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Citrus essential oil has many uses in food, cosmetics, and pharmaceuticals industries, especially the minor compounds present in the oil. This work aimed to evaluate the distillation patterns of the components of the peel essential oil ofCitrus sinensisusing vacuum fractional distillation. The performed tests show that the separation of the compounds occurs with the leaving of the lighter terpenes, followed by the heavier terpenes and part of the oxygenated functions. Most of the oxygenated functions remained in the bottom of the column, being concentrated in this fraction. Many trace compounds were found in the bottom products. The limonene mass percentage reduced from 96.68% to 52.81% in the bottom products. Linalool had its mass percentage increased from 0.37% in the raw oil to 4.22% in the bottom. It was also observed in the operation conditions of the column (10 kPa and 70°C), there was no evidence of thermal degradation of the raw oil, neither of the obtained fractions during the process. [ABSTRACT FROM PUBLISHER]

The "no reaction" lateral flow assay (nrLFA) uses a simplified LFA structure with no conjugate pad and no stored reagents. In the nrLFA, the capillary-based transport time or distance is the key indicator, rather than the outcome of a biochemical reaction. Hence, the calibration and reproducibility of the nrLFA device are critical. The capillary flow properties of several membrane types (nitrocellulose, nylon, cellulose acetate, polyethersulfone, and polyvinylidene difluoride) are evaluated. Flow rate evaluations of MilliporeSigma Hi-Flow™ Plus (HF075, HF135 and HF180) nitrocellulose membranes on nrLFA are performed using bodily fluids (whole blood, blood plasma, and artificial sweat). The results demonstrate that fluids with lower viscosity travel faster, and membranes with slower flow rate exhibit higher capability to distinguish fluids with different viscosities. Reproducibility tests of nrLFA are performed on HF075, demonstrating excellent reproducibility. The coefficient of variation for blood coagulation tests performed with the nrLFA using induced coagulation was 5% for the plasma front and 2% for the RBC front. The effects of variation in blood hematocrit and sample volume are also reported. The overall results indicate that the nrLFA approach has a high potential to be commercially developed as a blood monitoring point-of-care device with simple calibration capability and excellent reproducibility. [ABSTRACT FROM AUTHOR]

A simple approach to the evaluation of blood coagulation using a microfluidic paper-based lateral flow assay (LFA) device for point-of-care (POC) and self-monitoring screening is reported. The device utilizes whole blood, without the need for prior separation of plasma from red blood cells (RBC). Experiments were performed using animal (rabbit) blood treated with trisodium citrate to prevent coagulation. CaCl2 solutions of varying concentrations are added to citrated blood, producing Ca2+ ions to re-establish the coagulation cascade and mimic different blood coagulation abilities in vitro. Blood samples are dispensed into a paper-based LFA device consisting of sample pad, analytical membrane and wicking pad. The porous nature of the cellulose membrane separates the aqueous plasma component from the large blood cells. Since the viscosity of blood changes with its coagulation ability, the distance RBCs travel in the membrane in a given time can be related to the blood clotting time. The distance of the RBC front is found to decrease linearly with increasing CaCl2 concentration, with a travel rate decreasing from 3.25 mm min−1 for no added CaCl2 to 2.2 mm min−1 for 500 mM solution. Compared to conventional plasma clotting analyzers, the LFA device is much simpler and it provides a significantly larger linear range of measurement. Using the red colour of RBCs as a visible marker, this approach can be utilized to produce a simple and clear indicator of whether the blood condition is within the appropriate range for the patient's condition. [ABSTRACT FROM AUTHOR]

Copyright of Brazilian Journal of Biology is the property of Instituto Internacional de Ecologia and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Brazilian Journal of Biology is the property of Instituto Internacional de Ecologia and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Brazilian Journal of Biology is the property of Instituto Internacional de Ecologia and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Two patients aged 21 and 50 years presented with facial hemiatrophy and unilateral spasms of the masticatory muscles. Masticatory muscle biopsy showed normal findings in both patients and facial skin biopsy specimens only showed atrophy, although morphoea (localised facial scleroderma) had been diagnosed nine years previously in the second patient. The involuntary movements consisted of brief twitches and prolonged contractions clinically and electromyographically similar to those of hemifacial spasm and cramps. The jaw jerk and the silent periods were absent in the affected muscles. Direct stimulation of the muscle nerve and transcranial stimulation of the trigeminal root demonstrated slowing of conduction and after-activity due to autoexcitation. Observations in other reported cases and these two patients suggest that hemimasticatory spasm is produced by ectopic activity secondary to focal demyelination of the trigeminal motor nerve fibres. The proposed cause of the neuropathy is focal damage to the masticatory nerves caused by compression, possibly resulting from the deep tissue changes that occur in facial hemiatrophy. [ABSTRACT FROM AUTHOR]

Classical models of gene amplification envision chromosomally localized focal amplicons encompassing the immediate contiguous chromosomal regions flanking the driver gene. We present evidence that this is not the case for the HER2/neu amplicon in human breast cancer. Using a large panel of HER-2/neu positive cell lines, we observed that the HER- 2/neu amplicon is made up of DNA segments mapping at different positions along chromosome 17. Sub-regions were subsequently re-amplified with the creation of condensed array of shorter amplicons. In addition, we show involvement of other chromosomes. The massive inter and intra-chromosomal reshuffling associated with amplified HER-2/neu containing sequences not only shed new light on the mechanism of gene amplification in human cancer but also have significant clinical implications for developing new targeted therapies and for combating drug resistance in this important subtype of breast cancer. [ABSTRACT FROM AUTHOR]

Objective: Currently available contraceptive methods do not meet the needs of all users. We sought to explore preferences of potential end-users regarding an on-demand, non-hormonal female contraceptive currently under development, using a web-based survey., Study Design: We recruited respondents for an exploratory survey via web link on Amazon Mechanical Turk (MTurk). Individuals were eligible if they were 18-44 years of age, identified as cis-gender female, were English-speaking, not pregnant, and had used barrier contraception previously. Respondents provided demographic characteristics and a basic reproductive history. We then provided a brief description of the potential contraceptive. Respondents were asked about their interest in the proposed contraceptive and preferences for method attributes., Results: A total of 500 respondents completed the survey. Three-quarters of respondents were <35 years of age and 48.2% were currently using a barrier contraceptive method. Three-fourths of respondents (73.8%) expressed interest in using the contraceptive under development. The majority wanted the method to be small (≤2 inches), rod-shaped, and low cost (<$5 per use). More than half (59.4%) said it was important to be able to use the method without partners' knowledge. The most reported potential concerns were vaginal irritation (51.6%) and lack of effectiveness (46.4%). Sixty percent of respondents were confident they could use the method correctly., Discussion: Available contraceptive methods lack attributes preferred by some users. Development of new contraceptives frequently does not involve end-user input early in the development process. Individuals in this sample displayed interest in the proposed contraceptive and expressed preferences that can inform the further development of this method., Competing Interests: TM serves on a data safety monitoring board for phase 4 safety studies of Bayer contraceptive products. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor KN declared a past co-authorship with the author(s) MAT., (© 2023 Madden, Cohen, Paul, Hurley, Thomas and Pauletti.)

Background: Incident reporting (IR) is one of the most used systems to gain knowledge of adverse events (AEs) and to identify sources of risk. During COVID-19 pandemic, several organizational changes have been implemented to respond adequately and effectively to the emergency; this required the suspension of most deferrable activities., Objective: The aim of this study is to investigate whether IR attitude of health workers has been reduced during the pandemic event., Method: A retrospective analysis was conducted at the Azienda Ospedale - Università di Padova (Italy), considering IR of years 2019 and 2020. To standardize the effects of the decrease in admissions, we considered the number of incidents per 1000 admissions., Results: Data shows that during the first (March-May 2020) and second waves (October-December 2020) of the COVID-19 pandemic there was a statistically significant reduction in the rate of IR for every 1000 admissions (P = 0.001-Wilcoxon test), especially for AEs and in COVID-19 units., Conclusion: This study shows a reduction in IR especially during the first and second pandemic waves of COVID-19 in year 2020. Education and training interventions could be fundamental to raise awareness of the importance of IR in health workers, as this could provide opportunities to understand what is impacting on safety in a particular healthcare context and enable continuous improvement., (© The Author(s) 2022. Published by Oxford University Press on behalf of International Society for Quality in Health Care. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Historical antecedents of pharmaceutical sciences are sound on product orientation based on (analytical) chemistry, drug delivery and basic pharmacology. Over the last decades we have seen a transition towards a stronger disease orientation. This raises questions on whether, how and to what extent unmet medical need (UMN) is important in priority setting, funding and impact in pharmaceutical sciences. An online survey in 2020 collected perspectives of internationally recognised pharmaceutical scientists (N = 92), mainly from academia and industry, on drivers and influencing factors in pharmaceutical sciences. The study offers a unique global perspective, demonstrating a solid command of the global needs in pharmaceutical sciences. The survey revealed that UMN is currently seen as one of the three most important drivers, also in addition to emerging trends in science and opportunities driven by collaboration. There are expectations that UMN's impact becomes more influential. This was consistent for both industry and academic respondents. The majority of respondents also indicated that anticipated lessons learned from COVID-19 will strengthen the impact of UMN on science and leadership. This is important as prioritisation of research towards UMN can address the clinical needs where needed the most., Competing Interests: Declarations of Interest None. The views expressed in this article are the personal views of the authors and must not be understood or quoted as being made on behalf of the International Pharmaceutical Federation., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The increased demand for pesticide-free foods has also increased the search for healthier and environmentally friendly alternatives in agriculture. Essential oils are known to possess natural antifungal properties, becoming a reliable alternative for commercial fungicides, especially for postharvest decay control. However, essential oils are volatile and photodegradable, which reduces their long-term activities. This work presents the development of a lemongrass essential oil-containing poly(lactic acid) nanocapsules. They have shown in vitro antifungal activity against Colletotrichum acutatum and Colletotrichum gloeosporioides with a MIC dosage of 0.1% (v/v) for both phytopathogens. In the in vivo assay with postharvest apples, the ones treated with encapsulated essential oil showed bitter rot lesions three times smaller than the ones treated with non-encapsulated essential oil, or in comparison to the apples in positive control. The methodology led to stable nanocapsules with spherical morphology, a mean diameter of 96.4 nm, and with an encapsulation efficiency of 99%., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Low birth weight is associated with both short term problems and the fetal programming of adult onset diseases, including an increased risk of obesity, diabetes and cardiovascular disease. Placental insufficiency leading to intrauterine growth restriction (IUGR) contributes to the prevalence of diseases with developmental origins. Currently there are no therapies for IUGR or placental insufficiency. To address this and move towards development of an in utero therapy, we employ a nanostructure delivery system complexed with the IGF-1 gene to treat the placenta. IGF-1 is a growth factor critical to achieving appropriate placental and fetal growth. Delivery of genes to a model of human trophoblast and mouse placenta was achieved using a diblock copolymer (pHPMA-b-pDMAEMA) complexed to hIGF-1 plasmid DNA under the control of trophoblast-specific promoters (Cyp19a or PLAC1). Transfection efficiency of pEGFP-C1-containing nanocarriers in BeWo cells and non-trophoblast cells was visually assessed via fluorescence microscopy. In vivo transfection and functionality was assessed by direct placental-injection into a mouse model of IUGR. Complexes formed using pHPMA-b-pDMAEMA and CYP19a-923 or PLAC1-modified plasmids induce trophoblast-selective transgene expression in vitro, and placental injection of PLAC1-hIGF-1 produces measurable RNA expression and alleviates IUGR in our mouse model, consequently representing innovative building blocks towards human placental gene therapies.

Novel aptamer-functionalized polyethylene glycol-polylactic acid (PEG-PLA) (APP) micelles were developed with the objective to target the transferrin receptor on brain endothelial cells. Flurbiprofen, a potential drug for therapeutic management of Alzheimer's disease (AD), was loaded into the APP micelles using the co-solvent evaporation method. Results indicated that 9.03% (w/w) of flurbiprofen was entrapped in APP with good retention capacity in vitro. Targeting potential of APPs was investigated using the transferring receptor-expressing murine brain endothelial bEND5 cell line. APPs significantly enhanced surface association of micelles to bEND5 cells as quantified by fluorescence spectroscopy. Most importantly, APPs significantly enhanced intracellular flurbiprofen delivery when compared to unmodified micelles. These results suggest that APP micelles may offer an effective strategy to deliver therapeutically effective flurbiprofen concentrations into the brain for AD patients.

The treatment of dementias, which are currently incurable pathologies, requires an approach to care that involves both the patients and their families. The effect of alternative interventions, besides the pharmacological approach, therefore warrants evaluation. In this paper, we describe one such intervention, which was provided by our home care team for Alzheimer's Disease. Patients were granted a three-month period of home care assistance, which included physical and cognitive rehabilitation as well as interventions on the home environment and the family, such as psychological support for the main caregivers. The assistance was provided in thrice-weekly sessions, each lasting six hours. Twenty-two patients (age 78.4±6.5 yrs), all of whom had received a diagnosis of probable AD, were enrolled. There was a statistically significant improvement in the NPI score (p = 0.004), Barthel index (p = 0.01), Tinetti's scale (p = 0.013) and CBI score (p = 0.016) at the end of the 3-month treatment period. The patients' caregivers also reported a significant improvement in the physical and social burden at the CBI at the end of the period of home care assistance (p = 0.026 and p = 0.006). In a further evaluation performed 3 months after the end of the treatment period, the beneficial effect previously observed in both patients and caregivers was no longer present.

Purpose: Approximately 35% of HER2-amplified breast cancers have coamplification of the topoisomerase II-alpha (TOP2A) gene encoding an enzyme that is a major target of anthracyclines. This study was designed to evaluate whether TOP2A gene alterations may predict incremental responsiveness to anthracyclines in some breast cancers., Methods: A total of 4,943 breast cancers were analyzed for alterations in TOP2A and HER2. Primary tumor tissues from patients with metastatic breast cancer treated in a trial of chemotherapy plus/minus trastuzumab were studied for amplification/deletion of TOP2A and HER2 as a test set followed by evaluation of malignancies from two separate, large trials for changes in these same genes as a validation set. Association between these alterations and clinical outcomes was determined., Results: Test set cases containing HER2 amplification treated with doxorubicin and cyclophosphamide (AC) plus trastuzumab, demonstrated longer progression-free survival compared to those treated with AC alone (P = .0002). However, patients treated with AC alone whose tumors contain HER2/TOP2A coamplification experienced a similar improvement in survival (P = .004). Conversely, for patients treated with paclitaxel, HER2/TOP2A coamplification was not associated with improved outcomes. These observations were confirmed in a larger validation set, where HER2/TOP2A coamplification was again associated with longer survival when only anthracycline-containing chemotherapy was used for treatment compared with outcome in HER2-positive cancers lacking TOP2A coamplification., Conclusion: In a study involving nearly 5,000 breast malignancies, both test set and validation set demonstrate that TOP2A coamplification, not HER2 amplification, is the clinically useful predictive marker of an incremental response to anthracycline-based chemotherapy. Absence of HER2/TOP2A coamplification may indicate a more restricted efficacy advantage for breast cancers than previously thought.

Purpose: About 65% to 70% of melanomas harbor a mutation in v-raf murine sarcoma viral oncogene homolog B1 (BRAF) that causes the steady-state activation of extracellular signal-regulated kinase (ERK). We sought to investigate the efficacy of PLX4032 (BRAF inhibitor) to identify patterns/predictors of response/resistance and to study the effects of BRAF in melanoma., Experimental Design: Well-characterized melanoma cell lines, including several with acquired drug resistance, were exposed to PLX4032. Growth inhibition, phosphosignaling, cell cycle, apoptosis, and gene expression analyses were performed before and after exposure to drug., Results: Using a growth-adjusted inhibitory concentration of 50% cutoff of 1 microM, 13 of 35 cell lines were sensitive to PLX4032, 16 resistant, and 6 intermediate (37%, 46%, and 17% respectively). PLX4032 caused growth inhibition, G(0)/G(1) arrest, and restored apoptosis in the sensitive cell lines. A BRAF mutation predicted for but did not guarantee a response, whereas a neuroblastoma RAS viral oncogene homolog mutation or wild-type BRAF conferred resistance. Cells with concurrent BRAF mutations and melanocortin 1 receptor germ line variants and/or a more differentiated melanocyte genotype had a preferential response. Acquired PLX4032 resistance reestablishes ERK signaling, promotes a nonmelanocytic genotype, and is associated with an increase in the gene expression of certain metallothioneins and mediators of angiogenesis., Conclusions: PLX4032 has robust activity in BRAF mutated melanoma. The preclinical use of this molecule identifies criteria for its proper clinical application, describes patterns of and reasons for response/resistance, and affords insight into the role of a BRAF mutation in melanoma.

In breast cancer, recent studies suggest that the value of HER2 for predicting response to anthracycline-based chemotherapy may be more likely related to the concomitant amplification of the TOP2A gene. Here, we study the association between HER2 or TOP2A status and response to anthracycline-based preoperative chemotherapy and explore the interaction between HER2 or TOP2A status and intense dose-dense (IDD) chemotherapy. HER2 and TOP2A gene alterations were quantified by fluorescence in situ hybridization in primary tumor core biopsies from 373 high-risk primary breast cancer patients (tumors >/=3 cm or inflammatory) that received an IDD or conventionally scheduled anthracycline-based preoperative chemotherapy. HER2 was amplified in 94/350 tumors (27%) of which 40/94 (46%) demonstrated TOP2A amplification, and 17/94 (18%) TOP2A deletions. TOP2A gene alterations were not found in HER2 non-amplified cases. HER2 amplification was associated with a significantly higher pathologic complete response (pCR) rate only when TOP2A was co-amplified (30% vs. 11%, P = 0.002), but not when deleted (13% vs. 11%, P = 0.755), or normal (14% vs. 11%, P = 0.578) compared to HER2 non-amplified tumors. In multivariate analysis, TOP2A amplification (odds ratio [OR] 3.04, P = 0.021), but not HER2 amplification (OR 1.74, P = 0.170) was associated with a significantly higher pCR rate. No interaction was observed between HER2 or TOP2A status and IDD chemotherapy. TOP2A gene amplification may define a subset of HER2-amplified breast cancers that are responsible for the markedly improved chemosensitivity seen in HER2-positive breast cancer. However, added benefit of IDD chemotherapy itself was not associated with HER2 or TOP2A status.

Purpose: To evaluate the efficacy and safety of docetaxel, cisplatin, and trastuzumab as primary systemic therapy for human epidermal growth factor receptor 2 (HER2) -positive, locally advanced breast cancer (LABC)., Patients and Methods: Forty-eight patients with immunohistochemistry-confirmed HER2-positive LABC or inflammatory breast cancer received 12 weeks of docetaxel, cisplatin, and trastuzumab with filgrastim, followed by surgery, adjuvant doxorubicin and cyclophosphamide, and locoregional radiotherapy with or without tamoxifen. The primary end point was pathologic complete response (pCR) in breast., Results: Baseline mean tumor size was 9.2 cm (range, 4 to 32 cm). pCR occurred in breast in 11 patients (23%; 95% CI, 12% to 37%) and breast and axilla in eight patients (17%; 95% CI, 8% to 30%). pCR rates in breast (HER2 positive, seven of 30 patients, 23% v HER2 negative, four of 18 patients, 22%; P > .05) and breast and axilla (four of 30 patients, 13% v four of 18 patients, 22%, respectively; P > .05) were similar regardless of HER2 status by fluorescence in situ hybridization (FISH). At a median follow-up time of 43 months, 4-year progression-free survival (PFS) rate was 81% (95% CI, 64% to 90%); overall survival (OS) rate was 86% (95% CI, 71% to 94%). In patients with pCR in breast and axilla, PFS and OS rates were 100% (95% CI, inestimable). In patients without pCR, PFS rate was 76% (95% CI, 57% to 88%; P = .15, log-rank test), and OS rate was 83% (95% CI, 66% to 92%; P = .21). Survival rates were similar regardless of FISH status. There were only two grade 4 adverse events., Conclusion: Twelve weeks of docetaxel, cisplatin, and trastuzumab is clinically active and leads to excellent survival in patients with large, HER2-positive tumors.

Background: HER-2/neu overexpression appears to be associated with improved response to anthracycline-based chemotherapy, but its association with response to taxane-based chemotherapy is unclear. In this retrospective subset analysis of patients with metastatic breast cancer enrolled in a randomized treatment trial, we investigated the response of patients with known HER-2/neu status to treatment with taxane-based epirubicin-paclitaxel (ET) chemotherapy compared with treatment with epirubicin-cyclophosphamide (EC) chemotherapy., Methods: HER-2/neu status (positive [i.e., HER-2/neu amplification] or negative [i.e., no HER-2/neu amplification]) of archival specimens of primary tumors from 297 patients with metastatic breast cancer was determined by use of fluorescence in situ hybridization. Associations between HER-2/neu status and the efficacy of randomly assigned chemotherapy (ET versus EC) were investigated. All statistical tests were two-sided., Results: Patients with HER-2/neu-positive tumors had a statistically significantly greater objective response rate than patients with HER-2/neu-negative tumors to treatment with ET (76% versus 50%, respectively; P =.005) but not to treatment with EC (46% versus 33%; P =.130). The objective response rate associated with ET was greater than that associated with EC for both HER-2/neu-positive tumors (76% versus 46%; P =.004) and HER-2/neu-negative tumors (50% versus 33%; P =.002). However, the improvement in the objective response rate associated with ET, compared with that associated with EC, was greater for patients with HER-2/neu-positive tumors (adjusted odds ratio [OR] = 3.64, 95% confidence interval [CI] = 1.48 to 8.92; P=.005) than for patients with HER-2/neu-negative tumors (adjusted OR = 1.92, 95% CI = 1.01 to 3.64; P=.046). Among patients with HER-2/neu-positive tumors, those who received ET had better progression-free survival and overall survival than those who received EC (for progression-free survival, adjusted relative risk [RR] = 0.65, 95% CI = 0.42 to 1.02; P=.062; for overall survival, adjusted RR = 0.60, 95% CI = 0.36 to 1.02; P=.059). However, among patients with HER-2/neu-negative tumors, those who received ET and those who received EC had similar progression-free survival and overall survival., Conclusions: HER-2/neu amplification does not adversely influence response to first-line chemotherapy with either ET or EC. Furthermore, a taxane-containing regimen such as ET may provide a preferential benefit to patients with HER-2/neu-positive tumors.

Background: HER-2/neu, which encodes a receptor tyrosine kinase, is amplified and overexpressed in 20%-25% of human breast cancers. Such tumors are often resistant to hormone therapy. Despite a general inverse association between HER-2/neu amplification/overexpression and estrogen receptor (ER) and/or progesterone receptor (PR) expression, a fraction of patients are both HER-2/neu- and hormone receptor (HR)-positive. The efficacy of hormone therapy in this group is currently a matter of debate. To better understand the relationship between HER-2/neu positivity and HR expression, we analyzed HER-2/neu, ER, and PR as continuous variables in breast cancer cell lines and two cohorts of primary breast cancer patients., Methods: HER-2/neu and ER/PR expression was analyzed by enzyme-linked immunosorbent assay (ELISA) and enzyme immunoassay (EIA), respectively, in 14 human breast cancer cell lines, some of which had been transfected with the HER-2/neu gene. For the clinical study population, HER-2/neu protein levels were assessed by ELISA (cohort A, n = 665), and HER-2/neu gene copy number was determined using fluorescence in situ hybridization (cohort B, n = 894). ER/PR expression was analyzed by EIA (cohort A) or radioligand binding (cohort B). Associations between HER-2/neu and ER/PR expression were analyzed using Spearman's rho correlation and the chi-square test, and absolute levels were compared using the Mann-Whitney U test. All statistical tests were two-sided., Results: HR-positive human breast cancer cell lines transfected with the HER-2/neu gene expressed statistically significantly lower levels of ER and PR than parental lines. In the clinical cohorts, levels of HER-2/neu overexpression and gene amplification were inversely correlated with ER/PR levels (Cohort A [n = 112]: for ER, r = -0.34, P<.001; for PR, r = -0.24, P =.010. Cohort B [n = 188]: for ER, r = -0.39, P<.001; for PR, r = -0.26, P<.001). Among patients with HR-positive tumors, HER-2/neu-positive tumors had statistically significantly lower ER/PR levels than HER-2/neu-negative ones (Cohort A: for ER, median = 25 fmol/mg [interquartile range [IQR] = 13-78] versus median = 38.5 fmol/mg [IQR = 17-99] and P =.031; for PR, median = 35 fmol/mg [IQR = 12-119] versus median = 88.5 fmol/mg [IQR = 22-236] and P<.001. Cohort B: for ER, median = 44 fmol/mg [IQR = 13-156] versus median = 92 fmol/mg [IQR = 35-235] and P<.001; for PR, median = 36 fmol/mg [IQR = 13-108] versus median = 84 fmol/mg [IQR = 24-250] and P<.001). Patients with higher levels of HER-2/neu overexpression or amplification had statistically significantly lower levels of ER/PR than patients with lower levels of HER-2/neu overexpression or amplification., Conclusion: Because absolute HR levels are strongly related to response to hormone therapy in primary and advanced breast cancer, reduced ER/PR expression may be one mechanism to explain the relative resistance of HER-2/neu-positive:HR-positive tumors to hormone therapy.

Objectives/hypothesis: Cyclin D1, a cell cycle regulator localized to chromosome 11q13, is amplified in several human tumors including head and neck squamous cell carcinoma (HNSCC). Amplification and/or overexpression of cyclin D1 have been correlated to a poor prognosis. Deletion of the p16 gene, localized to 9p21, has also been observed in a significant proportion of HNSCC. The p16 gene regulates cyclin D1-CDK4 activity and prevents retinoblastoma tumor suppressor gene phosphorylation, thereby downregulating cellular proliferation. Detection of cyclin D1 amplification and p16 deletion using a simple and sensitive method will be valuable for the development of effective treatment modalities for head and neck cancer., Study Design: We have used fluorescence in situ hybridization (FISH) to study cyclin D1 amplification and p16 gene deletion in head and neck tumors. Both single- and dual-color FISH were performed., Methods: Paraffin-embedded tissues from 103 patients with HNSCC were analyzed using genomic DNA probes for cyclin D1 and p16. Dual-color FISH was performed with chromosome 11 or 9 centromeric probes as a control. Twenty-eight of these samples were analyzed for p16 expression by immunohistochemistry., Results: Cyclin D1 amplification was observed in 30% (31/103) of patients, and p16 deletion in 52% (54/103). Lack of p16 expression was observed in 64% (18/28) of patients. There was a good correlation between the deletion of p16 sequences and the loss of p16 expression (P = .008). Amplification of cyclin D1 had a statistically significant association with recurrence, distant metastasis, and survival at 36 months. There was a significant association between p16 deletion and the development of distant metastases. Cyclin D1 amplification and p16 deletion together correlated with recurrence, distant metastasis, and survival., Conclusions: We demonstrate that FISH is a simple and sensitive method for detecting cyclin D1 amplification and p16 deletion in head and neck cancer. Our results suggest that these two genetic aberrations together portend a poorer outcome than either of the abnormalities alone in head and neck cancer.

The essential oil composition and genetic variability of six commercial cultivars of thyme (Thymus vulgaris L.), a Mediterranean medicinal and aromatic plant, were analyzed by GC-MS and randomly amplified polymorphic DNA (RAPD), respectively. All evaluated cultivars belong to the thymol chemotype, with differences in the concentrations of thymol, gamma-terpinene, p-cymene, and other minor components. The comparison of the oil components concentration by multivariate analysis allowed separation of the cultivars into two groups. All of the cultivars exhibited characteristic RAPD patterns that allowed their identification. On the basis of the RAPD patterns, the cultivars could be divided into two clusters, which coincides with results obtained by oil GS-MS analysis, with a correlation coefficient of -0.779.

Purpose: To compare the efficacy of fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) in detecting the HER-2/neu alteration in human breast cancer., Patients and Methods: Unselected stage I, II, and III breast cancer patients (N = 900) were tested for HER-2/neu gene amplification by FISH in paraffin-embedded, formalin-fixed archival material. Of these samples, 856 were tested for HER-2/neu overexpression by non-antigen-retrieval IHC with the polyclonal antibody R60, the sensitivity and specificity of which was preliminarily compared with the United States Food and Drug Administration-approved HercepTest (Dako Corp, Carpinteria, CA). Patient survival was analyzed in relation to the presence of the HER-2/neu alteration as determined by these two methodologies., Results: A total of 189 (21%) of 900 patients were positive by FISH and 147 (17.2%) of 856 were positive by IHC. This discrepancy is consistent with expected loss of IHC sensitivity associated with tissue fixation/embedding. The HercepTest did not improve sensitivity and introduced false positives. Comparison of R60-based IHC with FISH demonstrates that patient survival is associated progressively to gene amplification level as determined by FISH, whereas for IHC an association is found only in the highest (3+) immunostaining group. Among FISH-negative tumors, 45 (6.6%) of 678 were IHC-positive, with a survival probability similar to that of FISH-negative/IHC-negative cases; FISH-positive/IHC-negative patients have a survival probability similar to that of FISH-positive/IHC-positive cases., Conclusion: IHC does not consistently discriminate patients likely to have a poor prognosis, whereas FISH provides superior prognostic information in segregating high-risk from lower-risk beast cancers. HER-2/neu protein overexpression in the absence of gene amplification occurs infrequently in breast cancer, in which case, patient outcome is similar to that of patients without the alteration.

One of the major obstacles to the development of biologically active peptides as clinically useful therapeutic agents has been their low permeation through biological barriers (e.g., intestinal mucosa, blood-brain barrier) and their metabolic lability (1,2). Overcoming these problems is a very contemporary issue for the development of peptide pharmaceuticals. In the preceding chapter, we have indicated that masking the C- and N-terminal polar functional groups of a peptide through cyclization with an acyloxyalkoxy linker can greatly enhance the membrane permeation and metabolic stability of the linear peptide (3). In this chapter, we wish to report a method for the preparation of esterase-sensitive cyclic prodrugs of peptides by taking advantage of a unique "trimethyl lock"-facilitated lactonization system (Fig. 1). Substituted phenol propionic acid derivatives such as 2, upon unmasking of the hydroxyl group, undergo a facile spontaneous intramolecular cyclization to release the moieties attached to the carboxyl functional group (Fig. 1) (4-6). The facile cyclization reaction is the result of the "trimethyl lock", which was shown earlier to increase the rate of the cyclization reaction in the order of 10(5-7) (4-7). The result of such facilitation is that compound 2 has a half-life of only approximately 100 s at room temperature in aqueous solution (8,9). Such systems have been used to develop prodrugs of amines and alcohols (8-10) and redox-sensitive protecting groups of amines (11). Fig. 1. The design of an esterase sensitive prodrug system for the cyclic deriva-tization of peptides.

Unlabelled: To evaluate the cellular permeation characteristics and the chemical and enzymatic stability of coumarinic acid-based cyclic prodrugs 1 and 2 of the opioid peptides [Leu5]-enkephalin (H-Tyr-Gly-Gly-Phe-Leu-OH) and DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH), respectively., Methods: The rates of conversion of the cyclic prodrugs 1 and 2 to [Leu5]-enkephalin and DADLE, respectively, in HBSS, pH 7.4 (Caco-2 cell transport buffer) and in various biological media having measurable esterase activity were determined by HPLC. The cell permeation characteristics of [Leu5]-enkephalin, DADLE and cyclic prodrugs 1 and 2 were measured using Caco-2 cell monolayers grown onto microporus membranes and monitored by HPLC., Results: In HBSS, pH 7.4, cyclic prodrugs 1 and 2 degraded chemically to intermediates that further degraded to [Leu5]-enkephalin and DADLE, respectively, in stoichiometric amounts. In 90% human plasma and rat liver homogenate, the disappearance of cyclic prodrugs 1 and 2 was significantly faster than in HBSS, pH 7.4. The half-lives in 90% human plasma and in rat liver homogenate were substantially longer after pretreatment with paraoxon, a known inhibitor of serine-dependent esterases. When applied to the AP side of a Caco-2 cell monolayer, cyclic prodrug 1 exhibited significantly greater stability against peptidase metabolism than did [Leu5]-enkephalin. Cyclic prodrug 2 and DADLE exhibited similar stability when applied to the AP side of the Caco-2 cell monolayer. Prodrug 1 was 665-fold more able to permeate the Caco-2 cell monolayers than was [Leu5]-enkephalin, in part because of its increased enzymatic stability. Prodrug 2 was shown to be approximately 31 fold more able to permeate a Caco-2 cell monolayer than was DADLE., Conclusions: Cyclic prodrugs 1 and 2, prepared with the coumarinic acid promoiety, were substantially more able to permeate Caco-2 cell monolayers than were the corresponding opioid peptides. Prodrug 1 exhibited increased stability to peptidase metabolism compared to [Leu5]-enkephalin. In various biological media, the opioid peptides were released from the prodrugs by an esterase-catalyzed reaction, which is sensitive to paraoxon inhibition.

The clinical development of orally active peptide drugs has been limited by their unfavorable physicochemical characteristics (e.g., charge, hydrogen bonding potential, size), which prevent them from permeating biological barriers such as the intestinal mucosa, and also their lack of stability against enzymatic degradation (1-12). Unfortunately, many of the structural features of peptides (i.e., the N-terminal amino group and C-terminal carboxyl group, and side chain carboxyl, amino, and hydroxyl groups) that bestow upon the molecule affinity and specificity for its pharmacological receptor severely restrict its ability to permeate biological barriers and make the molecules substrates for peptidases. Therefore, successful oral delivery of peptides depends on strategies designed to alter the physicochemical characteristics of these potential drugs without changing their biological activity in order to circumvent the intestinal epithelial cells.

Purpose: To compare the permeation characteristics of amide bond-containing HIV-1 protease inhibitors and their pyrrolinone-containing counterparts across Caco-2 cell monolayers, a model of the intestinal mucosa., Methods: Transepithelial transport and cellular uptake of three pairs of amide bond-containing and pyrrolinone-based peptidomimetics were assessed in the presence and absence of cyclosporin A using the Caco-2 cell culture model. The potential of the peptidomimetics to interact with biological membranes was estimated by IAM chromatography., Results: In the absence of cyclosporin A, apical (AP) to basolateral (BL) flux of all compounds studied was less than the flux determined in the opposite direction (i.e., BL-to-AP). The ratio of the apparent permeability coefficients (Papp) calculated for the BL-to-AP and AP-to-BL transport (P(BL-->AP)/P(AP-->BL)) varied between 1.7 and 36.2. When individual pairs were ompared, P(BL-->AP)/P(AP-BL) ratios of the pyrrolinone-containing compounds were 1.5 to 11.5 times greater than those determined for the amide bond-containing analogs. Addition of 25 microM cyclosporin A to the transport buffer reduced the P(BL-->AP)/P(AP-->BL) ratios for all protease inhibitors to a value close to unity. Under these conditions, the amide bond-containing peptidomimetics were at least 1.6 to 2.8 times more able to permeate Caco-2 cell monolayers than were the pyrrolinone-containing compounds. The intrinsic uptake characteristics into Caco-2 cells determined in the presence of 25 microM cyclosporin A were slightly greater for the amide bond-containing protease inhibitors than for the pyrrolinone-containing analogs. These uptake results are consistent with the transepithelial transport results determined across this in vitro model of the intestinal mucosa., Conclusions: The amide bond-containing and pyrrolinone-based peptidomimetics are substrates for apically polarized efflux systems present in Caco-2 cell monolayers. The intrinsic permeabilities of the amide bond-containing protease inhibitors are slightly greater than the intrinsic permeabilities of the pyrrolinone-based analogs through Caco-2 cell monolayers.

Amplification of the HER-2/neu (c-erbB-2) gene resulting in overexpression of the p185HER-2 growth factor receptor occurs in approximately 25% of early stage breast cancers. HER-2/neu has been established as an important independent prognostic factor in early stage breast cancer in large cohorts of patients and in cohorts with very long (30 year) follow-up duration. New data are emerging to suggest that HER-2/neu may be useful not only as a prognostic factor but also as a predictive marker for projecting response to chemotherapeutics, antiestrogens, and therapeutic anti-HER-2/neu monoclonal antibodies. In this review we highlight recent data on HER-2/neu as a predictive marker of response to breast cancer therapy and discuss the clinical implications of this information. The difficulty in comparing results from different data sets due to the wide variety of reagents and technologies used to detect HER-2/neu amplification/overexpression in clinical specimens is also discussed. Finally, we report results from experimental models of HER-2/neu overexpression which have been used in an effort to understand the relationship between HER-2/neu and response to chemotherapeutics and antiestrogens in breast cancer.

Purpose: To evaluate the effect of size and charge on the permeation characteristics of peptides across the intestinal mucosa., Methods: The lipophilicities of neutral, positively and negatively charged capped amino acids (Asn, Lys, Asp), tripeptides (Ac-Gly-X-Ala-NH2; X = Asn, Lys, Asp) and hexapeptides (Ac-Trp-Ala-Gly-Gly-X-Ala-NH2; X = Asn, Lys, Asp) were estimated using an immobilized artificial membrane. The diffusion coefficients used to calculate the molecular radii were measured by NMR. The transport characteristics of the model peptides were determined across Caco-2 cell monolayers., Results: When model compounds having the same charge were compared, permeation was highly size-dependent (capped amino acids > tripeptides > hexapeptides), suggesting transport predominantly via the paracellular route. For example, the flux of the negatively charged Asp amino acid (Papp = 10.04 +/- 0.43 x 10(-8) cm/s) was 3 times greater than that observed for the Asp-containing hexapeptide (Papp = 3.19 +/- 0.27 x 10(-8) cm/s). When model compounds of the same size were compared, permeation across the cell monolayer was charge-dependent (negative < positive < or = neutral). For example, the neutral, Asn-containing tripeptide (Papp = 25.79 +/- 4.86 x 10(-8) cm/s) was substantially more able to permeate the Caco-2 cell monolayer than the negatively charged Asp-containing tripeptide (Papp = 7.95 +/- 1.03 x 10(-8) cm/s) and the positively charged Lys-containing tripeptide (Papp = 9.86 +/- 0.18 x 10(-8) cm/s). The permeability of the cell monolayer to peptides became less sensitive to net charge as the size of the peptides increased., Conclusions: A positive net charge of hydrophilic peptides enhances their permeation across the intestinal mucosa via the paracellular pathway. With increasing molecular size, molecular sieving of the epithelial barrier dominates the transport of peptides, and the effect of the net charge becomes less significant.

Purpose: To determine how restricted conformational flexibility of hexapeptides influences their cellular permeation characteristics., Methods: Linear (Ac-Trp-Ala-Gly-Gly-X-Ala-NH2; X = Asp, Asn, Lys) and cyclic (cyclo[Trp-Ala-Gly-Gly-X-Ala]; X = Asp, Asn, Lys) hexapeptides were synthesized, and their transport characteristics were assessed using the Caco-2 cell culture model. The lipophilicities of the hexapeptides were determined using an immobilized artificial membrane. Diffusion coefficients used to calculate molecular radii were determined by NMR. Two-dimensional NMR spectroscopy, circular dichroism, and molecular dynamic simulations were used to elucidate the most favorable solution structure of the cyclic Asp-containing peptide., Results: The cyclic hexapeptides used in this study were 2-3 times more able to permeate (e.g., Papp = 9.3 +/- 0.3 x 10(-8) cm/sec, X = Asp) the Caco-2 cell monolayer than were their linear analogs (e.g., Papp = 3.2 +/- 0.3 x 10(-8) cm/sec, X = Asp). In contrast to the linear hexapeptides, the flux of the cyclic hexapeptides was independent of charge. The cyclic hexapeptides were shown to be more lipophilic than the linear hexapeptides as determined by their retention times on an immobilized phospholipid column. Determination of molecular radii by two different techniques suggests little or no difference in size between the linear and cyclic hexapeptides. Spectroscopic data indicate that the Asp-containing linear hexapeptide exists in a dynamic equilibrium between random coil and beta-turn structures while the cyclic Asp-containing hexapeptide exists in a well-defined compact amphophilic structure containing two beta-turns., Conclusions: Cyclization of the linear hexapeptides increased their lipophilicities. The increased permeation characteristics of the cyclic hexapeptides as compared to their linear analogs appears to be due to an increase in their flux via the transcellular route because of these increased lipophilicities. Structural analyses of the cyclic Asp-containing hexapeptide suggest that its well-defined solution structure and, specifically the existence of two beta-turns, explain its greater lipophilicity.

Purpose: To evaluate a cyclic phenylpropionic acid prodrug of a model hexapeptide (H-Trp-Ala-Gly-Gly-Asp-Ala-OH) as a novel approach to enhance the membrane permeation of a peptide and stabilize it to metabolism., Methods: Conversion to the linear hexapeptide was studied at 37 degrees C in HBSS, pH 7.4, and in various biological milieus having measurable esterase activities. Transport and metabolism characteristics were assessed using the Caco-2 cell culture model., Results: In aqueous buffered solution, pH 7.4, the cyclic prodrug degraded quantitatively (t1/2 = 1795 +/- 289 min) to the linear hexapeptide and the lactone. Substantially faster degradation of the cyclic prodrug was observed in 90% human plasma (t1/2 = 508 +/- 24 min), and in homogenates of Caco-2 cells (t1/2 = 940 +/- 13 min), the rat intestinal mucosa (t1/2 = 1286 +/- 32 min), and rat liver (t1/2 = 840 +/- 42 min). Pretreatment of these biological media with paraoxon significantly decreased the degradation rate of the prodrug. When applied to the apical side of Caco-2 cell monolayers, the cyclic prodrug was significantly more stable than the hexapeptide and at least 71-fold more able to permeate (P(app) = 1.21 +/- 0.12 X 10(-7) cm/s) than was the parent peptide (P(app) < or = 0.17 x 10(-8) cm/s). In the presence of 0.1 mM palmitoyl-DL-carnitine, the transport rate of the cyclic prodrug (P(app) = 2.19 X 10(-6) cm/s) was 1250-fold greater than that of the linear hexapeptide., Conclusions: Preparation of a cyclic peptide using a phenylpropionic acid promoiety reduced the lability of the peptide to peptidase metabolism and substantially increased its permeation through biological membranes. In various biological media the parent peptide was released from the prodrug by an apparent esterase-catalyzed reaction, sensitive to paraoxon inhibition.

Purpose: To evaluate a cyclic acyloxyalkoxycarbamate prodrug of a model hexapeptide (H-Trp-Ala-Gly-Gly-Asp-Ala-OH) as a novel approach to enhance the membrane permeation of the peptide and stabilize it to metabolism., Methods: Conversion to the linear hexapeptide was studied at 37 degrees C in aqueous buffered solutions and in various biological milieus having measurable esterase activities. Transport and metabolism characteristics were assessed using the Caco-2 cell culture model., Results: In buffered solutions the cyclic prodrug degraded chemically to the linear hexapeptide in stoichiometric amounts. Maximum stability was observed between pH 3-4. In 90% human plasma (t1/2 = 100 +/- 4 min) and in homogenates of the rat intestinal mucosa (t1/2 = 136 +/- 4 min) and rat liver (t1/2 = 65 +/- 3 min), the cyclic prodrug disappeared faster than in buffered solution, pH 7.4 (t1/2 = 206 +/- 11 min). Pretreatment of these media with paraoxon significantly decreased the degradation rate of the prodrug. When applied to the apical side of Caco-2 cell monolayers, the cyclic prodrug (t1/2 = 282 +/- 25 min) was significantly more stable than the hexapeptide (t1/2 = 14 min) and at least 76-fold more able to permeate (Papp = 1.30 +/- 0.15 x 10(-7) cm/s) than the parent peptide (Papp < or = 0.17 x 10(-8) cm/s)., Conclusions: Preparation of a cyclic peptide using an acyloxyalkoxy promoiety reduced the lability of the peptide to peptidase metabolism and substantially increased its permeation through biological membranes. In various biological media the parent peptide was released from the prodrug by an apparent esterase-catalyzed reaction, sensitive to paraoxon inhibition.

Amplification and overexpression of the HER-2/neu gene occurs in 25-30% of human breast cancers. This genetic alteration is associated with a poor clinical prognosis in women with either node negative or node positive breast cancers. The initial studies testing this association were somewhat controversial and this controversy was due in large part to significant heterogeneity in both the methods and/or reagents used in testing archival material for the presence of the alteration. These methods included a number of solid matrix blotting techniques for DNA, RNA and protein as well as immunohistochemistry. Fluorescence in situ hybridization (FISH) represents the newest methodologic approach for testing for this genetic alteration. In this study, FISH is compared to Southern, Northern and Western blot analyses as well as immunohistochemistry in a large cohort of archival human breast cancer specimens. FISH was found to be superior to all other methodologies tested in assessing formalin fixed, paraffin embedded material for HER-2/neu amplification. The results from this study also confirm that overexpression of HER-2/neu rarely occurs in the absence of gene amplification in breast cancer (approximately 3% of cases). This method of analysis is rapid, reproducible and extremely reliable in detecting presence of HER-2/neu gene amplification and should have clinical utility.

The excitatory and inhibitory interneuronal pathways in the brainstem are tested by examining the blink reflex and the masseter inhibitory reflex, respectively. We studied the R2 component of the blink reflex and the SP2 component of the masseter inhibitory reflex and their recovery cycle in 56 patients with various forms of dystonia. In patients with cranial, cervical, and generalized dystonia, but not in patients with extracranial segmental dystonia, the recovery cycle of both reflexes was enhanced. The recovery cycle of R2 and SP2 can demonstrate subclinical changes in excitability of brainstem interneurons. The degree of enhancement of the recovery cycles did not correlate, however, with the severity of clinical facial muscle impairment. In addition, the recovery cycles correlated positively with each other, showing that excitatory as well as inhibitory interneuronal pathways in the brainstem are perturbed in dystonia. Study of the trigemino-facial and trigemino-trigeminal reflexes provides an objective tool for assessing functional abnormalities in dystonia.

Evoked by electrical stimulation of the mental nerve, the masseter inhibitory reflex consists of an early and a late silent period (SP1 and SP2), which interrupt the voluntary electromyographic (EMG) activity in the masseter muscle. We recorded the masseter inhibitory reflex and measured its latency, depth of suppression, duration and recovery cycle to paired stimuli, in patients with Huntington's chorea. Parkinson's disease, dystonia, or unilateral masticatory spasm. In patients with Huntington's chorea the reflex data and recovery cycle were normal. In patients with Parkinson's disease or dystonia, although the reflex data were normal, SP2 recovered far more rapidly than it did in control subjects. This is possibly due to hypoactivity of an inhibitory control of the polysynaptic chain of ponto-medullary interneurons that mediate SP2. In patients with unilateral masticatory spasm, both SP1 and SP2 were absent. Suppression is probably absent because this involuntary movement originates at a point along the peripheral course of the nerve.

Submicroscopic extrachromosomal elements (amplisomes) containing amplified dihydrofolate reductase (DHFR) genes have been investigated in a methotrexate-resistant derivative of the human cell line HeLa BU25, 10B3, by field-inversion gel electrophoresis. The amount and kinetics of formation of these elements have been correlated with the level and time course of overall DHFR gene amplification. The amplisomes account for the great majority and possibly the totality of the amplified DHFR genes in 10B3 cells. They appear very early during the development of methotrexate resistance and increase in parallel with the amplified genes. These observations suggest that these elements are involved in an early event, possibly the first event, of gene amplification in this system. Amplisomes tend to be lost from 10B3 cells in the absence of selective pressure, although much more slowly than expected from simple dilution of nonreplicating elements. Surprisingly, under selective pressure, these elements have shown no tendency to become integrated into chromosomes or to generate minute chromosomes over a period of almost 1 year, in contrast to what has been described in other systems.