An ethanol-based fixation method for anatomical and micro-morphological characterization of leaves of various tree species.

The use of formalin constitutes serious health hazards for laboratory workers. We investigated the suitability and performance of the ethanol-based fixative, FineFIX, as a substitute for formalin for anatomical and cellular structure investigations of leaves by light microscopy and for leaf surface and ultrastructural analysis by scanning electron microscopy (SEM). We compared the anatomical features of leaf materials prepared using conventional formalin fixation with the FineFIX. Leaves were collected from ornamental tree species commonly used in urban areas. FineFIX was also compared with glutaraldehyde fixation and air drying normally used for scanning electron microscopy to develop a new method for evaluating leaf morphology and microstructure in three ornamental tree species. The cytological features of the samples processed for histological analysis were well preserved by both fixatives as demonstrated by the absence of nuclear swelling or shrinkage, cell wall detachment or tissue flaking, and good presentation of cytoplasmic vacuolization. In addition, good preservation of surface details and the absence of shrinkage artefacts confirmed the efficacy of FineFIX fixation for SEM analysis. Cuticular wax was preserved only in air dried samples. Samples treated with chemical substances during the fixation and dehydration phases showed various alterations of the wax structures. In some air dried samples a loss of turgidity of the cells was observed that caused general wrinkling of the epidermal surfaces. Commercial FineFIX is an adequate substitute for formalin in histology and it can be applied successfully also for SEM investigation, while reducing the health risks of glutaraldehyde or other toxic fixatives. To investigate the potential for plants to absorb and capture particulates in air, which requires preservation of the natural morphology of trichomes and epicuticular waxes, a combination of FineFIX fixation and air drying is recommended.

Clinical study of a noninvasive multimodal sono-contrast induced spectroscopy system for breast cancer diagnosis.

PURPOSE: To present a noninvasive multimodal sono-contrast induced spectroscopy (SCIS) system for breast cancer detection. METHODS: An IRB approved clinical study was carried out to evaluate its diagnostic power. A total of 66 subjects were enrolled with informed consent. The study data were grouped into healthy breast tissue (26), histologically proven cancer (14), and benign mass (26). The diffuse reflectance optical intensity and low intensity focused ultrasound (LIFU) signals, as well as ultrasound images, were collected during each study. The ratio of optical intensities at wavelengths 685 and 830 nm was analyzed using wavelet technique to compare the LIFU effects in cancer and noncancerous tissues. The ultrasound images were also processed to obtain tissue texture parameters, such as correlation, energy, contrast, homogeneity, etc. Backward stepwise regression method was performed to identify the statistically significant factors correlating to tissue types (cancer vs benign mass). RESULTS: Comparison of the optical signals showed that LIFU induced transitory fluctuation in noncancerous tissue, but not in malignant tissue, as quantified by the ratio of mean absolute deviation (RMAD) of the high frequency component. Statistical analysis revealed that the RMAD ratios were significantly different in tumor vs noncancerous masses (p ≪ 0.01). For tissue texture parameters, energy and correlation were found to statistically correlate with the tissue types. A cancer characterization model was developed using the weighted factors to differentiate the tumor from the benign mass. Trade-off between sensitivity and specificity was obtained by varying the threshold value that estimated the upper-bound of the cancer output factor, from which the receiver-operating characteristic (ROC) curve was generated. The characterization model was optimized using ten modeling datasets and verified using another ten validation datasets randomly generated from the database. The optimization results show that an AUC of 0.93 can be achieved. With threshold 0.3, sensitivity of 96.0%, specificity of 84.1%, and negative predictive value (NPV) of 97.3% can be achieved. CONCLUSIONS: The feasibility of the multimodal system in characterizing breast cancer vs benign mass is established.

Effectiveness of the photochemical reflectance index to track photosynthetic activity over a range of forest tree species and plant water statuses.

In this study, we investigated the potential of the photochemical resistance index (PRI) to track photosynthetic activity under water stress conditions by measuring PRI, leaf fluorescence, the xanthophyll cycle and photosynthetic activity in different forest tree species subjected to progressive drought. The PRI declined with pre-dawn water potential and a significant relationship between PRI and the xanthophyll de-epoxidation state (DEPS) was observed, although with large interspecific variability in the sensitivity of PRI to changes in DEPS. For single tree species, a strong relationship was observed on either PRI light saturated photosynthesis or PRI maximum photochemical efficiency of PSII (ΔF/Fm'); a larger variability in both relationships was apparent when data from different species were pooled together. However, an improved correlation was shown only in the former relationship by plotting the ΔPRI (dawn PRI minus the midday PRI values). Thus, we conclude that PRI is able to provide a good estimate of maximum CO2 assimilation at saturating light and ΔF/Fm' for single tree species, despite the severe drought conditions applied. PRI should be applied more cautiously when dealing with multispecific forests because of confounding factors such as the strong interspecific differences in the initial value of PRI and in the sensitivity of PRI to changes in DEPS in response to drought.

Ectopic expression of LEAFY COTYLEDON1-LIKE gene and localized auxin accumulation mark embryogenic competence in epiphyllous plants of Helianthus annuus x H. tuberosus.

BACKGROUND AND AIMS: The clone EMB-2 of the interspecific hybrid Helianthus annuus x H. tuberosus provides an interesting system to study molecular and physiological aspects of somatic embryogenesis. Namely, in addition to non-epiphyllous (NEP) leaves that expand normally, EMB-2 produces epiphyllous (EP) leaves bearing embryos on the adaxial surface. This clone was used to investigate if the ectopic expression of H. annuus LEAFY COTYLEDON1-LIKE (Ha-L1L) gene and auxin activity are correlated with the establishment of embryogenic competence. METHODS: Ha-L1L expression was evaluated by semi-quantitative RT-PCR and in situ hybridization. The endogenous level and spatial distribution of free indole-3-acetic acid (IAA) were estimated by a capillary gas chromatography-mass spectrometry-selected ion monitoring method and an immuno-cytochemical approach. KEY RESULTS: Ectopic expression of Ha-L1L was detected in specific cell domains of the adaxial epidermis of EP leaves prior to the development of ectopic embryos. Ha-L1L was expressed rapidly when NEP leaves were induced to regenerate somatic embryos by in vitro culture. Differences in auxin distribution pattern rather than in absolute level were observed between EP and A-2 leaves. More precisely, a strong IAA immuno-signal was detected in single cells or in small groups of cells along the epidermis of EP leaves and accompanied the early stages of embryo development. Changes in auxin level and distribution were observed in NEP leaves induced to regenerate by in vitro culture. Exogenous auxin treatments lightly influenced Ha-L1L transcript levels in spite of an enhancement of the regeneration frequency. CONCLUSIONS: In EP leaves, Ha-L1L activity marks the putative founder cells of ectopic embryos. Although the ectopic expression of Ha-L1L seems to be not directly mediated by auxin levels per se, it was demonstrated that localized Ha-L1L expression and IAA accumulation in leaf epidermis domains represent early events of somatic embryogenesis displayed by the epiphyllous EMB-2 clone.

Seasonal variation of monoterpene emission from Malus domestica and Prunus avium.

Emission rates of monoterpenes released by apple (Malus domestica Borkh) and cherry (Prunus avium L.) were estimated at different phenological stages. These measurements employed a dynamic flow-through Teflon chamber, sample collection onto cartridges filled with graphitized carbon and thermal desorption gas chromatography-mass spectrometry (GC-MS) for identification and quantification of the emitted volatiles. At full bloom the release of monoterpene hydrocarbons from cherry flowers was 1213 ng g(-1) dry weight (DW) h(-1), exceeding by approximately three-fold the emission rate of apple flowers (366 ng g(-1) DW h(-1)). Observed seasonal variations in biogenic volatile organic compound (VOC) emissions ranged over several order of magnitudes. At fruit-set and ripening stages, in fact, the hydrocarbon emission dramatically decreased reaching the lowest values at harvest time when leaves were fully mature (3-9 ng g(-1) DW h(-1)). Wide diversity in the composition of compounds from the species studied was also recorded. At blooming, linalool contributed significantly to the monoterpene emission from apple (94% of the emitted carbon) while alpha-pinene and camphene represented on average more than 60% of the total emitted volatiles from cherry flowers. Among the monoterpenes identified in flowers, alpha-pinene, camphene and limonene were also found in the foliage emission of both species. Fruit trees are relevant monoterpene emitters only at blooming and thus for a short period of the vegetative cycle. When leaves are fully developed, the carbon loss due to monoterpene emissions related to the photosynthetically carbon gain is negligible.

Hormonal influence on photocontrol of the protandry in the genus Helianthus.

Under natural photoperiodic conditions protandry in hermaphrodite disc flowers of sunflower (Helianthus annuus L.) is determined by the different elongation rates of the style and filaments. The elongation of the filament and style starts simultaneously after the daily dark period, but the style growth rate is slower. When plants close to anthesis are exposed to continuous white light (WL) a loss of protandry occurs: the filaments do not grow far enough to extrude the anthers from the corolla. The histological analyses show that the number of filament epidermal cells remains unaltered after organ elongation and that cells respond to photoperiod only by cell expansion. Emasculation does not substantially inhibit filament cell expansion, whereas isolation of the filament or stamen from the corolla suggests that this organ could be the perception site of the filament growth stimulus. In vitro treatments with auxin (indole-3-acetic acid, IAA or alpha-naphthaleneacetic acid, NAA) reverses the inhibition of cell expansion caused by continuous WL, whereas gibberellic acid (GA(3)) at high concentrations reproduces the effect of continuous WL. Experiments carried out on various Helianthus spp. show that all these plants have evolved the same photo- and hormonal-control of the protandry. In experiments in which the light treatments were continued for 24 h, the auxins drastically reduced the inhibiting effect of red light (R) and dichromatic treatments FR (far red)+R, whereas GA(3) repressed filament extension regardless of light quality. As far as auxins are concerned, the response of sunflower filaments does not appear to be connected with the polar transport of the hormone. Moreover, the promoting effect of darkness is not mediated by an increase of endogenous free IAA in disc flowers. However, sunflower filaments manifested a similar temporal pattern of response to the light/dark cycle and to auxin.

Advantages of spinal anesthesia in abdominal gynecologic surgery.

The advantages obtained in vaginal surgery and caesarean section using spinal anesthesia led us to test this anesthesia to verify feasibility, problems and advantages in abdominal surgery. Spinal anesthesia was performed in 60 patients between 21 and 87 years of age. Thirty-seven total abdominal hysterectomies with or without adnexectomy, 5 laparotomic miomectomies, 3 adnexectomies, 5 colposacropexies, 2 hysterectomies with lymphadenectomy, 7 Burch colposuspension with or without hysterectomy and 1 laparoscopy for sterilization were performed. No significant problems during surgery and the postoperative period were observed. Resumption of the different physiologic functions were more rapid, hospital stay shorter and compliance greater than with general anesthesia.

Partial detachment of round ligament found at laparoscopy for chronic pelvic pain in a female adolescent.

A 13-year-old female adolescent presented with pelvic pain localized in the right lower abdominal quadrant with diffusion to the groin and internal thigh and worsened by marked leg abduction. No symptoms were detected on physical or sonography examination. The patient reported pain for a further 4 months with no moderation. A laparoscopy was performed, which showed normal adnexa and pelvic hollow. No adhesions were found. The uterus was normal in size and appearance, but the right round ligament presented with a partial detachment at cornual insertion. An inflammation was present. The decision was made to perform no surgical repair with the belief that pain would worsen. The pain gradually lessened, and in 1998 she remains pain free.

Severe coital injury after Frank's creation of a neovagina.

A neovagina was created by the Frank's dilatation method in a girl with vaginal agenesis. At the first coital attempt complete laceration of the cul de sac with copious bleeding occurred. Daily application of estrogen cream was the only therapy until complete healing after four weeks. Possible causes and therapeutic approaches are discussed.

The value of postoperative ultrasound examination after laparoscopic laser cholecystectomy surgery.

Laparoscopic cholecystectomy surgery is rapidly becoming the procedure of choice for patients undergoing gallbladder removal. For the patient, this new minimally invasive surgery allows shorter hospitalization and faster recuperation than traditional open cholecystectomy surgery. Examinations were performed to determine the value of ultrasound in predicting morbidity of patients undergoing the laparoscopic cholecystectomy procedure. Examinations were performed immediately and 6-8 weeks postlaparoscopic surgery on 24 patients with acute or chronic gallbladder disease. All patients had ultrasound examinations prior to surgery. Of the 24 patients studied, 6 patients (25%) developed small fluid accumulations immediately postsurgery. All patients were asymptomatic clinically and remained asymptomatic 6-8 weeks after surgery. The fluid accumulation was thought to be insignificant on the immediate postsurgical studies. This percentage equals that found in open cholecystectomy surgeries. On the delayed ultrasound studies, no fluid deposits or other abnormalities were seen. Ultrasound examination of the asymptomatic patient postlaparoscopic cholecystectomy surgery is not of value for predicting complications in patients undergoing this procedure. Furthermore, immediately postlaparoscopic cholecystectomy surgery, 25% of patients will have small fluid collections, but will not develop complications.

Suppression of clonidine-induced release of growth hormone by thyrotropin releasing hormone in humans.

A single oral dose of clonidine (0.15 mg), a selective alpha-adrenergic stimulating agent, was able to increase plasma growth hormone (GH) levels (above 5 ng/ml) in 6 out of 7 normal men tested. This GH increase was independent of the hypotensive effect of the drug and was observed without any modification of plasma prolactin, thyroid stimulating hormone, gonadotropins and glycemia. When oral clonidine administration was associated to a slow thyrotropin releasing hormone (TRH) infusion (1 mg dissolved in 400 ml of 0.9% saline solution, at a constant rate during 150 min) the plasma GH response was significantly inhibited when compared with that observed after clonidine alone. These results suggest that in normal subjects TRH is capable of blocking the effect of an alpha-adrenergic stimulus which is conceivably acting at the level of central nervous system.