A plasma-derived extracellular vesicle mRNA classifier for the detection of breast cancer.

BACKGROUND: According to the global cancer burden data released in 2020, breast cancer (BC) has become the most common cancer in the world. Similar to those of other cancers, the present methods used in clinic for diagnosing early BC are invasive, inaccurate, and insensitive. Hence, new non-invasive methods capable of early diagnosis are needed. METHODS: We applied next-generation sequencing and analyzed the messenger RNA (mRNA) profiles of plasma extracellular vesicles (EVs) derived from 14 BC patients and 6 patients with benign breast lesions. We used 3 regression models, namely support vector machine (SVM), linear discriminate analysis (LDA), and logistic regression (LR), to develop classifiers for use in making predictive BC diagnoses; and used 259 plasma samples, including those obtained from 144 patients with BC, 72 patients with benign breast lesions, and 43 healthy women, which were divided into training groups and validation groups to verify their performances as classifiers by quantitative reverse transcription polymerase chain reaction (RT-qPCR). The area under the curve (AUC) and accuracy, sensitivity, and specificity of the classifiers were cross-validated with the leave-1-out cross-validation (LOOCV) method. RESULTS: Among all combinations assessed with the 3 different regression models, an 8-mRNA combination, named EXOBmRNA, exhibited high performance [accuracy =71.9% and AUC =0.718, 95% confidence interval (CI): 0.652 to 0.784] in the training cohort after LOOCV was performed, showing the largest AUC in the SVM model. The mRNAs in EXOBmRNA were HLA-DRB1, HAVCR1, ENPEP, TIMP1, CD36, MARCKS, DAB2, and CXCL14. In the validation cohort, the AUC of EXOBmRNA was 0.737 (95% CI: 0.636 to 0.837). In addition, gene function and pathway analyses revealed that different levels of gene expression were associated with cancer. CONCLUSIONS: We developed a high-performing predictive classifiers including 8 mRNAs from plasma extracellular vesicles for diagnosing breast cancer.

The rabbit vagina as an in vivo model for vaginal fenticonazole permeability and toxicity.

INTRODUCTION: Vaginal route is often used in topical antifungal formulations. Vaginal permeability assays are generally performed as in vitro tests. METHOD: An in vivo vaginal permeability assay was developed using female rabbits. Fenticonazole permeability was evaluated by assessing fenticonazole bioavailability in plasma by liquid chromatography coupled to tandem mass spectrometry (LC-MS-MS). Toxicity was monitored histopathologically after 8 consecutive days of antifungal treatment (20 mg/animal). RESULTS: The method of quantification was linear with a lower limit of quantification (LLOQ) of (0.1 ng/mL). The area-under-the-curves (AUC) of fenticonazole on day 1 and 8 of treatment were 280.3 ±â€¯86.1 ng/mL ∗ h and 805.7 ±â€¯252.4 ng/mL ∗ h, respectively. The calculated systemic bioavailability was 12.73% ±â€¯0.14%. No signs of toxicity were observed both macroscopically and histologically after 8 days fenticonazole treatment. DISCUSSION: The plasma levels of fenticonazole observed in rabbits are similar to that observed in human. Rabbit vagina may be a suitable model to evaluate vaginal antifungal formulations.

Predictable weathering of puparial hydrocarbons of necrophagous flies for determining the postmortem interval: a field experiment using Chrysomya rufifacies.

Preadult development of necrophagous flies is commonly recognized as an accurate method for estimating the minimum postmortem interval (PMImin). However, once the PMImin exceeds the duration of preadult development, the method is less accurate. Recently, fly puparial hydrocarbons were found to significantly change with weathering time in the field, indicating their potential use for PMImin estimates. However, additional studies are required to demonstrate how the weathering varies among species. In this study, the puparia of Chrysomya rufifacies were placed in the field to experience natural weathering to characterize hydrocarbon composition change over time. We found that weathering of the puparial hydrocarbons was regular and highly predictable in the field. For most of the hydrocarbons, the abundance decreased significantly and could be modeled using a modified exponent function. In addition, the weathering rate was significantly correlated with the hydrocarbon classes. The weathering rate of 2-methyl alkanes was significantly lower than that of alkenes and internal methyl alkanes, and alkenes were higher than the other two classes. For mono-methyl alkanes, the rate was significantly and positively associated with carbon chain length and branch position. These results indicate that puparial hydrocarbon weathering is highly predictable and can be used for estimating long-term PMImin.

Pharmacokinetic and pharmacodynamic evaluation of a nanotechnological topical formulation of lidocaine/prilocaine (nanorap) in healthy volunteers.

BACKGROUND: Nanorap is a new nanotechnological formulation for topical anesthesia composed of lidocaine (2.5%) and prilocaine (2.5%). This study evaluated the pharmacokinetics of Nanorap. For the determination of lidocaine and prilocaine in human plasma, a new method using high-performance liquid chromatography coupled with tandem mass spectrometry was developed. Nanorap pharmacodynamic (PD) and its physical proprieties were also evaluated. METHODS: Nanorap was administered by topical application of 2 g to healthy volunteers, and blood samples were collected for the pharmacokinetics analysis. The drugs were extracted from plasma by liquid-liquid extraction with ether/hexane (80/20, vol/vol). The chromatography separation was performed on a Genesis C18 analytical column 4 µm (100 × 2.1 mm i.d.) with a mobile phase of methanol/acetonitrile/water (40/30/30, for lidocaine, and 50/30/20, for prilocaine, vol/vol/vol) + 2 mM of ammonium acetate and ropivacaine as internal standard. The drugs were quantified using a mass spectrometer with an electrospray source in the electrospray ionization positive mode configured for multiple reaction monitoring. The PD of Nanorap was evaluated with the use of a visual analog scale. Nanorap was characterized by cryofracture. RESULTS: The chromatography run-time was 5.5 minutes for lidocaine and 3.3 minutes for prilocaine, and the lower limit of quantification was 0.05 ng/mL for both drugs. Mean Cmax was 6.62 and 1.72 ng/mL for lidocaine and prilocaine, respectively. Median Tmax was 6.5 hours for both drugs. Nanocapsules had a mean size of 88 nm and mean drug association of 92.5% and 89% for lidocaine and prilocaine, respectively. The PD study showed that Nanorap has a sufficient analgesic effect (>30% reduction in pain) after 10 minutes of application. CONCLUSIONS: A new simple, selective, and sensitive method for determination of lidocaine and prilocaine in human plasma was developed. Nanorap generated safe plasma levels of the drugs and satisfactory analgesic effect.

Quantification of dapaconazole in human plasma using high-performance liquid chromatography coupled to tandem mass spectrometry: application to a phase I study.

A simple, selective and sensitive method based on high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) has been developed for the determination of dapaconazole in human plasma using tioconazole as internal standard. The drugs were extracted from plasma by liquid-liquid extraction with ether/hexane (80/20, v/v). The chromatography separation was performed on a Genesis(®) C18 reversed phase analytical column 4µm (100×2.1mm i.d.) with a mobile phase of methanol/acetonitrile/water (80/10/10, v/v/v)+ammonium acetate (0.5mM). Dapaconazole was quantified using a mass spectrometer with an electrospray source in the ESI positive mode (ES+) configured for multiple reaction monitoring (MRM) to monitor the transitions 415.1>159.2 and 387.0>131.0 for dapaconazole and tioconazole, respectively. The method had a chromatography run time of 3.8min and a linear calibration curve over the range 0.2-100ng/mL (r=0.9998). The lower limit of quantification (LLOQ) was 0.2ng/mL. The precision and accuracy values of the assay were within ±10%. The stability tests indicate no significant degradation under the conditions of the experiment. This method was used for a phase I study of topical administration of dapaconazole tosylate in healthy human male volunteers.

[Morphological characteristics of hyoid bone and its gender difference].

OBJECTIVE: To understand and analyze the morphological characteristics of hyoid bone and its gender difference in order to find out its forensic significance. METHODS: The hyoid bones of 68 adult corpses were dissected from redundant soft tissues after heating. The connection status of body of hyoid, greater cornu and lesser cornu, the morphological characteristics of hyoid bone and the degree of ossification were observed by visual inspection. The height of hyoid bone and the arched height of hyoid bone were measured and compared the differences between male and female in order to deduce the analytic equation for gender estimation by hyoid bone. RESULTS: Five connection conditions of hyoid bone were identified by the morphological characteristics, including complete ossification in both sides, no ossification in both sides, partial ossification in both sides, complete ossification in one side (partial ossification in the other side), and complete ossification in one side (no ossification in the other side). The values of the arched height of hyoid bone (x1) and the height of hyoid bone (x2) in male were both higher than that in female (P < 0.01). The analytic equation for gender estimation (y) was y = 0.438 x1 + 1.042x2-12.979. The discriminant value was -0.272 5 and the resolution was 88.2%. CONCLUSION: According to the gender characteristics of hyoid bone, the data of hyoid bone can provide helps for forensic practices.

Quantification of cyproheptadine in human plasma by high-performance liquid chromatography coupled to electrospray tandem mass spectrometry in a bioequivalence study.

A rapid, sensitive and specific method to quantify cyproheptadine in human plasma using amitriptyline as the internal standard (IS) is described. The analyte and the IS were extracted from plasma by liquid-liquid extraction using a diethyl-ether/dichloromethane (70/30; v/v) solvent. After removing and drying the organic phase, the extracts were reconstituted with a fixed volume of acetonitrile/water (50/50 v/v)+0.1% of acetic acid. The extracts were analyzed by high performance liquid chromatography coupled to electrospray tandem mass spectrometry (LC-MS/MS). Chromatography was performed isocratically using an Alltech Prevail C18 5 µm analytical column, (150 mm x 4.6 mm I.D.). The method had a chromatographic run time of 4 min and a linear calibration curve ranging from 0.05 to 10 ng/mL (r2 > 0.99). The limit of quantification was 0.05 ng/mL. This HPLC/MS/MS procedure was used to assess the bioequivalence of cyproheptadine in two cyproheptadine + cobamamide (4 mg + 1 mg) tablet formulations (Cobactin® [cyproheptadine + cobamamide] test formulation supplied from Zambon Laboratórios Farmacêuticos Ltda. and Cobavital® from Solvay Farma (standard reference formulation)). A single 4 mg + 1 mg [cyproheptadine + cobamamide] dose of each formulation was administered to healthy volunteers. The study was conducted using an open, randomized, two-period crossover design with a 1-week washout interval. Since the 90% CI for Cmax and AUCs ratios were all within the 80-125% bioequivalence limit proposed by the US Food and Drug Administration, it was concluded that the cyproheptadine test formulation (Cobactin®) is bioequivalent to the Cobavital® formulation for both the rate and the extent of absorption of cyproheptadine.

Ciprofibrate quantification in human plasma by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry for pharmacokinetic studies.

A rapid, sensitive and specific method for quantifying ciprofibrate in human plasma using bezafibrate as the internal standard (IS) is described. The sample was acidified prior extraction with formic acid (88%). The analyte and the IS were extracted from plasma by liquid-liquid extraction using an organic solvent (diethyl ether/dichloromethane 70/30 (v/v)). The extracts were analyzed by high performance liquid chromatography coupled with electrospray tandem mass spectrometry (HPLC-MS/MS). Chromatography was performed using Genesis C18 4 µm analytical column (4.6 × 150 mm i.d.) and a mobile phase consisting of acetonitrile/water (70/30, v/v) and 1mM acetic acid. The method had a chromatographic run time of 3.4 min and a linear calibration curve over the range 0.1-60 µg/mL (r>0.99). The limit of quantification was 0.1 µg/mL. The intra- and interday accuracy and precision values of the assay were less than 13.5%. The stability tests indicated no significant degradation. The recovery of ciprofibrate was 81.2%, 73.3% and 76.2% for the 0.3, 5.0 and 48.0 ng/mL standard concentrations, respectively. For ciprofibrate, the optimized parameters of the declustering potential, collision energy and collision exit potential were -51 V, -16 eV and -5 V, respectively. The method was also validated without the use of the internal standard. This HPLC-MS/MS procedure was used to assess the bioequivalence of two ciprofibrate 100mg tablet formulations in healthy volunteers of both sexes. The following pharmacokinetic parameters were obtained from the ciprofibrate plasma concentration vs. time curves: AUC(last), AUC(0-168 h), C(max) and T(max). The geometric mean with corresponding 90% confidence interval (CI) for test/reference percent ratios were 93.80% (90% CI=88.16-99.79%) for C(max,) 98.31% (90% CI=94.91-101.83%) for AUC(last) and 97.67% (90% CI=94.45-101.01%) for AUC(0-168 h). Since the 90% CI for AUC(last), AUC(0-168 h) and C(max) ratios were within the 80-125% interval proposed by the US FDA, it was concluded that ciprofibrate (Lipless 100mg tablet) formulation manufactured by Biolab Sanus Farmacêutica Ltda. is bioequivalent to the Oroxadin (100 mg tablet) formulation for both the rate and the extent of absorption.

Comparative bioavailability of betahistine tablet formulations administered in healthy subjects.

OBJECTIVE: To assess the comparative bioavailability of two formulations (16 mg tablet) of betahistine (CAS 5579-84-0) in healthy volunteers of both sexes. METHODS: The study was conducted using an open, randomized, two-period crossover design with a 1-week washout interval. Plasma samples were obtained for up to 36 h post dose. Plasma 2-pyridylacetic acid concentrations were analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS) with positive ion electrospray ionization using multiple reaction monitoring (MRM). From the 2-pyridylacetic acid plasma concentration vs. time curves, the following pharmacokinetic parameters were obtained for AUCIast and Cmax. RESULTS: The limit of quantification was 4 ng/mL for plasma 2-pyridylacetic acid analysis. The geometric mean and 90% confidence interval (CI) of test/reference percent ratios were: 98.94% (92.21%-106.16%) for Cmax, 95.42% (91.74%-99.25%) for AUClast. CONCLUSION: Since the 90% CI for Cmax and AUCs ratios were all within the 80-125% interval proposed by the US Food and Drug Administration Agency, it was concluded that the test formulation is bioequivalent to the reference for both the rate and the extent of absorption.

[Estimation time of death by necrophagous flies life cycle].

OBJECTIVE: To study the nature of necrophagous flies, their developmental cycle and seasonal variation. METHODS: Animal corpse was used to be baiting. Eight kinds of necrophagous flies on their developmental cycle and the pattern of seasonal variation were analyzed. RESULTS: The community of necrophagous flies at high temperature in summer were more abundant than at low temperature in winter. Eight necrophagous flies through longer time at high temperature during every state than at low temperature. CONCLUSION: The life cycle and seasonal variation pattern of necrophagous flies could be used to estimate the time of death in practical cases.

[Determining postmortem interval by accumulated temperature method].

The experience of bug's growing and accumulated temperatures were important ways for determination of postmortem interval in forensic science. Here we used reverse accumulated temperature methods to estimate postmortem interval and made accordant result with their true time.

[Analysis of operative patterns of 2272 laparoscopic hysterectomies].

OBJECTIVE: To evaluate the clinical efficacy of four different operative patterns of laparoscopic hysterectomy: laparoscopically assisted vaginal hysterectomy (LAVH), laparoscopically introfasial subtotal hysterectomy (LISH), laparoscopically subtotal hysterectomy (LSH) and laparoscopically total hysterectomy (LTH). METHODS: A retrospective analysis on 2272 cases of laparoscopic hysterectomy was carried out, including operating time, blood loss, complication and postoperative recovery. RESULTS: For the two groups which preserved cervix, LISH was performed in 1323 cases. The operating time was (91 +/- 21) min, blood loss (93 +/- 23) ml, complication rate 4.1%. LSH was conducted in 229 cases, with an operating time (70 +/- 18) min, blood loss (69 +/- 17) ml, complication rate 0. The difference between the two groups was significant (all P < 0.01). For the two groups which excised cervix, LAVH was performed in 588 cases, with an operating time (119 +/- 28) min, blood loss (156 +/- 23) ml, complication rate 1.5%; while LTH was carried out in 132 cases, with an operating time (121 +/- 30) min, blood loss (193 +/- 38) ml, complication rate 1.2%. There were no significant differences between the two groups (all P > 0.05). All patients recovered well postoperatively. CONCLUSIONS: The four operative patterns are ideal for hysterectomy. Young patients should be operated with laparoscopic hysterectomy with preservation of cervix, old patients or patients with CIN should be operated with excision of cervix.

[Correlation between local hormones and CD36 transcription level in women with polycystic ovary].

OBJECTIVE: To detect CD(36) expressions in polycystic ovary (PCO), and to explore its correlation with local androgen and insulin at transcription level. METHODS: From August 2002 to February 2003, 12 patients with asymmetric PCO, 15 primary or secondary infertile patients without endocrine disorders and 8 polycystic ovary syndrome (PCOS) with bilateral PCO were recruited. Extraction of follicular fluid and detection of testosterone (T), dehydroepiandrosterone sulfate (DHEAS), insulin (INS) and androstenedione (A(2)) were performed. Relative CD(36) mRNA expression level of human ovarian inner thecal cells was analyzed by auto image analysis system (IAS) after RT-PCR. RESULTS: The level of CD(36) mRNA expression in thecal cells was 0.24 +/- 0.07 in polycystic ovary of PCO group and 0.21 +/- 0.05 in bilateral ovaries of PCOS group, respectively, which were significantly lower than 0.83 +/- 0.13 in normal ovaries (P < 0.01). T and INS levels of follicle fluid in PCO were significantly higher than that in normal ovaries (P < 0.01). T and INS levels of follicle fluid were negatively related to CD(36) mRNA expression of follicular theca interna (r = -0.6810, r = -0.6708, P < 0.01). CONCLUSION: Decrease of scavenger receptor gene CD(36) mRNA may play a role in the pathogenesis of PCO by increasing the level of T and INS in follicular fluid.