Long-term non-invasive interrogation of human dorsal root ganglion neuronal cultures on an integrated microfluidic multielectrode array platform.

Scientific studies in drug development and toxicology rely heavily on animal models, which often inaccurately predict the true response for human exposure. This may lead to unanticipated adverse effects or misidentified risks that result in, for example, drug candidate elimination. The utilization of human cells and tissues for in vitro physiological platforms has become a growing area of interest to bridge this gap and to more accurately predict human responses to drugs and toxins. The effects of new drugs and toxins on the peripheral nervous system are often investigated with neurons isolated from dorsal root ganglia (DRG), typically with one-time measurement techniques such as patch clamping. Here, we report the use of our multi-electrode array (MEA) platform for long-term noninvasive assessment of human DRG cell health and function. In this study, we acquired simultaneous optical and electrophysiological measurements from primary human DRG neurons upon chemical stimulation repeatedly through day in vitro (DIV) 23. Distinct chemical signatures were noted for the cellular responses evoked by each chemical stimulus. Additionally, the cell viability and function of the human DRG neurons were consistent through DIV 23. To the best of our knowledge, this is the first report on long-term measurements of the cell health and function of human DRG neurons on a MEA platform. Future generations will include higher electrode numbers in customized arrangements as well as integration with different tissue types on a single device. This platform will provide a valuable testing tool for both rodent and human cells, enabling a more comprehensive risk assessment for drug candidates and toxicants.

Color Doppler flow imaging for the early detection of nonpregnant cattle at 20 days after timed artificial insemination.

The objective was to determine the accuracy of a pregnancy test for predicting nonpregnant cattle based on the evaluation of corpus luteum (CL) blood flow at 20 d (CLBF-d20) after timed artificial insemination (TAI). Crossbred Holstein-Gir dairy heifers (n=209) and lactating cows (n=317) were synchronized for TAI using the following protocol: intravaginal implant (1.0 g of progesterone) and 2mg of estradiol benzoate i.m. on d -10, implant removal and 0.526 mg of sodium cloprostenol i.m. on d -2, 1mg of estradiol benzoate i.m. on d -1, and TAI on d 0. On d 20, animals underwent grayscale ultrasonography (US) to locate the CL and color flow Doppler to evaluate CLBF-d20 using a portable ultrasound equipped with a 7.5-MHz rectal transducer. Based only on a visual, subjective CLBF evaluation, the animals were classified as pregnant or not pregnant. On d 30 to 35, blinded from results of the previous diagnosis, the same operator performed a final pregnancy diagnosis using US to visualize the fetal heartbeat (gold standard; US-d30). A second evaluator also analyzed the CLBF-d20 in the same animals by watching 7-s recorded videos. Blood samples were collected from a subset of 171 females to determine, by RIA, plasma progesterone (P4) concentrations, which indicate CL function. The final pregnancy outcome (US-d30) was retrospectively compared with the CLBF-d20 diagnoses and then classified either as correct or incorrect. The number of true positive, true negative, false positive, and false negative decisions were inserted into a 2 × 2 decision matrix. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the CLBF-d20 test were calculated using specific equations. Binomial variables (pregnancy rate and proportions) were analyzed using Fisher's exact test for the effect of parity and to compare between evaluators and tests (CLBF-d20 vs. plasma P4). The kappa values were calculated to quantify the agreement between CLBF-d20 and the gold standard (US-d30) and between evaluators. The performance parameters of CLBF-d20 test were as follows: sensitivity=99.0%, specificity=53.7%, positive predictive value=65.1%, negative predictive value=98.5%, and accuracy=74.8%. False negatives represented only 0.4% of the exams. No differences existed in these parameters between evaluators (no. 1 vs. no. 2) and tests (CLBF-d20 vs. plasma P4). Moreover, a high level of agreement was observed between evaluators (0.91). In conclusion, visual evaluation of CLBF-d20 represents a quick, reliable, and consistent diagnostic test that enables the early detection of nonpregnant cattle.

Lifestyle-related risk factors for Parkinson's disease: a population-based study.

OBJECTIVES: To investigate the association of major lifestyle-related risk factors with the prevalent cases of Parkinson's disease (PD) identified by the Italian Longitudinal Study on Aging. METHODS: A total of 5632 individuals randomly selected from the population registers of eight centers were screened for parkinsonism using both a questionnaire and a neurologic examination. Screened positives underwent a structured clinical work-up for the diagnosis of parkinsonism and parkinsonism subtypes. RESULTS: We identified 113 prevalent cases of PD. Age, male gender, and pesticide-use license were significantly related to PD. Heavy smoking was inversely related to PD. Age (OR = 1.1; 95% CI, 1.06-1.15) and pesticide-use license (OR = 3.7; 95% CI, 1.6-8.6) kept their significant correlation with the disease in the multivariate analysis to adjust for all the variables under investigation. Multivariate analyses were made for men and women separately: pesticide exposure was positively associated with PD only in men. CONCLUSIONS: Pesticide exposure might represent a candidate for environmental factors involved in PD.

Pharmacological properties of the potent epileptogenic amino acid dysiherbaine, a novel glutamate receptor agonist isolated from the marine sponge Dysidea herbacea.

Dysiherbaine (DH) is a marine sponge-derived amino acid that causes seizures upon injection into mice. In this report we investigate the behavioral effects and characterize the pharmacological activity of DH. DH induced convulsive behaviors in mice with ED(50) values of 13 pmol/mouse, i.c.v. and 0.97 mg/kg, i.p. In rat brain synaptic membranes DH displaced binding of [3H]kainic acid (KA) and [3H]alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) with K(i) values of 26 and 153 nM, respectively; in contrast, DH did not displace the N-methyl-D-aspartic acid (NMDA) receptor ligand [3H]CGS-19755. DH displaced [3H]KA from recombinant GluR5 and GluR6 kainate receptor subunits expressed in HEK293 cells with K(i) values of 0.74 and 1.2 nM, respectively. In whole-cell voltage-clamp recordings from cultured rat hippocampal neurons, DH evoked inward currents from both AMPA and KA receptors with EC(50) values of 9.7 microM and 210 nM, respectively. AMPA receptor currents were blocked by GYKI 53655, whereas KA receptor currents were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Surprisingly, in calcium imaging experiments we found that DH also activated recombinant mGluR5 receptors but did not activate mGluR1 receptors. DH did not activate glutamate transporters or gamma-aminobutyric acid A (GABA(A)) receptors. These results indicate that DH is a potent non-NMDA-type agonist with very high affinity for KA receptors, as well as a subtype-selective mGluR agonist. DH possesses the most potent epileptogenic activity among the amino acids yet identified. This novel excitatory amino acid may prove useful for evaluating the physiological and pathological roles of non-NMDA receptors, especially KA receptors, in the central nervous system.

The role of RNA editing of kainate receptors in synaptic plasticity and seizures.

The ionotropic glutamate receptor subunit GluR6 undergoes developmentally and regionally regulated Q/R site RNA editing that reduces the calcium permeability of GluR6-containing kainate receptors. To investigate the functional significance of this editing in vivo, we engineered mice deficient in GluR6 Q/R site editing. In these mutant mice but not in wild types, NMDA receptor-independent long-term potentiation (LTP) could be induced at the medial perforant path-dentate gyrus synapse. This indicates that kainate receptors with unedited GluR6 subunits can mediate LTP. Behavioral analyses revealed no differences from wild types, but mutant mice were more vulnerable to kainate-induced seizures. Together, these results suggest that GluR6 Q/R site RNA editing may modulate synaptic plasticity and seizure vulnerability.

Characteristics, outcome, and care of stroke associated with atrial fibrillation in Europe: data from a multicenter multinational hospital-based registry (The European Community Stroke Project).

BACKGROUND AND PURPOSE: The role of atrial fibrillation (AF) as a determinant of stroke outcome is not well established. Studies focusing on this topic relied on relatively small samples of patients, scarcely representative of the older age groups. We aimed at evaluating clinical characteristics, care, and outcome of stroke associated with AF in a large European sample. METHODS: In a European Concerted Action involving 7 countries, 4462 patients hospitalized for first-in-a-lifetime stroke were evaluated for demographics, risk factors, clinical presentation, resource use, and 3-month survival, disability (Barthel Index), and handicap (Rankin scale). RESULTS: AF was present in 803 patients (18.0%). AF patients, compared with those without AF, were older, were more frequently female, and more often had experienced a previous myocardial infarction; they were less often diabetics, alcohol consumers, and smokers (all P:<0.001). At 3 months, 32.8% of the AF patients were dead compared with 19.9% of the non-AF patients (P:<0.001). With control for baseline variables, AF increased by almost 50% the probability of remaining disabled (multivariate odds ratio 1.43, 95% CI 1.13 to 1.80) or handicapped (multivariate odds ratio 1.51, 95% CI 1.13 to 2.02). Before stroke, only 8.4% of AF patients were on anticoagulants. The chance of being anticoagulated was reduced by 4% per year of increasing age. AF patients underwent CT scan and other diagnostic procedures less frequently and received less physiotherapy or occupational therapy. CONCLUSIONS: Stroke associated with AF has a poor prognosis in terms of death and function. Prevention and care of stroke with AF is a major challenge for European health systems.

NMDA-Dependent modulation of hippocampal kainate receptors by calcineurin and Ca(2+)/calmodulin-dependent protein kinase.

Neurotransmitter receptor function can be influenced by the phosphorylation state of the receptor or of associated proteins. Here we show that kainate receptors expressed in cultured hippocampal neurons can be modulated by Ca(2+)/calmodulin-dependent phosphatase (calcineurin) and Ca(2+)/calmodulin-dependent kinase (CaMK). Ca(2+) influx through NMDA receptor or voltage-sensitive calcium channels resulted in a transient depression of the kainate receptor current. This calcium-induced depression of the kainate receptor current depended on the activation of the phosphatase calcineurin. The amplitude of the kainate receptor currents returned to the baseline level in approximately 9 sec (tau = 3.6 sec), and the recovery of the current amplitude depended on CaMK activity. The effect on kainate receptor currents was dependent on the frequency of NMDA receptor activation. Although low-frequency (0.1 Hz) NMDA application induced depression followed by recovery of the kainate receptor currents, higher frequency (1 Hz) NMDA applications induced a more prolonged depression. Kainate receptors have been shown to modulate synaptic transmission by both presynaptic and postsynaptic mechanisms. Our results suggest that synaptic activity mediated by NMDA receptors, or other routes of Ca(2+) influx, may, in turn, modulate the function of kainate receptors.

[The role of radiology in assessing patients with pathological obesity submitted to modified Mason's vertical gastroplasty. A study of 322 cases]. / Ruolo della radiologia nella valutazione dei pazienti affetti da obesità patologica sottoposti a gastroplastica verticale secondo Mason modificata. Studio di 322 casi.

PURPOSE: Mason's vertical banded gastroplasty (VBG) is one the most popular surgical techniques for the treatment of morbid obesity because it is effective and easy to perform and presents a low incidence of long-term complications. We report our personal results in 322 Mason's VBG patients who were followed-up radiologically. MATERIAL AND METHODS: 322 patients submitted to modified Mason's VBG underwent radiological follow-up, preferably with single contrast, to assess the shape and volume of the pouch and the presence of postoperative complications. The patients were 272 women and 50 men whose average weight was 124 kg (range: 78 to 218 kg). The women's average age was 37 years (range: 17 to 69) and the men's 36 (range: 19 to 64). We performed a double contrast examination, adapting the dosage of effervescent powders to the gastric pouch capacity, only in particular cases where a more detailed study of pouch surface was required. The examinations were performed at 1 month and 12 months postoperatively to evaluate the pouch shape and volume, in order to correlate surgical results with weight loss. Premature or unscheduled examinations were necessary only in some cases due to suspected complications or unsatisfactory weight loss. First we perform right anterior oblique projections with the patient standing, with a direct preliminary study of the epigastric region to locate the two metal clips. The patient is given barium in small swallows because large amounts might obscure the pouch by filling the fundus of the excluded stomach. Then we take left anterior oblique views to depict the neopylorus and the staple lines, and left posterior oblique projections in lateral and supine recumbency. RESULTS: We observed early postoperative complications such as pseudopylorus edema (8 patients) and intragastric hemorrhage (1 patient), and late complications such as pseudopylorus adherence to the gallbladder bed (1 patient), pseudodiverticular extroversion (1 patient), and axial enlargement (4 patients) of the pouch. The only two cases of staple-line dehiscence were both identified radiographically. In 305 patients with satisfactory weight loss no complications were observed with a pouch size of 45-70 mL (at 1 month) and 58-70 mL (at 12 months). DISCUSSION AND CONCLUSIONS: Upper gastrointestinal radiological studies permit to detect both early and late postoperative complications in Mason's VBG patients. They also provide data on the neostomach and permit to correlate the pouch volume with weight loss.

Incidence and determinants of poststroke dementia as defined by an informant interview method in a hospital-based stroke registry.

BACKGROUND AND PURPOSE: Inconsistent information about incidence and determinants of poststroke dementia might be related to patient attrition, partly because of nonapplicability of formal neuropsychological testing to a large proportion of patients registered in a definite setting. METHODS: Using a proxy-informant interview based on ICD-10 criteria, we determined dementia at stroke onset and 1 year after stroke in the 339 patients who survived, were available for follow-up, and were not demented at stroke onset of 635 patients entered over a 1-year period in a stroke registry taken at 2 community hospitals in Florence, Italy. RESULTS: Of the 339 patients, 57 (16.8%) proved to have poststroke dementia. These patients were older, more frequently female, and more often (multivariate odds ratio, 2.35; 95% CI, 1.21 to 4.58) had atrial fibrillation than those without dementia. Aphasia and the clinical features expressing the severity of the stroke event in the acute phase predicted poststroke dementia. CONCLUSIONS: In a hospital-based nonselected series of stroke survivors, despite the use of a method with low sensitivity for defining dementia, our study confirms that dementia is a frequent sequela of stroke and is mainly predicted by stroke severity. Certain determinants could be controlled in the prestroke phase, thus reducing its risk.

[Surgery of inguinal hernia: our experience]. / Chirurgia dell'ernia inguinale: nostra esperienza.

The study is based on 848 patients that had surgery for inguinal hernia from January 1990 to May 1996. It compares the results obtained using the traditional plastic, called "direct suture", with the results of plastics with prosthesis. A special attention is given to tension-free techniques carried in local anesthesia, undoubtedly considered advantageous because of the technical simplicity in association with the total absence of postoperative complications and relapses, when correctly executed.

In vitro trans-splicing in Saccharomyces cerevisiae.

The interactions established at the 5'-splice site during spliceosome assembly are likely to be important for both precise recognition of the upstream intron boundary and for positioning this site in the active center of the spliceosome. Definition of the RNA-RNA and the RNA-protein interactions at the 5' splice site would be facilitated by the use of a small substrate amenable to modification during chemical synthesis. We describe a trans-splicing reaction performed in Saccharomyces cerevisiae extracts in which the 5' splice site and the 3' splice site are on separate molecules. The RNA contributing the 5' splice site is only 20 nucleotides long and was synthesized chemically. The trans-splicing reaction is accurate and has the same sequence, ATP, and Mg2+ requirements as cis-splicing. We also report how deoxy substitutions around the 5'-splice site affect trans-splicing efficiency.

Specificity of Prp24 binding to RNA: a role for Prp24 in the dynamic interaction of U4 and U6 snRNAs.

Prp24 was previously isolated as a suppressor of a cold-sensitive U4 mutation and is required for at least the first step of splicing in vitro. Our investigation of the in vitro RNA binding properties of the purified Prp24 protein shows that it binds preferentially to the U4/U6 hybrid snRNAs compared to other snRNAs. The interaction between Prp24 and the U4/U6 hybrid appears to involve two regions in the RNA: the 39-57 region of U6 and stem II of the U4/U6 hybrid. Interestingly, some U4 mutations, which destabilize stem II, increase the affinity of Prp24 for the U4/U6 RNAs compared to the wild type. This suggests that the binding of Prp24 to the U4/U6 RNAs may involve some destabilization of the RNA duplex. We also found that Prp24 can stimulate the annealing of U4 and U6, suggesting that Prp24 participates in both the formation and disassembly of the U4/U6 hybrid during splicing.

[The risk factors of atherosclerotic disease. Life style and primary prevention]. / Fattori di rischio della malattia aterosclerotica. Stile di vita e prevenzione primaria.

Atherosclerosis brings about cardiovascular diseases and is the world's largest cause of death. Risk factors are to be considered in their incidence and correlation to various pathologies. Cholesterolaemia, smoking, physical inactivity, alcohol are held to be the most important risk factors. Hypercholesterolaemia involves a greater incidence of coronary heart disease; studies on cholesterol reduction however did not give evidence of any reduction in overall mortality. Alcohol displays an u-shaped relation between levels of assumption and associated mortality, depending on the fact that moderate drinkers show decreasing mortality scores and even a decreasing incidence of cardiovascular diseases (while the curve inflects upwards for serious drinkers). A direct correlation appears instead between both smoking and sedentary life on the one hand and both cardiovascular pathologies and mortality on the other. In order to reduce the risk factors' influence, on the ground of available studies and published trials do the authors offer practical indications. Fats in diet should be less than 30% of total calories (where of 10% saturated, 10% monounsaturated, 10% polyunsaturated); abstention from smoking must be absolute; alcohol should not exceed 15 g/die; some physical activity should be practised for 40-60 minutes, twice a week.

[Crohn's disease of the appendix: a rare nosological entity]. / Il morbo di Crohn dell'appendice: una rara entità nosologica.

In this study, the authors describe a case of Crohn's disease confined to the vermiform appendix out of a total of 1,263 appendectomies performed over the past 10 years in a Clinical Surgery Department (Clinica Chirurgica III) of the Policlinico S. Orsola in Bologna. In agreement with other reports in the scientific literature, the essentially non-specific clinical picture prevents pre-operative identification of the condition, whereas a diagnosis of Crohn's disease can be reached only on the basis of histological characteristics. The extreme rarity of Crohn's disease confined to the appendix is, however, confirmed. Follow-up at 3 years revealed no involvement of other areas of the digestive tract.

hnRNP I, the polypyrimidine tract-binding protein: distinct nuclear localization and association with hnRNAs.

Many hnRNP proteins and snRNPs interact with hnRNA in the nucleus of eukaryotic cells and affect the fate of hnRNA and its processing into mRNA. There are at least 20 abundant proteins in vertebrate cell hnRNP complexes and their structure and arrangement on specific hnRNAs is likely to be important for the processing of pre-mRNAs. hnRNP I, a basic protein of ca. 58,000 daltons by SDS-PAGE, is one of the abundant hnRNA-binding proteins. Monoclonal antibodies to hnRNP I were produced and full length cDNA clones for hnRNP I were isolated and sequenced. The sequence of hnRNP I (59,632 daltons and pI 9.86) demonstrates that it is identical to the previously described polypyrimidine tract-binding protein (PTB) and shows that it is highly related to hnRNP L. The sequences of these two proteins, I and L, define a new family of hnRNP proteins within the large superfamily of the RNP consensus RNA-binding proteins. Here we describe experiments which reveal new and unique properties on the association of hnRNP I/PTB with hnRNP complexes and on its cellular localization. Micrococcal nuclease digestions show that hnRNP I, along with hnRNP S and P, is released from hnRNP complexes by nuclease digestion more readily than most other hnRNP proteins. This nuclease hypersensitivity suggests that hnRNP I is bound to hnRNA regions that are particularly exposed in the complexes. Immunofluorescence microscopy shows that hnRNP I is found in the nucleoplasm but in addition high concentrations are detected in a discrete perinucleolar structure. Thus, the PTB is one of the major proteins that bind pre-mRNAs; it is bound to nuclease-hypersensitive regions of the hnRNA-protein complexes and shows a novel pattern of nuclear localization.

Modeling by homology of RNA binding domain in A1 hnRNP protein.

Eukaryotic nuclear RNA binding proteins share a common sequence motif thought to be implicated in RNA binding. One of the two domains present in A1 hnRNP protein, has been modelled by homology in order to make a prediction of the main features of the RNA binding site. Acylphosphatase (EC 3.6.1.7) was selected as template for the modeling experiment. The predicted RNA binding site is a beta-sheet containing the two RNP consensus sequences as well as lysines and arginines conserved among the family.

Intermittent therapy with high-dose 5-aminosalicylic acid enemas for maintaining remission in ulcerative proctosigmoiditis.

Sixty patients who had presented recently with a relapse of mild to moderate ulcerative colitis with rectosigmoid involvement were randomly assigned to treatment with either 5-aminosalicylic acid enemas (N = 29) or oral sulfasalazine (N = 31). All patients were in remission, which was documented by clinical, histologic, and endoscopic criteria. Five-aminosalicylic acid treatment was administered on an intermittent schedule, consisting of 4 gm daily for the first seven days of each month; sulfasalazine was given as continuous therapy (2 gm daily as oral tablets). The study period was 2 years. Overall, 9 relapses occurred in the 5-aminosalicylic acid group and 12 occurred in the sulfasalazine group. The actuarial relapse rate at 12 months was 20 percent in the 5-aminosalicylic acid group and 24 percent in the sulfasalazine group; at 24 months, these rates were 37 and 43 percent, respectively. The actuarial relapse curves of the two groups were very similar. The relapse severity was also similar between the two groups. These results show that the authors proposed schedule of maintenance treatment with high-dose 5-aminosalicylic acid enemas is effective in subjects with rectosigmoiditis. This form of intermittent therapy may therefore be proposed for maintaining remission in patients who are refractory to oral and/or rectal treatment with sulfasalazine and steroids or who are intolerant or allergic to sulfasalazine. Treatment with 5-aminosalicylic acid enemas for seven days each month can also constitute an alternative for patients who favor the intermittent schedule over the classic continuous regimen of oral administrations.