Qualification of cardiac troponins for nonclinical use: a regulatory perspective.

The US Food and Drug Administration (FDA) Biomarker Qualification Review Team presents its perspective on the recent qualification of cardiac troponins for use in nonclinical safety assessment studies. The goal of this manuscript is to provide greater transparency into the qualification process and factors that were considered in reaching a regulatory decision. This manuscript includes an overview of the data that were submitted and a discussion of the strengths and shortcomings of these data supporting the qualification decision. The cardiac troponin submission is the first literature-based biomarker application to be reviewed by the FDA and insights gained from this experience may aid future submissions and help streamline the characterization and qualification of future biomarkers.

EARLY FLOWERING3 encodes a novel protein that regulates circadian clock function and flowering in Arabidopsis.

Higher plants use photoperiodic cues to regulate many aspects of development, including the transition from vegetative to floral development. The EARLY FLOWERING3 (ELF3) gene is required for photoperiodic flowering and normal circadian regulation in Arabidopsis. We have cloned ELF3 by positional methods and found that it encodes a novel 695-amino acid protein that may function as a transcriptional regulator. ELF3 transcript level is regulated in a circadian manner, as is expected of a zeitnehmer input pathway component. Overexpression of the LATE ELONGATED HYPOCOTYL gene, which has been proposed to function as a clock component, did not abolish circadian regulation of ELF3 transcription, providing further evidence that ELF3 is a circadian clock input pathway component.

The influence of a family program on adolescent tobacco and alcohol use.

OBJECTIVES: This study examined a family-directed program's effectiveness in preventing adolescent tobacco and alcohol use in a general population. METHODS: Adolescents aged 12 to 14 years and their families were identified by random-digit dialing throughout the contiguous United States. After providing baseline data by telephone interviews, they were randomly allocated to receive or not receive a family-directed program featuring mailed booklets and telephone contacts by health educators. Follow-up telephone interviews were conducted 3 and 12 months after program completion. RESULTS: The findings suggested that smoking onset was reduced by 16.4% at 1 year, with a 25.0% reduction for non-Hispanic Whites but no statistically significant program effect for other races/ethnicities. There were no statistically significant program effects for smokeless tobacco or alcohol use onset. CONCLUSIONS: The family-directed program was associated with reduced smoking onset for non-Hispanic Whites, suggesting that it is worthy of further application, development, and evaluation.

Conditional circadian dysfunction of the Arabidopsis early-flowering 3 mutant.

Photoperiodic responses, such as the daylength-dependent control of reproductive development, are associated with a circadian biological clock. The photoperiod-insensitive early-flowering 3 (elf3) mutant of Arabidopsis thaliana lacks rhythmicity in two distinct circadian-regulated processes. This defect was apparent only when plants were assayed under constant light conditions. elf3 mutants retain rhythmicity in constant dark and anticipate light/dark transitions under most light/dark regimes. The conditional arrhythmic phenotype suggests that the circadian pacemaker is intact in darkness in elf3 mutant plants, but the transduction of light signals to the circadian clock is impaired.

The Arabidopsis ELF3 gene regulates vegetative photomorphogenesis and the photoperiodic induction of flowering.

Flowering in Arabidopsis thaliana is promoted by longday (LD) photoperiods such that plants grown in LD flower earlier, and after the production of fewer leaves, than plants grown in short-day (SD) photoperiods. The early-flowering 3 (elf3) mutant of Arabidopsis, which is insensitive to photoperiod with regard to floral initiation has been characterized elf3 mutants are also altered in several aspects of vegetative photomorphogenesis, including hypocotyl elongation. When inhibition of hypocotyl elongation was measured, elf3 mutant seedlings were less responsive than wild-type to all wavelengths of light, and most notably defective in blue and green light-mediated inhibition. When analyzed for the flowering-time phenotype, elf3 was epistatic to mutant alleles of the blue-light receptor encoding gene, HY4. However, when elf3 mutants were made deficient for functional phytochrome by the introduction of hy2 mutant alleles, the elf3 hy2 double mutants displayed the novel phenotype of flowering earlier than either single mutant while still exhibiting photoperiod insensitivity, indicating that a phytochrome-mediated pathway regulating floral initiation remains functional in elf3 single mutants. In addition, the inflorescences of one allelic combination of elf3 hy2 double mutants form a terminal flower similar to the structure produced by tfk1 single mutants. These results suggest that one of the signal transduction pathways controlling photoperiodism in Arabidopsis is regulated, at least in part, by photoreceptors other than phytochrome, and that the activity of the Arabidopsis inflorescence and floral meristem identity genes may be regulated by this same pathway.

Echocardiographic evaluation of papillary fibroelastoma: a case report and review of the literature.

Papillary fibroelastomas comprise approximately 7.9% of benign primary cardiac tumors. Although papillary fibroelastomas were at first discovered incidentally at autopsy or during heart surgery, these tumors are increasingly being identified by echocardiography. This article reviews those papillary fibroelastomas detected by transthoracic or transesophageal echocardiography and discusses the echocardiographic features of these tumors, associated symptoms, and management. Echocardiography is important in influencing management decisions regarding excision, valve replacement, and valve repair.

Altering the level and regulation of the major sigma subunit of RNA polymerase affects gene expression and development in Bacillus subtilis.

In Bacillus subtilis, the major sigma factor, sigma-A (rpoD), and the minor sigma factor, sigma-H (spo0H), are present during growth and are required for the initiation of sporulation. Our experiments indicate that sigma-A and sigma-H compete for binding to core RNA polymerase. We used a fusion of rpoD to the LacI-repressible IPTG-inducible promoter, Pspac, to vary the levels of sigma-A in the cell. Increasing the amount of sigma-A caused a decrease in expression of genes controlled by sigma-H, and a delay in the production of heat-resistant spores. Decreasing the amount of sigma-A, in a strain deleted for the chromosomal rpoD, caused an increase in expression of genes controlled by sigma-H. As rpoD itself is controlled by at least two promoters recognized by RNA polymerase that contains sigma-H, the effect of sigma-A levels on expression of sigma-H-controlled promoters represents a feedback mechanism that might contribute to maintaining appropriate levels of sigma-A. While the level of sigma-A was important for efficient sporulation, our results indicate that the normal transcriptional control of rpoD, in the context of the rpoD operon and the numerous promoters in that operon, is not required for efficient sporulation or germination, provided that the sigma-A level from a heterologous promoter is comparable to that in wild-type cells.

Patterns of geriatric orthopaedic liaison in Scotland.

Despite increasing interest in collaboration between geriatricians and orthopaedic surgeons in the care of elderly trauma patients, there has been no systematic study to date of the extent of this collaboration in Scotland. This survey establishes the present patterns of such arrangements.

Characterization of csh203::Tn917lac, a mutation in Bacillus subtilis that makes the sporulation sigma factor sigma-H essential for normal vegetative growth.

spo0H encodes a sigma factor, sigma-H, of RNA polymerase that is required for sporulation in Bacillus subtilis. Null mutations in spo0H block the initiation of sporulation but have no obvious effect on vegetative growth. We have characterized an insertion mutation, csh203::Tn917lac, that makes spo0H essential for normal growth. In otherwise wild-type cells, the csh203::Tn917lac insertion mutation has no obvious effect on cell growth, viability, or sporulation. However, in combination with a mutation in spo0H, the csh203 mutation causes a defect in vegetative growth. The csh203::Tn917lac insertion mutation was found to be located within orf23, the first gene of the rpoD (sigma-A) operon. The transposon insertion separates the major vegetative promoters P1 and P2 from the coding regions of two essential genes, dnaG (encoding DNA primase) and rpoD (encoding the major sigma factor, sigma-A) and leaves these genes under the control of minor promoters, including P4, a promoter controlled by sigma-H. The chs203 insertion mutation caused a 2- to 10-fold increase in expression of promoters recognized by RNA polymerase containing sigma-H. The increased expression of genes controlled by sigma-H in the csh203 single mutant, as well as the growth defect of the csh203 spo0H double mutant, was due to effects on rpoD and not to a defect in orf23 or dnaG.

Heterogeneity of a herpes simplex virus clinical isolate exhibiting resistance to acyclovir and foscarnet.

Resistance of herpes simplex virus to acyclovir is a problem of growing clinical importance. Acyclovir-resistance can be due either to mutations in the viral thymidine kinase gene or in the viral DNA polymerase gene. Although clinical resistance has most frequently been associated with thymidine kinase alterations, heterogeneity in clinical isolates has not been addressed frequently. The potential for such heterogeneity has been emphasized by a report describing a pathogenic clinical isolate containing within its population at least one thymidine kinase-proficient DNA polymerase mutant as well as mutants exhibiting thymidine kinase-deficiency (Sacks, et al., 1989). We provide here additional characterization of this isolate and speculations regarding its significance.

Progressive esophagitis from acyclovir-resistant herpes simplex. Clinical roles for DNA polymerase mutants and viral heterogeneity?

Clinically acquired acyclovir resistance in herpes simplex has usually been associated with a deficiency in viral thymidine kinase, which, in turn, has been linked with attenuated virulence in animal models. Diminished pathogenicity in thymidine kinase-deficient isolates has been partly responsible for controversies about the clinical significance of antiviral resistance. We report on a series of resistant virus isolates from a patient who had severe, progressive esophagitis. These isolates had various thymidine kinase activities, ranging from 2.8% to 130% when compared with the activity of the isolate obtained before treatment; the resistant isolate 615 retained enzyme activity as well as neurovirulence in an encephalitis model. Plaque purification showed a heterogeneous mixture containing at least one acyclovir-resistant, foscarnet-resistant plaque isolate (615.8) fully able to phosphorylate acyclovir. The 3.3-kbp BamHI fragment containing most of the DNA polymerase gene from isolate 615.8 was purified and used to successfully transfer both acyclovir and foscarnet resistance. Acquisition of in-vitro acyclovir resistance was associated with progression of clinical disease, as well as with maintenance of pathogenicity in an animal model and at least one mutation in viral DNA polymerase. Patients with herpes simplex infections that progress during acyclovir therapy should be observed for acquisition of resistance in the setting of antiviral chemotherapy; future studies should also consider the presence of heterogeneous virus populations in such patients.

A deletion mutant of the latency-associated transcript of herpes simplex virus type 1 reactivates from the latent state with reduced frequency.

We have generated and characterized a deletion mutant of herpes simplex virus type-1, dlLAT1.8, which lacks the putative promoter region, transcriptional start site, and 1,015 base pairs of the DNA sequences specifying the latency-associated transcripts (LATs). When tested in a CD-1 mouse ocular model, dlLAT1.8 was replication competent in the eye and in ganglia during acute infection but reactivated from explant cultures of ganglia with reduced efficiency (49%) relative to those of wild-type and marker-rescued viruses (94 and 85%, respectively) despite the fact that levels of mutant viral DNA in ganglia during latent infection were comparable to wild-type levels. The neurovirulence of KOS was not significantly altered by the removal of sequences specifying the LATs, as judged by numbers of animals dying on or before 30 days postinfection. Examination of ganglia latently infected with dlLAT1.8 by in situ hybridization revealed no LAT expression. The genotype of reactivated virus was identical to that of input dlLAT1.8 virus as judged by Southern blot analysis. These studies suggest that although the LATs are not essential for the establishment and reactivation of latency in our model, they may play a role in determining the frequency of reactivation of virus from the latent state.

Immediate-early regulatory gene mutants define different stages in the establishment and reactivation of herpes simplex virus latency.

Using nonsense and deletion mutants of herpes simplex virus type 1, we investigated the roles of three immediate-early proteins (ICP4, ICP27 and ICP0) in the establishment and reactivation of ganglionic latency in a mouse ocular model. DNA hybridization, superinfection-rescue, and cocultivation techniques provided quantitative data that distinguished between the failure of a virus to establish latency in the ganglion and its failure to reactivate. Null mutants with lesions in the genes for ICP4 and ICP27 did not replicate in the eye or in ganglia and failed to establish reactivatable latent infections. Three ICP0 deletion mutants which could replicate in the eye and ganglia varied in their ability to establish and reactivate from the latent state, demonstrating that ICP0 plays a role both in the establishment and the reactivation of latency. The use of viral mutants and a variety of stage-specific assays allowed us to better define the stages in the establishment and reactivation of herpes simplex virus type 1 latency.

Inhibitory control of proximal colonic motility by the sympathetic nervous system.

The purpose of this study is to determine whether or not the sympathetic nervous system provides a tonic inhibitory input to the colon in chloralose-anesthetized cats. Proximal and midcolonic motility were monitored using extraluminal force transducers. An intravenous bolus injection of 5 mg of phentolamine in 14 animals elicited a pronounced increase in proximal colon contractility. The minute motility index changed from 0 +/- 0 to 26 +/- 4 after phentolamine administration. Midcolonic motility also increased in response to phentolamine. Specific blockade of alpha 2-receptors, but not alpha 1-receptors, caused the same response seen with phentolamine. alpha-Adrenergic blockade increased colon contractility after spinal cord transection but not after ganglionic blockade. Blockade of alpha-adrenergic receptors was also performed before vagal and pelvic nerve stimulation and in both cases increased colonic motility. Vagal stimulation alone had no effect on colonic contractility, while pelvic nerve stimulation increased motility at the midcolon. alpha-Receptor blockade did not alter the ineffectiveness of vagal stimulation but did unmask excitatory effects of pelvic nerve stimulation on the proximal colon. All excitatory colonic responses were prevented by blocking muscarinic cholinergic receptors. These data indicate that tonic sympathetic nervous system activity exerts an inhibitory effect on colonic motility. The inhibitory effect is mediated through alpha 2-adrenergic receptors. Based on these findings, we suggest that alterations in sympathetic nervous system activity may be extremely important for the regulation of circular muscle contractions in the colon.