Ingestion and effects of polystyrene nanoparticles in the silkworm Bombyx mori.

Information on the occurrence and effects of nanoplastics in ecosystems worldwide currently represent one of the main challenges from the ecotoxicological point of view. This is particularly true for terrestrial environments, in which nanoplastics are released directly by human activities or derive from the fragmentation of larger plastic items incorrectly disposed. Since insects can represent a target for these emerging contaminants in land-based community, the aim of this study was the evaluation of ingestion of 0.5 µm polystyrene nanoplastics and their effects in silkworm (Bombyx mori) larvae, a useful and well-studied insect model. The ingestion of nanoplastics, the possible infiltration in the tissues and organ accumulation were checked by confocal microscopy, while we evaluated the effects due to the administered nanoplastics through a multi-tier approach based on insect development and behaviour assessment, as endpoints at organism level, and the measurements of some biochemical responses associated with the imbalance of the redox status (superoxide dismutase, catalase, glutathione s-transferase, reactive oxygen species evaluation, lipid peroxidation) to investigate the cellular and molecular effects. We observed the presence of microplastics in the intestinal lumen, but also inside the larvae, specifically into the midgut epithelium, the Malpighian tubules and in the haemocytes. The behavioural observations revealed a significant (p < 0.05) increase of erratic movements and chemotaxis defects, potentially reflecting negative indirect effects on B. mori survival and fitness, while neither effect on insect development nor redox status imbalance were measured, with the exception of the significant (p < 0.05) inhibition of superoxide dismutase activity.

First evidence of protein modulation by polystyrene microplastics in a freshwater biological model.

Microplastics (MPs) are now one of the major environmental problems due to the large amount released in aquatic and terrestrial ecosystems, as well as their diffuse sources and potential impacts on organisms and human health. Still the molecular and cellular targets of microplastics' toxicity have not yet been identified and their mechanism of actions in aquatic organisms are largely unknown. In order to partially fill this gap, we used a mass spectrometry based functional proteomics to evaluate the modulation of protein profiling in zebra mussel (Dreissena polymorpha), one of the most useful freshwater biological model. Mussels were exposed for 6 days in static conditions to two different microplastic mixtures, composed by two types of virgin polystyrene microbeads (size = 1 and 10 µm) each one. The mixture at the lowest concentration contained 5 × 105 MP/L of 1 µm and 5 × 105 MP/L of 10 µm, while the higher one was arranged with 2 × 106 MP/L of 1 µm and 2 × 106 MP/L of 10 µm. Proteomics' analyses of gills showed the complete lack of proteins' modulation after the exposure to the low-concentrated mixture, while even 78 proteins were differentially modulated after the exposure to the high-concentrated one, suggesting the presence of an effect-threshold. The modulated proteins belong to 5 different classes mainly involved in the structure and function of ribosomes, energy metabolism, cellular trafficking, RNA-binding and cytoskeleton, all related to the response against the oxidative stress.

(+)-and (-)-Phenazocine enantiomers: Evaluation of their dual opioid agonist/σ1 antagonist properties and antinociceptive effects.

cis-N-Substituted N-normetazocine enantiomers possess peculiar pharmacological profiles. Indeed, dextro enantiomers bind with high affinity σ1 receptor while opposite enantiomers bind opioid receptors. In spite of their stereochemistry, cis-N-2-phenylethyl N-normetazocine (phenazocine) enantiomers showed mixed opioid/σ1 receptor profiles and a significant in vivo analgesia. To the best of our knowledge, there is no information available regarding the evaluation of σ1 pharmacological profile in the antinociceptive effects of (+)- and (-)-phenazocine. Therefore, the present study was designed to ascertain this component by in vitro and in vivo studies. In particular, we tested the σ1 affinity of both enantiomers by a predictive binding assay in absence or presence of phenytoin (DPH). Our results showed that DPH (1 mM) did not increase the σ1 receptor affinity of (+)-and (-)-phenazocine (Ki = 3.8 ± 0.4 nM, Ki = 85 ± 2.0 nM, respectively) suggesting a σ1 antagonist profile of both enantiomers. This σ1 antagonistic component of two phenazocine enantiomers was corroborated by in vivo studies in which the selective σ1 receptor agonist PRE-084, was able to unmask their σ1 antagonistic component associated with the opioid activity. The σ1 antagonistic component of (+)- and (-)-phenazocine may justify their analgesic activity and it suggests that they may constitute useful lead compounds to develop new ligands with this dual activity.

Ischemic tolerance modulates TRAIL expression and its receptors and generates a neuroprotected phenotype.

TNF-related apoptosis inducing ligand (TRAIL), a member of the TNF superfamily released by microglia, appears to be involved in the induction of apoptosis following focal brain ischemia. Indeed, brain ischemia is associated with progressive enlargement of damaged areas and prominent inflammation. As ischemic preconditioning reduces inflammatory response to brain ischemia and ameliorates brain damage, the purpose of the present study was to evaluate the role of TRAIL and its receptors in stroke and ischemic preconditioning and to propose, by modulating TRAIL pathway, a new therapeutic strategy in stroke. In order to achieve this aim a rat model of harmful focal ischemia, obtained by subjecting animals to 100 min of transient occlusion of middle cerebral artery followed by 24 h of reperfusion and a rat model of ischemic preconditioning in which the harmful ischemia was preceded by 30 mins of tMCAO, which represents the preconditioning protective stimulus, were used. Results show that the neuroprotection elicited by ischemic preconditioning occurs through both upregulation of TRAIL decoy receptors and downregulation of TRAIL itself and of its death receptors. As a counterproof, immunoneutralization of TRAIL in tMCAO animals resulted in significant restraint of tissue damage and in a marked functional recovery. Our data shed new light on the mechanisms that propagate ongoing neuronal damage after ischemia in the adult mammalian brain and provide new molecular targets for therapeutic intervention. Strategies aimed to repress the death-inducing ligands TRAIL, to antagonize the death receptors, or to activate the decoy receptors open new perspectives for the treatment of stroke.

Design and synthesis of new bifunctional sigma-1 selective ligands with antioxidant activity.

Herein we report the synthesis of new bifunctional sigma-1 (σ1)-selective ligands with antioxidant activity. To achieve this goal, we combined the structure of lipoic acid, a universal antioxidant, with an appropriate sigma aminic moiety. Ligands 14 and 26 displayed high affinity and selectivity for σ1 receptors (Kiσ1 = 1.8 and 5.5 nM; Kiσ2/σ1 = 354 and 414, respectively). Compound 26 exhibited in vivo antiopioid effects on kappa opioid (KOP) receptor-mediated analgesia. In rat liver and brain mitochondria (RLM, RBM), this compound significantly reduced the swelling and the oxidation of thiol groups induced by calcium ions. Our results demonstrate that the tested compound has protective effects against oxidative stress.

New benzomorphan derivatives of MPCB as MOP and KOP receptor ligands.

There is considerable interest in developing KOP Opioid receptor ligands as clinically useful analgesics. Moreover, compounds with mixed KOP receptor and mu-opioid peptide (MOP) receptor agonist/antagonist properties could have a better therapeutic potential. The benzomorphan-based synthetic ligands MPCB and CCB have been shown to bind KOP receptors with high affinity and selectivity. We report here a series of compounds synthesized to perform structure-affinity relationship (SAR) studies on MPCB. The aim of this study was to optimise KOP receptor-ligand interaction and to modulate MOP receptor selectivity. In the benzylamide analogue of MPCB (compound 9) the presence of a third aromatic nucleus, at an appropriate distance and conformation with respect to aromatic pharmacophoric residues, increased KOP receptor affinity by about 6-fold compared to MPCB (Ki = 35 nM and Ki = 240 nM, respectively). Instead, compound 28 with a tertiary amino group in the nitrogen substituent displayed a comparable KOP receptor affinity (Ki = 179 nM) but also high MOP receptor affinity (Ki = 45 nM). Thus, the present study shows that in benzomorphan-based ligands the presence of different functional groups in the nitrogen substituent, ranging from a positive charged amine to an additional aromatic ring, is able to promote the correct aligment of aromatic pharmacophoric residues with MOP and KOP receptor types. Evaluation of docking simulations of compounds 9 and 28 into the KOP and MOP receptor displayed selective ligand interactions with the important amino acid residues Tyr320 (TMVII) and Trp318 (TMVII), respectively.

Detection of levodopa, dopamine and its metabolites in rat striatum dialysates following peripheral administration of L-DOPA prodrugs by mean of HPLC-EC.

A high performance liquid chromatography (HPLC) method was developed to detected simultaneously L-dihydroxyphenylalanine (L-DOPA), dopamine (DA), dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in rat striatum dilaysates following oral administration of L-DOPA or its prodrugs. The chromatographic system uses a reversed-phase C18 column with electrochemical detection at +0.30 V. Mobile phase consisted of 0.05 M citric acid, sodium EDTA 50 microM, sodium octylsulphonate 0.4 nM at pH of 2.9 and 8% methanol (v/v) at a flow rate of 1 ml/min. The calibration curves were linear over the concentration range of 10nm to 100 microM and the lower limits of detections were 125 fmol for L-DOPA, 50 fmol for DOPAC, 250 fmol for DA and 150 fmol for HVA at signal noise to ratio of 3. The repeatability (or intra-day precision), expressed by the relative standard deviation, were better than 4%. The construction of microdialysis probes has been described. The in vitro relative recoveries of each microdialysis probe were evaluated and the results show that they are similar and reproducible for all the analytes with CVs from 1 to 4%. The HPLC-EC method was applied to detect the extracellular levels of L-DOPA, DA, DOPAC and HVA in the striatum dialysates of freely moving rats after oral administration of six new potential L-DOPA prodrugs.

Different in vitro activity of flurbiprofen and its enantiomers on human articular cartilage.

The 2-arylpropionic acid derivatives or 'profens' are an important group of non-steroidal anti-inflammatory drugs that have been used for the symptomatic treatment of various forms of arthritis. These compounds are chiral and the majority of them are still marketed as racemate although it is known that the (S)- form is the principal effective in the cyclooxygenase inhibition. However, recent findings suggest that certain pharmacological effect of 2-arylpropionic acids cannot be attributed exclusively to the (S)-(+) enantiomer. To obtain further insights into the pharmacological effect of profens, the present study investigated the influence of racemic and pure enantiomers of flurbiprofen on the production of nitric oxide and glycosaminoglycans, key molecules involved in cartilage destruction. The culture of human articular cartilage stimulated by interleukin-1beta (IL-1beta), which plays an important role in the degradation of cartilage, has been established, as a profit experimental model, for reproducing the mechanisms involved in the pathophysiology of arthritic diseases. Our results show that mainly (S)-(+)-flurbiprofen decreases, at therapeutically concentrations, the IL-1beta induced cartilage destruction.

High-performance liquid chromatographic method for the quantification of anthranilic and 3-hydroxyanthranilic acid in rat brain dialysate.

Anthranilic acid (ANA) and 3-hydroxyanthranilic acid (3-HANA) have attracted considerable attention as two of the L-tryptophan kynurenine pathway metabolites in the central nervous system. In this study, a highly sensitive and accurate method for the quantification of ANA and 3-HANA has been developed using reversed-phase high performance liquid chromatography (HPLC) with fluorimetric detection. The HPLC assay was carried out using a C(18) column (5 microm, 250 x 4.6 mm i.d.). The mobile phase consisted of a mixture of 25 mM sodium/acetic acid buffer (pH 5.5) and methanol (90:10 v/v). Fluorimetric detection at lambda(ex)=316 nm and lambda(em)=420 nm was used. The assay was applied to the measurement of ANA and 3-HANA acid in rat brain dialysate following administration of L-tryptophan or L-kynurenine. 3-HANA and ANA levels were progressively increased during 90 min following administration of L-tryptophan, then decreased progressively to basal levels. 3-HANA levels were significantly higher than ANA levels after L-kynurenine administration. These findings suggest that the assay developed should provide an improved means for investigation of neurobiology of kynurenine pathway.

Changes in kynurenic, anthranilic, and quinolinic acid concentrations in rat brain tissue during development.

Kynurenic, anthranilic, and quinolinic acid, brain tissue concentrations and indoleamine 2,3-dioxygenase [EC 1 13.11.17] activity were determined in rat brain, during pre- and postnatal development. Quinolinic acid brain tissue concentration was significantly increased at birth as compared with the prenatal level, then it declined rapidly in the postnatal period. By the contrary, kynurenic and anthranilic acids brain tissue concentrations in rat brain were significantly lower at birth as compared with those found prenatally; then kynurenic acid concentration decreased in the first postnatal week and increased thereafter, while anthranilic acid concentration increased in the first postnatal week and decreased thereafter. Indoleamine 2,3-dioxygenase [EC 1 13.11.17] activity were found unchanged in pre and post natal rat brain. The described opposite changes in quinolinic and kynurenic acids concentrations, occurring in pre- and postnatal period, despite the lack of knowledge on the precise role played by these compounds on the different neurotransmitter systems in the brain, could be involved in brain ontogenetic development.

How physicians diagnose urinary tract infections: the potential influence of laboratory regulations on test availability and use.

OBJECTIVE: Physician office laboratory regulations may decrease test availability. We examined the potential effects of regulations on test availability and whether the use of tests in diagnosing uncomplicated urinary tract infections is related to availability. STUDY DESIGN: We performed an analysis of a cross-sectional survey conducted in 1994 and 1995. Test availability and use were determined by physicians' reports. POPULATION: The survey respondents included practicing physicians in 3 specialties (family medicine, general internal medicine, and obstetrics and gynecology) from 4 states: Pennsylvania (which had longstanding office laboratory regulations), and Alabama, Minnesota, and Nebraska (states that were not regulated until the implementation of the Clinical Laboratory Improvement Amendment of 1988). OUTCOMES MEASURED: We determined whether 4 specific tests were available in the office and how the tests were used to diagnose uncomplicated urinary tract infections. RESULTS: Our analysis was based on the responses from the 1898 respondents to the survey. All tests were less commonly available in Pennsylvania; this included the dipstick, microscopic urinalysis, wet prep, and urine culture (odds ratio [OR]=0.20-0.34; all P values < .05). The availability of the microscopic urinalysis and culture increased their use (OR = 4.37 and 2.03, respectively; P=.001). The availability of microscopic urinalysis was associated with a decrease in ordering urine cultures (OR=0.42; P=.001), and the availability of the dipstick was associated with a decrease in the use of both the microscopic urinalysis (OR=0.36; P=.02) and the culture (OR=0.48; P=.05). CONCLUSIONS: We found lower test availability in the state with office laboratory regulations and a decrease in testing when availability is reduced, suggesting that laboratory regulations may influence physicians&rsquo diagnostic approach to urinary tract infections. Further study will be required to determine the level of testing that maximizes patient welfare.

Studies of enantiomerization of chiral 3,4-dihydro-1,2,4-benzothiadiazine 1,1-dioxide type compounds.

An on-column HPLC procedure using a chiral stationary phase (CSP) was developed for the determination of rate constants and free energy barriers of enantiomerization of (+/-)IDRA21. Subsequently, the HPLC method was applied for investigation of two structurally related chiral compounds. The individual enantiomers of the studied compounds were isolated in parallel by preparative HPLC and rate constants and free energy barriers of enantiomerization were determined in different solvents. The on-column enantiomerization data revealed that CSP induces different rate constants for the two enantiomers. The results generated off-line were used to determine the influence of solvents on the racemization of (+) and (-) IDRA21 and to gain further insight into the enantiomerization mechanism of chiral 3,4-dihydro-1,2,4-benzothiadiazine 1,1-dioxide type compounds.

Withdrawal of chronic systemic corticosteroids in patients with COPD: a randomized trial.

The benefits of chronic systemic corticosteroids for patients with chronic obstructive pulmonary disease (COPD) are not well established. To determine whether chronic corticosteroid treatment can be safely withdrawn in "steroid-dependent""COPD patients, we performed a double-blind, placebo-controlled study of 38 patients with steroid-dependent COPD. Patients were randomly assigned to receive their usual maintenance prednisone dose for 6 mo (continuous group) or to be withdrawn from prednisone at a rate of 5 mg per week (demand group). The number of COPD exacerbations per patient (primary outcome) was 2.5 +/- 2.7 (mean +/- SD) in the continuous group and 2.7 +/- 2.5 in the demand group (p = 0.60, 95% confidence interval for the difference: -1.1 to 1.7). Spirometric results, dyspnea, and health-related quality of life did not differ significantly in the two groups. The average daily corticosteroid dose was 10.7 +/- 5.2 mg in the continuous group and 6.3 +/- 6.4 mg in the demand group (p = 0.003). Weight decreased in the demand group by 4.8 +/- 2.0 kg, compared with an increase in the continuous group of 0.5 +/- 3.5 kg (p = 0.007). Discontinuation of chronic systemic corticosteroid treatment in steroid-dependent COPD patients did not cause a significant increase in COPD exacerbations, but did reduce total systemic corticosteroid use and body weight. Larger studies may be warranted to establish the relative risks and benefits of chronic corticosteroid treatment of patients with COPD.

Chiral resolution of the enantiomers of 7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide using high-performance liquid chromatography on cellulose-based chiral stationary phases.

Analytical high-performance liquid chromatography (HPLC) methods using derivatized cellulose chiral stationary phases (CSPs) were developed for the separation of the enantiomers of 7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide ((+/-) IDRA21). In previous studies, (+/-) IDRA21 has been found to have an interesting inhibitory effect on the desensitization of alpha-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropanoic acid (AMPA) receptor and improve cognition in animals. This compound possess one chiral carbon atom, but very little information has been reported on the stereoselectivity of his activity. Therefore resolution of the enantiomers of this compound and subsequent identification of stereospecificity in his pharmacological actions are clearly matters of interest. The resolution were made under normal- and reversed-phase conditions using a mobile phase consisting of n-hexane:2-propanol (70/30, v/v) and water:acetonitrile (60/40, v/v) respectively, and a CSP of silica-based cellulose tris-3,5-dimethyl-phenylcarbamate (Chiralcel OD and Chiracel OD-R). The enantiomeric nature of eluates was confirmed by circular dichroism (CD) spectra. A baseline separation (R(S) > 1.5) was obtained in both cases. Furthermore the isolation of optical isomers of (+/-) IDRA21 was performed using a semipreparative column packed with the same cellulose OD CSP.

Variation by specialty in the treatment of urinary tract infection in women.

To determine practicing physicians' strategies for diagnosing and managing uncomplicated urinary tract infection, we surveyed physicians in general internal medicine, family practice, obstetrics and gynecology, and emergency medicine in four states. Responses differed significantly by respondents' specialty. For example, nitrofurantoin was the antibiotic of first choice for 46% of obstetricians, while over 80% in the other specialties chose trimethoprim-sulfamethoxazole. Most surveyed said they do not usually order urine culture, but the percentage who do varied by specialty. Most use a colony count of 10(5) colony-forming units or more for diagnosis although evidence favors a lower threshold, and 70% continue antibiotic therapy even if the culture result is negative. This survey found considerable variation by specialty and also among individual physicians regarding diagnosis and treatment of urinary tract infection and also suggests that some of the new information from the literature has not been translated to clinical practice.

Physician risk assessment and APACHE scores in cardiac care units.

BACKGROUND: The need to correct outcome data for case mix is well recognized, but risk assessment for coronary care unit (CCU) patients remains problematic. HYPOTHESIS: This study determined the feasibility of using physicians' opinions to predict mortality for CCU patients and compared their results to Acute Physiology and Chronic Health Evaluation II (APACHE II) scores. METHODS: A prospective observational study was performed on consecutive patients admitted to a university-affiliated Veterans Affairs Medical Center CCU over a 2-month period. Physician assessment of likely mortality during hospitalization, obtained using an MD Prognosis Score ranging from 1 (best) to 7 (worst), was compared with APACHE II scores. RESULTS: MD Prognosis Scores were obtained on 122 of the 237 eligible patients (51% response rate) and averaged 2.3 +/- 1.4 (mean +/- standard deviation). APACHE II scores on these patients averaged 9.9 +/- 4.8 (range 2-29) with very poor correlation between the two methods (r = 0.3). Of the four patients who died, three had MD prognosis scores of 7. None of the survivors had scores of 7 and only three had scores of 6. APACHE II did not predict a high likelihood that any of the patients would die (none with > 90% likelihood of mortality). CONCLUSIONS: APACHE scores are inadequate for cardiac patients. Although physicians can identify CCU patients most likely to die, reliance on physician scoring systems is limited by difficulties in obtaining their opinion. A new method of risk assessment for acutely ill cardiac patients is needed if CCU outcomes are to be compared across institutions.

Central and peripheral involvement of endogenous opioid peptides in gastric protection in stressed rats.

The effects of intraperitoneal and intracerebroventricular administration of the inhibitor of endopeptidase EC 24.11 (enkephalinase), thiorphan, and the synthetic enkephalin analogue [D-Ala2-Met5]enkephalinamide (DALA) were investigated in cold-restraint-stressed rats. Drugs were administered alone or after pretreatment with naloxone or naloxone methiodide given 20 min prior to the drugs. Thiorphan and DALA, administered centrally (4 micrograms i.c.v./rat) or peripherally (400 micrograms/kg), induced a significant gastric protection. Prior treatment with naloxone s.c. (1 mg/kg) inhibited the effect induced by i.c.v. or i.p. injections of thiorphan or DALA. In contrast, s.c. administration of naloxone methiodide (1 mg/kg) did not affect the response induced by central administration of thiorphan or DALA, but was able to prevent that of thiorphan or DALA when they were administered i.p. These data strongly support the hypothesis of a central and peripheral involvement of endogenous opioid peptides in gastric protection in stressed rats.

Training internal medicine residents in the community: the Minnesota experience.

The University of Minnesota Internal Medicine Residency Program has developed ambulatory general medicine rotations in rural community settings and urban managed care settings in Minnesota. Based on what had been learned from community focus groups, from discussions with residents about what they perceived to be training holes in the traditional curriculum, and from resident evaluations of pilot rotations, an educational framework for the rotations was established. The authors describe the process of developing these rotations, their educational rationale and objectives, the structure of the rotations, teaching strategies, faculty development, and the evaluation system, and outline the important elements of the successful implementation of these new rotations. The community practice setting has offered a unique, important, and previously untapped resource for residency training, and the community rotations have been highly valued both by housestaff and by community preceptors. As residency programs begin to offer more community-based ambulatory care opportunities for their trainees, the impact of this trend on quality of training, residents' career choices, and patient outcomes will need to be evaluated.

The kaleidoscope of general internist careers: a challenge for internal medicine training.

As internal medicine residency programs struggle to produce general internists in greater numbers and assure that they are adequately prepared for practice, it is imperative that the graduate medical education system have a clear picture of what competencies will be expected of those entering general internist careers. Feedback from the practicing community and large managed care organizations in Minnesota has made it clear that general internists in that state are functioning in a variety of positions requiring a wide range of skills depending on the practice description, choice of practice setting, and the complement of other primary care providers. General internists functioning in nontraditional careers have special curricular needs. It is imperative that training programs constantly monitor the changing practice environment and stay current on the variety of new generalist career choices to adequately prepare their residents for generalist careers. The graduate medical education enterprise needs to be involved in determining the best teaching strategies for the broad range of ambulatory general medicine competencies and in determining how best to preserve the richness of the medical subspecialty experience critical to the training of excellent general internists.