Oriented imaging of 3D subcellular structures in bacterial cells using optical tweezers.

Using oscillating optical tweezers, we show that controlled alignment of rod-shaped bacterial cells allows imaging fluorescently labeled three-dimensional (3D) subcellular structures from different, optimized viewpoints. To illustrate our method, we analyze the Z ring of E. coli. We obtain that the radial width of the Z ring in unconstricted cells is about 120 nm. This result suggests that the Z ring consists of an extremely sparse network of FtsZ filaments.

Case series: 2q33.1 microdeletion syndrome--further delineation of the phenotype.

Recurrent deletions of 2q32q33 have recently been reported as a new microdeletion syndrome, clinical features of which include significant learning difficulties, growth retardation, dysmorphic features, thin and sparse hair, feeding difficulties, and cleft or high palate. Haploinsufficiency of one gene within the deleted region, SATB2, has been suggested to be responsible for most of the features of the syndrome. This article describes seven previously unreported patients with deletions at 2q33.1, all partially overlapping the previously described critical region for the 2q33.1 microdeletion syndrome. The deletions ranged in size from 35 kb to 10.4 Mb, with the smallest deletion entirely within the SATB2 gene. Patients demonstrated significant developmental delay and challenging behaviour, a particular behavioural phenotype that seems to be emerging with more reported patients with this condition. One patient in this cohort has a deletion entirely within SATB2 and has a cleft palate, whereas several patients with larger deletions have a high arched palate. In addition, one other patient has significant orthopaedic problems with ligamentous laxity. Interestingly, this patient has a deletion that lies just distal to SATB2. The orthopaedic problems have not been reported previously and are possibly an additional feature of this syndrome. Overall, this report provides further evidence that the SATB2 gene is the critical gene in this microdeletion syndrome. In addition, because the individuals in this study range in age from 3-19 years, these patients will help define the natural progression of the phenotype in patients with this microdeletion.

Timing of Z-ring localization in Escherichia coli.

Bacterial cell division takes place in three phases: Z-ring formation at midcell, followed by divisome assembly and building of the septum per se. Using time-lapse microscopy of live bacteria and a high-precision cell edge detection method, we have previously found the true time for the onset of septation, τ(c), and the time between consecutive divisions, τ(g). Here, we combine the above method with measuring the dynamics of the FtsZ-GFP distribution in individual Escherichia coli cells to determine the Z-ring positioning time, τ(z). To analyze the FtsZ-GFP distribution along the cell, we used the integral fluorescence profile (IFP), which was obtained by integrating the fluorescence intensity across the cell width. We showed that the IFP may be approximated by an exponential peak and followed the peak evolution throughout the cell cycle, to find a quantitative criterion for the positioning of the Z-ring and hence the value of τ(z). We defined τ(z) as the transition from oscillatory to stable behavior of the mean IFP position. This criterion was corroborated by comparison of the experimental results to a theoretical model for the FtsZ dynamics, driven by Min oscillations. We found that τ(z) < τ(c) for all the cells that were analyzed. Moreover, our data suggested that τ(z) is independent of τ(c), τ(g) and the cell length at birth, L(0). These results are consistent with the current understanding of the Z-ring positioning and cell septation processes.

Rotation of single bacterial cells relative to the optical axis using optical tweezers.

Using a single-beam, oscillating optical tweezers, we demonstrate trapping and rotation of rod-shaped bacterial cells with respect to the optical axis. The angle of rotation, θ, is determined by the amplitude of the oscillation. It is shown that θ can be measured from the longitudinal cell intensity profiles in the corresponding phase-contrast images. The technique allows viewing the cell from different perspectives and can provide a useful tool in fluorescence microscopy for the analysis of three-dimensional subcellular structures.

Timing the start of division in E. coli: a single-cell study.

We monitor the shape dynamics of individual E. coli cells using time-lapse microscopy together with accurate image analysis. This allows measuring the dynamics of single-cell parameters throughout the cell cycle. In previous work, we have used this approach to characterize the main features of single-cell morphogenesis between successive divisions. Here, we focus on the behavior of the parameters that are related to cell division and study their variation over a population of 30 cells. In particular, we show that the single-cell data for the constriction width dynamics collapse onto a unique curve following appropriate rescaling of the corresponding variables. This suggests the presence of an underlying time scale that determines the rate at which the cell cycle advances in each individual cell. For the case of cell length dynamics a similar rescaling of variables emphasizes the presence of a breakpoint in the growth rate at the time when division starts, tau(c). We also find that the tau(c) of individual cells is correlated with their generation time, tau(g), and inversely correlated with the corresponding length at birth, L(0). Moreover, the extent of the T-period, tau(g) - tau(c), is apparently independent of tau(g). The relations between tau(c), tau(g) and L(0) indicate possible compensation mechanisms that maintain cell length variability at about 10%. Similar behavior was observed for both fast-growing cells in a rich medium (LB) and for slower growth in a minimal medium (M9-glucose). To reveal the molecular mechanisms that lead to the observed organization of the cell cycle, we should further extend our approach to monitor the formation of the divisome.

Shape of nonseptated Escherichia coli is asymmetric.

The shape of Escherichia coli is approximately that of a cylinder with hemispherical caps. Since its size is not much larger than optical resolution, it has been difficult to quantify deviations from this approximation. We show that one can bypass this limitation and obtain the cell shape with subpixel accuracy. The resulting contours are shown to deviate from the hemisphere-cylinder-hemisphere shape. In particular, the cell is weakly asymmetric. Its two caps are different from each other and the sides are slightly curved. Most cells have convex sides. We discuss our results in light of several mechanisms that are involved in determining the shape of cells.

Relaxation dynamics of a single DNA molecule.

The relaxation of a single DNA molecule is studied. The experimental system consists of optical tweezers and a micron-sized bead that is tethered to the bottom of the sample by a single double-stranded DNA molecule. The bead slows down the DNA relaxation from a strongly stretched configuration such that it is passing through stretched equilibrium states. This allows for a theoretical description of the relaxation trajectory, which is in good agreement with experiment.

Composite index of skeletal mass: principal components analysis of regional bone mineral densities.

Principal components analysis is a statistical method that is used to reduce and explore data to facilitate further analyses. This method was applied to bone mineral densities measured at seven sites in 109 black and 44 white women, ages 22-80, at an internal medicine clinic in urban Detroit. We excluded subjects with a history of diseases or drugs known to affect bone metabolism. Principal components analysis was used to summarize the interrelationship of the densities and yielded two major results. First, the seven site measurements were reduced to a single, composite index (PC1) of skeletal mass that accounted for 73% of the variation in density among subjects. PC1 had roughly equal weights among the sites. A second combination of the seven sites indicated that the contrast between axial and appendicular regional densities accounted for another 10% of the variation among subjects. In investigating the relationship of density to age, body mass index, and ethnic group, we found that the principal components composite index had a stronger correlation with age (r = -0.58) and with body mass index (r = 0.34) than almost all of the regional densities. Black-white differences were larger for the composite index than for any single site density. A multiple regression of the composite index on ethnicity, body mass index, and age yielded a larger R2 (0.46) than any of the individual site densities. The second principal component, although of theoretical interest, showed a minimal ability to discriminate among subjects using the three independent variables of this study.

Expression of Wilms' tumor suppressor gene (WT1) in term human trophoblast: regulation by cyclic adenosine 3',5'-monophosphate.

The Wilms' tumor suppressor gene (WT1), which is deleted in some Wilms' tumors, encodes a zinc finger transcription factor. We studied WT1 messenger ribonucleic acid (mRNA) in human term placenta and cytotrophoblasts differentiating into syncytiotrophoblasts in vitro by RT-PCR. The results suggest that WT1 mRNA is expressed in the trophoblasts in a cell-specific fashion. WT1 mRNA expression has been observed to decline remarkably in trophoblast cells after 72 h, when these cells are morphologically differentiated into multinucleated syncytiotrophoblasts. As it is well known that cAMP as a second messenger plays a significant role in cellular proliferation and differentiation of placental cells, we examined the effect of 8-bromo-cAMP on WT1 mRNA expression in undifferentiated cytotrophoblasts and differentiated syncytiotrophoblasts. We observed that cAMP enhanced WT1 mRNA expression in cytotrophoblasts, but remained ineffective in altering WT1 mRNA in syncytiotrophoblasts. In summary, the results of this investigation demonstrate that the WT1 gene is developmentally regulated during trophoblast differentiation. An involvement of the cAMP-mediated system in regulating the WT1 gene in the trophoblast is suggested.

Microcephaly, lymphedema, and chorioretinal dysplasia: a distinct syndrome?

We report on 2 unrelated patients with microcephaly, lymphedema, and chorioretinal changes. They are compared with previously reported patients with microcephaly and lymphedema and microcephaly with chorioretinal changes. The question is raised whether all of these patients represent one entity or are separate syndromes. Until more data are available we propose that our patients represent a single entity.

Familial Brachmann-de Lange syndrome: further evidence for autosomal dominant inheritance and review of the literature.

We report on a mother and daughter with the Brachmann-de Lange syndrome which supports the view that in some families this disorder is due to autosomal dominant inheritance. A review of the literature concerning autosomal and recessive inheritance of this syndrome is presented.

Syndrome of microcephaly, facial and hand abnormalities, tracheoesophageal fistula, duodenal atresia, and developmental delay.

We report on six new families (12 new patients) with the syndrome of microcephaly, facial and hand abnormalities, tracheoesophageal fistula, duodenal atresia, and developmental delay. The most common findings were hand abnormalities, microcephaly, short and/or narrow palpebral fissures, broad nasal bridge, anteverted nostrils, ear abnormalities, and micrognathia. Inheritance is autosomal dominant. There is a significant amount of intrafamilial variability especially as it relates to the gastrointestinal findings. Although the first patients reported, who were very young, did not exhibit any developmental delay, they subsequently did develop learning problems, and 87% of our 12 patients had mental retardation or learning difficulties.

Isolated hemihyperplasia (hemihypertrophy): report of a prospective multicenter study of the incidence of neoplasia and review.

Hemihyperplasia is characterized by asymmetric growth of cranium, face, trunk, limbs, and/or digits, with or without visceral involvement. It may be an isolated finding in an otherwise normal individual, or it may occur in several syndromes. Although isolated hemihyperplasia (IHH) is of unknown cause, it may represent one end of the clinical spectrum of the Wiedemann-Beckwith syndrome (WBS). Uniparental paternal disomy of 11p15.5 or altered expression of insulin-like growth factor 2 (IGF2) from the normally silent maternal allele have been implicated as causes of some cases of WBS. IHH and other mild manifestations of WBS may represent patchy overexpression of the IGF2 gene following defective imprinting in a mosaic fashion. The natural history of IHH varies markedly. An association among many overgrowth syndromes and a predisposition to neoplasia is well recognized. Heretofore the risk for tumor development in children with IHH was unknown. We report on the results of a prospective multicenter clinical study of the incidence and nature of neoplasia in children evaluated because of IHH. One hundred sixty-eight patients were ascertained. A total of 10 tumors developed in nine patients, for an overall incidence of 5.9%. Tumors were of embryonal origin (similar to those noted in other overgrowth disorders), including Wilms tumor, hepatoblastoma, adrenal cell carcinoma, and leiomyosarcoma of the small bowel in one case. These data support a tumor surveillance protocol for children with IHH similar to that performed in other syndromes associated with overgrowth.

Aortic pressure during human cardiac arrest. Identification of pseudo-electromechanical dissociation.

We measured aortic pressure during clinically apparent cardiac electromechanical dissociation (EMD). Patients with pulse pressures were designated as having pseudo-EMD; those without, as having true EMD. Of the 200 patients studied, 54 presented with EMD, and 40 others developed it during resuscitation. Of the 94 with EMD, 39 were found to have pseudo-EMD. We compared the two types of EMD for electrocardiographic duration, return of palpable pulses, and response to standard- and high-dose epinephrine. The mean resting aortic pressure was 18 +/- 11 mm Hg in patients with true EMD and 28 +/- 11 mm Hg in those with pseudo-EMD. The mean pulse pressure in patients with pseudo-EMD was 6.3 +/- 3.5 mm Hg. Patients with pseudo-EMD had a higher proportion of witnessed arrests, higher PaO2, and lower PaCO2 than patients with true EMD. Patients with pseudo-EMD had shorter QR and QRS durations than patients with true EMD. They had a better response to standard- and high-dose epinephrine than patients with true EMD. Many patients diagnosed clinically to be in EMD have mechanical cardiac activity; this should be considered when interpreting the results of cardiac arrest research.

The plantar compartments of the foot: a roentgen approach I. Experimental observations.

The integrity of the plantar muscle compartments of the human foot were studied utilizing radiographic and anatomic techniques in an attempt to determine potential pathways for spread of soft tissue infection. One of three major plantar muscle compartments--medial, intermediate or lateral--in 24 unembalmed cadaveric feet was injected percutaneously under fluoroscopy with radiopaque Methyl-methacrylate using both manual and automatic injection techniques with pressure callibration. Following freezing, specimens were sectioned, radiographed, and dissected or partially macerated. Frequency and patterns of extravasation of contrast material were documented and recorded. The results obtained support the concepts that: a) there are three distinct plantar muscle compartments that are maintained by intermuscular septae; and b) there are potential communicating pathways provided by specific anatomic structures.

Fetal neuroblastoma and catecholamine-induced maternal hypertension.

A case of congenital neuroblastoma is presented. The interesting features are maternal symptomatology consistent with fetal production of catecholamines and the dilemmas of differential diagnosis. Observations are made on the frequency and diagnosis of fetal neoplasia. Antepartum detection of catecholamine metabolites may be the basis for the diagnosis of fetal neuroblastoma.

Effect of mode of delivery on morbidity and mortality of infants at early gestational age.

The effect of mode of delivery on the mortality and morbidity of 26- to 32-week neonates was studied. Five hundred six consecutive deliveries at 26 to 32 weeks' gestation were reviewed. The populations were divided into high-risk and low-risk neonates by evaluation of antepartum variables known to increase neonatal risk, ie, abruptio placenta. One hundred ninety-six infants were classified as low risk. In this group, 124 vaginal and 72 cesarean section deliveries were compared using demographic, peripartum, and neonatal variables. Cesarean delivery was associated with highly significant maternal morbidity, including a 30% incidence of vertical uterine incision. No difference in neonatal mortality was shown. Cesarean delivery was associated with lower one-minute Apgar scores and a greater incidence and severity of hyaline membrane disease. No neonatal differences were shown in the incidence of trauma, intraventricular hemorrhage, or seizures. This study does not support cesarean delivery of all tiny neonates.

Bulimia nervosa in pregnancy: a case report.

Bulimia nervosa is an eating disorder characterized by secretive binge eating and purging with induced vomiting, laxatives, and diuretics. The disorder primarily afflicts young white women between 18-35 years of age. We report the case of a 30-year-old pregnant woman with a 17-year history of bulimia that involved up to six episodes of binging and purging daily. A multidisciplinary approach enabled outpatient management throughout gestation. The pregnancy resulted in the delivery of a normal 3000-g female infant at term. The ramifications for maternal and fetal well-being and the goals of therapeutic management are discussed.

Diagnostic and prognostic value of retinal hemorrhages in the neonate.

Ocular fundi of 410 newborns were examined for retinal hemorrhages within 24 hours after delivery. The purpose of this study was to investigate whether the course of pregnancy and management of delivery influence the incidence of neonatal retinal hemorrhage (RH) and whether this incidence could be reduced by electronic monitoring of delivery. To assess the prognostic value of neonatal RH for future development of the child, the physical, motor, and psychologic states of 252 infants were examined 6 to 18 months following delivery. The incidence of RH was 37.3% and was not influenced by maternal age, parity, gestational age, course of pregnancy, birth weight, or electronic monitoring of delivery. The mode of delivery did influence the incidence of RH. Gesell developmental schedule showed no correlation between ocular findings and later development of the child.