Site-specific fluorescent labeling of DNA using Staudinger ligation

We report the site-specific fluorescent labeling of DNA using Staudinger ligation with high efficiency and high selectivity. An oligonucleotide modified at its 5' end by an azido group was selectively reacted with 5-[(N-(3'-diphenylphosphinyl-4'-methoxycarbonyl)phenylcarbonyl)aminoacetamido]fluorescein (Fam) under aqueous conditions to produce a Fam-labeled oligonucleotide with a high yield (approximately 90%). The fluorescent oligonucleotide was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Because of the relatively high yield of the Staudinger ligation, simple purification of the product by size-exclusion chromatography and desalting is sufficient for the resulting fluorescent oligonucleotide to be used as a primer in a Sanger dideoxy sequencing reaction to produce fluorescent DNA extension fragments, which are analyzed by a fluorescent electrophoresis DNA sequencer. The results indicate that the Staudinger ligation can be used successfully and site-specifically to prepare fluorescent oligonucleotides to produce DNA sequencing products, which are detected with single base resolution in a capillary electrophoresis DNA sequencer using laser-induced fluorescence detection.     

Sensitive determination of carnosine in urine by high-performance liquid chromatography using 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride as a fluorescent labeling reagent

A simple and highly sensitive high-performance liquid chromatography procedure was developed for the determination of carnosine in urine. Carnosine was derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride at 70 °C for 15 min in borate buffer (20 mmol l^?1, pH 9.0) to produce fluorescent sulfonamides. After hydrolysis of the reaction mixture with formic acid at 100 °C for 15 min, the fluorescent derivative of carnosine was separated on a reversed-phase column with a linear gradient elution using solvents of (A) acetate buffer (0.1 mmol l^?1, pH 7.0) and (B) acetonitrile at a flow-rate of 1.0 ml/min and was detected at excitation and emission wavelengths of 318 and 400 nm, respectively. The detection limit of carnosine was 4 fmol at a signal-to-noise ratio of 3. The within-day and day-to-day relative standard deviations were 2.7–4.6% and 0.4–5.2%, respectively. The concentration of carnosine in normal human urine was found to be 4.6–125 nmol (mg creatinine)^?1 (mean ± SD: 21.6 ± 26.6 nmol (mg creatinine)^?1, n = 20).     

A mathematical model of the adaptive control of human arm motions

This paper discusses similarities between models of adaptive motor control suggested by recent experiments with human and animal subjects, and the structure of a new control law derived mathematically from nonlinear stability theory. In both models, the control actions required to track a specified trajectory are adaptively assembled from a large collection of simple computational elements. By adaptively recombining these elements, the controllers develop complex internal models which are used to compensate for the effects of externally imposed forces or changes in the physical properties of the system. On a motor learning task involving planar, multi-joint arm motions, the simulated performance of the mathematical model is shown to be qualitatively similar to observed human performance, suggesting that the model captures some of the interesting features of the dynamics of low-level motor adaptation. Received: 20 September 1994 / Accepted in revised form: 18 November 1998     

The psychosis spectrum in a young U.S. community sample: findings from the Philadelphia Neurodevelopmental Cohort

Little is known about the occurrence and predictors of the psychosis spectrum in large non‐clinical community samples of U.S. youths. We aimed to bridge this gap through assessment of psychosis spectrum symptoms in the Philadelphia Neurodevelopmental Cohort, a collaborative investigation of clinical and neurobehavioral phenotypes in a prospectively accrued cohort of youths, funded by the National Institute of Mental Health. Youths (age 11‐21; N=7,054) and collateral informants (caregiver/legal guardian) were recruited through the Children's Hospital of Philadelphia and administered structured screens of psychosis spectrum symptoms, other major psychopathology domains, and substance use. Youths were also administered a computerized neurocognitive battery assessing five neurobehavioral domains. Predictors of psychosis spectrum status in physically healthy participants (N=4,848) were examined using logistic regression. Among medically healthy youths, 3.7% reported threshold psychotic symptoms (delusions and/or hallucinations). An additional 12.3% reported significant sub‐psychotic positive symptoms, with odd/unusual thoughts and auditory perceptions, followed by reality confusion, being the most discriminating and widely endorsed attenuated symptoms. A minority of youths (2.3%) endorsed subclinical negative/disorganized symptoms in the absence of positive symptoms. Caregivers reported lower symptom levels than their children. Male gender, younger age, and non‐European American ethnicity were significant predictors of spectrum status. Youths with spectrum symptoms had reduced accuracy across neurocognitive domains, reduced global functioning, and increased odds of depression, anxiety, behavioral disorders, substance use and suicidal ideation. These findings have public health relevance for prevention and early intervention.     

Robust spatial extent inference with a semiparametric bootstrap joint inference procedure

Spatial extent inference (SEI) is widely used across neuroimaging modalities to adjust for multiple comparisons when studying brain‐phenotype associations that inform our understanding of disease. Recent studies have shown that Gaussian random field (GRF)‐based tools can have inflated family‐wise error rates (FWERs). This has led to substantial controversy as to which processing choices are necessary to control the FWER using GRF‐based SEI. The failure of GRF‐based methods is due to unrealistic assumptions about the spatial covariance function of the imaging data. A permutation procedure is the most robust SEI tool because it estimates the spatial covariance function from the imaging data. However, the permutation procedure can fail because its assumption of exchangeability is violated in many imaging modalities. Here, we propose the (semi‐) parametric bootstrap joint (PBJ; sPBJ) testing procedures that are designed for SEI of multilevel imaging data. The sPBJ procedure uses a robust estimate of the spatial covariance function, which yields consistent estimates of standard errors, even if the covariance model is misspecified. We use the methods to study the association between performance and executive functioning in a working memory functional magnetic resonance imaging study. The sPBJ has similar or greater power to the PBJ and permutation procedures while maintaining the nominal type 1 error rate in reasonable sample sizes. We provide an R package to perform inference using the PBJ and sPBJ procedures.     

<Emphasis Type="Italic">HFE</Emphasis> mRNA expression is responsive to intracellular and extracellular iron loading: short communication

In liver hepatocytes, the HFE gene regulates cellular and systemic iron homeostasis by modulating cellular iron-uptake and producing the iron-hormone hepcidin in response to systemic iron elevation. However, the mechanism of iron-sensing in hepatocytes remain enigmatic. Therefore, to study the effect of iron on HFE and hepcidin (HAMP) expressions under distinct extracellular and intracellular iron-loading, we examined the effect of holotransferrin treatment (1, 2, 5 and 8 g/L for 6 h) on intracellular iron levels, and mRNA expressions of HFE and HAMP in wild-type HepG2 and previously characterized iron-loaded recombinant-TfR1 HepG2 cells. Gene expression was analyzed by real-time PCR and intracellular iron was measured by ferrozine assay. Data showed that in the wild-type cells, where intracellular iron content remained unchanged, HFE expression remained unaltered at low holotransferrin treatments but was upregulated upon 5 g/L (p?<?0.04) and 8 g/L (p?=?0.05) treatments. HAMP expression showed alternating elevations and increased upon 1 g/L (p?<?0.05) and 5 g/L (p?<?0.05). However, in the recombinant cells that showed higher intracellular iron levels than wild-type cells, HFE and HAMP expressions were elevated only at low 1 g/L treatment (p?<?0.03) and were repressed at 2 g/L treatment (p?<?0.03). Under holotransferrin-untreated conditions, the iron-loaded recombinant cells showed higher expressions of HFE (p?<?0.03) and HAMP (p?=?0.05) than wild-type cells. HFE mRNA was independently elevated by extracellular and intracellular iron-excess. Thus, it may be involved in sensing both, extracellular and intracellular iron. Repression of HAMP expression under simultaneous intracellular and extracellular iron-loading resembles non-hereditary iron-excess pathologies.     

Persistence of psychosis spectrum symptoms in the Philadelphia Neurodevelopmental Cohort: a prospective two‐year follow‐up

Prospective evaluation of youths with early psychotic‐like experiences can enrich our knowledge of clinical, biobehavioral and environmental risk and protective factors associated with the development of psychotic disorders. We aimed to investigate the predictors of persistence or worsening of psychosis spectrum features among US youth through the first large systematic study to evaluate subclinical symptoms in the community. Based on Time 1 screen of 9,498 youth (age 8‐21) from the Philadelphia Neurodevelopmental Cohort, a subsample of participants was enrolled based on the presence (N=249) or absence (N=254) of baseline psychosis spectrum symptoms, prior participation in neuroimaging, and current neuroimaging eligibility. They were invited to participate in a Time 2 assessment two years on average following Time 1. Participants were administered the Structured Interview for Prodromal Syndromes, conducted blind to initial screen status, along with the Schizotypal Personality Questionnaire and other clinical measures, computerized neurocognitive testing, and neuroimaging. Clinical and demographic predictors of symptom persistence were examined using logistic regression. At Time 2, psychosis spectrum features persisted or worsened in 51.4% of youths. Symptom persistence was predicted by higher severity of subclinical psychosis, lower global functioning, and prior psychiatric medication at baseline. Youths classified as having psychosis spectrum symptoms at baseline but not at follow‐up nonetheless exhibited comparatively higher symptom levels and lower functioning at both baseline and follow‐up than typically developing youths. In addition, psychosis spectrum features emerged in a small number of young people who previously had not reported significant symptoms but who had exhibited early clinical warning signs. Together, our findings indicate that varying courses of psychosis spectrum symptoms are evident early in US youth, supporting the importance of investigating psychosis risk as a dynamic developmental process. Neurocognition, brain structure and function, and genomics may be integrated with clinical data to provide early indices of symptom persistence and worsening in youths at risk for psychosis.     

Hepcidin secretion was not directly proportional to intracellular iron-loading in recombinant-TfR1 HepG2 cells: short communication

Molecular and Cellular Biochemistry - Hepcidin is the master regulator of systemic iron homeostasis and its dysregulation is observed in several chronic liver diseases. Unlike the extracellular...     

Digital genotyping using molecular affinity and mass spectrometry

The goal of DNA sequencing and genotyping is to efficiently generate accurate high-throughput digital genetic information that unambiguously identifies sources of genetic variation and clearly distinguishes heterozygous from homozygous variants. Recent advances in mass-spectrometry-based DNA sequencing and genotyping bode well for meeting these criteria. Pilot studies show that these recently developed approaches allow unambiguous multiplex detection of heterozygous variants and the identification of deletion and insertion variants.