Case Report: Esthesioneuroblastoma Involving the Optic Pathways.

Olfactory neuroblastoma, or esthesioneuroblastoma, is an uncommon malignant tumor originating from the neural crest that commonly occurs in the upper nasal cavity. Its ectopic origin is extremely rare, especially when located in the optical pathways. This paper reports the case of a giant ectopic esthesioneuroblastoma of the optic pathways that were surgically treated through a cranio-orbital-zygomatic (COZ) craniotomy with extensive resection, in addition to a literature review. The patient is a 46-year-old female presenting with a 4-month history of visual loss in the left eye. Since she was previously blind in the right eye from a traumatic injury, it was evolving to loss of bilateral vision. Imaging depicted an expansive infiltrating lesion involving the entire path of the right optic nerve, extending to the optic chiasm, cisternal portion of the left optic nerve, bilateral optic tract, and hypothalamus. Investigation of pituitary function was unremarkable. Esthesioneuroblastoma is a rare tumor with poorly defined standard clinical management. Its ectopic presentation makes the diagnosis even more challenging, making it difficult to manage these cases properly. Surgeons should be aware of this rare possibility, as early aggressive treatment is likely to be associated with better results.

Dual indexed library design enables compatibility of in-Drop single-cell RNA-sequencing with exAMP chemistry sequencing platforms.

BACKGROUND: The increasing demand of single-cell RNA-sequencing (scRNA-seq) experiments, such as the number of experiments and cells queried per experiment, necessitates higher sequencing depth coupled to high data quality. New high-throughput sequencers, such as the Illumina NovaSeq 6000, enables this demand to be filled in a cost-effective manner. However, current scRNA-seq library designs present compatibility challenges with newer sequencing technologies, such as index-hopping, and their ability to generate high quality data has yet to be systematically evaluated. RESULTS: Here, we engineered a dual-indexed library structure, called TruDrop, on top of the inDrop scRNA-seq platform to solve these compatibility challenges, such that TruDrop libraries and standard Illumina libraries can be sequenced alongside each other on the NovaSeq. On scRNA-seq libraries, we implemented a previously-documented countermeasure to the well-described problem of index-hopping, demonstrated significant improvements in base-calling accuracy on the NovaSeq, and provided an example of multiplexing twenty-four scRNA-seq libraries simultaneously. We showed favorable comparisons in transcriptional diversity of TruDrop compared with prior inDrop libraries. CONCLUSIONS: Our approach enables cost-effective, high throughput generation of sequencing data with high quality, which should enable more routine use of scRNA-seq technologies.

Recurrent RNA motifs as scaffolds for genetically encodable small-molecule biosensors.

Allosteric RNA devices are increasingly being viewed as important tools capable of monitoring enzyme evolution, optimizing engineered metabolic pathways, facilitating gene discovery and regulators of nucleic acid-based therapeutics. A key bottleneck in the development of these platforms is the availability of small-molecule-binding RNA aptamers that robustly function in the cellular environment. Although aptamers can be raised against nearly any desired target through in vitro selection, many cannot easily be integrated into devices or do not reliably function in a cellular context. Here, we describe a new approach using secondary- and tertiary-structural scaffolds derived from biologically active riboswitches and small ribozymes. When applied to the neurotransmitter precursors 5-hydroxytryptophan and 3,4-dihydroxyphenylalanine, this approach yielded easily identifiable and characterizable aptamers predisposed for coupling to readout domains to allow engineering of nucleic acid-sensory devices that function in vitro and in the cellular context.

The purine riboswitch as a model system for exploring RNA biology and chemistry.

Over the past decade the purine riboswitch, and in particular its nucleobase-binding aptamer domain, has emerged as an important model system for exploring various aspects of RNA structure and function. Its relatively small size, structural simplicity and readily observable activity enable application of a wide variety of experimental approaches towards the study of this RNA. These analyses have yielded important insights into small molecule recognition, co-transcriptional folding and secondary structural switching, and conformational dynamics that serve as a paradigm for other RNAs. In this article, the current state of understanding of the purine riboswitch family and how this growing knowledge base is starting to be exploited in the creation of novel RNA devices are examined. This article is part of a Special Issue entitled: Riboswitches.

Higher capecitabine AUC in elderly patients with advanced colorectal cancer (SWOGS0030).

BACKGROUND: The aging process is accompanied by physiological changes including reduced glomerular filtration and hepatic function, as well as changes in gastric secretions. To investigate what effect would aging have on the disposition of capecitabine and its metabolites, the pharmacokinetics between patients ≥70 years and <60 years were compared in SWOG0030. METHODS: Twenty-nine unresectable colorectal cancer patients were stratified to either ≥70 or <60 years of age, where the disposition of capecitabine and its metabolites were compared. RESULTS: Notable increase in capecitabine area under the curve (AUC) was accompanied by reduction in capecitabine clearance in ≥70 years patients (P<0.05). No difference in 5'-deoxy-5-fluorocytidine, 5'-deoxy-5-fluorouridine (DFUR), and 5-fluorouracil (5FU) AUCs between the two age groups, suggesting that carboxylesterase and cytidine deaminase (CDA) activity was similar between the two age groups. These results suggest that metabolic enzymes involved in converting capecitabine metabolites are not altered by age. An elevation in capecitabine Cmax and reduction in clearance was seen in females, where capecitabine AUC was 40.3% higher in women. Elevation of DFUR Cmax (45%) and AUC (46%) (P<0.05) was also noted, suggesting that CDA activity may be higher in females. CONCLUSION: Increases in capecitabine Cmax and AUC was observed in patients ≥70 years when compared with younger patients who were >60 years.

Insights into the regulatory landscape of the lysine riboswitch.

A prevalent means of regulating gene expression in bacteria is by riboswitches found within mRNA leader sequences. Like protein repressors, these RNA elements must bind an effector molecule with high specificity against a background of other cellular metabolites of similar chemical structure to elicit the appropriate regulatory response. Current crystal structures of the lysine riboswitch do not provide a complete understanding of selectivity as recognition is substantially mediated through main-chain atoms of the amino acid. Using a directed set of lysine analogs and other amino acids, we have determined the relative contributions of the polar functional groups to binding affinity and the regulatory response. Our results reveal that the lysine riboswitch has >1000-fold specificity for lysine over other amino acids. The aptamer is highly sensitive to the precise placement of the ε-amino group and relatively tolerant of alterations to the main-chain functional groups in order to achieve this specificity. At low nucleotide triphosphate (NTP) concentrations, we observe good agreement between the half-maximal regulatory activity (T(50)) and the affinity of the receptor for lysine (K(d)), as well as many of its analogs. However, above 400 µM [NTP], the concentration of lysine required to elicit transcription termination rises, moving into the riboswitch into a kinetic control regime. These data demonstrate that, under physiologically relevant conditions, riboswitches can integrate both effector and NTP concentrations to generate a regulatory response appropriate for global metabolic state of the cell.

Evaluation of the Megaduct sweat collector for mineral analysis.

Accurate measurement of sweat mineral loss is important for whole body mineral balance estimates and dietary reference intake formulation. Currently, common localized sweat collection methods such as the pouch and patch techniques may be limited by skin encapsulation and/or hidromeiosis, which may alter sweat mineral concentrations. The design of the newly developed Megaduct sweat collector may avoid these possible limitations. Therefore, the purpose of this study was to evaluate the utility of the Megaduct sweat collector for mineral analysis. Megaduct sweat collectors were affixed to ten volunteers on the final day of a heat acclimation protocol; collection time, sweat volume, and mineral concentrations of calcium, copper, iron, potassium, sodium, and zinc were measured. Megaduct filling required a collection period of 62 ± 3 min due to a small collection surface (22.1 cm(2)). The mineral content of the sweat was 0.3 ± 0.1 mmol L(-1), 1.5 ± 1.5 µmol L(-1), 8.5 ± 2.1 mmol L(-1), 43.2 ± 15.0 mmol L(-1), and 10.1 ± 5.7 µmol L(-1) for Ca, Cu, K, Na, and Zn, respectively. The Megaduct sweat collector appears to avoid skin encapsulation and hidromeiosis, and captures sweat with similar mineral concentrations as reported in the literature for pouches. However, the filling time of the Megaduct (>60 min) may not capture possible changes in sweat mineral concentrations that are documented to occur in as little as 15 to 30 min.

Evolution of an MHC class Ia gene fragment in four North American Morone species.

A nucleotide sequence analysis of a fragment of a Morone MHC class Ia gene detected high levels of polymorphism in striped bass Morone saxatilis, white perch Morone americana and yellow bass Morone mississippiensis. Extremely low levels of MHC diversity, however, were detected in white bass Morone chrysops, suggesting the possibility of a severe population bottleneck for this species.

Skin temperature modifies the impact of hypohydration on aerobic performance.

This study determined the effects of hypohydration on aerobic performance in compensable [evaporative cooling requirement (E(req)) < maximal evaporative cooling (E(max))] conditions of 10 degrees C [7 degrees C wet bulb globe temperature (WBGT)], 20 degrees C (16 degrees C WBGT), 30 degrees C (22 degrees C WBGT), and 40 degrees C (27 degrees C WBGT) ambient temperature (T(a)). Our hypothesis was that 4% hypohydration would impair aerobic performance to a greater extent with increasing heat stress. Thirty-two men [22 +/- 4 yr old, 45 +/- 8 ml.kg(-1).min(-1) peak O(2) uptake (Vo(2 peak))] were divided into four matched cohorts (n = 8) and tested at one of four T(a) in euhydrated (EU) and hypohydrated (HYPO, -4% body mass) conditions. Subjects completed 30 min of preload exercise (cycle ergometer, 50% Vo(2 peak)) followed by a 15 min self-paced time trial. Time-trial performance (total work, change from EU) was -3% (P = 0.1), -5% (P = 0.06), -12% (P < 0.05), and -23% (P < 0.05) in 10 degrees C, 20 degrees C, 30 degrees C, and 40 degrees C T(a), respectively. During preload exercise, skin temperature (T(sk)) increased by approximately 4 degrees C per 10 degrees C T(a), while core (rectal) temperature (T(re)) values were similar within EU and HYPO conditions across all T(a). A significant relationship (P < 0.05, r = 0.61) was found between T(sk) and the percent decrement in time-trial performance. During preload exercise, hypohydration generally blunted the increases in cardiac output and blood pressure while reducing blood volume over time in 30 degrees C and 40 degrees C T(a). Our conclusions are as follows: 1) hypohydration degrades aerobic performance to a greater extent with increasing heat stress; 2) when T(sk) is >29 degrees C, 4% hypohydration degrades aerobic performance by approximately 1.6% for each additional 1 degrees C T(sk); and 3) cardiovascular strain from high skin blood flow requirements combined with blood volume reductions induced by hypohydration is an important contributor to impaired performance.

Complex evolution of a highly conserved microsatellite locus in several fish species.

The evolutionary dynamics of a highly conserved microsatellite locus (Dla 11) were studied in several fish species. The data indicated that multiple types of compound microsatellites arose through point mutations that were sometimes followed by expansion of the derived motif. Furthermore, extensive length variation was detected among species in the regions immediately flanking the repeat region.

Analyses of nuclear ldhA gene and mtDNA control region sequences of Atlantic northern bluefin tuna populations.

There has been considerable debate about whether the Atlantic northern bluefin tuna exist as a single panmictic unit. We have addressed this issue by examining both mitochondrial DNA control region nucleotide sequences and nuclear gene ldhA allele frequencies in replicate size or year class samples of northern bluefin tuna from the Mediterranean Sea and the northwestern Atlantic Ocean. Pairwise comparisons of multiple year class samples from the 2 regions provided no evidence for population subdivision. Similarly, analyses of molecular variance of both mitochondrial and ldhA data revealed no significant differences among or between samples from the 2 regions. These results demonstrate the importance of analyzing multiple year classes and large sample sizes to obtain accurate estimates when using allele frequencies to characterize a population. It is important to note that the absence of genetic evidence for population substructure does not unilaterally constitute evidence of a single panmictic population, as genetic differentiation can be prevented by large population sizes and by migration.

Complete genome sequence of Caulobacter crescentus.

The complete genome sequence of Caulobacter crescentus was determined to be 4,016,942 base pairs in a single circular chromosome encoding 3,767 genes. This organism, which grows in a dilute aquatic environment, coordinates the cell division cycle and multiple cell differentiation events. With the annotated genome sequence, a full description of the genetic network that controls bacterial differentiation, cell growth, and cell cycle progression is within reach. Two-component signal transduction proteins are known to play a significant role in cell cycle progression. Genome analysis revealed that the C. crescentus genome encodes a significantly higher number of these signaling proteins (105) than any bacterial genome sequenced thus far. Another regulatory mechanism involved in cell cycle progression is DNA methylation. The occurrence of the recognition sequence for an essential DNA methylating enzyme that is required for cell cycle regulation is severely limited and shows a bias to intergenic regions. The genome contains multiple clusters of genes encoding proteins essential for survival in a nutrient poor habitat. Included are those involved in chemotaxis, outer membrane channel function, degradation of aromatic ring compounds, and the breakdown of plant-derived carbon sources, in addition to many extracytoplasmic function sigma factors, providing the organism with the ability to respond to a wide range of environmental fluctuations. C. crescentus is, to our knowledge, the first free-living alpha-class proteobacterium to be sequenced and will serve as a foundation for exploring the biology of this group of bacteria, which includes the obligate endosymbiont and human pathogen Rickettsia prowazekii, the plant pathogen Agrobacterium tumefaciens, and the bovine and human pathogen Brucella abortus.

Pediatric nurses' pain management practice: barriers to change.

A qualitative, descriptive design was used to examine factors that influence pediatric nurses' pain management practices. Staff nurses from one pediatric unit (N = 16) at a community-based hospital attended from one to six discussion groups. Detailed content analysis of transcripts from each discussion group was conducted. Several themes emerged in the analysis. Some themes are based on the nurses clinical practice of pain assessment and management, while others reflect the organizational issues and changes staff experienced. Themes identified included: barriers/solutions to clinical practice change, organizational barriers to practice change, and staff commitment toward pain management. Staff attendance and commitment to participation in this research was commendable considering the external pressures they felt and expressed about job insecurity and poor unit morale. It seemed that their commitment to attend and participate was, in part, based on the value they placed upon clinical skills and knowledge. The opportunity to talk about the effects of organizational change, both individually and collectively, on their work environment was also considered beneficial.

Amphitrite ornata, a marine worm, contains two dehaloperoxidase genes.

Amphitrite ornata, a terebellid polychaete, inhabits marine environments that are contaminated by biogenically produced halometabolites. These halogenated organic compounds are toxic and quite diverse. To survive in this environment, A. ornata produces a novel dehaloperoxidase (DHP I) that detoxifies haloaromatic compounds. In this study we identified and characterized two dehaloperoxidase genes, designated dhpA and dhpB, from an A. ornata complementary DNA library. The deduced amino acid sequences (DHP A and DHP B) of the two dhp genes both contain 137 amino acid residues, but they differ at 5 amino acid positions. Allelic variation was observed for both genes as well. Polymerase chain reaction-restriction fragment length polymorphism assays of genomic DNA from 19 in individuals showed that each individual contains both the dhpA and the dhpB genes. Therefore, the two types of DHP are encoded by separate genes and are not alleles of a single gene. Furthermore, DHP A and DHP B may have different substrate specificities since they have amino acid differences in the active site.

Expression, purification, and characterization of gp160e, the soluble, trimeric ectodomain of the simian immunodeficiency virus envelope glycoprotein, gp160.

The envelope glycoprotein, gp160, of simian immunodeficiency virus (SIV) shares approximately 25% sequence identity with gp160 from the human immunodeficiency virus, type I, indicating a close structural similarity. As a result of binding to cell surface CD4 and co-receptor (e.g. CCR5 and CXCR4), both SIV and human immunodeficiency virus gp160 mediate viral entry by membrane fusion. We report here the characterization of gp160e, the soluble ectodomain of SIV gp160. The ectodomain has been expressed in both insect cells and Chinese hamster ovary (CHO)-Lec3.2.8.1 cells, deficient in enzymes necessary for synthesizing complex oligosaccharides. Both the primary and a secondary proteolytic cleavage sites between the gp120 and gp41 subunits of gp160 were mutated to prevent cleavage and shedding of gp120. The purified, soluble glycoprotein is shown to be trimeric by chemical cross-linking, gel filtration chromatography, and analytical ultracentrifugation. It forms soluble, tight complexes with soluble CD4 and a number of Fab fragments from neutralizing monoclonal antibodies. Soluble complexes were also produced of enzymatically deglycosylated gp160e and of gp160e variants with deletions in the variable segments.

A family of six flagellin genes contributes to the Caulobacter crescentus flagellar filament.

The Caulobacter crescentus flagellar filament is assembled from multiple flagellin proteins that are encoded by six genes. The amino acid sequences of the FljJ and FljL flagellins are divergent from those of the other four flagellins. Since these flagellins are the first to be assembled in the flagellar filament, one or both might have specialized to facilitate the initiation of filament assembly.

The growth inhibitory effect of p21 adenovirus on human bladder cancer cells.

PURPOSE: To evaluate whether p21 (WAF-1/CIP1) should be considered a potential candidate for human bladder cancer gene therapy, we determined: (1) the basal level of p21 expression in bladder cancer cell lines, (2) the response of bladder cancer cells to increased p21 expression following p21 adenovirus infection, and (3) the mechanism of growth inhibition produced by p21 overexpression. MATERIALS AND METHODS: Five established human bladder cancer cell lines and one primary culture derived from an invasive transitional cell carcinoma were used in this study. To examine the effect of p21 protein on the growth of human bladder cancer cells, a recombinant adenovirus vector system containing p21 cDNA, under the control of cytomegalovirus promoter, was constructed. A control virus containing p21 in an antisense orientation was used to eliminate potential artifacts caused by viral toxicity. RESULTS: Human bladder cancer cell lines exhibit variable endogenous p21 levels which correlate with the in vitro growth status. Significant, but highly variable increases in the steady-state level of p21 were detected in p21 adenovirus infected cells. Human bladder cancer cell lines responded heterogeneously to p21 adenovirus infection. Growth of the WH cell line was substantially inhibited in a dose and time-course dependent fashion. The mechanism of p21 growth inhibition was found to be due to G0/G1 arrest and not the induction of apoptosis. In contrast, p21 adenovirus failed to inhibit the growth of T24 bladder cancer cells because T24 cells were resistant to viral infection. The 253J bladder cancer cells exhibited marked sensitivity to adenovirus; substantial growth inhibition was seen with both sense and antisense p21 very early in the time course of infection. CONCLUSIONS: We found significant variation in the basal level of p21 protein expression in several human bladder cancer cell lines. Increased p21 expression as a result of adenoviral infection may be a potent growth suppressor in some human bladder cancer because it elicits cell cycle arrest in G0/G1 stage, but not the induction of apoptosis. Bladder cancer cells exhibit a wide spectrum of sensitivity to adenoviral infection that may be caused by the presence of viral receptor heterogeneity. This wide spectrum of sensitivity has significant basic scientific and clinical implications and warrants further study.

PCR-based gene synthesis as an efficient approach for expression of the A+T-rich malaria genome.

The A+T-rich genome of the human malaria parasite Plasmodium falciparum encodes genes of biological importance that cannot be expressed efficiently in heterologous eukaryotic systems, owing to an extremely biased codon usage and the presence of numerous cryptic polyadenylation sites. In this work we have optimized an assembly polymerase chain reaction (PCR) method for the fast and extremely accurate synthesis of a 2.1 kb Plasmodium falciparum gene (pfsub-1) encoding a subtilisin-like protease. A total of 104 oligonucleotides, designed with the aid of dedicated computer software, were assembled in a single-step PCR. The assembly was then further amplified by PCR to produce a synthetic gene which has been cloned and successfully expressed in both Pichia pastoris and recombinant baculovirus-infected High Five(TM) cells. We believe this strategy to be of special interest as it is simple, accessible and has no limitation with respect to the size of the gene to be synthesized. Used as a systematic approach for the malarial genome or any other A + T-rich organism, the method allows the rapid synthesis of a nucleotide sequence optimized for expression in the system of choice and production of sufficiently large amounts of biological material for complete molecular and structural characterization.

The role of DOC-2/DAB2 protein phosphorylation in the inhibition of AP-1 activity. An underlying mechanism of its tumor-suppressive function in prostate cancer.

DOC-2/DAB2, a novel phosphoprotein with signal-transducing capability, inhibits human prostatic cancer cells (Tseng, C.-P., Ely, B. D., Li, Y., Pong, R.-C., and Hsieh, J.-T. (1998) Endocrinology 139, 3542-3553). However, its mechanism of action is not understood completely. This study delineates the functional significance of DOC-2/DAB2 protein phosphorylation and demonstrates that in vivo activation of protein kinase C (PKC) by 12-O-tetradecanoylphorbol-13-acetate (TPA) induces DOC-2/DAB2 phosphorylation, including a serine residue at position 24. Mutation of Ser(24) to Ala reduced DOC-2/DAB2 phosphorylation by PKC. Using a synthetic Ser(24) peptide (APS(24)KKEKKKGSEKTD) or recombinant DOC-2/DAB2 as substrates, PKCbetaII, PKCgamma, and PKCdelta (but not casein kinase II) directly phosphorylated Ser(24) in vitro. This indicates that DOC-2/DAB2 is a PKC-specific substrate. Since expression of wild-type DOC-2/DAB2, but not the S24A mutant, inhibited TPA-induced AP-1 activity in prostatic epithelial cells, phosphorylation of Ser(24) appears to play a critical role in modulating TPA-induced AP-1 activity. Taken together, these data suggest that PKC-regulated phosphorylation of DOC-2/DAB2 protein may help its growth inhibitory function.

FlbT couples flagellum assembly to gene expression in Caulobacter crescentus.

The biogenesis of the polar flagellum of Caulobacter crescentus is regulated by the cell cycle as well as by a trans-acting regulatory hierarchy that functions to couple flagellum assembly to gene expression. The assembly of early flagellar structures (MS ring, switch, and flagellum-specific secretory system) is required for the transcription of class III genes, which encode the remainder of the basal body and the external hook structure. Similarly, the assembly of class III gene-encoded structures is required for the expression of the class IV flagellins, which are incorporated into the flagellar filament. Here, we demonstrate that mutations in flbT, a flagellar gene of unknown function, can restore flagellin protein synthesis and the expression of fljK::lacZ (25-kDa flagellin) protein fusions in class III flagellar mutants. These results suggest that FlbT functions to negatively regulate flagellin expression in the absence of flagellum assembly. Deletion analysis shows that sequences within the 5' untranslated region of the fljK transcript are sufficient for FlbT regulation. To determine the mechanism of FlbT-mediated regulation, we assayed the stability of fljK mRNA. The half-life (t(1/2)) of fljK mRNA in wild-type cells was approximately 11 min and was reduced to less than 1.5 min in a flgE (hook) mutant. A flgE flbT double mutant exhibited an mRNA t(1/2) of greater than 30 min. This suggests that the primary effect of FlbT regulation is an increased turnover of flagellin mRNA. The increased t(1/2) of fljK mRNA in a flbT mutant has consequences for the temporal expression of fljK. In contrast to the case for wild-type cells, fljK::lacZ protein fusions in the mutant are expressed almost continuously throughout the C. crescentus cell cycle, suggesting that coupling of flagellin gene expression to assembly has a critical influence on regulating cell cycle expression.