Analysis of human biological samples using porous graphitic carbon columns and liquid chromatography-mass spectrometry: a review.

Liquid chromatography-mass spectrometry (LC-MS) has emerged as a powerful analytical technique for analyzing complex biological samples. Among various chromatographic stationary phases, porous graphitic carbon (PGC) columns have attracted significant attention due to their unique properties-such as the ability to separate both polar and non-polar compounds and their stability through all pH ranges and to high temperatures-besides the compatibility with LC-MS. This review discusses the applicability of PGC for SPE and separation in LC-MS-based analyses of human biological samples, highlighting the diverse applications of PGC-LC-MS in analyzing endogenous metabolites, pharmaceuticals, and biomarkers, such as glycans, proteins, oligosaccharides, sugar phosphates, and nucleotides. Additionally, the fundamental principles underlying PGC column chemistry and its advantages, challenges, and advances in method development are explored. This comprehensive review aims to provide researchers and practitioners with a valuable resource for understanding the capabilities and limitations of PGC columns in LC-MS-based analysis of human biological samples, thereby facilitating advancements in analytical methodologies and biomedical research.

Enrichment and quantification of 18 polycyclic aromatic hydrocarbons from intermediates for plastics production by a generic liquid chromatography column switching.

The polycyclic aromatic hydrocarbon concentration in plastic products is regulated in (European Union) No. 1272/2013. However, this only covers the end products and not intermediate substances. Therefore, a generic method was developed to analyze the polycyclic aromatic hydrocarbons listed by the Environmental Protection Agency and the European Union. This method is based on direct large volume injection from solutions of plastic additives followed by liquid chromatography coupled to fluorescence detection. The additives Irganox 1010, ureido methacrylate, and cetyl methacrylate 1618F were used as examples for method development. Two serially coupled columns allowed the matrix to be removed on the first column and the analytes to be separated on the second column. The columns were connected by an intermediate valve. The valve allowed the matrix to be diverted after the first column and water to be dosed upstream of the second column via an additional pump. This allowed samples in aqueous or organic media to be focused at the column head. An injection volume of 100 µl and online aqueous dilution of 1:3 led to a limit of detection below 1 ng/ml for 15 polycyclic aromatic hydrocarbons. Moreover, concentrations between 1.6 and 10.3 ng/ml were found in the three plastic additives.

Dissolution of adhesive resins present in plastic waste to recover polyolefin by sink-float separation processes.

This study investigated the dissolution of adhesive resins present in polyolefin films that cause plastic materials to adhere to each other. The process of dissolution was made by the use of ethyl acetate and followed by separation through the sink-float process. The objective was to separate and characterize polyolefin films from plastic solid waste derived from recycled post-consumer paper. Through these procedures, 6% polyethylene of high-density (HDPE), 14% polyethylene of low-density (LDPE) and 39% polypropylene (PP) were separated and recovered from plastic waste. Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analyzes (TGA), differential scanning calorimetry (DSC), and dynamic mechanical analysis (DMA) were conducted to determine the chemical, thermal and mechanical properties of the recovered polymers and to establish a comparison with standard commercial polymers. It demonstrated that recovered material kept their chemical, thermal, and mechanical properties. This process indicates possible economic viability considering the demand, the market value of the PP, and the required investment to be implemented in the recycling process that could be amortized in a short period of time. Moreover, the organic solvent used in the dissolution process can be easily recovered by distillation.

Development of a column switching for direct online enrichment and separation of polar and nonpolar analytes from aqueous matrices.

Trace substances in surface waters may threaten health and pose a risk for the aquatic environment. Moreover, separation and detection by instrumental analysis is challenging due to the low concentration and the wide range of polarities. Separation of polar and nonpolar analytes can be achieved by using stationary phases with different selectivity. Lower limits of detection of trace substances can be obtained by offline enrichment on solid phase materials. However, these practices require substantial effort and are time consuming and costly. Therefore, in this study, a column switching was developed to enrich and separate both polar and nonpolar analytes by an on-column large volume injection of aqueous samples. The column switching can significantly reduce the effort and time for analyzing trace substances without compromising on separation and detection. A reversed phase (RP) column is used to trap the nonpolar analytes. The polar analytes are enriched on a porous graphitized carbon column (PGC) coupled serially behind the RP column. A novel valve switching system is implemented to enable elution of the nonpolar analytes from the RP column and, subsequently, elution of polar analytes from the PGC column and separation on a hydrophilic interaction liquid chromatography (HILIC) column. To enable separation of polar analytes dissolved in an aqueous matrix by HILIC, the water plug that is flushed from the PGC column is diluted by dosing organic solvent directly upstream of the HILIC column. The developed method was tested by applying target analysis and non-target screening, highlighting the advantage to effectively separate and detect both polar and nonpolar compounds in a single chromatographic run. In the target analysis, the analytes, with a logD at pH 3 ranging from -2.8 to + 4.5, could be enriched and separated. Besides the 965 features in the RP phase, 572 features from real wastewater were observed in the HILIC phase which would otherwise elute in the void time in conventional one-dimensional RP methods.

Illicit drugs in Brazil: environmental consequences and consumption patterns.

This article reviews the literature on the consumption, street drug analysis, distribution, and main environmental impacts of illicit drugs in Brazil and analyzes the III National Survey on Drug Use by the Brazilian Population. The literature review is based on articles published in national and international journals between 2018 and 2023. This review consists of two analyses, the first of which addresses publications from the last 6 years on the monitoring of illicit drugs in Brazil and a second analysis based on the III National Survey on Drug Use that addresses the different possibilities of contact with drugs. The results revealed that the Southeast region of Brazil has the highest number of studies on the subject, especially in the state of São Paulo, while the North and Northeast regions have the lowest number of studies. The Midwest regions only have studies in the federal capital city, Brasília, while no studies were found in states bordering countries that produce illicit drugs, such as Paraguay and Bolivia. Analytical methods that use the concept of miniaturization, green chemistry, and the adoption of acceptance methods are frequent in most articles. Chemometric and statistical tools are widely used for the analysis, development, and conclusion of identification and quantification methods. Among the articles studied, there was a predominance in the analysis of cocaine metabolites and cannabis metabolites in the aquatic environment, where their concentrations ranged from 0.01 to 2000 ng L-1. Studies also reported bioaccumulation in marine biota with concentrations of up to 4.58 µg kg-1 for mussels and sediments, posing a risk to algae, crustaceans, and fish. Furthermore, the data show that the consumption of illicit drugs is increasing in Brazil, especially among young people.

An overview of New Psychoactive Substances (NPS) in northeast Brazil: NMR-based identification and analysis of ecstasy tablets by GC-MS.

The actual illicit market for synthetic drugs is characterized by a wide variety of psychoactive substances of different chemical and pharmacological classes, such as amphetamine-type stimulants and new psychoactive substances. The knowledge about its chemical composition, as well as the nature and quantity of the active substances present, is important for emergency care in intoxication cases by these substances and to establish adequate chemical and toxicological analysis procedures in forensic laboratories. The aim of this work was to study the prevalence of amphetamine-type stimulants and new psychoactive substances in the states of Bahia and Sergipe, in the northeast region of Brazil, involving samples of drugs seized by the local police forces from 2014 to 2019. In a total of 121 seized and analyzed samples, in which ecstasy tablets predominated (n = 101), nineteen substances were identified using GC-MS and 1D NMR techniques, comprising classical synthetic drugs and new psychoactive substances (NPS). In order to determine the composition of ecstasy tablets, an analytical method based on GC-MS was applied after validation. Analyzes of 101 ecstasy tablets showed that MDMA was the main substance, being found in 57% of the samples, in amounts between 27.3 and 187.1 mg per tablet. In addition, mixtures of MDMA, MDA, synthetic cathinones and caffeine were observed in 34 samples. These results demonstrate that the variety of substances found and the composition of seized materials in northeast Brazil is similar to other studies carried out previously in other Brazilian regions.

A green method for the determination of illicit drugs in wastewater and surface waters-based on a semi-automated liquid-liquid microextraction device.

Liquid-phase microextraction (LPME) is a simple, low-cost, and eco-friendly technique that enables the detection of trace concentrations of organic contaminants in water samples. In this work, a novel customized microextraction device was developed for the LPME extraction and preconcentration of nine illicit drugs in surface water and influent and effluent wastewater samples, followed by analysis by GC-MS without derivatization. The customized device was semi-automated by coupling it with a peristaltic pump to perform the collection of the upper layer of the organic phase. The extraction parameters affecting the LPME efficiency were optimized. The optimized conditions were: 100 µL of a toluene/DCM/EtAc mixture as extractor solvent; 30min of extraction time under vortex agitation (500rpm) and a solution pH of 11.6. The limits of detection and quantification ranged from 10.5ng L-1 (ethylone) to 22.0ng L-1 (methylone), and from 34.9ng L-1 to 73.3ng L-1 for these same compounds, respectively. The enrichment factors ranged from 39.7 (MDMA) to 117 (cocaethylene) and the relative recoveries ranged from 80.4% (N-ethylpentylone) to 120% (cocaine and cocaine-d3). The method was applied to real surface water, effluent, and influent wastewater samples collected in Salvador City, Bahia, Brazil. Cocaine was the main drug detected and quantified in wastewater samples, and its concentration ranged from 312ng L-1 to 1,847ng L-1. Finally, the AGREE metrics were applied to verify the greenness of the proposed method, and an overall score of 0.56 was achieved, which was considered environmentally friendly.

Worker honeybee brain proteome.

A large-scale mapping of the worker honeybee brain proteome was achieved by MudPIT. We identified 2742 proteins from forager and nurse honeybee brain samples; 17% of the total proteins were found to be differentially expressed by spectral count sampling statistics and a G-test. Sequences were compared with the EuKaryotic Orthologous Groups (KOG) catalog set using BLASTX and then categorized into the major KOG categories of most similar sequences. According to this categorization, nurse brain showed increased expression of proteins implicated in translation, ribosomal structure, and biogenesis (14.5%) compared with forager (1.8%). Experienced foragers overexpressed proteins involved in energy production and conversion, showing an extensive difference in this set of proteins (17%) in relation to the nurse subcaste (0.6%). Examples of proteins selectively expressed in each subcaste were analyzed. A comparison between these MudPIT experiments and previous 2-DE experiments revealed nine coincident proteins differentially expressed in both methodologies.

Adaptation to stress in football athletes: The importance of cognitive appraisal.

This study analysed the adaptation of football athletes to competitive stressors regarding the upcoming match. For that, the study adopted a cross-sectional methodology using a critical incident approach. The participants were 352 young male football athletes, aged between 15 and 19 years (M = 16.91, SD = 0.99), who were competing in the national football championship. The results indicated that cognitive appraisal partially mediated the relationship between competitive stressors and emotions: athletes who perceived stressors as a challenge, tended to feel more control over the situation and more resourceful (coping perception), leading to a more positive emotional experience, while those perceiving the stressors as a threat were more prone to experience less control and more negative emotions. This mediation model was moderated by athletes' competitive level (U17 or U19), as the role of challenge perception was more pronounced in the U19 team, while the relationship between threat perception and less control was only observed for the U17 team. In sum, the data reveals the importance of cognitive appraisal in young football athletes' adaptation to competitive stressors, bolstering the theoretical models in this area and the importance of psychologists to consider these variables during intervention, particularly cognitive appraisal.

Chitosan as a matrix of nanocomposites: A review on nanostructures, processes, properties, and applications.

Chitosan is a biopolymer that is natural, biodegradable, and relatively low price. Chitosan has been attracting interest as a matrix of nanocomposites due to new properties for various applications. This study presents a comprehensive overview of common and recent advances using chitosan as a nanocomposite matrix. The focus is to present alternative processes to produce embedded or coated nanoparticles, and the shaping techniques that have been employed (3D printing, electrospinning), as well as the nanocomposites emerging applications in medicine, tissue engineering, wastewater treatment, corrosion inhibition, among others. There are several reviews about single chitosan material and derivatives for diverse applications. However, there is not a study that focuses on chitosan as a nanocomposite matrix, explaining the possibility of nanomaterial additions, the interaction of the attached species, and the applications possibility following the techniques to combine chitosan with nanostructures. Finally, future directions are presented for expanding the applications of chitosan nanocomposites.

Development and Validation of a Method for Quantification of 28 Psychotropic Drugs in Postmortem Blood Samples by Modified Micro-QuEChERS and LC-MS-MS.

The development of new sample preparation alternatives in analytical toxicology leading to quick, effective, automated and environmentally friendly procedures is growing in importance. One of these alternatives is the QuEChERS, originally developed for the analysis of pesticide residues, producing cleaner extracts than liquid-liquid extraction, and easier separation of aqueous and organic phases. However, there are few published studies on the miniaturization of this technique for forensic toxicology, especially in postmortem analysis. We developed and validated a modified micro-QuEChERS and LC-MS-MS assay to quantify 16 antidepressants, 7 antipsychotics and 3 metabolites and semi-quantify norfluoxetine and norsertraline in postmortem blood. The calibration curve was linear from 1 to 500 ng/mL, achieved an r > 0.99, with all standards quantifying within ±15% of target except ±20% at the limit of quantification of 1 ng/mL for 26 substances. The F test was applied to evaluate if the variance between replicates remained constant for all calibrators. Six weighting factors were analyzed (1/x, 1/x2, 1/x0,5, 1/y, 1/y2 and 1/y0,5), with the weighting factor with the lowest sum of residual regression errors (1/x2) selected. No endogenous or exogenous interferences were observed. Method imprecision and bias were <19.0% and 19.7%, respectively. Advantages of this method include a low sample volume of 100 µL, simple but effective sample preparation and a rapid 8.5-min run time. The validated analytical method was successfully applied to the analysis of 100 authentic postmortem samples.

(E)-2-styrylchromones as potential anti-norovirus agents.

Human noroviruses (NoV) are now recognized as the most frequent cause of outbreaks and sporadic cases of acute gastroenteritis. Despite the significant economic impact and considerable morbidity of norovirus disease, no drug or vaccine is currently available to treat or prevent this disease, therefore the discovery of anti-norovirus drugs is urgent. In the present work, a total of 12 structure related chromone and (E)-2-styrylchromones were evaluated for their potential anti-norovirus activity using the murine norovirus (MNV) as a surrogate model for human NoV. From the 12 compounds studied, six (E)-2-styrylchromones were found to have with interesting anti-norovirus activity. The best compounds of the series were (E)-5-hydroxy-2-styrylchromone and (E)-4'-methoxy-2-styrylchromone with an IC(50) approximately 7muM. A first insight into the mechanism of action of these compounds was possible. An interesting relationship between the anti-norovirus activity and the chemical structure was observed. The present study points out that the (E)-2-styrylchromones skeleton is an important one which deserves to be developed and further explored as new antiviral drugs against NoV.

Staphylococcus aureus mastitis in a white-eared opossum (Didelphis albiventris) in the Atlantic Forest of northeast Brazil.

This is the first brief communication of clinical mastitis caused by Staphylococcus aureus occurring in a white-eared opossum (Didelphis albiventris) captured in a remnant of the Atlantic Forest in northeast Brazil. The bacterium was cultured from a milk sample by using sheep blood and Levine agars. Phenotyping of S aureus was performed with coagulase biochemical tests, DNase agar, mannitol agar, and anaerobic glucose and mannitol fermentation.

Acellular Human Amniotic Membrane Scaffold with 15d-PGJ2 Nanoparticles in Postinfarct Rat Model.

The difficulty in the regeneration of cardiomyocytes after myocardial infarction is a major cause of heart failure. Together, the amniotic membrane and 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) can help in the recovery of cardiomyocyte, as they present many growth factors and anti-inflammatory effect, respectively. The objective of this study is to compare the efficacy of Human Decellularized Amniotic Membrane Scaffold (AHAS) loaded with 15d-PGJ2 in improving ventricular function in a rat model of postinfarct ventricular dysfunction. Myocardial infarction was induced in 24 rats by left coronary occlusion. After a week, the animals were subjected to echocardiography for evaluation of left ventricle ejection fraction (LVEF), left ventricle end diastolic volume (LVEDV), and left ventricle end systolic volume (LVESV). Animals with ejection fraction <40% were included in the study and were randomized into three groups: control (n = 8), AHAS (n = 8) and AHAS +15d-PGJ2 (n = 8). In the AHAS group only the membrane was implanted, whereas in the AHAS +15d-PGJ2 the membrane +15d-PGJ2 was implanted on myocardial infarction. Echocardiographic evaluation was performed after 1 month. For histological analysis, heart tissue was stained with Gomori trichome, Sirius Red, the antibody against CD31 and connexin 43 (Cx43). There were no significant differences in the baseline LVEF, LVEDV, and LVESV in all groups. After 1 month, ejection fraction decreased in the control group but increased in the AHAS group and in the AHAS +15d-PGJ2 group in comparison with the control group. The LVEDV and LVESV in the AHAS and AHAS +15d-PGJ2 groups decreased compared with the control group, featuring a ventricular antiremodeling effect. Histopathology of the infarcted area identified the reduction of infarct size and collagen type 1 in the AHAS and AHAS +15d-PGJ2 groups. New blood vessels and cardiomyocytes have been identified in an infarcted area by CD31 and Cx43. AHAS +15d-PGJ2 provided an increase in the ejection fraction and prevented ventricular dilation in this postinfarction ventricular dysfunction model. Impact Statement Our study demonstrated reduction of myocardial fibrosis, proliferation of cardiomyocytes and increase in ejection fraction in rats after experimental acellular amniotic membrane scaffold (AHAS) carrying nanoparticles of 15d-PGJ2 scaffold engraftment in infarcted myocardium. AHAS grafts facilitated colonization of fibrotic myocardium regions with new contractile cells, in addition to preventing reduction of left ventricle wall thickness. This contribution is theoretically and practically relevant as current literature describes experimental studies performed on cardiac ischemic models which present conflicting results concerning cell types used in a research model.

A study on electrocardiographic changes secondary to the use of tricyclic antidepressants in patients with chronic pain.

BACKGROUND AND OBJECTIVES: Tricyclic antidepressants (TCAs) are widely used as analgesics in chronic lumbar pain and neuropathic pain. The objective of this study was to evaluate the electrocardiographic changes in patients with chronic pain treated with amitriptyline or imipramine. METHODS: Forty patients, ages 26 to 81 years (57.27 +/- 13.65 years) of both genders (female 19, male 21), with neuropathic syndromes (lumbosciatalgia, postlaminectomy syndromes, and postherpetic neuritis, among others) participated in this study; 60% had cardiovascular diseases; 30% had changes in the ECG (RBBB, LBBB, first-degree AVB, LAHB, or PVCs). Three ECGs were done in each patient: one ECG was done before beginning treatment, and 30 and 60 days after beginning treatment evaluating PR, QRS, QT QTc, DQT DQTc, and HR. Thirty-two patients were on amitriptyline and eight on imipramine. The mean dose at the end of the study was 54.29 mg of amitriptyline and 46.87 mg of imipramine. RESULTS: Analysis of electrocardiographic parameters after the use of TCAs showed that amitriptyline caused a transitory increase in heart rate in females (p = 0.049), and the duration of the QRS in patients 60 years or older and patients with cardiopathies (p = 0.01). In patients who received 75 mg of amitriptyline, the QTc interval was greater when compared to that of patients who received 25 mg of the drug (p = 0.0044). The increase in those parameters demonstrated the effects of amitriptyline on cardiac conduction; however, clinical compromise was not seen, since they remained within normal limits (QRS < 110 msec and QTc < 470 msec). CONCLUSIONS: The chronic use of TACs proved to be safe and effective, and it did not show changes in cardiac conduction with clinical repercussion.

Analytical quantification, intoxication case series, and pharmacological mechanism of action for N-ethylnorpentylone (N-ethylpentylone or ephylone).

Synthetic cathinones continue to proliferate in clandestine drug markets worldwide. N-ethylnorpentylone (also known as N-ethylpentylone or ephylone) is a popular emergent cathinone, yet little information is available about its toxicology and pharmacology. Here we characterize the analytical quantification, clinical presentation, and pharmacological mechanism of action for N-ethylnorpentylone. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was used to quantify N-ethylnorpentylone in blood obtained from human cases. Clinical features exhibited by the intoxicated individuals are described. The activity of N-ethylnorpentylone at plasma membrane transporters for dopamine (DAT), norepinephrine (NET) and 5-HT (SERT) was assessed using in vitro assays measuring uptake inhibition and evoked release of [3 H] neurotransmitters in rat brain synaptosomes. Our LC-MS/MS method assayed N-ethylnorpentylone concentrations with limits of detection and quantification of 1 and 5 ng/mL, respectively. Quantitation was linear from 5 to 500 ng/mL, and the method displayed specificity and reproducibility. Circulating concentrations of N-ethylnorpentylone ranged from 7 to 170 ng/mL in clinical cases, and the associated symptoms included palpitations, tachycardia, agitation, hallucinations, coma and death. N-Ethylnorpentylone was a potent inhibitor at DAT (IC50  = 37 nM), NET (IC50  = 105 nM) and SERT (IC50  = 383 nM) but displayed no transporter releasing activity. We present a validated method for quantifying N-ethylnorpentylone in human case work. The drug is a psychomotor stimulant capable of inducing serious cardiovascular and neurological side-effects which can be fatal. In vitro findings indicate that N-ethylnorpentylone exerts its effects by potent blockade of DAT and NET, thereby elevating extracellular levels of dopamine and norepinephrine in the brain and periphery.

Characterization of a beta-glucanase produced by Rhizopus microsporus var. microsporus, and its potential for application in the brewing industry.

BACKGROUND: In the barley malting process, partial hydrolysis of beta-glucans begins with seed germination. However, the endogenous 1,3-1,4-beta-glucanases are heat inactivated, and the remaining high molecular weight beta-glucans may cause severe problems such as increased brewer mash viscosity and turbidity. Increased viscosity impairs pumping and filtration, resulting in lower efficiency, reduced yields of extracts, and lower filtration rates, as well as the appearance of gelatinous precipitates in the finished beer. Therefore, the use of exogenous beta-glucanases to reduce the beta-glucans already present in the malt barley is highly desirable. RESULTS: The zygomycete microfungus Rhizopus microsporus var. microsporus secreted substantial amounts of beta-glucanase in liquid culture medium containing 0.5% chitin. An active protein was isolated by gel filtration and ion exchange chromatographies of the beta-glucanase activity-containing culture supernatant. This isolated protein hydrolyzed 1,3-1,4-beta-glucan (barley beta-glucan), but showed only residual activity against 1,3-beta-glucan (laminarin), or no activity at all against 1,4-beta-glucan (cellulose), indicating that the R. microsporus var. microsporus enzyme is a member of the EC 3.2.1.73 category. The purified protein had a molecular mass of 33.7 kDa, as determined by mass spectrometry. The optimal pH and temperature for hydrolysis of 1,3-1,4-beta-glucan were in the ranges of 4-5, and 50-60 degrees C, respectively. The Km and Vmax values for hydrolysis of beta-glucan at pH 5.0 and 50 degrees C were 22.39 mg.mL-1 and 16.46 mg.min-1, respectively. The purified enzyme was highly sensitive to Cu+2, but showed less or no sensitivity to other divalent ions, and was able to reduce both the viscosity and the filtration time of a sample of brewer mash. In comparison to the values determined for the mash treated with two commercial glucanases, the relative viscosity value for the mash treated with the 1,3-1,4-beta-glucanase produced by R. microsporus var. microsporus. was determined to be consistently lower. CONCLUSION: The zygomycete microfungus R. microsporus var. microsporus produced a 1,3-1,4-beta-D-glucan 4-glucanhydrolase (EC 3.2.1.73) which is able to hydrolyze beta-D-glucan that contains both the 1,3- and 1,4-bonds (barley beta-glucans). Its molecular mass was 33.7 kDa. Maximum activity was detected at pH values in the range of 4-5, and temperatures in the range of 50-60 degrees C. The enzyme was able to reduce both the viscosity of the brewer mash and the filtration time, indicating its potential value for the brewing industry.

Human Adipose-Derived Mesenchymal Stem Cells Cryopreservation and Thawing Decrease α4-Integrin Expression.

Aim. The effects of cryopreservation on adipose tissue-derived mesenchymal stem cells are not clearly documented, as there is a growing body of evidence about the importance of adipose-derived mesenchymal stem cells for regenerative therapies. The aim of this study was to analyze human adipose tissue-derived mesenchymal stem cells phenotypic expression (CD34, CD45, CD73, CD90, CD105, and CD49d), colony forming unit ability, viability, and differentiation potential before and after cryopreservation. Materials and Methods. 12 samples of the adipose tissue were collected from a healthy donor using the liposuction technique. The cell isolation was performed by enzymatic digestion and then the cells were cultured up to passage 2. Before and after cryopreservation the immunophenotype, cellular viability analysis by flow cytometer, colony forming units ability, differentiation potential into adipocytes and osteoblasts as demonstrated by Oil Red O and Alizarin Red staining, respectively. Results. The immunophenotypic markers expression was largely preserved, and their multipotency was maintained. However, after cryopreservation, the cells decreased α4-integrin expression (CD49d), cell viability, and number of colony forming units. Conclusions. These findings suggest that ADMSC transplanted after cryopreservation might compromise the retention of transplanted cells in the host tissue. Therefore, further studies are warranted to standardize protocols related to cryopreservation to attain full benefits of stem cell therapy.

Primary structure, behavioral and electroencephalographic effects of an epileptogenic peptide from the sea anemone Bunodosoma cangicum.

The primary structure of cangitoxin (CGX), a 4958 Da peptide from the sea anemone Bunodosoma cangicum, was determined: GVACRCDSDGPTVRGNSLSGTLWLTGGCPSGWHNCRGSGPFIGYCCKK. CGX contains all the 11 residues that are conserved and the 5 that are conservatively substituted within or between the type 1 and type 2 sequences of sea anemone peptides with specific action on voltage-sensitive sodium channels. Furthermore, it also has 6 identities (Asp9, Arg14, Asn16, Leu18, Trp33 and Lys48) and 1 homology (Arg36) in the 8 residues of the pharmacophore of the sea anemone ApB which are essential for interaction with mammalian sodium channels. The intrahippocampal injection of CGX induces several sequential behavioral alterations--episodes of akinesia alternating with facial automatisms and head tremor, salivation, rearing, jumping, barrel-rolling, wet dog shakes and forelimb clonic movements--and the electroencephalography analysis shows that they were followed by important seizure periods that gradually evolved to status epilepticus that lasted 8-12 h, similar to that observed in the acute phase of the pilocarpine model of epilepsy. These results suggest that CGX may be an important tool to develop a new experimental model of status epilepticus which may contribute to understanding the etiology of epilepsy and to test the effects of new antiepileptic drugs.