Recognizing drug‐facilitated crimes: Detection and quantification of benzodiazepines in beverages using fast liquid–liquid extraction with low temperature partitioning and paper spray mass spectrometry

A novel methodology using liquid–liquid extraction with low temperature partitioning (LLE–LTP) and paper spray mass spectrometry (PS–MS) was developed to identify and quantify benzodiazepines in beverages. Four types of alcoholic beverages usually consumed in parties and bars were spiked with 5 distinct benzodiazepines (diazepam, alprazolam, bromazepam, clonazepam, and cloxazolam) simulating a drug‐facilitated crime occurrence. The direct PS–MS analysis of the spiked beverages revealed a remarkable matrix effect with an unclear detection of protonated benzodiazepines. However, by the application of the LLE–LTP using liquid nitrogen, a prompt and doubtless detection of such compounds was achieved. The quantification potential of the LLE–LTP/PS–MS methodology was demonstrated by using beer as matrix, diazepam as target analyte and cloxazolam as an internal standard. Figures of merit (linearity, limit of detection, linear dynamic range, relative standard deviation, and recovery) were determined and adequate values were obtained. In conclusion, we demonstrated herein that the LLE–LTP/PS–MS methodology has potential to be applied directly at the crime scene through of a portable mass spectrometer and a thermal container for the transport of liquid nitrogen.     

Antimicrobial activity of Buchenavia tetraphylla against Candida albicans strains isolated from vaginal secretions

Context: Buchenavia tetraphylla (Aubl.) RA Howard (Combretaceae: Combretoideae) is an ethnomedicinal plant with reported antifungal action.

Objective: This study evaluates the antimicrobial activity of B. tetraphylla leaf extracts against clinical isolates of Candida albicans. The morphological alterations, combinatory effects with fluconazole and the cytotoxicity of the active extract were analyzed.

Materials and methods: Extracts were obtained using different solvents (hexane: BTHE; chloroform: BTCE; ethyl acetate: BTEE; and methanol: BTME). Antimicrobial activity was determined by the broth microdilution method using nine strains of C. albicans isolated from vaginal secretions and one standard strain (UFPEDA 1007).

Results: All extracts showed anti-C. albicans activity, including against the azole-resistant strains. The MIC values ranged from 156 to 2500?μg/mL for the BTHE; 156 to 1250?μg/mL for the BTCE; 625 to 1250?μg/mL for the BTME and 625?μg/mL to 2500?μg/mL for the BTEE. BTME showed the best anti-C. albicans activity. This extract demonstrated additive/synergistic interactions with fluconazole. Scanning electron microscopy analysis suggested that the BTME interferes with the cell division and development of C. albicans. BTME showed IC₅₀ values of 981 and 3935?μg/mL, against J774 macrophages and human erythrocytes, respectively. This extract also enhanced the production of nitric oxide by J774 macrophages.

Discussion and conclusion: Buchenavia tetraphylla methanolic extract (BTME) is a great source of antimicrobial compounds that are able to enhance the action of fluconazole against different C. albicans strains; this action seems related to inhibition of cell division.     

Follicular fluid lipid peroxidation levels in women with endometriosis during controlled ovarian hyperstimulation

This observational study aimed to establishing a relationship between lipid peroxidation and endometriosis in women undergoing controlled ovarian hyperstimulation. A total of 79 women were divided into two groups: (i) controls (tubal or male factor); and (ii) endometriosis (stages III/IV). The endometriosis diagnosis was confirmed by videolaparoscopy and the controlled ovarian stimulation protocol was similar to all patients. Follicular fluid (FF) lipid peroxidation levels were determined through the quantification of malondialdehyde. Statistical analysis was performed using parametric and non-parametric tests, logistic regression was performed to estimate the chance of achieving a pregnancy in each group and a moving average was calculated for the endometriosis group. Peroxidation levels in the endometriosis group were significantly higher when compared to controls. The moving average showed a decrease of MDA levels in the endometriosis group with increasing female age. Moreover, women with endometriosis who were under 33 years of age were 4.3 times more likely to achieve a pregnancy than women above that age. In conclusion, endometriosis is associated with increased FF oxidative stress (OS) in patients undergoing in vitro fertilization (IVF). Also, increasing age is associated with a decrease in severity of the oxidative status, but a decreased chance of pregnancy.     

Reestablishment of the hyperglycemia to the normal levels seems not to be essential to the anxiolytic-like effect induced by insulin

Diabetes is a chronic metabolic disease accompanied by several comorbidities, including neuropsychiatric conditions. Since the hyperglycemia appears to be the primary factor involved in diabetic conditions, we examined the effect of insulin treatment in diabetic rats on behavioral responses related to anxiety and aversive memory extinction. For this, normoglycemic (NGL) or streptozotocin-diabetic (DBT) rats were submitted to the elevated T maze (ETM) and the contextual conditioned fear (CCF) tests. Therefore, animals were subjected to the prolonged treatment with insulin (6 IU/day, s.c.) to investigate the effect of the treatment on distinct behaviors. When anxiety-like responses such as the inhibitory avoidance (IA) on the ETM and the time of freezing in the first session of the CCF test were evaluated, our data showed a more pronounced anxiogenic-like behavior in DBT animals when compared to NGL ones. In addition, an increased freezing time was observed in DBT animals exposed to the CCF test (sessions 2 and 3) when compared to the NGL group, suggestive of an impairment in the extinction of aversive memory. Insulin treatment induced an anxiolytic-like effect when IA and freezing time (session 1) was evaluated, but did not alter the impaired extinction of aversive memory (sessions 2 and 3). To better understand the involvement of a rigorous control of glycaemia, we also investigated the effect of a lower dose of insulin (3 IU/day, s.c.), unable to reestablish the hyperglycemia to the normal levels, on the same behavioral parameters. Our data show that independent of the dose of insulin, the same effects were observed when animals were evaluated in the ETM and CCF tests. However, only the highest dose of insulin was able to reduce the hyperglycemia to the normal levels. To conclude, our data suggest that a severe glycemic control by insulin treatment seems to be important, but not essential in improving diabetes-induced anxiety.     

Integrative study of cell damage and cancer risk in gas station attendants

Gas station attendants are potentially exposed to carcinogenic substances. This study aimed to evaluate chronic occupational exposed gas station attendant’s according to genetic parameters and its relationship with cancer predisposition. Forty gas station attendants were recruited and paired with 40 control subjects. Thousand cells from each subject were analyzed counting the number of pyknotic (PYC), karyolitic (KYL), karyorrhetic (KHC), condensed chromatin (CC), binucleated (BN), basal cells (BC), Nuclear buds (Nbud), and differentiated cells (DIFF). More 2.000 differentiated cells were analyzed counting micronucleated (MNi) and nuclear buds (NBud). We observed a statistical increase in BC and decrease in DIFF (p < 0.05). PYC, CC, KYL, MN, NBud were statistically increased (p < 0.05). All in all, our data showed elevated DNA damage, chromosomal instability, and cell cycle disturbing in our group of gas station attendants. It is a clear risk for their health in future and could be classified as a cancer predisposition.     

Updates in biological therapies for knee injuries: menisci

The preservation of meniscal tissue is paramount for long-term joint function, especially in younger patients who are athletically active. Many studies have reported encouraging results following the repair of meniscus tears, including both simple longitudinal tears located in the periphery and complex multiplanar tears that extend into the central third avascular region. However, most types of meniscal lesions are managed with a partial meniscectomy. Options to restore the meniscus range from an allograft transplantation to the use of synthetic and biological technologies. Recent studies have demonstrated good long-term outcomes with meniscal allograft transplantation, although the indications and techniques continue to evolve, and the long-term chondroprotective potential of this approach has yet to be determined. Several synthetic implants, most of which are approved in the European market, have shown some promise for replacing part of or the entire meniscus, including collagen meniscal implants, hydrogels, and polymer scaffolds. Currently, there is no ideal implant generated by means of tissue engineering. However, meniscus tissue engineering is a fast developing field that promises to develop an implant that mimics the histologic and biomechanical properties of a native meniscus.     

Validation of a reference control for an SYBR-Green fluorescence assay-based real-time PCR for detection of bovine herpesvirus 5 in experimentally exposed bovine embryos

The objective of this study was to optimize an internal control to improve SYBR-Green-based qPCR to amplify/detect the BoHV-5 US9 gene in bovine embryos produced in vitro and experimentally exposed to the virus. We designed an SYBR-Green-based binding assay that is quick to perform, reliable, easily optimized and compares well with the published assay. Herein we demonstrated its general applicability to detect BoHV-5 US9 gene in bovine embryos produced in vitro experimentally exposed to BoHV-5. In order to validate the assay, three different reference genes were tested; and the histone 2a gene was shown to be the most adequate for normalizing the qPCR reaction, by considering melting and standard curves (p < 0.05). On the other hand, no differences were found in the development of bovine embryos in vitro whether they were exposed to BoHV-5 reference and field strains comparing to unexposed embryos. The developed qPCR assay may have important field applications as it provides an accurate BoHV-5 US9 gene detection using a proven reference gene and is considerably less expensive than the TaqMan qPCR currently employed in sanitary programs.     

Tooth sensitivity and efficacy of in-office bleaching in restored teeth

ObjectivesThe aim of this clinical trial was to evaluate efficacy (BE) and tooth sensitivity (TS) of in-office bleaching with a 35% hydrogen peroxide (HP) in patients with aesthetic restorations.MethodsHydrogen peroxide 35% was applied in two sessions, of three 15 min applications, in 15 patients with upper anterior sound teeth (S) and 15 with aesthetic restorations (R). The colour was recorded at baseline, one week and 6 months after treatment completion. Patients recorded TS on a 0–4 scale. The BE was evaluated by two-way ANOVA and Tukey's tests (α = 0.05). The percentage of patients with TS was evaluated by Fisher's exact test and TS intensity of treatments was compared with Mann–Whitney U-test (α = 0.05).ResultsAll participants experienced TS at least once during treatment. Higher TS intensity was observed in R (1.5 [1/1.75]) compared to S (0.5 [0/1.25]) during the bleaching (p < 0.05). S and R demonstrated similar tooth colour enhancement compared to baseline (p < 0.05) and both presented colour stability after 6 months of evaluation (p > 0.05).ConclusionsThe in-office bleaching with 35% HP was effective in patients with aesthetic restorations, however, a higher intensity of TS was observed during the bleaching protocol.Clinical relevanceIn-office dental bleaching can be performed in patients with adhesive restorations promoting satisfactory results; however, it can promote higher intensity of sensitivity compared to patients with sound teeth.