Role of miRNAs and alternative mRNA 3'-end cleavage and polyadenylation of their mRNA targets in cardiomyocyte hypertrophy.

miRNAs play critical roles in heart disease. In addition to differential miRNA expression, miRNA-mediated control is also affected by variable miRNA processing or alternative 3'-end cleavage and polyadenylation (APA) of their mRNA targets. To what extent these phenomena play a role in the heart remains unclear. We sought to explore miRNA processing and mRNA APA in cardiomyocytes, and whether these change during cardiac hypertrophy. Thoracic aortic constriction (TAC) was performed to induce hypertrophy in C57BL/6J mice. RNA extracted from cardiomyocytes of sham-treated, pre-hypertrophic (2 days post-TAC), and hypertrophic (7 days post-TAC) mice was subjected to small RNA- and poly(A)-test sequencing (PAT-Seq). Differential expression analysis matched expectations; nevertheless we identified ~400 mRNAs and hundreds of noncoding RNA loci as altered with hypertrophy for the first time. Although multiple processing variants were observed for many miRNAs, there was little change in their relative proportions during hypertrophy. PAT-Seq mapped ~48,000 mRNA 3'-ends, identifying novel 3' untranslated regions (3'UTRs) for over 7000 genes. Importantly, hypertrophy was associated with marked changes in APA with a net shift from distal to more proximal mRNA 3'-ends, which is predicted to decrease overall miRNA repression strength. We independently validated several examples of 3'UTR proportion change and showed that alternative 3'UTRs associate with differences in mRNA translation. Our work suggests that APA contributes to altered gene expression with the development of cardiomyocyte hypertrophy and provides a rich resource for a systems-level understanding of miRNA-mediated regulation in physiological and pathological states of the heart.

Temporal parameters of post-stress prophylactic glucose treatment in rats.

Acute trauma can lead to life-long changes in susceptibility to psychiatric disease, such as post-traumatic stress disorder (PTSD). Rats given free access to a concentrated glucose solution for 24 h beginning immediately after trauma failed to show stress-related pathology in the learned helplessness model of PTSD and comorbid major depression. We assessed effective dosing and temporal constraints of the glucose intervention in three experiments. We exposed 120 male Sprague-Dawley rats to 100, 1 mA, 3-15 s, inescapable and unpredictable electric tail shocks (over a 110-min period) or simple restraint in the learned helplessness procedure. Rats in each stress condition had access to a 40% glucose solution or water. We measured fluid consumption under 18-h free access conditions, or limited access (1, 3, 6, 18 h) beginning immediately after trauma, or 3-h access with delayed availability of the glucose solution (0, 1, 3, 6 h). We hypothesized that longer and earlier access following acute stress would improve shuttle-escape performance. Rats exposed to traumatic shock and given 18-h access to glucose failed to show exaggerated fearfulness and showed normal reactivity to foot shock during testing as compared to their water-treated counterparts. At least 3 h of immediate post-stress access to glucose were necessary to see these improvements in test performance. Moreover, delaying access to glucose for more than 3 h post-trauma yielded no beneficial effects. These data clearly identify limits on the post-stress glucose intervention. In conclusion, glucose should be administered almost immediately and at the highest dose after trauma.

Timeline to dysphagia resolution after endoscopic intervention of an interarytenoid defect based on Video Fluoroscopic Swallow Study dysphagia severity.

INTRODUCTION: We previously reported that endoscopic repair of a Type 1 Laryngeal Cleft (LC1) or Deep Interarytenoid Groove (DIG) improves swallowing function postoperatively. However, caregivers often ask about the timeline to resolution of the need for thickening. This study re-examines this cohort to answer this important caregiver-centered question. METHODS: We reassessed a 3-year retrospective, single-center dataset of children with dysphagia found to have a LC-1 or DIG on endoscopic exam. The primary outcome was rate of complete resolution of dysphagia at 2, 6, and 12 months after endoscopic intervention. A sub-group analysis was made based on severity of dysphagia prior to intervention and by type of endoscopic repair. RESULTS: Thirty-nine patients with mean age 1.35 years that had a LC-1 or DIG met criteria for inclusion. Rate of complete dysphagia resolution increased over time. Those with mild dysphagia (flow-reducing nipple and/or IDDSI consistency 1 or 2) had brisker resolution than those with moderate dysphagia (IDDSI consistency 3 or 4) at 2 months (67% vs 5%, p < 0.01) and at 6 months (80% vs 18%, p < 0.01) after endoscopic repair. There was no difference in dysphagia resolution between patients grouped by type of endoscopic repair. CONCLUSION: Addressing an interarytenoid defect in patients will not result in immediate, complete dysphagia resolution in most patients. However, patients that only require a flow-reducing nipple and/or thickening to an IDDSI consistency 1 or 2 have brisker resolution of the need for thickening than those that require an IDSSI consistency 3 or 4 prior to intervention. These results inform pre-operative discussions of the timeline to resolution based upon severity of dysphagia and help manage caregiver expectations.

The impact of the two-week wait referral pathway on rectal cancer survival.

AIM: The aim of this study was to compare the outcome of patients with rectal cancer referred through the two-week wait (TWW) system with those identified by routine referral pathways (non-TWW). METHOD: A prospective study was carried out of 125 consecutive patients diagnosed with rectal cancer between January 2000 and December 2005 (6 years) in one district general hospital. Data were recorded prospectively in a local clinicopathological registry. The patients were divided into two groups: group 1 (TWW) and group 2 (routine referral pathway). RESULTS: Fifty-two (41%) of the 125 patients were diagnosed through the TWW (group 1). There was no significant difference in patient demographics, including baseline tumour characteristics, between the two groups. There was no difference in preoperative or postoperative T stage between the two groups (P = 0.63). There was no significant difference in circumferential margin positivity (five of 52 in group 1 vs four of 73 in group 2; P = 0.52) or local recurrence rates (P = 0.37). The 5-year all-cause mortality was 49% for group 1 and 52% for group 2 (P = 0.3). The overall disease-free survival was similar in the two groups (1521 days for group 1 vs 1591 days for group 1, P = 0.29). CONCLUSION: Referral under the TWW strategy does not translate into improved survival in rectal cancer.

Angiotensin1-9 antagonises pro-hypertrophic signalling in cardiomyocytes via the angiotensin type 2 receptor.

The renin­angiotensin system (RAS) regulates blood pressure mainly via the actions of angiotensin (Ang)II, generated via angiotensin converting enzyme (ACE). The ACE homologue ACE2 metabolises AngII to Ang1-7, decreasing AngII and increasing Ang1-7, which counteracts AngII activity via the Mas receptor. However, ACE2 also converts AngI to Ang1-9, a poorly characterised peptide which can be further converted to Ang1-7 via ACE. Ang1-9 stimulates bradykinin release in endothelium and has antihypertrophic actions in the heart, attributed to its being a competitive inhibitor of ACE, leading to decreased AngII, rather than increased Ang1-7. To date no direct receptor-mediated effects of Ang1-9 have been described. To further understand the role of Ang1-9 in RAS function we assessed its action in cardiomyocyte hypertrophy in rat neonatal H9c2 and primary adult rabbit left ventricular cardiomyocytes, compared to Ang1-7. Cardiomyocyte hypertrophy was stimulated with AngII or vasopressin, significantly increasing cell size by approximately 1.2-fold (P < 0.05) as well as stimulating expression of the hypertrophy gene markers atrial natriuretic peptide, brain natriuretic peptide, ß-myosin heavy chain and myosin light chain (2- to 5-fold, P < 0.05). Both Ang1-9 and Ang1-7 were able to block hypertrophy induced by either agonist (control, 186.4 µm; AngII, 232.8 µm; AngII+Ang1-7, 198.3 µm; AngII+Ang1-9, 195.9 µm; P < 0.05). The effects of Ang1-9 were not inhibited by captopril, supporting previous evidence that Ang1-9 acts independently of Ang1-7. Next, we investigated receptor signalling via angiotensin type 1 and type 2 receptors (AT1R, AT2R) and Mas. The AT1R antagonist losartan blocked AngII-induced, but not vasopressin-induced, hypertrophy. Losartan did not block the antihypertrophic effects of Ang1-9, or Ang1-7 on vasopressin-stimulated cardiomyocytes. The Mas antagonist A779 efficiently blocked the antihypertrophic effects of Ang1-7, without affecting Ang1-9. Furthermore, Ang1-7 activity was also inhibited in the presence of the bradykinin type 2 receptor antagonist HOE140, without affecting Ang1-9. Moreover, we observed that the AT2R antagonist PD123,319 abolished the antihypertrophic effects of Ang1-9, without affecting Ang1-7, suggesting Ang1-9 signals via the AT2R. Radioligand binding assays demonstrated that Ang1-9 was able to bind the AT2R (pKi = 6.28 ± 0.1). In summary, we ascribe a direct biological role for Ang1-9 acting via the AT2R. This has implications for RAS function and identifying new therapeutic targets in cardiovascular disease.

Diffuse large B-cell lymphoma of the central nervous system in mycophenolate mofetil-treated patients with systemic lupus erythematosus.

Patients with systemic lupus erythematosus (SLE) are susceptible to the development of lymphoproliferative disorders and postulated causes include intrinsic defects in immune surveillance and iatrogenic administration of immunosuppressants. Since the introduction of mycophenolate mofetil (MMF) to the immunosuppressive regimen for the management of post-organ transplantation, there have been reports of primary lymphoma of the central nervous system (PCNSL). MMF has been widely used to treat active SLE patients with Class IV lupus nephritis. In addition to two previously reported cases of PCNSL among SLE patients on long-term MMF, we report a third patient who has been on treatment with MMF for 8 years. The histology showed features compatible with diffuse large B-cell lymphoma with strong immunohistochemical staining for CD20 and positive signal for Epstein-Barr virus (EBV)-encoded RNA by in-situ hybridization that is similar to other case reports, suggesting EBV driven B-cell lymphoproliferative disease. The patient responded to withdrawal of MMF, intravenous methotrexate, rituximab and whole brain radiotherapy. With the increasing use of MMF in active renal as well as non-renal exacerbations of SLE, PCNSL should be included in the differential diagnosis in patients who present with gradual onset of focal neurological deficit.

Limits on the spin-dependent WIMP-nucleon cross sections from the first science run of the ZEPLIN-III experiment.

We present new experimental constraints on the WIMP-nucleon spin-dependent elastic cross sections using data from the first science run of ZEPLIN-III, a two-phase xenon experiment searching for galactic dark matter weakly interacting massive particles based at the Boulby mine. Analysis of approximately 450 kg x days fiducial exposure allow us to place a 90%-confidence upper limit on the pure WIMP-neutron cross section of sigma(n)=1.9x10(-2) pb at 55 GeV/c(2) WIMP mass. Recent calculations of the nuclear spin structure based on the Bonn charge-dependent nucleon-nucleon potential were used for the odd-neutron isotopes 129Xe and 131Xe. These indicate that the sensitivity of xenon targets to the spin-dependent WIMP-proton interaction could be much lower than implied by previous calculations, whereas the WIMP-neutron sensitivity is impaired only by a factor of approximately 2.

Colonization lessons from a tropical forest.

The decade-old Transamazon Highway provides a useful stage for examining some of the major issues related to frontier conquest and the impact of pioneer settlement on one of the world's richest biomes. The highway project is an ambitious colonization scheme and the lessons that can be drawn from it, ranging from the environmental effects of stripping back the tree cover to the spread of diseases, will be useful in guiding development policy in other tropical regions.

Agricultural research and Third World food production.

By the close of this century the world may have to feed as many as 2 billion additional people. Most of them will be born in developing countries, especially in marginal lands ill-suited for food production. This article focuses on efforts by the International Agricultural Research Centers to increase food production in the Third World and addresses the social and ecological issues raised by the introduction of high-yielding varieties into fertile Third World lands and describe how varieties are being tailored for introduction into marginal areas.

Networking in international agricultural research.

Informal and structured collaboration is becoming increasingly common in international agricultural research. A network approach to research generally reduces costs, minimizes duplication, and boosts efficiency. Collaborative teams, sometimes involving hundreds of scientists in dozens of countries, have been formed to tackle numerous constraints to boosting food production. Networks have been established to test crop germplasm over a broad range of environments, explore ways of boosting the efficiency of fertilizer use, upgrade disease resistance in livestock, and identify socioeconomic obstacles to improved agricultural output. The benefits of networking are especially valuable to countries with limited funds and scientific manpower.

Crop germplasm conservation and developing countries.

Loss of the genetic diversity of some of the world's crops has accelerated in recent decades, with many crops becoming increasingly susceptible to diseases, pests, and environmental stresses. A global network of gene banks has therefore been established to provide plant breeders with the genetic resources necessary for developing more resistant crops that will enable farmers to maintain high yields. Most of these gene banks now store the germplasm of only the major crops such as cereals, potatoes, and grain legumes. Cultivated varieties of these crops are conserved as well as wild species that might otherwise become extinct. Tropical cash crops such as bananas and coconuts are also important food crops in many Third World countries, and more effort needs to be made to conserve the germplasm of these crops as well as of other important plants such as plantation crops, medicinal herbs, and fruit and timber trees.

Vulvar basal cell carcinoma in China: a 13-year review.

OBJECTIVE: We conducted a 12-year retrospective review of vulvar basal cell carcinoma (BCC) in a Chinese population. STUDY DESIGN: Medical records and histopathologic reports were examined from 5 major Hospitals in Hong Kong to list all patients diagnosed with vulvar BCC. Clinical data and histologic materials were reviewed. RESULTS: Sixteen vulvar BCCs were diagnosed. Most of them were pigmented. They were removed by simple excision or wide local excision. All the carcinomas were identified in the reticular dermis. The predominant histologic pattern was nodular, which may be mistaken as adenoid cystic carcinoma. CONCLUSION: The high proportion of pigmented vulvar BCCs suggested that biopsy should be performed for any pigmented lesion in a Chinese patient. The BCCs are superficial and tissue-preserving treatment approach is recommended. The tumor depth estimation is difficult and intraoperative frozen section consultation may be helpful. Formal histopathologic assessment should be used to reach an objective diagnosis.

Prognostic significance of magnetic resonance imaging-detected extramural vascular invasion in rectal cancer.

BACKGROUND: Extramural vascular invasion (EMVI) is a poor prognostic feature in colorectal cancer. The accuracy of magnetic resonance imaging (MRI) in detecting EMVI and predicting relapse-free survival (RFS) was compared retrospectively with the histological reference standard. METHODS: Preoperative magnetic resonance images from patients diagnosed with rectal and sigmoid cancer were reviewed and an MRI-EMVI score (range 0 to 4) was assigned. Comparison was made with histology and clinical outcome. RESULTS: Some 142 patients with a median follow-up of 3.3 (range 0.9-5.7) years were reviewed. Histological EMVI was reported in a quarter of patients. The sensitivity and specificity of MRI detection of EMVI in 94 patients undergoing primary surgery were 62 and 88 per cent respectively. On univariable analysis, RFS at 3 years was 35 per cent for patients with an MRI-EMVI score of 3-4, compared with 74 per cent for those with a score of 0-2 (P < 0.001), similar to values in patients with positive and negative histological EMVI status respectively (34 versus 73.7 per cent; P < 0.001). CONCLUSION: High MRI-EMVI scores may help in predicting disease relapse.

Penile lichen sclerosus after allogeneic stem cell transplantation.

Graft-versus-host disease (GVHD) often complicates allogeneic stem cell transplantation (SCT) and affects mainly the gut, liver, lung and skin. The microscopic morphological features of late-phase sclerodermatous chronic GVHD in the skin, namely epidermal atrophy, lymphoplasmacytic infiltration, dense dermal fibrosis and adnexal atrophy, are histologically indistinguishable from those in sporadic systemic sclerosis, morphoea and the related condition of lichen sclerosus. Mucosal orifices including those of the genitourinary system may be severely affected. We present three SCT recipients with chronic GVHD and severe posthitis leading to phimosis requiring surgery. The excised prepuces showed features of lichen sclerosus including epidermal atrophy and a subepidermal zone of eosinophilic, homogeneous and hyalinized collagen above a band-like lymphoplasmacytic infiltrate. These cases add further evidence to support the notion that penile lichen sclerosus should be included within the expanding sclerodermoid spectrum of late-stage cutaneous chronic GVHD.

NVP-QBE170: an inhaled blocker of the epithelial sodium channel with a reduced potential to induce hyperkalaemia.

BACKGROUND AND PURPOSE: Inhaled amiloride, a blocker of the epithelial sodium channel (ENaC), enhances mucociliary clearance (MCC) in cystic fibrosis (CF) patients. However, the dose of amiloride is limited by the mechanism-based side effect of hyperkalaemia resulting from renal ENaC blockade. Inhaled ENaC blockers with a reduced potential to induce hyperkalaemia provide a therapeutic strategy to improve mucosal hydration and MCC in the lungs of CF patients. The present study describes the preclinical profile of a novel ENaC blocker, NVP-QBE170, designed for inhaled delivery, with a reduced potential to induce hyperkalaemia. EXPERIMENTAL APPROACH: The in vitro potency and duration of action of NVP-QBE170 were compared with amiloride and a newer ENaC blocker, P552-02, in primary human bronchial epithelial cells (HBECs) by short-circuit current. In vivo efficacy and safety were assessed in guinea pig (tracheal potential difference/hyperkalaemia), rat (hyperkalaemia) and sheep (MCC). KEY RESULTS: In vitro, NVP-QBE170 potently inhibited ENaC function in HBEC and showed a longer duration of action to comparator molecules. In vivo, intratracheal (i.t.) instillation of NVP-QBE170 attenuated ENaC activity in the guinea pig airways with greater potency and duration of action than that of amiloride without inducing hyperkalaemia in either guinea pig or rat. Dry powder inhalation of NVP-QBE170 by conscious sheep increased MCC and was better than inhaled hypertonic saline in terms of efficacy and duration of action. CONCLUSIONS AND IMPLICATIONS: NVP-QBE170 highlights the potential for inhaled ENaC blockers to exhibit efficacy in the airways with a reduced risk of hyperkalaemia, relative to existing compounds.

ICAM-1 expression by vascular smooth muscle cells is phenotype-dependent.

Atherosclerosis is an inflammatory disease characterised by increased expression of adhesion molecules for leukocytes on both the surface of dysfunctional endothelium and on smooth muscle cells (SMC) within the lesion. It is also characterised by altered SMC phenotypic expression, indicated by a decreased volume fraction of myofilaments (V(v)myo) [1,2] and changes in gene expression [3]. The present study used an in vitro model to investigate, by immunofluorescence staining and flow cytometry, the influence of phenotype on vascular SMC expression of the adhesion molecule for leukocytes, intracellular adhesion molecule-1 (ICAM-1), and the regulatory mechanisms involved in this process. Smooth muscle cells with a high V(v)myo, freshly isolated from rat aortic media, expressed little or no ICAM-1 and this could not be induced by interleukin-1beta (IL-1beta). As SMC modulated phenotype, indicated by decreasing V(v)myo over the first 5 days of culture, there was a concomitant increase in ICAM-1 expression. At day 9 of primary culture, when SMC cultures had returned to the high V(v)myo phenotype, ICAM-1 expression was markedly lower. However, these cells retained the capacity to express ICAM-1 in response to IL-1beta. After several passages in culture, cells (with a low V(v)myo) constitutively expressed ICAM-1, with levels further up-regulated in response to IL-1beta. These changes in ICAM-1 expression were not related to proliferative state, since similar results were obtained with growth arrested SMC. Investigation of signalling pathways involved in regulating ICAM-1 expression by primary vascular SMC suggested a complex regulatory mechanism. Activation of adenyl cyclase (with forskolin) caused a significant increase in cells expressing ICAM-1. Treatment with inhibitors of protein kinase C (chelerythrine chloride), protein tyrosine kinase (genistein), or the transcription factor NF-kappaB (PDTC) had no significant effect on IL-1-induced ICAM-1 expression. However, in the presence of serum, both genistein and PDTC caused a significant increase in basal expression. The results indicate that ICAM-1 expression by SMC is phenotype-dependent, with expression evident only after cells have modulated to a low V(v)myo phenotype. They also indicate the existence of complex regulatory mechanisms, possibly involving the SMC cytoskeleton.

Relationship of maternal and infant iron stores as assessed by determination of plasma ferritin.

Using plasma ferritin derminations, iron stores have been evaluated at the end of pregnancy in 26 women and followed sequentially in their healthy, full-term infants. It has been shown that the iron storage status of the mother does not affect the iron status of the infant. Determinations of plasma ferritin in the infants have demonstrated that by 6 months of age the iron status of the infant reflects the adequacy of the dietary intake of iron. In the absence of effective iron supplementation during the first six months, plasma ferritin levels fall to low levels, indicating the depletion of iron stores in the infant.