A hyperthermal energy ion beamline for probing hot electron chemistry at surfaces.

An ultrahigh vacuum ion beamline and chamber have been assembled to produce hyperthermal (<400 eV) energy ions for studying hot electron chemistry at surfaces. The specific design requirements for this modified instrument were chosen to enable the exposure of a metal-oxide-semiconductor (MOS) device to monoenergtic, well-collimated beams of alkali ions while monitoring both the scattered beam flux and the device characteristics. Our goal is to explore the role that hot electrons injected toward the MOS device surface play in the neutralization of scattered ions. To illustrate the functionality of our system, we present energy-resolved spectra for Na+, K+, and Cs+ ions scattered from the surface of a Ag(001) single crystal for a range of incident energies. In addition, we show MOS device current-voltage characteristics measured in situ in a new rapid-turnaround load lock and sample translation stage.

foxD5a, a Xenopus winged helix gene, maintains an immature neural ectoderm via transcriptional repression that is dependent on the C-terminal domain.

Xenopus foxD5a, the full-length fork head gene previously described as a PCR fragment (XFLIP), is first detectable at stage II of oogenesis. Low-abundance maternal transcripts are localized to the animal hemisphere of the cleavage embryo, and protein can be translocated to the nucleus prior to the onset of zygotic transcription. Zygotic expression is strongest in the presumptive neural ectoderm at gastrula and neural plate stages, but there is minor paraxial mesodermal expression during primary gastrulation that becomes significant in the tail bud during secondary gastrulation. Expression of foxD5a in animal cap explants induces elongation and expression of mesodermal, neural-inducing, and early neural-specifying genes, indicating a role in dorsal axis formation. Zygotic foxD5a expression is induced strongly by siamois, moderately by cerberus, weakly by Wnt8 and noggin, and not by chordin in animal cap explants. Expression of foxD5a in whole embryos has differential dorsal and ventral effects. Ventral mRNA injection induces partial secondary axes composed of expanded mesodermal and epidermal tissues, but does not induce ectopic neural tissues. Dorsal mRNA injection causes hypertrophy of the neural plate and expansion of early neural genes (sox3 and otx2), but this is not the result of increased proliferation or expanded neural-inducing mesoderm. The neural plate appears to be maintained in an immature state because otx2 expression is expanded and expression of en2, Krox20, proneural genes (Xnrgn1, neuroD) and a neural differentiation gene (n-tubulin) is repressed in foxD5a-expressing cells. These results indicate that foxD5a maintains an undifferentiated neural ectoderm after neural induction. Expression of foxD5a constructs fused with the engrailed repressor domain or with the VP16 activation domain demonstrates that FoxD5a acts as a transcriptional repressor in axis formation and neural plate expansion. Deletion constructs indicate that this activity requires the C-terminal domain of the protein.

Transcription factors of the anterior neural plate alter cell movements of epidermal progenitors to specify a retinal fate.

The embryonic progenitors that give rise to the vertebrate retina acquire their cell fate identity through a series of transitions that ultimately determine their final, differentiated retinal cell fates. In Xenopus, these transitions have been broadly defined as competence, specification, and determination. The expression of several transcription factors within the anterior neural plate at the time when the presumptive eye field separates from other neural derivatives suggests that these genes function to specify competent embryonic progenitors toward a retinal fate. In support of this, we demonstrate that some transcription factors expressed in the anterior neural ectoderm and/or presumptive eye field (otx2, pax6, and rx1) change the fate of competent, ventral progenitors, which normally do not contribute to the retina, from an epidermal to a retinal fate. Furthermore, the expression of these factors changes the morphogenetic movements of progenitors during gastrulation, causing ventral cells to populate the native anterior neural plate. In addition, we experimentally demonstrate that the efficacy of pax6 to specify retinal cells depends on the position of the affected cell relative to the field of neural induction. Thereby, otx2, pax6, and rx1 mediate early steps of retinal specification, including the regulation of morphogenetic cell movements, that are dependent on the level of neural-inductive signaling.

Xenopus Six1 gene is expressed in neurogenic cranial placodes and maintained in the differentiating lateral lines.

Six genes are homeobox-containing transcription factors, many of which are expressed in head structures. We isolated a full-length cDNA of a previously unknown Xenopus member of this family. It shares a high sequence homology with mouse and human Six1, which during development are expressed in mesoderm and muscle. In contrast, XSix1 is prominently expressed in all neurogenic cephalic placodes and lateral line primordia from neurula to tadpole stages. The neurons derived from these placodes do not express XSix1, but the lateral line mechanoreceptors maintain expression. XSix1 is weakly expressed in muscle later in development.

Squamous cell carcinoma of the external auditory canal: an evaluation of a staging system.

OBJECTIVE: The study was conducted to review a staging system proposed by the University of Pittsburgh for temporal bone cancer and to evaluate survival status according to stage, treatment, and certain prognostic factors. STUDY DESIGN: The study was a retrospective case review. SETTING: The study was conducted at a tertiary care medical center and specialty hospital. PATIENTS: Thirty-two patients with primary squamous cell carcinoma of the external auditory canal were studied. INTERVENTION: All patients underwent surgery of the temporal bone. Radiotherapy was given depending on tumor stage and histopathologic findings. MAIN OUTCOME MEASURES: The 2-year survival rates of patients undergoing surgical resection with or without adjuvant radiotherapy. RESULTS: The 2-year survival rates for primary squamous cell carcinoma of the temporal bone were as follows: T1 lesions 100%, T2 80%, T3 50%, and T4 7%. Survival for T3 tumors was 75% with postoperative radiotherapy, compared with 0% with surgery alone. CONCLUSIONS: The 2-year survival data directly correlated with the staging system. The use of adjuvant radiotherapy increased survival rate in patients with a T3 lesion.

Intrinsic bias and lineage restriction in the phenotype determination of dopamine and neuropeptide Y amacrine cells.

Blastomere lineages are differentially biased to produce different neurotransmitter subtypes of amacrine cells (Huang and Moody, 1995, 1997,). To elucidate when this bias is acquired, we examined amacrine lineages at different early developmental times. Our experiments demonstrate that the bias to express dopamine and neuropeptide Y amacrine fates involves several steps before the formation of the definitive optic cup. At cleavage stages, a retinal progenitor that contributes large numbers of cells is already biased to produce its normal repertoire of dopamine amacrine cells, as revealed by transplantation to a new location, whereas the amacrine fate of a progenitor that contributes fewer cells is modified by its new position. At neural plate stages, not all retinal progenitors are multipotent. Nearly one-half populate only the inner nuclear layer and are enriched in amacrine cells. During early optic vesicle stages, an appropriate mitotic tree is required for dopamine and neuropeptide Y, but not serotonin, amacrine cell clusters to form. Thus, the acquisition of amacrine fate bias involves intrinsic maternal factors at cleavage, fate restriction in the neural plate, and specified mitotic patterns in the optic vesicle. At each of these steps only a subset of the embryonic retinal progenitors contributing to amacrine subtypes is biased; the remaining progenitors maintain multipotency. Thus, from the earliest embryonic stages, progenitors of the retina are a dynamic mosaic. This is the first experimental demonstration of amacrine fate decisions that occur during early embryonic periods in advance of the events described in the later, committed retina.

A novel fork head gene mediates early steps during Xenopus lens formation.

Xlens1 is a novel Xenopus member of the fork head gene family, named for its nearly restricted expression in the anterior ectodermal placode, presumptive lens ectoderm (PLE), and anterior epithelium of the differentiated lens. The temporal and spatial restriction of its expression suggests that: (1) Xlens1 is transcribed initially at neural plate stages in response to putative signals from the anterior neural plate that transform lens-competent ectoderm to lens-biased ectoderm; (2) further steps in the process of lens-forming bias restrict Xlens1 expression to the presumptive lens ectoderm (PLE) during later neural plate stages; (3) interactions with the optic vesicle maintain Xlens1 expression in the lens placode; and (4) Xlens1 expression is downregulated as committed lens cells undergo terminal differentiation. Induction assays demonstrate that pax6 induces Xlens1 expression, but unlike pax6, Xlens1 cannot induce the expression of the lens differentiation marker beta-crystallin. In the whole embryo, overexpression of Xlens1 in the lens ectoderm causes it to thicken and maintain gene expression characteristics of the PLE. Also, this overexpression suppresses differentiation in the lens ectoderm, suggesting that Xlens1 functions to maintain specified lens ectoderm in an undifferentiated state. Misexpression of Xlens1 in other regions causes hypertrophy of restricted tissues but only occasionally leads ectopic sites of gamma-crystallin protein expression in select anterior head regions. These results indicate that Xlens1 expression alone does not specify lens ectoderm. Lens specification and differentiation likely depends on a combination of other gene products and an appropriate level of Xlens1 activity.

Animal-vegetal asymmetries influence the earliest steps in retina fate commitment in Xenopus.

An individual retina descends from a restricted and invariant group of nine animal blastomeres at the 32-cell stage. We tested which molecular signaling pathways are responsible for the competence of animal blastomeres to contribute to the retina. Inactivation of activin/Vg1 or fibroblast growth factor (FGF) signaling by expression of dominant-negative receptors does not prevent an animal blastomere from contributing to the retina. However, increasing bone morphogenetic protein (BMP) signaling in the retina-producing blastomeres significantly reduces their contribution. Conversely, reducing BMP signaling by expression of a dominant-negative BMP receptor or Noggin allows other animal blastomeres to contribute to the retina. Thus, the initial step in the retinal lineage is regulated by position within the BMP/Noggin field of epidermal versus neural induction. Vegetal tier blastomeres, in contrast, cannot contribute to the retina even when given access to the appropriate position and signaling fields by transplantation to the dorsal animal pole. We tested whether expression of molecules within the mesoderm inducing (activin, FGF), mesoderm-modifying (Wnt), or neural-inducing (BMP, Noggin) pathways impart a retinal fate on vegetal cell descendants. None of these, several of which induce secondary head structures, caused vegetal cells to contribute to retina. This was true even if the injected blastomeres were transplanted to the dorsal animal pole. Two pathways that specifically induce head tissues also were investigated. The simultaneous blockade of Wnt and BMP signaling, which results in the formation of a complete secondary axis with head and eyes, did not cause the vegetal clone to give rise to retina. However, Cerberus, a secreted protein that also induces an ectopic head with eyes, redirected vegetal progeny into the retina. These experiments indicate that vegetal blastomere incompetence to express a retinal fate is not due to a lack of components of known signaling pathways, but relies on a specific pathway of head induction.

Illness and otological changes during upper respiratory virus infection.

OBJECTIVES: Upper respiratory virus infection is associated with the expression of symptoms and signs of illness, and with the development of complications in anatomically contiguous structures. In most epidemiological studies, the frequency of the various complications is expressed as a fraction of the total population judged to be ill by report, signs, or symptoms. Because not all infected subjects become ill and because infected non-ill subjects may develop complications, such risk estimates could be inaccurate. The objective of this study was to estimate the magnitude of the presentation bias during controlled, experimental infections. STUDY DESIGN: This was a prospective, experimental study of the relationship between illness and otological complications during experimental upper respiratory virus infection in 316 adult volunteers. METHODS: The data for illness and for abnormal middle ear underpressure in adult (18-54 y) volunteers experimentally infected with one of three viruses (rhinovirus type 39, rhinovirus strain hanks, influenza A virus) were analyzed and expressed as the relative frequencies of infected subjects reporting illness, developing abnormal middle ear pressure, and developing abnormal middle ear pressure in the absence of illness. RESULTS: For all three viruses, illness was documented in approximately 50% of the infected subjects. While the frequency of persons developing abnormal middle ear underpressure was greater in the infected-ill subjects, approximately one third of all infected subjects developing that complication did not report illness. CONCLUSIONS: These results support a large presentation bias in epidemiological surveys of viral upper respiratory infections, and infer that those surveys underestimate the true frequency of complications resulting from such infections.

Daily tympanometry in children during the cold season: association of otitis media with upper respiratory tract infections.

The causal association between otitis media and viral upper respiratory tract infections (URI) suggests that early intervention during the course of a viral URI could prevent many episodes of otitis media. However, the feasibility of this approach can not be assessed at present since many aspects of the epidemiology and natural history of URI-associated otitis media are undefined. To address this deficiency, daily monitoring of middle ear status (tympanometry) and cold symptoms and weekly pneumatic otoscopy were done on a pilot cohort of 20 children. These children, between the ages of 2 and 6, were followed from November 1996 to April 1997. Compliance with symptom diaries was 85%, with tympanograms was 90%, and with weekly physician visits was 70%. During the study period, there were 53 'colds' (average 2.65 per child) and 28 new episodes of middle ear effusion (10 unilateral and 9 bilateral). Overall, 47.3% of the tympanograms were Type A, 17.2% Type C1, 9.4% Type C2, and 21.8% Type B. Children who developed MEE during the study spent more time with abnormal MEP (either MEP < -150 or flat) during both cold and healthy days than children who did not develop MEE. Temporally, during colds, high negative pressures preceded the development of Type B tympanograms in children who developed middle ear effusions. Children who did not develop effusions still had high negative pressures during colds, but recovered to a normal pressure within days. These observations document the feasibility of this investigational format for study of the sequential changes in middle ear status before, during and after a URI.

Dual expression of GABA or serotonin and dopamine in Xenopus amacrine cells is transient and may be regulated by laminar cues.

Both local cell-cell interactions and lineage bias have roles in determining the different retina cell phenotypes. In this study, subpopulations of amacrine cells that dually express GABA or serotonin (5-HT) and dopamine (DA) are identified in the early Xenopus tadpole (stages 42-48) retina. GABA is first detected by immunocytochemistry in amacrine cells at stage 35/36, 5-HT at stage 39, and DA at stage 41. As the number of these subtypes of amacrine cells increases by differentiation, a subset of them transiently express two neurotransmitters. GABA/DA double-labeled amacrine cells are detected first at stage 42, at which time they constitute 52% of the DA-containing population; this percentage decreases to only 3% by stage 48. 5-HT/DA amacrine cells are detected only at stage 44, constituting about 20% of the DA-containing cells and 4% of the small-dim 5-HT-containing cells. Regional location does not differentially affect the differentiation of these three types of amacrine cells (DA only, GABA/DA, and 5-HT/DA cells); each type is found more in the anterior and dorsal than the posterior and ventral quadrants, and their overall distribution patterns are statistically indistinguishable. However, these subtypes of amacrine cells reside in different sublamina of the inner nuclear layer. DA-only amacrine cells are located predominantly in the inner sublayer of the 2-3 cell thick amacrine cell layer, closest to the inner plexiform and the ganglion cell layers. Both types of double-labeled cells are located mostly in the outer sublayer of the amacrine cell layer, closest to other interneurons in the inner nuclear layer. This distinct sublaminar location of different neurotransmitter phenotypes suggests that local laminar cues influence the coexpression of neurotransmitters in amacrine cells.

Timing and mechanisms of mesodermal and neural determination revealed by secondary embryo formation in Cynops and Xenopus.

We examined the timing and mechanisms of mesodermal and neural determination in Cynops, using the secondary embryo induced by transplantation of the prechordal endomesoderm. Two unique approaches were used: one was to observe gastrulation movements induced by the graft, and the other to measure the volumes of formed tissues. Transplanted graft pulled host animal cap cells inside to form a new notochord and other mesoderm of the secondary embryo, showing determination of mesoderm during gastrulation. The graft attained a certain width beneath the host ectoderm and moved near to the animal pole of the host by late gastrula, and a neural plate, which had a similar width to the graft, was formed covering the graft. The volume of neural tissues of the secondary embryo at tail-bud stages was about half that of the normal embryo, while the volumes of notochord were comparable in each case. These data suggest that prechordal endomesoderm, rather than notochord, determines the limit of neural plate in the overlying ectoderm. Similar dorsal grafts were transplanted at early gastrula in Xenopus but did not form well developed secondary embryos, demonstrating that the timing and mechanisms of mesoderm formation in Xenopus are different from those in Cynops.

Effect of rimantadine treatment on clinical manifestations and otologic complications in adults experimentally infected with influenza A (H1N1) virus.

Susceptible adults (n = 105) were enrolled into a randomized double-blind study of rimantadine treatment of experimental influenza A infection. Subjects were cloistered for 8 days and challenged with a rimantadine-sensitive strain of influenza A H1N1 virus at the end of the first day. Forty-eight hours after challenge and for 8 days, 54 subjects received placebo and 51 received rimantadine (100 mg orally, twice a day). Symptoms, signs, and pathophysiologies were monitored. Nine subjects were not infected. Seventeen subjects (38%) in the rimantadine and 26 (53%) in the placebo group became ill. A beneficial effect of rimantadine was documented for virus shedding, symptom load, and sinus pain. Rimantadine had no effect on nasal patency, mucociliary clearance, nasal signs, or on symptoms and signs of otologic complications. These results do not support a preventive effect of rimantadine on the development of otologic manifestations of influenza A infection in adults.

Three types of serotonin-containing amacrine cells in tadpole retina have distinct clonal origins.

In the Xenopus tadpole there are three different serotonin-containing amacrine cells: large, brightly fluorescent (LB), and small, dimly fluorescent (SD) cells in the inner nuclear layer and displaced (DIS) cells in the ganglion cell layer. To reveal the potential roles of regional cues and lineage factors in their determination, quantitative maps were made of the spatial distribution and blastomere origin of each cell type. LB and SD cells were evenly distributed across the four retinal quadrants, arguing against a hypothesis that these cells are induced differentially by quadrant-specific cues. Blastomere progenitors of the 32-cell embryo are biased to produce only subsets of serotonin amacrine cells: 1) all nine progenitors of one retina produced some SD cells, but only eight produced LB, and only five produced DIS cells; and 2) there are overlapping but distinct subsets of blastomere progenitors for each serotonin subtype. This bias is not simply a reflection of the size of a clone in the retina; significant quantitative differences were observed between the proportion of serotonin progeny and the proportion of the entire retina produced by six of the nine retinal progenitors. This bias also is not simply a reflection of the spatial distribution of a blastomere clone in the retina; the number of LB descendants in each retinal quadrant was statistically different from its progenitor's total contribution to the quadrant. These results indicate that the development of the three different serotonin-containing amacrine cells in the retina is biased by membership in specific blastomere clones.

A contact-dependent animal-to-vegetal signal biases neural lineages during Xenopus cleavage stages.

The central nervous system (CNS) of Xenopus is derived from three of four tiers of blastomeres of the 32-cell embryo, and each blastomere in these tiers produces a characteristic number of primary spinal neurons. The C-tier blastomeres constitute the boundary between those that contribute to the CNS (A-, B-, and C-tiers) and those that do not (D-tier). To test whether the neural lineages descended from the C-tier are established by intrinsic information or by cell-cell interactions, single B-tier blastomeres were deleted and the lineage of their C-tier neighbors mapped. The contributions of C-tier blastomeres to subdivisions of the CNS and to specific spinal neurons were significantly reduced. Contributions of these blastomeres to other tissues were mostly normal, indicating that those C-tier progeny that no longer contribute to CNS are distributed in small numbers throughout the rest of the clone. To test whether the changes in neural lineages after B-tier deletions were the result of the C-tier blastomere changing position, contacting new neighbors, or losing contact with inductive B-tier neighbors, intact embryos were transiently dissociated within their vitelline membranes at different time points prior to the midblastula transition. This treatment disrupted cell-cell contact, but not gap junction-mediated dye coupling or the positions of neighboring cells. C-tier CNS lineages were reduced as after deletion of the B-tier neighbor, suggesting that the neural fate of C-tier cells depends upon specific B-tier interactions. To determine whether these interactions occurred specifically between B-tier and C-tier neighbors, barriers were inserted transiently between individual B/C pairs; similar reductions in C-tier CNS lineages were observed. These data demonstrate that an animal-to-vegetal, contact-dependent signal passes from B-tier to C-tier blastomeres and is required for the normal C-tier contribution to the CNS. This cell-cell interaction occurs many hours before the onset of zygotic transcription or neural induction and may bias the field of cells that can respond to neural induction.

Ventricular septal aneurysm after atrioventricular septal repair with pericardium.

A 2-month-old infant underwent a two-patch repair of a type C atrioventricular septal defect using autologous pericardium. Several months later a large, symptomatic aneurysm of the ventricular septal patch developed, requiring resection. The use of untreated autologous pericardium for large, congenital ventricular septal defects is unpredictable and should be avoided.

Characterization of the Xenopus rhodopsin gene.

The abundant Xenopus rhodopsin gene and cDNA have been cloned and characterized. The gene is composed of five exons spanning 3.5 kilobase pairs of genomic DNA and codes for a protein 82% identical to the bovine rhodopsin. The cDNA was expressed in COS1 cells and regenerated with 11-cis-retinal, forming a light-sensitive pigment with maximal absorbance at 500 nm. Both Southern blots and polymerase chain reaction amplification of intron 1 revealed multiple products, indicating more than one allele for the rhodopsin gene. Comparisons with other vertebrate rhodopsin 5 upstream sequences showed significant nucleotide homologies in the 200 nucleotides proximal to the transcription initiation site. This homology included the TATA box region, Ret 1/PCE1 core sequence (CCAATTA), and surrounding nucleotides. To functionally characterize the rhodopsin promoter, transient embryo transfections were used to assay transcriptional control elements in the 5 upstream region using a luciferase reporter. DNA sequences encompassing -5500 to +41 were able to direct luciferase expression in embryo heads. Reporter gene expression was also observed in embryos microinjected with reporter plasmids during early blastomere stages. These results locate transcriptional control elements upstream of the Xenopus rhodopsin gene and show the feasibility of embryo transfections for promoter analysis of rod-specific genes.