, which lead to insulin resistance and metabolic derangements like cardiovascular diseases (CVDs) mediated by oxidative stress. The mortality rate due to CVDs is on the rise in developing countries. Insulin resistance (IR) leads to micro or macro angiopathy, peripheral arterial dysfunction, hampered blood flow, hypertension, as well as the cardiomyocyte and the endothelial cell dysfunctions, thus increasing risk factors for coronary artery blockage, stroke and heart failure suggesting that there is a strong association between IR and CVDs. The plausible linkages between these two pathophysiological conditions are altered levels of insulin signaling proteins such as IR-β, IRS-1, PI3K, Akt, Glut4 and PGC-1α that hamper insulin-mediated glucose uptake as well as other functions of insulin in the cardiomyocytes and the endothelial cells of the heart. Reduced AMPK, PFK-2 and elevated levels of NADP(H)-dependent oxidases produced by activated M1 macrophages of the adipose tissue and elevated levels of circulating angiotensin are also cause of CVD in diabetes mellitus condition. Insulin sensitizers, angiotensin blockers, superoxide scavengers are used as therapeutics in the amelioration of CVD. It evidently becomes important to unravel the mechanisms of the association between IR and CVDs in order to formulate novel efficient drugs to treat patients suffering from insulin resistance-mediated cardiovascular diseases. The possible associations between insulin resistance and cardiovascular diseases are reviewed here.


Neuropeptide S receptor gene is associated with cortisol responses to social stress in humans.
The neuropeptide S (NPS) and its receptor NPSR represent a transmitter system critically involved in the modulation of anxiety and arousal in rodents. Initial human studies indicate that the T-allele of the functional NPSR gene (NPSR1) polymorphism (rs324981), which increases NPS potency at NPSR, is associated with anxiety-related phenotypes. Since stress is critically involved in the pathogenesis of anxiety disorders, we tested the association between rs324981 and stress reactivity in 196 healthy males. Participants were exposed to the Trier Social Stress Test for Groups (TSST-G), a standardized laboratory protocol for stress exposure in a group format. Salivary cortisol and subjective stress responses were assessed. A significant genotype by time interaction and a main effect of genotype were shown, with T-allele carriers displaying larger cortisol and subjective stress responses. This is the first report to show involvement of the NPS system in the regulation of the neuroendocrine stress response in humans.


Neuronal chromatin dynamics of imprinting in development and disease.
Epigenetic mechanisms play essential roles in mammalian neurodevelopment and genetic mutations or chromosomal deletions or duplications of epigenetically regulated loci or pathways result in several important human neurodevelopmental disorders. Postnatal mammalian neurons have among the most structured and dynamic nuclear organization of any cell type. Human chromosome 15q11-13 is an imprinted locus required for normal neurodevelopment and is regulated by a plethora of epigenetic mechanisms in neurons, including multiple noncoding RNAs, parentally imprinted transcription and histone modifications, large-scale chromatin decondensation, and homologous pairing in mature neurons of the mammalian brain. Here, we describe the multiple epigenetic layers regulating 15q11-13 gene expression and chromatin dynamics in neurons and propose a model of how noncoding RNAs may influence the unusual neuronal chromatin structure and dynamics at this locus. We also discuss the need for improved neuronal cell culture systems that model human 15q11-13 and other neurodevelopmental disorders with epigenetic bases in order to test the mechanisms of chromatin dynamics and nuclear organization in neurons. Induced pluripotent stem cells and other stem cell technologies hold promise for improved understanding of and therapeutic interventions for multiple human neurodevelopmental disorders.


Ethylene response factor 1 mediates Arabidopsis resistance to the soilborne fungus Fusarium oxysporum.
Ethylene response factor 1 (ERF1) is a transcriptional factor from Arabidopsis thaliana that regulates plant resistance to the necrotrophic fungi Botrytis cinerea and Plectosphaerella cucumerina and whose overexpression enhances resistance to these fungi. Here, we show that ERF1 also mediates Arabidopsis resistance to the soilborne fungi Fusarium oxysporum sp. conglutinans and F. oxysporum f. sp. lycopersici, because its constitutive expression in Arabidopsis confers enhanced resistance to these pathogens. Expression of ERF1 was upregulated after inoculation with F. oxysporum f. sp. conglutinans, and this response was blocked in ein2-5 and coi1-1 mutants, impaired in the ethylene (ET) and jasmonic acid (JA) signal pathways, respectively, which further indicates that ERF1 is a downstream component of ET and JA defense responses. The signal transduction network controlling resistance to F. oxysporum fungi was explored using signaling-defective mutants in ET (ein2-5), JA (jar1-1), and salicylic acid (SA) (NahG, sid2-1, eds5-1, npr1-1, pad4-1, eds1-1, and pad2-1) transduction pathways. This analysis revealed that Arabidopsis resistance to F. oxysporum requires the ET, JA, and SA signaling pathways and the NPR1 gene, although it is independent of the PAD4 and EDS1 functions.


Anatomical Sciences in Chiropractic Education: A Survey of Chiropractic Programs in Australia.
Human anatomy knowledge is a core requirement for all health care clinicians. There is a paucity of information relating to anatomy content and delivery in Australian chiropractic programs. The aim of this study was to describe anatomy teaching in Australian chiropractic programs, utilizing a survey which was distributed to all four programs, requesting information on: anatomy program structure, delivery methods, assessment, teaching resources, and academic staff profile at their institution. The survey was undertaken in 2016 and documented practices in that academic year. All four institutions responded. There was a reported difference in the teaching hours, content, delivery and assessment of anatomy utilized in Australian chiropractic programs. Anatomy was compulsory at all four institutions with the mean total of 214 (SD ± 100.2) teaching hours. Teaching was undertaken by permanent ongoing (30%) and sessional academic staff, and student to teacher ratio varied from 15:1 to 12:1. A variety of teaching resources were utilized, including human tissue access, either as prosected cadavers or plastinated body parts. The results of this survey confirm that anatomy has an established place in chiropractic education programs in Australia and while curricular variations exist, all programs had similar course design, delivery, and assessment methods. This study confirmed the provision of a strong foundation in topographical anatomy and neuroanatomy, while other anatomical sciences, such as histology and embryology were not consistently delivered. Formalization of a core anatomy curriculum together with competency standards is needed to assist program evaluation and development, and for accreditation purposes.


First Report of Plectosphaerella cucumerina on Greenhouse Cultured Wild Rocket (Diplotaxis tenuifolia) in Italy.
During spring 2012, symptoms of an unusual leaf spot disease were observed in several commercial greenhouses near Salerno (southern Italy) on plants of Diplotaxis tenuifolia (cv Selvatica). The first symptoms on leaves of affected plants consisted of small (1 mm) black-brown spots of irregular shape, later coalescing into larger spots, 1 cm in diameter. Spots were surrounded by a yellow halo, and were mostly located on the foliar limb, rib, and petiole. Affected leaves were often distorted, appearing hook-like. The disease was severe under 75 to 90% RH, at air temperature of 20 to 26°C, and caused severe production losses on about 50 ha. Particularly, affected tissues rotted quickly after packaging and during transit and commercialization of processed rocket. Diseased tissue was excised, immersed in a solution containing 1% sodium hypochlorite for 60 s, rinsed in water, then placed on potato dextrose agar (PDA) medium, containing 25 mg/l of streptomycin sulphate. After 5 days, a fungus developed producing a whitish-orange mycelium when incubated under 12 h/day of fluorescent light at 22°C. The isolates obtained were purified on PDA. On this medium, they produced hyaline elliptical and ovoid conidia, sometimes one-septate, measuring 4.5 to 9.2 × 1.7 to 3.5 (average 6.8 × 2.6) μm. Conidia were born on phialides, measuring 6.8 to 20.2 × 1.3 to 3.1 (average 16.5 × 2.1) μm. Such characteristics are typical of Plectosphaerella sp. (2). The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS1/ITS4 (3) and sequenced. BLAST analysis of the 519-bp segment showed a 98% similarity with the sequence of Plectosphaerella cucumerina (GenBank Accession No. AB469880). The nucleotide sequence has been assigned the GenBank Accession JX185769. To confirm pathogenicity, tests were conducted on 45-day-old D. tenuifolia plants. Plants (21/treatment), grown in 15 liter pots (7 plants/pot) were inoculated by spraying a 1 × 106 CFU/ml conidial suspension of one isolate of P. cucumerina, prepared from 10-day-old cultures, grown on PDA. Inoculated plants were maintained in a growth chamber at 23 ± 1°C, at 90% RH for 4 days. Non-inoculated plants served as control. Inoculated plants showed the typical first leaf spots 6 days after the artificial inoculation. Four days later, spots enlarged and leaves became distorted, showing chlorosis. Non-inoculated plants remained healthy. P. cucumerina was reisolated from inoculated plants. The pathogenicity test was conducted twice with identical results. This is, to our knowledge, the first report of P. cucumerina on D. tenuifolia in Italy as well as worldwide. P. cucumerina has been described as associated with root and collar rots of other horticultural crops in southern Italy (1). Due to the importance of the crop in Italy, this disease can cause serious economic losses. References: (1) A. Carlucci et al., Persoonia, 28:34, 2012. (2) M. E. Palm et al. Mycologia, 87:397, 1995. (3) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.


Male and female bees show large differences in floral preference.
Intraspecific variation in foraging niche can drive food web dynamics and ecosystem processes. In particular, male and female animals can exhibit different, often cascading, impacts on their interaction partners. Despite this, studies of plant-pollinator interaction networks have focused on the partitioning of the floral community between pollinator species, with little attention paid to intraspecific variation in plant preference between male and female bees. We designed a field study to evaluate the strength and prevalence of sexually dimorphic foraging, and particularly resource preferences, in bees. We observed bees visiting flowers in semi-natural meadows in New Jersey, USA. To detect differences in flower use against a shared background of resource (flower) availability, we maximized the number of interactions observed within narrow spatio-temporal windows. To distinguish observed differences in bee use of flower species, which can reflect abundance patterns and sampling effects, from underlying differences in bee preferences, we analyzed our data with both a permutation-based null model and random effects models. We found that the diets of male and female bees of the same species were often dissimilar as the diets of different species of bees. Furthermore, we demonstrate differences in preference between male and female bees. We show that intraspecific differences in preference can be robustly identified among hundreds of unique species-species interactions, without precisely quantifying resource availability, and despite high phenological turnover of both bees and plant bloom. Given the large differences in both flower use and preferences between male and female bees, ecological sex differences should be integrated into studies of bee demography, plant pollination, and coevolutionary relationships between flowers and insects.


Transgenic mouse model of kidney disease: insertional inactivation of ubiquitously expressed gene leads to nephrotic syndrome.
Transgenic mouse strains carrying proviruses were generated by exposing mouse embryos to a recombinant retrovirus. Animals carrying a single provirus were intercrossed to derive mice homozygous for a given proviral insertion. Adult mice homozygous for the Mpv17 integration developed nephrotic syndrome and chronic renal failure. Histologically, affected kidneys showed progressive glomerular sclerosis. Similar lesions are seen in patients with progressive renal function deterioration. A probe to DNA sequences flanking the provirus detected a 1.7 kb RNA ubiquitously expressed during embryogenesis and in adults with high levels in kidney, brain, and heart. This RNA was not detected in tissues of homozygous animals, suggesting that the provirus interferes with RNA expression. Sequence analysis of the cDNA revealed that the gene encodes a 176 amino acid peptide containing hydrophobic regions, suggesting membrane association of the putative protein. The Mpv17 mutant is a potentially useful experimental system for studying mechanisms leading to renal disorders in man.


Time-Dependent Changes of Cell Proliferation After Laser Photocoagulation in Mouse Chorioretinal Tissue.
This study investigated the time course of cell proliferation after laser photocoagulation and analyzed the cell types of proliferating cells. C57BL/6J mice received unilateral laser photocoagulation. Intraperitoneal bromodeoxyuridine (BrdU) injection was performed, and mice were divided into two groups according to the injection paradigm: group 1 with continuous injection and group 2 with periodic injection. Each group was again divided into four subgroups according to injection period: 0 to 3 days (n = 11), 0 to 7 days (n = 14), 0 to 14 days (n = 6), and 0 to 28 days (n = 6) after laser photocoagulation for group 1; and 0 to 3 days (n = 11), 4 to 7 days (n = 6), 8 to 14 days (n = 6), and 15 to 28 days (n = 6) after laser photocoagulation for group 2. The eyes were examined with immunohistochemistry using anti-BrdU antibody and other various antibodies for identification of proliferating cells. Manual cell counting and flow cytometry were performed for quantification. In group 1, the number of BrdU+ cells showed marked increase during the first 3 days of laser lesioning, reaching its maximum after 7 days (P < 0.05). Group 2 also demonstrated peak proliferation during the first 3 days, but a significantly reduced number of BrdU+ cells were detected during 4 to 7 days, 8 to 14 days, and 15 to 28 days of laser treatment (P < 0.05). BrdU+ cells colocalized with CD11b, F4/80, iba1, RPE65, CD31, and glial fibrillary acidic protein (GFAP) labeling, and CD11b+, F4/80+, and iba1+ cells constituted the main fraction of BrdU+ cells. Laser photocoagulation induced cell proliferation mostly during the first 3 days, and many proliferating cells were identified as inflammatory cells, RPE cells, endothelial cells, and Müller cells.


Antibody-dependent SARS coronavirus infection is mediated by antibodies against spike proteins.
The severe acute respiratory syndrome coronavirus (SARS-CoV) still carries the potential for reemergence, therefore efforts are being made to create a vaccine as a prophylactic strategy for control and prevention. Antibody-dependent enhancement (ADE) is a mechanism through which dengue viruses, feline coronaviruses, and HIV viruses take advantage of anti-viral humoral immune responses to infect host target cells. Here we describe our observations of SARS-CoV using ADE to enhance the infectivity of a HL-CZ human promonocyte cell line. Quantitative-PCR and immunofluorescence staining results indicate that SARS-CoV is capable of replication in HL-CZ cells, and of displaying virus-induced cytopathic effects and increased levels of TNF-α, IL-4 and IL-6 two days post-infection. According to flow cytometry data, the HL-CZ cells also expressed angiotensin converting enzyme 2 (ACE2, a SARS-CoV receptor) and higher levels of the FcγRII receptor. We found that higher concentrations of anti-sera against SARS-CoV neutralized SARS-CoV infection, while highly diluted anti-sera significantly increased SARS-CoV infection and induced higher levels of apoptosis. Results from infectivity assays indicate that SARS-CoV ADE is primarily mediated by diluted antibodies against envelope spike proteins rather than nucleocapsid proteins. We also generated monoclonal antibodies against SARS-CoV spike proteins and observed that most of them promoted SARS-CoV infection. Combined, our results suggest that antibodies against SARS-CoV spike proteins may trigger ADE effects. The data raise new questions regarding a potential SARS-CoV vaccine, while shedding light on mechanisms involved in SARS pathogenesis.


Massage provision by physiotherapists at major athletics events between 1987 and 1998.
The equivocal findings in the literature on efficacy of massage makes it difficult to assess the requirement for, or justify the use of, specialist massage personnel at major athletics events. However, the use of massage by athletes during training and competition remains popular. To quantify the amount of their time that physiotherapists devote to massage treatment at major athletics events in an attempt to determine the importance of this treatment modality, and to examine whether the use of massage at athletics events is changing over time. Data recorded by the head team physiotherapist from 12 major athletics events (national and international events) between 1987 and 1998 were examined. For each event, the data included: total number of treatments administered by the physiotherapist, the treatment modalities used, and the number of attendances for treatment. The amount of massage provided was expressed as a percentage of the total number of treatments for each athletic event, and the pattern of change in use of massage treatment over time was evaluated. The percentage of time spent providing massage treatment ranged from 24.0% to 52.2% of the total number of treatments made. The overall median percentage of total treatments in the form of massage was 45.2%. No significant increase or decrease in the use of massage as a treatment modality was observed between 1987 and 1998 in the athletics events examined (p = 0.95). A significant proportion of physiotherapists' time is devoted to the delivery of massage treatment at athletics events. The demand for massage treatment has been steady over the time period, in the events for which data are available, indicating a consistent use of this treatment modality. Given the popularity of massage among athletes, consideration should be given to the use of specialist sports massage staff at major athletics events. Furthermore, it would seem prudent to further investigate the efficacy of the treatment.


Participation of the inositol 1,4,5-trisphosphate-gated calcium channel in the zona pellucida- and progesterone-induced acrosome reaction and calcium influx in human spermatozoa.
The acrosome reaction is a prerequisite for fertilization, and its signaling pathway has been investigated for decades. Regardless of the type of inducers present, the acrosome reaction is ultimately mediated by the elevation of cytosolic calcium. Inositol 1,4,5-trisphosphate-gated calcium channels are important components of the acrosome reaction signaling pathway and have been confirmed by several researchers. In this study, we used a novel permeabilization tool BioPORTER® and first demonstrated its effectiveness in spermatozoa. The inositol 1,4,5-trisphosphate type-1 receptor antibody was introduced into spermatozoa by BioPORTER® and significantly reduced the calcium influx and acrosome reaction induced by progesterone, solubilized zona pellucida, and the calcium ionophore A23187. This finding indicates that the inositol 1,4,5-trisphosphate type-1 receptor antibody is a valid inositol 1,4,5-trisphosphate receptor inhibitor and provides evidence of inositol 1,4,5-trisphosphate-gated calcium channel involvement in the acrosome reaction in human spermatozoa. Moreover, we demonstrated that the transfer of 1,4,5-trisphosphate into spermatozoa induced acrosome reactions, which provides more reliable evidence for this process. In addition, by treating the spermatozoa with inositol 1,4,5-trisphosphate/BioPORTER® in the presence or absence of calcium in the culture medium, we showed that the opening of inositol 1,4,5-trisphosphate-gated calcium channels led to extracellular calcium influx. This particular extracellular calcium influx may be the major process of the final step of the acrosome reaction signaling pathway.


Generation of achromatic, uniform-phase, radially polarized beams.
Axially symmetric half-wave plates have been used to generate radially polarized beams that have constant phase in the plane transverse to propagation. However, since the retardance introduced by these waveplates depends on the wavelength, it is difficult to generate radially polarized beams achromatically. This paper describes a technique suitable for the generation of achromatic, radially polarized beams with uniform phase. The generation system contains, among other optical components, an achromatic, axially symmetric quarter-wave plate based on total internal reflection. For an incident beam with a constant phase distribution, the system generates a beam with an extra geometrical phase term. To generate a beam with the correct phase distribution, it is therefore necessary to have an incident optical vortex with an azimuthally varying phase distribution of the form exp( + iθ). We show theoretically that the phase component of radially polarized beam is canceled out by the phase component of the incident optical vortex, resulting in a radially polarized beam with uniform phase. Additionally, we present an experimental setup able to generate the achromatic, uniform-phase, radially polarized beam and experimental results that confirm that the generated beam has the correct phase distribution.


Factors associated with self and informant ratings of the quality of life of people with dementia living in care facilities: a cross sectional study.
There is no consensus regarding the optimal approach to assessment of the quality of life of people with dementia. We undertook the present study to describe and determine the factors associated with ratings of the quality of life of a cohort of people with dementia living in a residential care facility. 351 people with dementia living in residential care facilities, and their staff and family informants participated in this cross sectional observational study. Quality of life was measured using self (Quality of Life in Alzheimer's Disease [QoL-AD] scale), and informant (QoL-AD and Alzheimer's Disease Related QoL Scale) reports. 226 people (64%) with dementia (median MMSE 17; 12-21) were able to self rate the QoL-AD scale and these subjects' ratings were compared to ratings by staff and family. Both staff and family informant ratings of the QoL-AD underestimated self ratings (mean difference -7.8, 95% CI -8.8, -6.7 for staff rated QoL-AD; and mean difference -7.2, 95% CI -8.5, -6.0 for family rated QoL-AD). Self ratings of QoL were lower among people who were restrained, had fallen or had pain. Informant ratings of the QoL of the participants with dementia were consistently and significantly lower for people with severe cognitive impairment, who had fallen, had presence of neuropsychiatric symptoms, or where care giver distress was present. Documented restraint, reported pain and neuropsychiatric symptoms were independently associated with lower self rating of the QoL-AD in multivariate models. Cognitive impairment, case conferencing, hospitalizations and neuropsychiatric symptoms were found to be independently associated with staff rated ADRQL. The majority of people with dementia living in residential care facilities can rate their own QoL. Informant ratings underestimate self ratings of QoL of people with dementia, and appear to be associated with factors which are not associated with self ratings.


3D reconstruction of the cerebellar germinal layer reveals tunneling connections between developing granule cells.
The difficulty of retrieving high-resolution, in vivo evidence of the proliferative and migratory processes occurring in neural germinal zones has limited our understanding of neurodevelopmental mechanisms. Here, we used a connectomic approach using a high-resolution, serial-sectioning scanning electron microscopy volume to investigate the laminar cytoarchitecture of the transient external granular layer (EGL) of the developing cerebellum, where granule cells coordinate a series of mitotic and migratory events. By integrating image segmentation, three-dimensional reconstruction, and deep-learning approaches, we found and characterized anatomically complex intercellular connections bridging pairs of cerebellar granule cells throughout the EGL. Connected cells were either mitotic, migratory, or transitioning between these two cell stages, displaying a chronological continuum of proliferative and migratory events never previously observed in vivo at this resolution. This unprecedented ultrastructural characterization poses intriguing hypotheses about intercellular connectivity between developing progenitors and its possible role in the development of the central nervous system.


Desmopressin for treatment of platelet dysfunction and reversal of antiplatelet agents: a systematic review and meta-analysis of randomized controlled trials.
Essentials The optimal management of patients with platelet dysfunction undergoing surgery is unclear. This meta-analysis compared perioperative administration of desmopressin to placebo. Desmopressin reduced red cell transfusions, blood loss and risk of re-operation due to bleeding. There were too few events to determine if there was a change in the risk of thrombotic events. Background Platelet dysfunction, including that caused by antiplatelet agents, increases the risk of perioperative bleeding. The optimal management of patients with platelet dysfunction undergoing surgery is unclear. Objectives To assess whether desmopressin reduces perioperative allogeneic red cell transfusion and bleeding in patients with platelet dysfunction. Patients/Methods We searched for randomized controlled trials in The Cochrane Central Register of Controlled Trials, MEDLINE, PubMed, Embase, the Transfusion Evidence Library and the ISI Web of Science to 7th July 2016. Data were pooled using mean difference (MD), relative risks or Peto odds ratios (pOR) using a random-effects model. Results Ten trials with 596 participants were identified, all in the setting of cardiac surgery. Platelet dysfunction was due to antiplatelet agents in six trials and cardiopulmonary bypass in four trials. Patients treated with desmopressin were transfused with fewer red cells (MD, -0.65 units; 95% Confidence Interval [CI], -1.16 to -0.13 units), lost less blood (MD, -253.93 mL; 95% CI, -408.01 to -99.85 mL) and had a lower risk of re-operation due to bleeding (pOR, 0.39; 95% CI, 0.18-0.84). The GRADE quality of evidence was very low to moderate, suggesting considerable uncertainty over the results Conclusions Desmopressin may be a useful agent to reduce bleeding and transfusion requirements for people with platelet dysfunction or with a history of recent antiplatelet drug administration undergoing cardiac surgery.


Trans-activation of transcription, from promoters containing immunoglobulin gene octamer sequences, by myeloma cell mRNA in Xenopus oocytes.
To study factors required for immunoglobulin gene transcription hybrid promoters were made by linking octamer elements to a Xenopus albumin gene construct containing only 50bp of the albumin gene promoter. When injected into oocytes these hybrid promoters directed transcription far less efficiently than the unmodified 50bp albumin gene promoter fragment. Activity of the hybrid promoter, but not the unmodified albumin promoter, could be stimulated by preinjection of poly(A)+ RNA from NS1 myeloma cells. This stimulation may be caused by translation of the NS1 poly(A)+ RNA into transcription factors that act on the octamer. Both the reduction in transcription caused by octamer insertion and the extent of the inducibility by NS1 RNA are greater when two, rather than one, octamers are inserted.


Protective Effects of Ellagitannin-Rich Strawberry Extracts on Biochemical and Metabolic Disturbances in Rats Fed a Diet High in Fructose.
The present study compares the effects of two dietary strawberry extracts rich in monomeric (ME) or dimeric (DE) ellagitannins (ETs) on gastrointestinal, blood and tissue biomarkers in Wistar rats fed high-fructose diets. Both strawberry extracts beneficially affect the antioxidant status and lipid profile of the liver and serum. The ME extract shows a greater ability to inhibit lipid peroxidation in kidneys, more effectively decreases serum and liver triglycerides, and exerts greater anti-inflammatory effects in blood serum than the DE extract. The DE extract significantly reduces the activity of microbial enzymes in the cecum. These effects might be associated with higher cecum and urine levels of ET metabolites in rats fed with ME than in rats fed with DE. In conclusion, the diet-induced fructose-related disturbances observed in biochemical parameters are regulated by both extracts; nevertheless, the beneficial effects of the ME extract are mostly associated with systemic parameters, while those of the DE extracts are associated with local microbial activity.


A Weakly Supervised Deep Learning Framework for Sorghum Head Detection and Counting.
The yield of cereal crops such as sorghum (Sorghum bicolor L. Moench) depends on the distribution of crop-heads in varying branching arrangements. Therefore, counting the head number per unit area is critical for plant breeders to correlate with the genotypic variation in a specific breeding field. However, measuring such phenotypic traits manually is an extremely labor-intensive process and suffers from low efficiency and human errors. Moreover, the process is almost infeasible for large-scale breeding plantations or experiments. Machine learning-based approaches like deep convolutional neural network (CNN) based object detectors are promising tools for efficient object detection and counting. However, a significant limitation of such deep learning-based approaches is that they typically require a massive amount of hand-labeled images for training, which is still a tedious process. Here, we propose an active learning inspired weakly supervised deep learning framework for sorghum head detection and counting from UAV-based images. We demonstrate that it is possible to significantly reduce human labeling effort without compromising final model performance (R 2 between human count and machine count is 0.88) by using a semitrained CNN model (i.e., trained with limited labeled data) to perform synthetic annotation. In addition, we also visualize key features that the network learns. This improves trustworthiness by enabling users to better understand and trust the decisions that the trained deep learning model makes.


The genome sequence of Caenorhabditis briggsae: a platform for comparative genomics.
The soil nematodes Caenorhabditis briggsae and Caenorhabditis elegans diverged from a common ancestor roughly 100 million years ago and yet are almost indistinguishable by eye. They have the same chromosome number and genome sizes, and they occupy the same ecological niche. To explore the basis for this striking conservation of structure and function, we have sequenced the C. briggsae genome to a high-quality draft stage and compared it to the finished C. elegans sequence. We predict approximately 19,500 protein-coding genes in the C. briggsae genome, roughly the same as in C. elegans. Of these, 12,200 have clear C. elegans orthologs, a further 6,500 have one or more clearly detectable C. elegans homologs, and approximately 800 C. briggsae genes have no detectable matches in C. elegans. Almost all of the noncoding RNAs (ncRNAs) known are shared between the two species. The two genomes exhibit extensive colinearity, and the rate of divergence appears to be higher in the chromosomal arms than in the centers. Operons, a distinctive feature of C. elegans, are highly conserved in C. briggsae, with the arrangement of genes being preserved in 96% of cases. The difference in size between the C. briggsae (estimated at approximately 104 Mbp) and C. elegans (100.3 Mbp) genomes is almost entirely due to repetitive sequence, which accounts for 22.4% of the C. briggsae genome in contrast to 16.5% of the C. elegans genome. Few, if any, repeat families are shared, suggesting that most were acquired after the two species diverged or are undergoing rapid evolution. Coclustering the C. elegans and C. briggsae proteins reveals 2,169 protein families of two or more members. Most of these are shared between the two species, but some appear to be expanding or contracting, and there seem to be as many as several hundred novel C. briggsae gene families. The C. briggsae draft sequence will greatly improve the annotation of the C. elegans genome. Based on similarity to C. briggsae, we found strong evidence for 1,300 new C. elegans genes. In addition, comparisons of the two genomes will help to understand the evolutionary forces that mold nematode genomes.


Low-dose dobutamine stress echocardiography to assess left ventricular contractile reserve for cardiac resynchronization therapy: data from the Low-Dose Dobutamine Stress Echocardiography to Predict Cardiac Resynchronization Therapy Response (LODO-CRT) trial.
Cardiac resynchronization therapy (CRT) is an effective methodology indicated in selected heart failure patients. Identifying responders to the therapy is still challenging. Most studies report that at least 30% of the patients are nonresponders. Baseline characteristics of the Low-Dose Dobutamine Stress Echocardiography to Predict Cardiac Resynchronization Therapy Response (LODO-CRT) trial population are presented. The study investigates dobutamine stress echocardiography's role in predicting CRT response. Two hundred seventy-one CRT candidates were studied. Mean age was 67+/-10 years, 69% were male, 96% had New York Heart Association class III disease, and 39% had heart failure of ischemic etiology. Mean QRS and left ventricular ejection fraction were 146+/-24 ms and 26%+/-6%, respectively. Seventy-seven percent of participants showed contractile reserve. Left ventricular end-diastolic volume was shown to be independently associated with contractile reserve presence. In particular, more dilated ventricles are associated with a lower chance of having contractile reserve. The LODO-CRT trial enrolled a cohort of patients fulfilling criteria for CRT. Dobutamine stress echocardiography was highly feasible and safe in this population. Contractile reserve was associated with healthier ventricles.


Performance tuning non-uniform sampling for sensitivity enhancement of signal-limited biological NMR.
Non-uniform sampling (NUS) has been established as a route to obtaining true sensitivity enhancements when recording indirect dimensions of decaying signals in the same total experimental time as traditional uniform incrementation of the indirect evolution period. Theory and experiments have shown that NUS can yield up to two-fold improvements in the intrinsic signal-to-noise ratio (SNR) of each dimension, while even conservative protocols can yield 20-40% improvements in the intrinsic SNR of NMR data. Applications of biological NMR that can benefit from these improvements are emerging, and in this work we develop some practical aspects of applying NUS nD-NMR to studies that approach the traditional detection limit of nD-NMR spectroscopy. Conditions for obtaining high NUS sensitivity enhancements are considered here in the context of enabling (1)H,(15)N-HSQC experiments on natural abundance protein samples and (1)H,(13)C-HMBC experiments on a challenging natural product. Through systematic studies we arrive at more precise guidelines to contrast sensitivity enhancements with reduced line shape constraints, and report an alternative sampling density based on a quarter-wave sinusoidal distribution that returns the highest fidelity we have seen to date in line shapes obtained by maximum entropy processing of non-uniformly sampled data.


Prediction of calcium-binding sites by combining loop-modeling with machine learning.
Protein ligand-binding sites in the apo state exhibit structural flexibility. This flexibility often frustrates methods for structure-based recognition of these sites because it leads to the absence of electron density for these critical regions, particularly when they are in surface loops. Methods for recognizing functional sites in these missing loops would be useful for recovering additional functional information. We report a hybrid approach for recognizing calcium-binding sites in disordered regions. Our approach combines loop modeling with a machine learning method (FEATURE) for structure-based site recognition. For validation, we compared the performance of our method on known calcium-binding sites for which there are both holo and apo structures. When loops in the apo structures are rebuilt using modeling methods, FEATURE identifies 14 out of 20 crystallographically proven calcium-binding sites. It only recognizes 7 out of 20 calcium-binding sites in the initial apo crystal structures.We applied our method to unstructured loops in proteins from SCOP families known to bind calcium in order to discover potential cryptic calcium binding sites. We built 2745 missing loops and evaluated them for potential calcium binding. We made 102 predictions of calcium-binding sites. Ten predictions are consistent with independent experimental verifications. We found indirect experimental evidence for 14 other predictions. The remaining 78 predictions are novel predictions, some with intriguing potential biological significance. In particular, we see an enrichment of beta-sheet folds with predicted calcium binding sites in the connecting loops on the surface that may be important for calcium-mediated function switches. Protein crystal structures are a potentially rich source of functional information. When loops are missing in these structures, we may be losing important information about binding sites and active sites. We have shown that limited loop modeling (e.g. loops less than 17 residues) combined with pattern matching algorithms can recover functions and propose putative conformations associated with these functions.


Semi-analytic impedance modeling of three-dimensional photonic and metamaterial structures.
We define the concept of an impedance matrix for three-dimensional (3D) photonic and metamaterial structures relative to a reference medium and show that it satisfies a matrix generalization of the basic algebraic properties of the wave impedance between homogeneous media. This definition of the impedance matrix is motivated by the structure of the Fresnel reflection and transmission matrices at the interface between the media. In the derivation of the Fresnel scattering matrices, the field in each medium is expressed by a Bloch mode expansion, with field matching at the interface being undertaken in a least-squares manner by exploiting a biorthogonality relation between primal and adjoint Bloch modes. A semi-analytic technique, based on the impedance matrix, is developed for modeling the scattering of light by 3D periodic photonic and metamaterial structures. The advantages (in design and intuition) of the formalism are demonstrated through two applications.


Subjective and functional deterioration in recurrences of neovascular AMD are often preceded by morphologic changes in optic coherence tomography.
Different tests were applied to test the sensitivity of patient self-control; Amsler grid and visual acuity (VA) assessment, as well as fundus examinations to reveal recurrent choroidal neovascularisation (CNV) activity in age-related macular degeneration as detected by spectral domain optical coherence tomography (SD-OCT) in monthly controls. A prospective interventional case series of patients with exudative age-related macular degeneration was examined, which received ranibizumab injections until complete resolution of fluid in SD-OCT. Analysis of changes in subjective perception, Amsler grid, early treatment diabetic retinopathy study (ETDRS) VA, Radner reading VA and fundus examination was conducted in the case of OCT-confirmed CNV recurrences. Out of 40 morphological recurrences determined by SD-OCT, six (15%) were noticed by subjective patient perception. Amsler grid testing revealed deterioration in 12 cases (30%); 11 recurrences (28%) were accompanied by loss of ≥5 letters in ETDRS VA and/or ≥ 1 line in Radner VA; fundus examination showed signs of novel CNV activity in 10 out of 40 recurrences (25%). The combined sensitivity of all diagnostic methods compared to SD-OCT for recurrence detection was 67.5% (27 out of 40 recurrences). Subjective patient perception, Amsler grid, VA as well as fundus examination lead to pronounced underestimations of CNV recurrences. Morphologic recurrences can be detected prior to functional deterioration. As any delay of treatment can result in irreversible vision loss, attempts should be made to provide monthly OCT controls to detect recurrences as early as possible.


Metabolic engineering of tomato fruit enriched in L-DOPA.
L-DOPA, also known as Levodopa or L-3,4-dihydroxyphenylalanine, is a non-standard amino acid, and the gold standard drug for the treatment for Parkinson's Disease (PD). Recently, a gene encoding the enzyme that is responsible for its synthesis, as a precursor of the coloured pigment group betalains, was identified in beetroot, BvCYP76AD6. We have engineered tomato fruit enriched in L-DOPA through overexpression of BvCYP76AD6 in a fruit specific manner. Analysis of the transgenic fruit revealed the feasibility of accumulating L-DOPA in a non-naturally betalain-producing plant. Fruit accumulating L-DOPA also showed major effects on the fruit metabolome. Some of these changes included elevation of amino acids levels, changes in the levels of intermediates of the TCA and glycolysis pathways and reductions in the levels of phenolic compounds and nitrogen-containing specialised metabolites. Furthermore, we were able to increase the L-DOPA levels further by elevating the expression of the metabolic master regulator, MYB12, specifically in tomato fruit, together with BvCYP76AD6. Our study elucidated new roles for L-DOPA in plants, because it impacted fruit quality parameters including antioxidant capacity and firmness. The L-DOPA levels achieved in tomato fruit were comparable to the levels in other non-seed organs of L-DOPA - accumulating plants, offering an opportunity to develop new biological sources of L-DOPA by widening the repertoire of L-DOPA-accumulating plants. These tomato fruit could be used as an alternative source of this important pharmaceutical.


Rhodapentalenes: Pincer Complexes with Internal Aromaticity.
Pincer complexes are a remarkably versatile family benefited from their stability, diversity, and tunability. Many of them contain aromatic organic rings at the periphery, and aromaticity plays an important role in their stability and properties, whereas their metallacyclic cores are not aromatic. Herein, we report rhodapentalenes, which can be viewed as pincer complexes in which the metallacyclic cores exhibit considerable aromatic character. Rhodapentalenes show good thermal stability, although the rhodium-carbon bonds in such compounds are fragile. Experimental and computational studies suggest that the stabilization of rigid CCC pincer architectures together with an intrinsic aromaticity is vital for these metallacyclic rhodium species. Dearomatization-aromatization reactions, corresponding to metal-ligand cooperation of classical aromatic pincer complexes, were observed in this system. These findings suggest a new concept for pincer chemistry, the internal aromaticity involving metal d-orbitals, which would be useful for exploiting the nature of construction motif and inspire further applications.


A comparative analysis of marine mammal tracheas.
In 1940, Scholander suggested that stiffened upper airways remained open and received air from highly compressible alveoli during marine mammal diving. There are few data available on the structural and functional adaptations of the marine mammal respiratory system. The aim of this research was to investigate the anatomical (gross) and structural (compliance) characteristics of excised marine mammal tracheas. Here, we defined different types of tracheal structures, categorizing pinniped tracheas by varying degrees of continuity of cartilage (categories 1-4) and cetacean tracheas by varying compliance values (categories 5A and 5B). Some tracheas fell into more than one category along their length; for example, the harbor seal (Phoca vitulina) demonstrated complete rings cranially, and as the trachea progressed caudally, tracheal rings changed morphology. Dolphins and porpoises had less stiff, more compliant spiraling rings while beaked whales had very stiff, less compliant spiraling rings. The pressure-volume (P-V) relationships of isolated tracheas from different species were measured to assess structural differences between species. These findings lend evidence for pressure-induced collapse and re-inflation of lungs, perhaps influencing variability in dive depth or ventilation rates of the species investigated.


Future of endemic flora of biodiversity hotspots in India.
India is one of the 12 mega biodiversity countries of the world, which represents 11% of world's flora in about 2.4% of global land mass. Approximately 28% of the total Indian flora and 33% of angiosperms occurring in India are endemic. Higher human population density in biodiversity hotspots in India puts undue pressure on these sensitive eco-regions. In the present study, we predict the future distribution of 637 endemic plant species from three biodiversity hotspots in India; Himalaya, Western Ghats, Indo-Burma, based on A1B scenario for year 2050 and 2080. We develop individual variable based models as well as mixed models in MaxEnt by combining ten least co-related bioclimatic variables, two disturbance variables and one physiography variable as predictor variables. The projected changes suggest that the endemic flora will be adversely impacted, even under such a moderate climate scenario. The future distribution is predicted to shift in northern and north-eastern direction in Himalaya and Indo-Burma, while in southern and south-western direction in Western Ghats, due to cooler climatic conditions in these regions. In the future distribution of endemic plants, we observe a significant shift and reduction in the distribution range compared to the present distribution. The model predicts a 23.99% range reduction and a 7.70% range expansion in future distribution by 2050, while a 41.34% range reduction and a 24.10% range expansion by 2080. Integration of disturbance and physiography variables along with bioclimatic variables in the models improved the prediction accuracy. Mixed models provide most accurate results for most of the combinations of climatic and non-climatic variables as compared to individual variable based models. We conclude that a) regions with cooler climates and higher moisture availability could serve as refugia for endemic plants in future climatic conditions; b) mixed models provide more accurate results, compared to single variable based models.


Production of ribosome components in effector CD4+ T cells is accelerated by TCR stimulation and coordinated by ERK-MAPK.
Effector CD4+ T cells rapidly activate high-level cytokine expression following TCR stimulation. Consistent with accelerated protein production in these cells, global mRNA profiles revealed that, after cytokines, the most impressive cluster of activated genes encode rRNA-maturation factors. Activation of these genes was ERK-MAPK dependent, accompanied by increased rRNA transcription and faster maturation kinetics, and much greater in effector CD4+ T cells than in naive cells. Ribosomal protein subunit (RPS) synthesis was also ERK-MAPK dependent and increased to match rRNA production, but without evident increase in RPS mRNA. Instead, stimulation promoted polysome loading of RPS mRNA via cis-acting, 5'-terminal oligopyrimidines. These results demonstrate how, in response to extracellular signals, effector CD4+ T cells coordinately increase multiple ribosomal components to accommodate burgeoning cytokine production.


Essential oil from Cymbopogon flexuosus as the potential inhibitor for HSP90.
The essential oil of Cymbopogon flexuosus or lemongrass oil (LO) is reported to have antibacterial, antifungal and anticancerous effects. HSP90 is one of the major chaperones responsible for the proper folding of cancer proteins. In this paper we show that the essential oil of C. flexuosus significantly suppresses the HSP90 gene expression. The cytotoxicity of the compounds was tested by MTT assay and the gene expression studies were carried out using HEK-293 and MCF-7 cells. Also we tested the efficacy of the major component of this essential oil viz. citral and geraniol in inhibiting the HSP90 expression. The oil was found to be more cytotoxic to MCF-7 cells with different IC50 values for the oil (69.33 μg/mL), citral (140.7 μg/mL) and geraniol (117 μg/mL). The fold change of expression was calculated by RT-qPCR using ΔΔCt (2^-ΔΔCt) method and it was 0.1 and 0.03 in MCF-7 cells at 80 μg/mL and 160 μg/mL of LO. Western blot results showed suppression of HSP90 protein expression and HSP90 - ATPase activity inhibition was also observed using LO. This study shows the anticancer mechanism exhibited by the essential oil of C. flexuosus is by the inhibition of the important chaperone protein HSP90.


Molecular hepatoprotective effects of lipoic acid against carbon tetrachloride-induced liver fibrosis in rats: Hepatoprotection at molecular level.
Liver fibrosis is a noteworthy well-being issue that can prompt the progression of liver cirrhosis and hepatocellular carcinoma. Prominently, many antioxidants have been shown to have defensive impacts against liver fibrosis. Subsequently, in the present study, the viability of alpha-lipoic acid (α-LA) in ensuring against carbon tetrachloride (CCl4)-actuated liver fibrosis and the mechanism(s) involved in this defensive impact were considered in rats. The present results uncovered that in the CCl4-treated group, the expression of antioxidant enzymes and matrix metalloproteinase-13 (MMP-13) messenger RNA (mRNA) was downregulated ( p < 0.05), and the levels of lipid peroxide and nitric oxide were increased ( p < 0.05) in the treated rat livers along with increased collagen deposition compared to that of the control group. Also, the gene expression levels of the proinflammatory factors interleukin-6 and tumor necrosis factor-alpha, nuclear factor-kappa B (NF-κB) p65, transforming growth factor-alpha, and inducible nitric oxide synthase (iNOS) were upregulated significantly ( p < 0.05) in the CCl4 group. These negative impacts were all restrained by α-LA. These outcomes show that α-LA might be compelling at forestalling collagen deposition and hepatic oxidative stress as well as downregulating the expression of hepatic proinflammatory cytokines, iNOS, and NF-κB and upregulating MMP-13 expression.


Selective gene expression induced by ecdysterone in cultured fat bodies of Drosophila.
Expression of the LSP-2 and P1 genes was induced in cultured fat bodies of Drosophila third-instar larvae by supplementing the culture medium with ecdysterone. The fat bodies were isolated from ecdysterone-deficient larvae of the temperature-sensitive mutant ecd1, which were shifted from the permissive to the restrictive temperature either at the beginning of the third instar for the detection of LSP-2 induction or several hours later for the detection of P1 induction. During normal larval development, the LSP-2 gene is expressed before the P1 gene, and this order is also observed in the cultured fat bodies. Induction was demonstrated by increased amounts of LSP-2 and P1 transcripts in the ecdysterone-supplemented fat bodies. The amount of P1 transcript was determined by two methods: one involved measuring the hybridization of a labeled P1 DNA probe to total fat body RNA; the other involved labeling the newly synthesized RNA in nuclei isolated from cultured fat bodies and measuring the hybridization of the labeled RNA to P1 DNA. Only the second method was used for the LSP-2 transcript because the earlier expression of the LSP-2 gene results in a measurable accumulation of the transcript in the fat bodies before ecdysterone supplementation. The maximal level of P1 induction was reached within 2 hr after supplementation, and the induction was not affected by a concentration of cycloheximide that strongly inhibited total protein synthesis, suggesting that ecdysterone acts directly on the P1 gene rather than indirectly by inducing formation of proteins required for the subsequent induction of P1. Ecdysterone appears to function normally in the cultured fat body system because the LSP-2 and P1 genes are induced in culture in the same order as in vivo and a third gene, G12, that is not induced in fat bodies in vivo also is not induced in culture.


Chronic Phenotype Characterization of a Large-Animal Model of Hereditary Tyrosinemia Type 1.
Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disease caused by deficiency in fumarylacetoacetate hydrolase, the last enzyme in the tyrosine catabolic pathway. In this study, we investigated whether fumarylacetoacetate hydrolase deficient (FAH-/-) pigs, a novel large-animal model of HT1, develop fibrosis and cirrhosis characteristic of the human disease. FAH-/- pigs were treated with the protective drug 2-(2-nitro-4-trifluoromethylbenzoyl)-1, 3 cyclohexandione (NTBC) at a dose of 1 mg/kg per day initially after birth. After 30 days, they were assigned to one of three groups based on dosing of NTBC. Group 1 received ≥0.2 mg/kg per day, group 2 cycled on/off NTBC (0.05 mg/kg per day × 1 week/0 mg/kg per day × 3 weeks), and group 3 received no NTBC thereafter. Pigs were monitored for features of liver disease. Animals in group 1 continued to have weight gain and biochemical analyses comparable to wild-type pigs. Animals in group 2 had significant cessation of weight gain, abnormal biochemical test results, and various grades of fibrosis and cirrhosis. No evidence of hepatocellular carcinoma was detected. Group 3 animals declined rapidly, with acute liver failure. In conclusion, the FAH-/- pig is a large-animal model of HT1 with clinical characteristics that resemble the human phenotype. Under conditions of low-dose NTBC, FAH-/- pigs developed liver fibrosis and portal hypertension, and thus may serve as a large-animal model of chronic liver disease.


Effect of Notch and PARP Pathways' Inhibition in Leukemic Cells.
Differentiation of blood cells is one of the most complex processes in the body. It is regulated by the action of transcription factors in time and space which creates a specific signaling network. In the hematopoietic signaling system, Notch is one of the main regulators of lymphocyte development. The aim of this study was to get insight into the regulation of Notch signalization and the influence of poly(ADP-ribose)polymerase (PARP) activity on this process in three leukemia cell lines obtained from B and T cells. PARP1 is an enzyme involved in posttranslational protein modification and chromatin structure changes. B and T leukemia cells were treated with Notch and PARP inhibitors, alone or in combination, for a prolonged period. The cells did not show cell proliferation arrest or apoptosis. Analysis of gene and protein expression set involved in Notch and PARP pathways revealed increase in JAGGED1 expression after PARP1 inhibition in B cell lines and changes in Ikaros family members in both B and T cell lines after γ-secretase inhibition. These data indicate that Notch and PARP inhibition, although not inducing differentiation in leukemia cells, induce changes in signaling circuits and chromatin modelling factors.


Intracranial 3D and 4D MR Angiography Using Arterial Spin Labeling: Technical Considerations.
In the 1980's some of the earliest studies of arterial spin labeling (ASL) MRI have demonstrated its ability to generate MR angiography (MRA) images. Thanks to many technical improvements, ASL has been successfully moving its position from the realm of research into the clinical area, albeit more known as perfusion imaging than as MRA. For MRA imaging, other techniques such as time-of-flight, phase contrast MRA and contrast-enhanced (CE) MRA are more popular choices for clinical applications. In the last decade, however, ASL-MRA has been experiencing a remarkable revival, especially because of its non-invasive nature, i.e. the fact that it does not rely on the use of contrast agent. Very importantly, there are additional benefits of using ASL for MRA. For example, its higher flexibility to achieve both high spatial and temporal resolution than CE dynamic MRA, and the capability of vessel specific visualization, in which the vascular tree arising from a selected artery can be exclusively visualized. In this article, the implementation and recent developments of ASL-based MRA are discussed; not only focusing on the basic sequences based upon pulsed ASL or pseudo-continuous ASL, but also including more recent labeling approaches, such as vessel-selective labeling, velocity-selective ASL, vessel-encoded ASL and time-encoded ASL. Although these ASL techniques have been already utilized in perfusion imaging and their usefulness has been suggested by many studies, some additional considerations should be made when employing them for MRA, since there is something more than the difference of the spatial resolution of the readout sequence. Moreover, extensive discussion is included on what readout sequence to use, especially by highlighting how to achieve high spatial resolution while keeping scan-time reasonable such that the ASL-MRA sequence can easily be included into a clinical examination.


Efficacy of individual and group cognitive processing therapy for military personnel with and without child abuse histories.
Objective: Many clinicians question whether patients with a history of childhood trauma will benefit from trauma-focused treatment. In this secondary analysis, we examined whether reports of childhood abuse moderated the efficacy of cognitive processing therapy (CPT) for active-duty military with posttraumatic stress disorder (PTSD). Methods: Service members (N = 254, mean age 33.11 years, 91% male, 41% Caucasian) were randomized to receive individual or group CPT (n = 106 endorsing and n = 148 not endorsing history of childhood abuse). Outcomes included baseline cognitive-emotional characteristics [Posttraumatic Cognitions Inventory (PTCI), Trauma-Related Guilt Inventory (TRGI), Cognitive Emotion Regulation Questionnaire-Short Form (CERQ)], treatment completion, and symptom outcome (PTSD Checklist, Beck Depression Inventory-II). We predicted participants endorsing childhood abuse would have higher scores on the PTCI, TRGI, and CERQ at baseline, but be noninferior on treatment completion and change in PTSD and depression symptoms. We also predicted those endorsing childhood abuse would do better in individual CPT than those not endorsing abuse. Results: Those endorsing childhood abuse primarily experienced physical abuse. There were no baseline differences between service members with and without a history of childhood abuse (all p ≥ .07). Collapsed across treatment arms, treatment completion and symptom reduction were within the noninferiority margins for those endorsing versus not endorsing childhood abuse. History of abuse did not moderate response to individual versus group CPT. Conclusions: In this primarily male, primarily physically abused sample, active-duty military personnel with PTSD who endorsed childhood abuse benefitted as much as those who did not endorse abuse. (PsycInfo Database Record (c) 2021 APA, all rights reserved).


Sodium nitroprusside, a nitric oxide donor, fails to bypass the block of neuronal differentiation in PC12 cells imposed by a dominant negative Ras protein.
Nitric oxide (NO) is a mediator of a diverse array of inter- and intracellular signal transduction processes. The aim of the present study was to analyze its possible role as a second messenger in the process of neuronal differentiation of PC12 pheochromocytoma cells. Upon NGF treatment wildtype PC12 cells stop dividing and develop neurites. In contrast, a PC12 subclone (designated M-M17-26) expressing a dominant-negative mutant Ras protein keeps proliferating and fails to grow neurites after NGF treatment. Sodium nitroprusside (SNP), an NO donor, was found to induce the p53 protein and to inhibit proliferation of both PC12 and M-M17-26 cells, but failed to induce neuronal differentiation in these cell lines. Key signaling pathways (the ERK and Akt pathways) were also not affected by SNP treatment, and the phosphorylation of CREB transcription factor was only slightly stimulated. It is thus concluded from the results presented in this paper that NO is unable to activate signaling proteins acting downstream or independent of Ras that are required for neuronal differentiation.


Novel transcriptional targets of the SRY-HMG box transcription factor SOX4 link its expression to the development of small cell lung cancer.
The HMG box transcription factor SOX4 involved in neuronal development is amplified and overexpressed in a subset of lung cancers, suggesting that it may be a driver oncogene. In this study, we sought to develop this hypothesis including by defining targets of SOX4 that may mediate its involvement in lung cancer. Ablating SOX4 expression in SOX4-amplified lung cancer cells revealed a gene expression signature that included genes involved in neuronal development such as PCDHB, MYB, RBP1, and TEAD2. Direct recruitment of SOX4 to gene promoters was associated with their upregulation upon ectopic overexpression of SOX4. We confirmed upregulation of the SOX4 expression signature in a panel of primary lung tumors, validating their specific response by a comparison using embryonic fibroblasts from Sox4-deficient mice. Interestingly, we found that small cell lung cancer (SCLC), a subtype of lung cancer with neuroendocrine characteristics, was generally characterized by high levels of SOX2, SOX4, and SOX11 along with the SOX4-specific gene expression signature identified. Taken together, our findings identify a functional role for SOX genes in SCLC, particularly for SOX4 and several novel targets defined in this study.


Identification of Robust Pathway Markers for Cancer through Rank-Based Pathway Activity Inference.
One important problem in translational genomics is the identification of reliable and reproducible markers that can be used to discriminate between different classes of a complex disease, such as cancer. The typical small sample setting makes the prediction of such markers very challenging, and various approaches have been proposed to address this problem. For example, it has been shown that pathway markers, which aggregate the gene activities in the same pathway, tend to be more robust than gene markers. Furthermore, the use of gene expression ranking has been demonstrated to be robust to batch effects and that it can lead to more interpretable results. In this paper, we propose an enhanced pathway activity inference method that uses gene ranking to predict the pathway activity in a probabilistic manner. The main focus of this work is on identifying robust pathway markers that can ultimately lead to robust classifiers with reproducible performance across datasets. Simulation results based on multiple breast cancer datasets show that the proposed inference method identifies better pathway markers that can predict breast cancer metastasis with higher accuracy. Moreover, the identified pathway markers can lead to better classifiers with more consistent classification performance across independent datasets.


Inhibitors of protein aggregation and toxicity.
The aggregation of numerous peptides or proteins has been linked to the onset of disease, including Abeta (amyloid beta-peptide) in AD (Alzheimer's disease), asyn (alpha-synuclein) in Parkinson's disease and amylin in Type 2 diabetes. Diverse amyloidogenic proteins can often be cut down to an SRE (self-recognition element) of as few as five residues that retains the ability to aggregate. SREs can be used as a starting point for aggregation inhibitors. In particular, N-methylated SREs can bind to a target on one side, but have hydrogen-bonding blocked on their methylated face, interfering with further assembly. We applied this strategy to develop Abeta toxicity inhibitors. Our compounds, and a range of compounds from the literature, were compared under the same conditions, using biophysical and toxicity assays. Two N-methylated D-peptide inhibitors with unnatural side chains were the most effective and can reverse Abeta-induced inhibition of LTP (long-term potentiation) at concentrations as low as 10 nM. An SRE in asyn (VAQKTV) was identified using solid-state NMR. When VAQKTV was N-methylated, it was able to disrupt asyn aggregation. N-methylated derivatives of the SRE of amylin are also able to inhibit amylin aggregation.


[The foundation of the Dutch Society for Tropical Medicine: an affair of national importance].
The 'Nederlandsche Vereeniging voor Tropische Geneeskunde' (Dutch Society for Tropical Medicine--in short: NVTG) was founded in 1907, a time not only of colonial expansion, but also a time in which the economic interest of the Dutch East-Indies for The Netherlands grew exceptionally. This had its effect on the motivations behind medical aid. Hygienic measures were financially backed because healthy workers had a positive effect on profits. This atmosphere of economic interest and political-military expansion had its effect on the foundation of the NVTG as well. Colonialism was generally approved of, and the goal of the society apart from sociability motives--was to support research into illnesses torturing mainly the Dutch colonies. This was in line with the reasoning that the colonies were rightfully governed by the Netherlands, because the peoples living there had proven not to be able to look after themselves in a proper manner. The foundation of the Society should have been a part of the foundation of an international society for tropical medicine, but this failed. Although internationalism generally was seen as an important feature of science and certainly of medical science, it is not strange this was not seen as a major problem. For it was national and not international reasons that had led to founding the Dutch society. It was the Dutch position as a colonial power that had convinced Dutch tropical doctors a society should be called into being. A generally shared spirit of internationalism had little to do with this unless internationalism is defined as: what others have, we need to have as well, and let us see in international conferences what could be of national benefit. That nevertheless this spirit of internationalism--and not nationalism--is commonly hailed as the main reason behind the foundation, is probably due to the fact that around 1900 nationalism was such a normal state of mind and so fully incorporated in man's way of thinking and acting, that, as for instance was the case with racism, it was hardy noticed and recognised as such.


[Promoter methylation and microsatellite mutation reveals the clonal relationship of multiple urothelial carcinomas with mutator phenotype].
The clonality of multiple urothelial carcinomas (UC) is subject to debate and affects treatment. Evidence derived from X-chromosome mosaicism and patterns of molecular alterations supports both a mono- and polyclonal relationship. In contrast to most UC, tumours with the mutator phenotype have frequent mutations in repetitive sequences (MSI) and promoter methylation. The aim of this study was to investigate the clonality of multifocal UC with MSI. We have screened 400 UC for MSI and found it to occur in 1% of bladder and 15% of upper tract UC. Of these, 9 patients, whose tumours had MSI, developed or presented with multiple UC. A total of 32 UC (occurring over 0-6 years, 2-12 TCC per patient), 2 cases of CIS and 9 normal urothelial samples were screened for MSI at 17 loci and aberrant promoter methylation at 7 genes. In 8 of 9 patients, the pattern of microsatellite mutation and promoter methylation suggested that the multiple tumours had a clonal origin. Patterns of aberrant methylation in multiple tumours were more similar than microsatellite mutations, suggesting an earlier carcinogenic timing. MSI and promoter methylation were present in macroscopically normal urothelium from these patients. Aberrant promoter methylation occurs before microsatellite alteration in UC with mutator phenotype. The majority of recurrent UC with MSI are monoclonal in origin and macroscopically normal urothelium harbours multiple molecular abnormalities. Thus, at the time of apparently successful treatment, there is molecular evidence of residual tumour that subsequently develops into recurrent disease.


Human immune responses to the highly repetitive Plasmodium falciparum antigen Pf332.
The B and T cell responses to EB200, a repetitive part of the Plasmodium falciparum antigen Pf332, were examined in malaria-exposed Senegalese adults. Most donors had high levels of antibodies to recombinant EB200 and 17 overlapping peptides spanning EB200. Taking proliferation and/or cytokine (interferon-gamma and interleukin-4) production as a measure of T cell activation, eight of the EB200-derived peptides induced responses in > 40% of the donors tested. There was no general association between the different types of T cell responses measured, emphasizing the importance of including multiple parameters when analyzing T cell responses and suggesting that EB200 induces functionally distinct T cell responses. The most efficient peptide for induction of proliferative responses was one previously shown to induce T cell responses in five different H-2 congenic mouse strains primed with EB200, suggesting that this is a universal T cell epitope. The presence of multiple B and T cell epitopes in EB200, widely recognized by humans, is important since EB200 has been shown to elicit protective antibody responses in monkeys and may be considered for inclusion in malaria subunit vaccines.


Genetic factors in moderately severe and mild affective disorders.
The aim of this study was to investigate the contribution of hereditary factors in the development of affective and depressive adjustment disorders. I interviewed 151 index twins with moderately severe and mild affective illness, as well as their co-twins. The analysis of concordance rates indicates that hereditary factors may be important in the development of bipolar disorder and in major depression, except in non-psychotic, hysterical individuals. Furthermore, hereditary factors may not play any role in dysthymic disorder and depressive adjustment disorder. These findings are tentative and should be viewed against the methodologic limitations of this study, which include small sample size of the subgroups and the use of the Present State Examination as the basis for the DSM-III diagnoses.


Histology-guided protein digestion/extraction from formalin-fixed and paraffin-embedded pressure ulcer biopsies.
Herein we present a simple, reproducible and versatile approach for in situ protein digestion and identification on formalin-fixed and paraffin-embedded (FFPE) tissues. This adaptation is based on the use of an enzyme delivery platform (hydrogel discs) that can be positioned on the surface of a tissue section. By simultaneous deposition of multiple hydrogels over select regions of interest within the same tissue section, multiple peptide extracts can be obtained from discrete histological areas. After enzymatic digestion, the hydrogel extracts are submitted for LC-MS/MS analysis followed by database inquiry for protein identification. Further, imaging mass spectrometry (IMS) is used to reveal the spatial distribution of the identified peptides within a serial tissue section. Optimization was achieved using cutaneous tissue from surgically excised pressure ulcers that were subdivided into two prime regions of interest: the wound bed and the adjacent dermal area. The robust display of tryptic peptides within these spectral analyses of histologically defined tissue regions suggests that LC-MS/MS in combination with IMS can serve as useful exploratory tools.


Changes in subjective symptoms of craniomandibular disorders in children and adolescents during a 10-year period.
An epidemiologic sample of 293 subjects in three age groups, now 17, 21, and 25 years of age, were followed longitudinally with respect to symptoms of craniomandibular dysfunction during a 10-year period. Reports of one or more such symptoms increased in all three age groups during the 10 years. At the follow-up, 1 in 3 individuals in all three groups had noticed such symptoms occasionally and 10% had them frequently. Reports of oral parafunctions such as bruxing and clenching also increased, while other parafunctions such as nail, lip, cheek, and tongue biting increased from the age of 7 to 11 but then decreased with age. Despite the high incidence of subjective symptoms of craniomandibular disorders, only a few subjects had had any kind of functional treatment performed during the 10-year period, and only 7 had an actual demand for treatment at present.


Effects of ethacrynic acid and furosemide on respiration of isolated kidney tubules: the role of ion transport and the source of metabolic energy.
In order to investigate the mechanism of action of ethacrynic acid and furosemide, experiments were designed to determine whether these drugs directly inhibit active transport or energy metabolism. The effects of these diuretics on the respiration of tubule suspensions isolated from renal cortex (of rats and rabbits) and outer medulla (of rabbits) were measured. The respiration of tubules prepared from renal outer medulla was stimulated by the presence of chloride in the incubation medium, whereas the respiration of cortical tubules was unaffected by chloride. Both ethacrynic acid and furosemide produced the greatest inhibition of respiration on tubules from outer medulla suspended in chloride-containing media; this result suggests that the diuretics directly inhibit chloride transport. The source of metabolic energy for ion transport was varied by using substrates which donate electrons to the respiratory chain at different phosphorylation sites. Both ethacrynic acid and furosemide inhibit respiration supported by beta-hydroxybutyrate, but there was little or no inhibition of respiration with succinate or tetramethylphenylenediamine ascorbate. Similarly, ouabain inhibited respiration with beta-hydroxybutyrate, but not with the other substrates. Therefore, both diuretics inhibited respiration in a fashion similar to ouabain. It is concluded from both types of experiments that ethacrynic acid and furosemide may directly inhibit active chloride transport.


Novel conserved group A streptococcal proteins identified by the antigenome technology as vaccine candidates for a non-M protein-based vaccine.
Group A streptococci (GAS) can cause a wide variety of human infections ranging from asymptomatic colonization to life-threatening invasive diseases. Although antibiotic treatment is very effective, when left untreated, Streptococcus pyogenes infections can lead to poststreptococcal sequelae and severe disease causing significant morbidity and mortality worldwide. To aid the development of a non-M protein-based prophylactic vaccine for the prevention of group A streptococcal infections, we identified novel immunogenic proteins using genomic surface display libraries and human serum antibodies from donors exposed to or infected by S. pyogenes. Vaccine candidate antigens were further selected based on animal protection in murine lethal-sepsis models with intranasal or intravenous challenge with two different M serotype strains. The nine protective antigens identified are highly conserved; eight of them show more than 97% sequence identity in 13 published genomes as well as in approximately 50 clinical isolates tested. Since the functions of the selected vaccine candidates are largely unknown, we generated deletion mutants for three of the protective antigens and observed that deletion of the gene encoding Spy1536 drastically reduced binding of GAS cells to host extracellular matrix proteins, due to reduced surface expression of GAS proteins such as Spy0269 and M protein. The protective, highly conserved antigens identified in this study are promising candidates for the development of an M-type-independent, protein-based vaccine to prevent infection by S. pyogenes.


Evolution of antigenic diversity in the tick-transmitted bacterium Borrelia afzelii: a role for host specialization?
Antigenic diversity in pathogenic microbes can be a result of at least three different processes: diversifying selection by acquired immunity, host-pathogen coevolution and/or host specialization. Here, we investigate whether host specialization drives diversity at ospC (which encodes an immunodominant surface protein) in the tick-transmitted bacterium Borrelia afzelii. We determined prevalence and infection intensity of ospC strains in naturally infected wild mammals (rodents and shrews) by 454 amplicon sequencing in combination with qPCR. Neither prevalence nor infection intensity of specific ospC strains varied in a species-specific manner (i.e. there were no significant ospC × host species interactions). Rankings of ospC prevalences were strongly positively correlated across host species. Rankings of ospC infection intensities were correlated more weakly, but only in one case significantly < 1. ospC prevalences in the studied mammals were similar to those in ticks sampled at the study site, indicating that we did not miss any mammal species that are important hosts for specific ospC strains. Based on this, we conclude that there is at best limited host specialization in B. afzelii and that other processes are likely the main drivers of ospC diversity.


Polymorphisms in the renin-angiotensin system and endothelium-dependent vasodilation in normotensive subjects.
Our aim was to test the hypothesis that genes encoding components in the renin-angiotensin system influence endothelial vasodilatory function. In 59 apparently healthy, normotensive individuals, endothelium-dependent vasodilation (EDV) and endothelial-independent vasodilation (EIDV) was evaluated by infusing metacholine and sodium nitroprusside into the brachial artery. Forearm blood flow was measured by venous occlusion plethysmography. The ACE insertion (I)/deletion (D) polymorphism, the T174M and M235T angiotensinogen restriction fragments length polymorphisms, the angiotensin II receptor type 1 (AT1R) A1166C, and the aldosterone synthase gene (CYP11B2) C-344T polymorphisms were analysed. When analysing the ACE, the two angiotensinogen and the aldosterone synthase CYP11B2 genotypes independently, no significant association with endothelial vasodilatory function was found. However, a significant reduction in endothelium-dependent vasodilation was observed in the subjects (n=9) with the ACE D allele and the angiotensinogen T174M genotype (P<0.05). Subjects with the AT1R genotype AC showed a reduction in both EDV (P=0.05) and EIDV (P=0.04) when compared with those with the AA genotype. The subjects with the ACE D allele in combination with the angiotensinogen T174M genotype are associated with a reduced EDV. This together with the observation that the AC AT1R genotype is associated with a reduction in both EDV and EIDV, supports the hypothesis that endothelial vasodilatory function is influenced by genes in the renin-angiotensinogen system.


An evidence-based approach to prescribing NSAIDs in musculoskeletal disease: a Canadian consensus. Canadian NSAID Consensus Participants.
To make recommendations for the long-term use of nonsteroidal anti-inflammatory drugs (NSAIDs) in primary care practice, particularly for patients at high risk for NSAID-induced complications. The use of misoprostol to prevent gastrointestinal ulceration and other unwanted NSAIDs effects was considered. The role of cyclooxygenase-2 (COX-2) versus COX-1 inhibiting agents was also examined. Reduction of complications associated with long-term use of NSAIDs. Evidence was gathered in late 1995 from published research studies and reviews. Position papers were prepared by faculty and advisory board members and discussed at the Canadian NSAID Consensus Symposium in Cambridge, Ont., Jan. 26 and 27, 1996. Recommendations were based on randomized, placebo-controlled clinical trials (level I evidence) and case-control studies (level II evidence) involving NSAID use when such evidence was available. When the scientific literature was incomplete or inconsistent in a particular area, recommendations reflect the consensus of the participants at the symposium (level III evidence). Physicians were recruited from across Canada for their expertise in rheumatology, gastroenterology, epidemiology, gerontology, family practice, and clinical and basic scientific research. Although a reduction in complications due to inappropriate NSAID use should reduce costs of additional investigations, admissions to hospital and time lost from work, definitive cost analysis studies are not yet available. Currently, no NSAID is available that lacks potential for serious toxicity; therefore, long-term use of NSAIDs should be avoided whenever possible, particularly in high-risk patients (e.g., those who are elderly, suffer from hypertension, congestive heart failure, renal or hepatic impairment or volume depletion, take certain concomitant medications or have a history of peptic ulcer disease) (level I evidence). If NSAIDs are to be used in patients with gastric or nephrotoxic risk factors, the lowest effective dose of NSAID should be used (level III evidence); NSAIDs that are weak COX-1 inhibitors may be preferred (level II evidence). In addition, concomitant administration of misoprostol is recommended in patients at increased risk for upper gastrointestinal complications (level I evidence). However, the clinical judgement of the practising clinician must always be part of any therapeutic decision. These recommendations are based on the consensus of Canadian experts in rheumatology, gastroenterology and epidemiology, and have been subjected to external peer review.


Maternal Transcripts of Hox Genes Are Found in Oocytes of Platynereis dumerilii (Annelida, Nereididae).
Hox genes are some of the best studied developmental control genes. In the overwhelming majority of bilateral animals, these genes are sequentially activated along the main body axis during the establishment of the ground plane, i.e., at the moment of gastrulation. Their activation is necessary for the correct differentiation of cell lines, but at the same time it reduces the level of stemness. That is why the chromatin of Hox loci in the pre-gastrulating embryo is in a bivalent state. It carries both repressive and permissive epigenetic markers at H3 histone residues, leading to transcriptional repression. There is a paradox that maternal RNAs, and in some cases the proteins of the Hox genes, are present in oocytes and preimplantation embryos in mammals. Their functions should be different from the zygotic ones and have not been studied to date. Our object is the errant annelid Platynereis dumerilii. This model is convenient for studying new functions and mechanisms of regulation of Hox genes, because it is incomparably simpler than laboratory vertebrates. Using a standard RT-PCR on cDNA template which was obtained by reverse transcription using random primers, we found that maternal transcripts of almost all Hox genes are present in unfertilized oocytes of worm. We assessed the localization of these transcripts using WMISH.


A fat-enriched, glucose-enriched diet markedly attenuates adiponectin mRNA levels in rat epididymal adipose tissue.
Adiponectin levels are decreased in subjects with obesity, diabetes and coronary artery disease. In the present study, we have investigated whether the decrease in the levels and mRNA expression of adiponectin is due to obesity or to the diet itself. Wistar rats were either fed standard laboratory chow throughout (controls) or given a fat-enriched, glucose-enriched diet (diet-fed) for 2 days or 16 weeks. After 2 days of diet feeding, total body weight, fat pad masses and the plasma levels of glucose, insulin and leptin were all comparable between the two groups, while plasma NEFA (non-esterified fatty acid) and triacylglycerol levels were increased in the diet-fed animals (P<0.01 for both). There was a marked (P<0.01) decrease in plasma adiponectin levels. After 16 weeks of diet feeding, diet-fed rats had significantly higher body weight, fat pad mass and plasma levels of leptin, adiponectin, NEFA and triacylglycerol (P<0.001 for all) compared with chow-fed controls, whereas plasma levels of glucose and insulin were similar in the two groups. After 2 days of diet feeding, there were no significant changes in Ob mRNA levels in epididymal fat, whereas there was a marked decrease in adiponectin mRNA levels. After 16 weeks of diet feeding, rats had significantly increased levels of Ob mRNA, but decreased adiponectin mRNA levels, in epididymal fat compared with the chow-fed group (P<0.001 for both). These findings suggest that obesity per se is not a factor in the decreased adiponectin levels observed in obese subjects. We propose that the lipid profile of the plasma and/or the constituents of the diet consumed by rats may contribute to adiponectin levels more than obesity per se.


Genetically selected cardiomyocytes from differentiating embronic stem cells form stable intracardiac grafts.
This study describes a simple approach to generate relatively pure cultures of cardiomyocytes from differentiating murine embryonic stem (ES) cells. A fusion gene consisting of the alpha-cardiac myosin heavy chain promoter and a cDNA encoding aminoglycoside phosphotransferase was stably transfected into pluripotent ES cells. The resulting cell lines were differentiated in vitro and subjected to G418 selection. Immunocytological and ultrastructural analyses demonstrated that the selected cardiomyocyte cultures (> 99% pure) were highly differentiated. G418 selected cardiomyocytes were tested for their ability to form grafts in the hearts of adult dystrophic mice. The fate of the engrafted cells was monitored by antidystrophin immunohistology, as well as by PCR analysis with primers specific for the myosin heavy chain-aminoglycoside phosphotransferase transgene. Both analyses revealed the presence of ES-derived cardiomyocyte grafts for as long as 7 wk after implantation, the latest time point analyzed. These studies indicate that a simple genetic manipulation can be used to select essentially pure cultures of cardiomyocytes from differentiating ES cells. Moreover, the resulting cardiomyocytes are suitable for the formation of intracardiac grafts. This selection approach should be applicable to all ES-derived cell lineages.