Immunohistochemical assessment of glucagon-like peptide 1 receptor (GLP-1R) expression in the pancreas of patients with type 2 diabetes.

AIMS: Glucagon-like peptide-1 (GLP-1) is an incretin hormone which stimulates insulin release and inhibits glucagon secretion from the pancreas in a glucose-dependent manner. Incretin-based therapies, consisting of GLP-1 receptor (GLP-1R) agonists and dipeptidyl peptidase-4 (DPP-4) inhibitors, are used for the treatment of type 2 diabetes (T2D). Immunohistochemical studies for GLP-1R expression have been hampered previously by the use of unspecific polyclonal antibodies. This study aimed to assess the expression levels of GLP-1R in a set of T2D donor samples obtained via nPOD. METHODS: This study used a new monoclonal antibody to assess GLP-1R expression in pancreatic tissue from 23 patients with T2D, including 7 with a DPP-4 inhibitor and 1 with a history of GLP-1R agonist treatment. A software-based automated image analysis algorithm was used for quantitating intensities and area fractions of GLP-1R positive compartments. RESULTS: The highest intensity GLP-1R immunostaining was seen in beta-cells in islets (average signal intensity, 76.1 [±8.1]). GLP-1R/insulin double-labelled single cells or small clusters of cells were also frequently located within or in close vicinity of ductal epithelium in all samples and with the same GLP-1R immunostaining intensity as found in beta-cells in islets. In the exocrine pancreas a large proportion of acinar cells expressed GLP-1R with a 3-fold lower intensity of immunoreactivity as compared to beta-cells (average signal intensity 25.5 [±3,3]). Our studies did not unequivocally demonstrate GLP-1R immunoreactivity on normal-appearing ductal epithelium. Pancreatic intraepithelial neoplasia (PanINs; a form of non-invasive pancreatic ductular neoplasia) was seen in most samples, and a minority of these expressed low levels of GLP-1R. CONCLUSION: These data confirm the ubiquity of early stage PanIN lesions in patients with T2D and do not support the hypothesis that incretin-based therapies are associated with progression towards the more advanced stage PanIN lesions.

Functional domains of the PETAL LOSS protein, a trihelix transcription factor that represses regional growth in Arabidopsis thaliana.

PETAL LOSS (PTL) is a trihelix transcription factor that represses growth, especially between sepal primordia. As one of 30 trihelix proteins in Arabidopsis, it falls in the GT2 clade with duplicated trihelix DNA-binding domains and a long α-helical central domain. PTL orthologs occur in all angiosperm genomes examined except grasses, and sequence comparisons reveal that there are two further short conserved domains at each end. GT2 itself carries two nuclear localization sequences, but PTL has an additional nuclear localization sequence (NLS). We show that PTL can act as a transcriptional activator in yeast and in planta, with the latter tested by two different functional assays. Specific deletions revealed that the activation region is C-terminal. Site-directed mutagenesis of the DNA-binding domains has shown that a conserved tryptophan and two downstream acidic amino acids in the second trihelix, predicted to promote folding, are each required for PTL function. Also, three basic residues in the third helix, near the DNA interaction sites, support its function. PTL was found to dimerize in yeast. This was confirmed and extended by jointly expressing differentially tagged forms of PTL in a transient expression system in Nicotiana benthamiana leaves. Cytoplasmic PTL (with mutant NLS sequences) was carried into the nucleus upon binding with nuclear-localized PTL, providing each partner carried intact central domains. As this 90-amino acid domain is conserved in most trihelix family members, it seems likely that they all function in dimeric form.

PETAL LOSS, a trihelix transcription factor that represses growth in Arabidopsis thaliana, binds the energy-sensing SnRK1 kinase AKIN10.

Organogenesis in plants involves differential growth. Rapidly growing primordia are distinguished from the meristem and each other by slower growing boundaries. PETAL LOSS (PTL) is a trihelix transcription factor of Arabidopsis that represses growth in boundaries between newly arising sepals. To identify partners involved in this growth limitation, a young inflorescence cDNA library was screened by yeast two-hybrid technology with PTL as bait. The most frequent prey identified was AKIN10, the catalytic α-subunit of the Snf1-related kinase1 (SnRK1). Interaction was mapped to the C-terminal (non-kinase) half of AKIN10 and the N-terminal portion of PTL. Binding of PTL was specific to AKIN10 as there was little binding to the related AKIN11. The interaction was confirmed by co-immunoprecipitation in vitro. Fluorescently tagged products of 35S:YFP-AKIN10 and 35S:CFP-PTL also interacted when transiently expressed together in leaf cells of Nicotiana benthamiana. In this case, most of the cytoplasmic AKIN10 was preferentially moved to the nucleus where PTL accumulated, possibly because a nuclear export sequence in AKIN10 was now masked. During these experiments, we observed that AKIN10 could variably accumulate in the Golgi, shown by its co-localization with a tagged Golgi marker and through its dispersal by brefeldin A. Tests of phosphorylation of PTL by AKIN10 gave negative results. The functional significance of the PTL-AKIN10 interaction remains open, although a testable hypothesis is that AKIN10 senses lower energy levels in inter-sepal zones and, in association with PTL, promotes reduced cell division.

Genome-wide data (ChIP-seq) enabled identification of cell wall-related and aquaporin genes as targets of tomato ASR1, a drought stress-responsive transcription factor.

BACKGROUND: Identifying the target genes of transcription factors is important for unraveling regulatory networks in all types of organisms. Our interest was precisely to uncover the spectrum of loci regulated by a widespread plant transcription factor involved in physiological adaptation to drought, a type of stress that plants have encountered since the colonization of land habitats 400 MYA. The regulator under study, named ASR1, is exclusive to the plant kingdom (albeit absent in Arabidopsis) and known to alleviate the stress caused by restricted water availability. As its target genes are still unknown despite the original cloning of Asr1 cDNA 20 years ago, we examined the tomato genome for specific loci interacting in vivo with this conspicuous protein. RESULTS: We performed ChIP followed by high throughput DNA sequencing (ChIP-seq) on leaves from stressed tomato plants, using a high-quality anti-ASR1 antibody. In this way, we unraveled a novel repertoire of target genes, some of which are clearly involved in the response to drought stress. Many of the ASR1-enriched genomic loci we found encode enzymes involved in cell wall synthesis and remodeling as well as channels implicated in water and solute flux, such as aquaporins. In addition, we were able to determine a robust consensus ASR1-binding DNA motif. CONCLUSIONS: The finding of cell wall synthesis and aquaporin genes as targets of ASR1 is consistent with their suggested role in the physiological adaptation of plants to water loss. The results gain insight into the environmental stress-sensing pathways leading to plant tolerance of drought.

Differentiating Arabidopsis shoots from leaves by combined YABBY activities.

In seed plants, leaves are born on radial shoots, but unlike shoots, they are determinate dorsiventral organs made of flat lamina. YABBY genes are found only in seed plants and in all cases studied are expressed primarily in lateral organs and in a polar manner. Despite their simple expression, Arabidopsis thaliana plants lacking all YABBY gene activities have a wide range of morphological defects in all lateral organs as well as the shoot apical meristem (SAM). Here, we show that leaves lacking all YABBY activities are initiated as dorsiventral appendages but fail to properly activate lamina programs. In particular, the activation of most CINCINNATA-class TCP genes does not commence, SAM-specific programs are reactivated, and a marginal leaf domain is not established. Altered distribution of auxin signaling and the auxin efflux carrier PIN1, highly reduced venation, initiation of multiple cotyledons, and gradual loss of the SAM accompany these defects. We suggest that YABBY functions were recruited to mold modified shoot systems into flat plant appendages by translating organ polarity into lamina-specific programs that include marginal auxin flow and activation of a maturation schedule directing determinate growth.

The Arabidopsis glutathione transferase gene family displays complex stress regulation and co-silencing multiple genes results in altered metabolic sensitivity to oxidative stress.

Plant glutathione transferases (GSTs) are induced by diverse biotic and abiotic stimuli, and are important for protecting plants against oxidative damage. We have studied the primary transcriptional stress response of the entire Arabidopsis GST family to seven stresses, including both biotic and abiotic stimuli, with a focus on early changes in gene expression. Our results indicate that individual GST genes are highly specific in their induction patterns. Furthermore, we have been able to link individual GSTs to particular stress stimuli. Using RNAi, we successfully co-silenced a group of four phi GSTs that represent some of the most highly expressed GST genes. Despite a marked reduction in total phi GST protein levels, the transgenic plants showed no reduction in GST activity as measured using the model substrate 1-chloro-2,4-dinitrobenzene (CDNB), and appeared to have surprisingly robust physical phenotypes during stress. However, analysis of metabolite pools showed oxidation of the glutathione pool in the RNAi lines, and we observed alterations in carbon and nitrogen compounds following salicylic acid and hydrogen peroxide stress treatments, indicative of oxidative modification of primary metabolism. Thus, there appears to be a high degree of functional redundancy within the Arabidopsis GST family, with extensive disruption being required to reveal the roles of phi GSTs in protection against oxidative stress.

Quantitative Outcomes of a One Health approach to Study Global Health Challenges.

Having gained momentum in the last decade, the One Health initiative promotes a holistic approach to address complex global health issues. Before recommending its adoption to stakeholders, however, it is paramount to first compile quantitative evidence of the benefit of such an approach. The aim of this scoping review was to identify and summarize primary research that describes monetary and non-monetary outcomes following adoption of a One Health approach. An extensive literature search yielded a total of 42,167 references, of which 85 were included in the final analysis. The top two biotic health issues addressed in these studies were rabies and malaria; the top abiotic health issue was air pollution. Most studies described collaborations between human and animal (n = 42), or human and environmental disciplines (n = 41); commonly reported interventions included vector control and animal vaccination. Monetary outcomes were commonly expressed as cost-benefit or cost-utility ratios; non-monetary outcomes were described using disease frequency or disease burden measurements. The majority of the studies reported positive or partially positive outcomes. This paper illustrates the variety of health challenges that can be addressed using a One Health approach, and provides tangible quantitative measures that can be used to evaluate future implementations of the One Health approach.

[Portosystemic shunt encephalopathy. Clinical and radiological evaluation by using multidetector-row CT angiography]. / Encefalopatia da shunt portosistemico. Valutazione clinica e radiologica con angio-TC multidetettore.

We describe the case of a 72-year-old woman who presented a symptomatology characterized by recurrent episodes of confusion, weakness with cerebral vasculopathy. The high values of ammonium were correctly defined thanks to the diagnostic multidetector-row CT information and we referred the symptoms to porto-systemic shunts with the exclusion of hepatic vascularization due to an inferior vena cava stenosis.

Quality in emergency departments: a study on 3,285,440 admissions.

INTRODUCTION: A multi-centre study has been conducted, during 2005, by means of a questionnaire posted on the Italian Society of Emergency Medicine (SIMEU) web page. Our intention was to carry out an organisational and functional analysis of Italian Emergency Departments (ED) in order to pick out some macro-indicators of the activities performed. Participation was good, in that 69 ED (3,285,440 admissions to emergency services) responded to the questionnaire. METHODS: The study was based on 18 questions: 3 regarding the personnel of the ED, 2 regarding organisational and functional aspects, 5 on the activity of the ED, 7 on triage and 1 on the assessment of the quality perceived by the users of the ED. RESULTS AND CONCLUSION: The replies revealed that 91.30% of the ED were equipped with data-processing software, which, in 96.83% of cases, tracked the entire itinerary of the patient. About 48,000 patients/year used the ED: 76.72% were discharged and 18.31% were hospitalised. Observation Units were active in 81.16% of the ED examined. Triage programmes were in place in 92.75% of ED: in 75.81% of these, triage was performed throughout the entire itinerary of the patient; in 16.13% it was performed only symptom-based, and in 8.06% only on-call. Of the patients arriving at the ED, 24.19% were assigned a non-urgent triage code, 60.01% a urgent code, 14.30% a emergent code and 1.49% a life-threatening code. Waiting times were: 52.39 min for non-urgent patients, 40.26 min for urgent, 12.08 for emergent, and 1.19 for life-threatening patients.

PPARgamma agonist induced cardiac enlargement is associated with reduced fatty acid and increased glucose utilization in myocardium of Wistar rats.

In toxicological studies, high doses of peroxisome proliferator-activated receptor-gamma (PPARgamma) agonists cause cardiac enlargement. To investigate whether this could be explained by a large shift from free fatty acid to glucose utilization by the heart, Wistar rats were treated for 2-3 weeks with a potent, selective PPARgamma agonist (X334, 3 micromol/kg/d), or vehicle. X334 treatment increased body-weight gain and ventricular mass. Treatment lowered plasma triglycerides by 61%, free fatty acid levels by 72%, insulin levels by 45%, and reduced total plasma protein concentration by 7% (indicating plasma volume expansion) compared to vehicle animals. Fasting plasma glucose levels were unaltered. To assess cardiac free fatty acid and glucose utilization in vivo we used simultaneous infusions of non-beta-oxidizable free fatty acid analogue, [9,10-(3)H](R)-2-bromopalmitate and [U-(14)C]2-deoxy-d-glucose tracers, which yield indices of local free fatty acid and glucose utilization. In anesthetized, 7 h fasted animals, left ventricular glucose utilization was increased to 182% while free fatty acid utilization was reduced by 28% (P<0.05) compared to vehicle. In separate studies we attempted to prevent the X334-induced hypolipidemia. Various dietary fat supplements were unsuccessful. By contrast, restricting the time during which the treated animals had access to food (promoting endogenous lipolysis), restored plasma free fatty acid from 27% to 72% of vehicle control levels and prevented the cardiac enlargement. Body-weight gain in these treated-food restricted rats was not different from vehicle controls. In conclusion, the cardiac enlargement caused by intense PPARgamma activation in normal animals is associated with marked changes in free fatty acid/glucose utilization and the enlargement can be prevented by restoring free fatty acid availability.

Untangling multi-gene families in plants by integrating proteomics into functional genomics.

The classification and study of gene families is emerging as a constructive tool for fast tracking the elucidation of gene function. A multitude of technologies can be employed to undertake this task including comparative genomics, gene expression studies, sub-cellular localisation studies and proteomic analysis. Here we focus on the growing role of proteomics in untangling gene families in model plant species. Proteomics can specifically identify the products of closely related genes, can determine their abundance, and coupled to affinity chromatography and sub-cellular fractionation studies, it can even provide location within cells and functional assessment of specific proteins. Furthermore global gene expression analysis can then be used to place a specific family member in the context of a cohort of co-expressed genes. In model plants with established reverse genetic resources, such as catalogued T-DNA insertion lines, this gene specific information can also be readily used for a wider assessment of specific protein function or its capacity for compensation through assessing whole plant phenotypes. In combination, these resources can explore partitioning of function between members and assess the level of redundancy within gene families.

Evaluation of the efficacy and toxicity of the cyclosporine A-flucortolone-methotrexate combination in the treatment of sarcoidosis.

Eleven patients with chronic sarcoidosis resistant to high-dose corticosteroids and other immunosuppressive treatments were treated with cyclosporine A at the initial daily dose of 5 mg per kg body weight (ideal weight in the case of overweight subjects) combined with flucortolone and methotrexate. A complete and lasting remission of the disease was obtained in all patients with total disappearance of pulmonary and extrapulmonary manifestations. In addition, the disease activity indexes normalized and remained normal for the rest of the follow-up period (24.82 +/- 8.22 months, range 12-33). No renal or hepatic toxicity was observed in any patient. Two of them presented hypertrichosis and one nausea.

Long-term efficacy and toxicity of cyclosporin A + fluocortolone + methotrexate in the treatment of rheumatoid arthritis.

OBJECTIVE: The therapeutic efficacy and tolerability of the combination of cyclosporin A, methotrexate and fluocortolone was evaluated after 96 months of treatment in 140 patients with rheumatoid arthritis. METHODS: The initial dose of CyA was 5 mg/kg per day and was subsequently modified on the basis of the individual clinical response. Fluocortolone was initially administered at a dose that was sufficient to control disease activity (80-130 mg/week) and then was gradually tapered down to a maintenance dose of 15-20 mg/week. MTX was given intravenously at a dose of 15 mg once weekly for 4 consecutive weeks and then, after a 2-week interval, every 2 weeks or every month depending on the evolution of the disease. RESULTS: At the end of the study a statistically significant improvement was observed in both clinical (VAS, grip-strength, duration of morning stiffness, number of swollen joints, number of painful joints, Ritchie's index and Lee's functional index) and laboratory parameters: ESR (p = 0.000); alpha 2 globulins (p = 0.000); hemoglobin (p = 0.000); CRP (p < 0.001); and rheumatoid factor (p = 0.000). Radiological evaluation revealed little progression in anatomic lesions (Larsen score p = 0.699; number of erosions p = 0.344), thus suggesting that our protocol may be capable of showing down both bone resorption and cartilage loss. Renal toxicity, defined as an increase in plasma creatinine concentrations of more than 50% of the baseline value, was observed in 12 patients (8.5%), but the drug was discontinued in only one, who simultaneously presented high blood pressure. CONCLUSION: The positive results so far achieved in our study must be interpreted as being due to the combined action of the individual drugs, which made it possible for them to be used at relatively low dosages that minimised the onset of their side effects while maintaining the efficacy of their suppressive action.

Hypoechoic images in hepatic steatosis: a recent ultrasound finding in differential diagnosis of space-occupying lesions of the liver.

The Authors described a particular ultrasound finding which can sometimes be observed in the echographic picture of diffused hepatic steatosis and which was seen by these Authors in 117 out of 312 patients affected by steatosis. This finding consists of the presence of one or more hypoechoic focal areas situated at the IV-V and/or II-III segment level surrounded by wide-spread increase in echogenicity of remaining parenchyma, which is typical of "bright liver". These areas, which can easily be mistaken for neoplastic formations, were instead seen to be limited areas of normal liver parenchyma free of fatty infiltration. In those patients presenting valid reasons for suspecting the presence of primary or secondary neoplastic formations, the Authors believe that echographic examination of these areas should always be accompanied by more invasive methods in order to confirm or exclude the possible presence of neoplastic formations with certainty. On the contrary, in those patients where no such diagnosis is suspected, it should prove sufficient to monitor these ares by means of ultrasound.

[Detectable hypoechogenic images in diffuse hepatic steatosis: a new ultrasonic finding in the differential diagnosis of expansive lesions of the liver]. / Immagini ipoecogene rilevabili nella steatosi epatica diffusa: un nuovo reperto ultrasonografico nella diagnosi differenziale delle lesioni espansive del fegato.

The authors describe a particular ultrasound finding which can sometimes be observed in the echographic picture of diffused hepatic steatosis and which was seen by these Authors in 117 out of 312 patients affected by steatosis. This finding consists of the presence of one or more hypoechoic focal areas situated at the IV-V and/or II-III segment level surrounded by widespread increase in echogenicity of remaining parenchyma, which is typical of "bright liver". These areas, which can easily be mistaken for neoplastic formations, were instead seen to be limited areas of normal liver parenchyma free of fatty infiltration. In those patients presenting valid reasons for suspecting the presence of primary or secondary neoplastic formations, the Authors believe that echographic examination of these areas should always be accompanied by more invasive methods in order to confirm or exclude the possible presence of neoplastic formations with certainty. On the contrary, in those patients where no such diagnosis is suspected, it should prove sufficient to monitor these areas by means of ultrasounds.

[Mebendazole in the therapy of human hydatidosis. Evaluation of the results obtained in 9 patients with pulmonary localization]. / Il mebendazolo nella terapia dell'idatidosi umana. Valutazione dei risultati ottenuti su nove pazienti con localizzazione polmonare.

Nine patients with pulmonary cysts of echinococcus varying in size have been treated with Mebendazole. When the cysts were small or medium in size a complete disappearance or an important reduction was observed, whereas in four patients in which the cyst was big in size the treatment did not cause any decrease in size. The Authors point out the efficacy of the Mebendazole in the small or medium size cysts if the drug is given at high doses and for a long period. Nevertheless the use of this drug may be suitable also in the treatment of big size cysts together with surgical treatment to avoid possible secondary local or distant implant and in all cases of human inoperable echinococcal cyst.

[Radiosensitivity of lymphocyte subpopulations in subjects with systemic lupus erythematosus. An in vitro preliminary study]. / Radiosensibilità delle sottopopolazioni linfocitarie in soggetti affetti da lupus eritematodes sistemico. Studio preliminare "in vitro".

The Authors have studied the radiosensitivity of lymphocyte cultures of patients affected by systemic lupus erythematosus in the active phase, and in the remissive phase after treatment with cyclosporine and fluocortolone, compared to a group of normal subjects. Total lymphocyte cultures and cultures of T, B, NK lymphocytes, T helper/inducer and T suppressor cytotoxic obtained with specific monoclonal antibodies were used and then irradiated with Co-60 gamma photons at scale doses between 0 and 10 Gy. Damage due to irradiation was evaluated using 3H-TdR. Patients in the clinically active phase of the disease showed an increased lymphocytic sensibility of the total lymphocytic population as well as of the various populations and T subpopulations studied. Radiosensitivity tended to become normal in patients in the remissive phase of the disease. The Authors conclude that a preliminary study of the lymphocytic sensitivity should be carried out in all patients treated with radiotherapy with autoimmune pathologies in order to avoid damages due to irradiation. Moreover, the Authors retain that a test for lymphocytic sensitivity to radiation could apply with regard to the evaluation of the clinical remissive conditions of patients affected by systemic lupus erithematosus.

[Behavior of blood copper, ceruloplasmin and procollagen III in acute myocardial infarction]. / Sul comportamento della cupremia, della ceruloplasminemia e del procollagene III sierico nell'infarto acuto del miocardio.

The authors report the determination of copper, ceruloplasmin and procollagen III peptide serum levels (sPIIIP) in patients affected by acute myocardial infarction. These measurements were taken daily during the first ten days after diagnosis and subsequently every five days until the fortieth after the acute event. A significant increase above the normal values was observed, proportioned to the extension of the infarctuated area, reaching a peak respectively for cupraemia, ceruloplasminaemia and sPIIIP on the fourth, sixth and eighth day after the acute event and still being evident in varying proportion till the 25th-35th day and even further, again in relation to the extension of the infarctuated area. The Authors relate this phenomenon to the involvement of the homeostatic mechanism supposed to face necrotic and critical processes as those which take place during acute myocardial infarction and subsequent riparation. The Authors suggest the clinical use of cupraemia, ceruloplasminaemia and sPIIIP determinations as diagnostic and prognostic indexes of acute myocardial infarction.

Short term treatment with ketoconazole: effects on gonadal and adrenal steroidogenesis in women.

Ketoconazole, an imidazole derivative, is a large spectrum antifungal agent. The drug is known to cause a decrease in plasma androgens and adrenal steroids in normal men; it is also an active drug in the treatment of malignant tumors of the prostate. To examine the antiandrogenic action of this drug in women, we measured several gonadal and adrenal steroids in 21 normally menstruating women before and after receiving oral ketoconazole (200 mg twice daily) for 5 days. Plasma testosterone (T) decreased from a basal level of 0.35 to 0.25 ng/ml (+/- SEM) (P less than 0.001); dihydrotestosterone (DHT) from a basal level of 190.62 +/- 23.2 to 159.75 +/- 19.43 pg/ml (P less than 0.02); dehydroepiandrosterone sulphate (DHEA-S) from 1.42 +/- 0.44 to 1.15 +/- 0.19 micron/ml (P less than 0.02). Plasma 17 beta-estradiol (E2) decreased from a basal level of 97.42 +/- 29.37 to 54.32 +/- 9.9 pg/ml (P less than 0.05). In contrast, plasma 17-OH-progesterone (17-OHP) levels increased from a basal level of 44.81 +/- 8.21 to 71.81 +/- 15.81 ng/100 ml (P less than 0.05). These results confirm that the ketoconazole blocks the conversion of progestins into androgens. The decrease in the plasma concentration of E2 suggest a direct effect of the ketoconazole on the ovary. It is likely that the effect of the drug, both at the level of the ovaries and of the adrenal gland, is dose-dependent.(ABSTRACT TRUNCATED AT 250 WORDS)