Tracing apoptosis and stimulation in individual cells by fluorescence intensity and anisotropy decay

Presented is the use of fluorescence lifetime (FLT), anisotropy decay, and associated parameters as differential indicators of cellular activity. A specially designed combination of a frequency mode based time resolved microscope and a picoliter well-per-cell array have been used to perform temporal measurements in individual cells under various biological conditions. Two biological models have been examined: mitogenic activation of peripheral blood mononuclear cells (PBMC) and induction of programmed cell death (apoptosis) in Jurkat T cells (JTC). The FLT of fluorescein stained PBMC was found to increase from 4+/-0.02 to 4.5+/-0.025 ns due to mitogenic activation, whereas during apoptosis in fluorescein stained JTC, the FLT remained constant. Notably, the rotational correlation times changed in both models: decreased in PBMC from 2.5+/-0.08 to 2+/-0.1 ns, and increased in JTC from 2.1+/-0.07 to 3.3+/-0.09 ns. FLT and rotational correlation time were used to calculate the steady state fluorescence anisotropy (FA) which was compared to directly measured FA values. The present study suggests that in addition to bioindication, the said parameters can provide valuable information about cellular mechanisms that may involve complex molecular diffusion dynamics, as well as information about structural changes that a cellular fluorophore undergoes in the course of cell activation.     

Gastric residual in growing preterm infants: effect of body position

Studies of the effect of body position during and after bolus feeding upon gastric emptying or gastric residual have not had consistent results. We tested the hypotheses that right lateral decubitus leads to less gastric residual than left lateral decubitus and that the prone position leads to less gastric residual than the supine position. A prospective randomized clinical trial with triple crossover of healthy growing, appropriate for gestational age preterm infants. Each infant was successively studied while fed in the four different positions. Gastric residuals were measured at 1 and 3 hours after initiation of feeding and returned. Thirty-one patients were studied. At 1 hour, right lateral decubitus led to less significant residuals than the left lateral decubitus and the prone position led to less residual than the left lateral decubitus. The amount of gastric residuals 1 hour after a meal appears to be in the following decreasing order: left, supine, prone, right.     

Realignment arthrodesis of the rearfoot and ankle: a comprehensive evaluation

Ankle and tibiotalocalcaneal arthrodeses are performed for the treatment of painful, arthritic, unstable, and deformed rearfoot and ankle joints. Surgical complications are not uncommon (approximately 30%); some can be attributed to poor preoperative planning and inadequate intraoperative position. Several authors have attempted to define the optimal position for ankle arthrodesis without objective multiplanar radiographic analysis and consistent reference points. This investigation explored the effects of ankle and tibiotalocalcaneal realignment arthrodeses on static lower-extremity position in 20 patients. The most common preoperative diagnosis was severe degenerative joint disease following ankle fractures and ankle instability. Seven tibiotalocalcaneal arthrodeses and 13 isolated ankle arthrodeses were performed (mean follow-up, 22 months). Average time to radiographic osseous union of the isolated ankle and tibiotalocalcaneal arthrodeses was 11 and 7 weeks, respectively. Medical complications occurred in 2 patients (10%). There were no statistically significant differences between preoperative and postoperative angular relationships. This study objectively quantifies multiplanar foot-to-leg realignment and defines the optimal clinical and radiographic positions for ankle and tibiotalocalcaneal realignment arthrodeses.     

Mutation analysis of SBDS in pediatric acute myeloblastic leukemia

BACKGROUND: Shwachman-Diamond syndrome (SDS) is associated with a high risk of myelodysplasia, acute myeloid leukemia (AML), and chromosome 7 abnormalities. Ninety percent of SDS patients have mutations in SBDS on 7q11. Herein, we studied the role of genetic alterations in SBDS in AML. PROCEDURE: DNA was extracted from marrows of SDS patients with AML, as well as from children with de novo AML. Direct sequencing of PCR amplified genomic DNA was performed using specific primers flanking each exon. To study whether SBDS heterozygosity confers a risk for MDS/AML, data on family members of SDS patients on the Canadian Inherited Marrow Failure Registry (CIMFR) was analyzed. RESULTS: Of two SDS patients with SDS/AML one was homozygous 258 + 2T > C, and one was compound heterozygous 183-184TA > CT/258 + 2T > C. To determine whether a subset of patients with SDS can present with AML, we analyzed 48 AML samples at remission, but no mutations were identified. To address whether acquired mutated SBDS gene is associated with leukemic transformation in de novo AML, we analyzed 77 AML samples at diagnosis or relapse (4 with -7 and 7q-) for SBDS mutations; no alterations were detected. Also, among the relatives of an SDS patient cohort on the registry no cases of MDS/AML were reported. CONCLUSIONS: Common mutations occurred in our SDS patients who develop AML, and thus, AML is not confined to a rare genetic subgroup of SDS. Newly diagnosed patients with AML are unlikely to have an underlying undiagnosed SDS. Acquired SBDS gene mutations also would appear unlikely to play a mechanistic role in de novo AML, and might not be involved in the pathogenesis of chromosome 7 abnormalities as well.     

Nucleated red blood cells in preterm infants who have necrotizing enterocolitis

Absolute nucleated red blood cell counts for 23 preterm newborn infants who subsequently had development of necrotizing enterocolitis were significantly higher than for 23 control infants. These infants may have been exposed to relative intrauterine hypoxemia, a possible risk factor for the development of necrotizing enterocolitis.     

Ratiometric fluorescence polarization as a cytometric functional parameter: theory and practice

The use of ratiometric fluorescence polarization (RFP) as a functional parameter in monitoring cellular activation is suggested, based on the physical phenomenon of fluorescence polarization dependency on emission wavelengths in multiple (at least binary) solutions. The theoretical basis of this dependency is thoroughly discussed and examined via simulation. For simulation, aimed to imitate a fluorophore-stained cell, real values of the fluorescence spectrum and polarization of different single fluorophore solutions were used. The simulation as well as the experimentally obtained values of RFP indicated the high sensitivity of this measure. Finally, the RFP parameter was utilized as a cytometric measure in three exemplary cellular bioassays. In the first, the apoptotic effect of oxLDL in a human Jurkat FDA-stained T cell line was monitored by RFP. In the second, the interaction between cell surface membrane receptors of human T lymphocyte cells was monitored by RFP measurements as a complementary means to the fluorescence resonance energy transfer (FRET) technique. In the third bioassay, cellular thiol level of FDA- and CMFDA-labelled Jurkat T cells was monitored via RFP.     

Sutureless thyroidectomy using electrothermal system: a new technique

Bleeding is a dangerous complication of thyroid surgery. We describe a new technique of vessel and tissue sealing and bleeding control using an electrothermal sealer. The technique has some potential advantages over the current methods used for this purpose.     

Vestibular neuronitis in pilots: follow-up results and implications for flight safety

OBJECTIVES To report our experience over the past 12 years with the evaluation and follow-up of pilots with vestibular neuronitis and to discuss points relevant to flight safety and the resumption of flying duties.STUDY DESIGN A retrospective, consecutive case series.METHODS Eighteen military pilots with vestibular neuronitis were examined and followed up. A complete otoneurological workup was performed, including both physical examination and laboratory evaluation. The latter included electro-oculography (EOG) and a rotatory chair test using the smooth harmonic acceleration protocol.RESULTS The mean patient age was 35 +/- 6 years (range, 23 to 42 y), and the average follow-up period was 20.5 +/- 12.8 months (mean +/- standard deviation [SD]; (range, 11 to 48 mo). Electro-oculography caloric test on presentation documented significant unilateral hypofunction in all patients. Thirteen of the 18 patients (72%) had abnormal smooth harmonic acceleration test results. None of the pilots reported any symptoms on follow-up. However, five (28%) had positive otoneurological examination findings, and eight (44%) still had significant caloric lateralization (>25%). The average caloric hypofunction was reduced from 67.8% +/- 29.3% at onset to 40% +/- 16% (mean +/- SD, <.05, paired test). Seven of the patients (39%) had additional electro-oculography findings beyond caloric hypofunction. These included spontaneous, positional, and positioning nystagmus. Smooth harmonic acceleration disease on follow-up was documented in eight patients (44%), five of whom had canal paresis. Eleven patients (61%) demonstrated residual vestibular damage on follow-up. In 6 of these 11 cases (55%), the laboratory evaluation revealed vestibular deficits otherwise undiagnosed by the bedside test battery.CONCLUSIONS The vestibular system plays a central role in orientation awareness and is often challenged by flying conditions. The finding that approximately 60% of pilots who have had vestibular neuronitis continue to show signs of vestibular malfunction, despite apparent clinical recovery, emphasizes the need for a complete vestibular evaluation, including specific bedside testing and laboratory examinations, before flying duties can be resumed.