Prediction of m6A and m5C at single-molecule resolution reveals a transcriptome-wide co-occurrence of RNA modifications.

The epitranscriptome embodies many new and largely unexplored functions of RNA. A significant roadblock hindering progress in epitranscriptomics is the identification of more than one modification in individual transcript molecules. We address this with CHEUI (CH3 (methylation) Estimation Using Ionic current). CHEUI predicts N6-methyladenosine (m6A) and 5-methylcytosine (m5C) in individual molecules from the same sample, the stoichiometry at transcript reference sites, and differential methylation between any two conditions. CHEUI processes observed and expected nanopore direct RNA sequencing signals to achieve high single-molecule, transcript-site, and stoichiometry accuracies in multiple tests using synthetic RNA standards and cell line data. CHEUI's capability to identify two modification types in the same sample reveals a co-occurrence of m6A and m5C in individual mRNAs in cell line and tissue transcriptomes. CHEUI provides new avenues to discover and study the function of the epitranscriptome.

Rhizosphere metabolism and its effect on phosphorus pools in the root zone of a submerged macrophyte, Isoëtes kirkii.

The presence of oxygen in lake sediments reduces sediment oxygen demand, and potentially improves sediment phosphorus retention and coupled nitrification/denitrification. However, the release of oxygen from the roots of macrophytes has not previously been measured in highly reducing sediments. Here, in the highly reducing environments of a commercial garden soil and sediment from a hyper-eutrophic lake, we used nine oxygen optodes, placed onto scintillation vials to detect oxygen in the rhizosphere of Isoetes kirkii referred to as quillworts. We calculated rhizosphere metabolism using "night-time regression" a method designed to estimate stream metabolism at the reach scale. After the incubations, sediment was collected from each vial (with and without macrophytes) and was subjected to sequential phosphorus extractions. A lag period between light availability and increasing oxygen concentration, that varied between individual optodes, was used to improve the accuracy of metabolism estimates as it was postulated to represent the distance between the root and the optode. Higher sediment oxygen demand in the lake sediment caused I. kirkii to have higher root oxygen release than those plants grown in the garden soil and may have pushed plants in lake sediment close to their ability to survive. This was evident as a significant, negative relationship between root oxygen release and increasing sediment oxygen demand, indicating that if photosynthesis decreased or sediment oxygen demand increased, the plants would no longer being able to oxygenate the sediment surrounding their roots, which would likely lead to death. Finally, the presence of quillworts in lake sediments significantly increased stores of metal oxide and recalcitrant phosphorus in the lake sediment but not the garden soil.

Heritability of plasma cholinesterase activity in inbred mouse strains.

The activity of cholinesterase was measured in the plasma of 543 mice of both sexes from 23 inbred mouse strains. Differences between strains were highly significant. The heritability of this characteristic was 0.70 in females and 0.67 in males.

Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by the widespread development of distinctive tumors termed hamartomas. TSC-determining loci have been mapped to chromosomes 9q34 (TSC1) and 16p13 (TSC2). The TSC1 gene was identified from a 900-kilobase region containing at least 30 genes. The 8.6-kilobase TSC1 transcript is widely expressed and encodes a protein of 130 kilodaltons (hamartin) that has homology to a putative yeast protein of unknown function. Thirty-two distinct mutations were identified in TSC1, 30 of which were truncating, and a single mutation (2105delAAAG) was seen in six apparently unrelated patients. In one of these six, a somatic mutation in the wild-type allele was found in a TSC-associated renal carcinoma, which suggests that hamartin acts as a tumor suppressor.

Sensory-motor network functional connectivity in children with unilateral cerebral palsy secondary to perinatal stroke.

BACKGROUND: Perinatal stroke is the most common cause of unilateral cerebral palsy. Mechanisms of post-stroke developmental plasticity in children are poorly understood. To better understand the relationship between functional connectivity and disability, we used resting-state fMRI to compare sensorimotor connectivity with clinical dysfunction. METHODS: School-aged children with periventricular venous infarction (PVI) and unilateral cerebral palsy were compared to controls. Resting-state BOLD signal was acquired on 3 T MRI and analyzed using CONN in SPM12. Functional connectivity was computed between S1, M1, supplementary motor area (SMA), and thalamus of the left/non-lesioned and right/lesioned hemisphere. Primary outcome was connectivity expressed as a Fisher-transformed correlation coefficient. Motor function was measured using the Assisting Hand Assessment (AHA), and Melbourne Assessment (MA). Proprioceptive function was measured using a robotic position matching task (VarXY). RESULTS: Participants included 15 PVI and 21 controls. AHA and MA in stroke patients were negatively correlated with connectivity (increased connectivity = poorer performance). Position sense was inversely correlated with connectivity (increased connectivity = improved performance) between the non-lesioned S1 and thalamus/SMA. In controls, VarXY was positively correlated with connectivity between the thalamus and bilateral sensorimotor regions. CONCLUSIONS: Resting state fMRI measures of sensorimotor connectivity are associated with clinical sensorimotor function in children with unilateral cerebral palsy secondary to PVI. Greater insight into understanding reorganization of brain networks following perinatal stroke may facilitate personalized rehabilitation.

First detection of bovine noroviruses and detection of bovine coronavirus in Australian dairy cattle.

BACKGROUND AND OBJECTIVE: Noroviruses have been recognised as a significant cause of neonatal enteritis in calves in many countries, but there has been no investigation of their occurrence in Australian cattle. This study aimed to establish whether bovine noroviruses could be detected in faecal samples from Australian dairy cattle. It also sought to determine whether bovine coronaviruses, also associated with neonatal enteritis in calves, could be detected in the same faecal samples. METHODS: A selection of faecal samples that were negative for rotaviruses from dairy farms located in three geographically distinct regions of Victoria were pooled and tested by reverse transcription-PCR for the presence of noroviruses (genogroup III), neboviruses and bovine coronaviruses. RESULTS AND CONCLUSION: Genetically distinct genogroup III noroviruses were detected in two sample pools from different geographic regions and bovine coronavirus was detected in a third pool of samples. This is the first report of bovine norovirus infection in Australian cattle and suggests that future work is required to determine the significance of these agents as a cause of bovine enteric disease in Australia.

Proteolipid protein gene: Pelizaeus-Merzbacher disease in humans and neurodegeneration in mice.

The dosage of the myelin gene and mutant forms of the protein can affect the CNS and PNS. Pelizaeus-Merzbacher disease (PMD) is a myelin disorder of the CNS that arises from both mutational mechanisms. Investigating the molecular basis of PMD in patients and animal models is furthering our understanding of the disease, dosage sensitivity and proteolipid protein function during myelinogenesis.

Inhibition of phorbol ester-receptor binding by a factor from human serum.

The inhibition of receptor binding of [3H]phorbol-12,13-dibutyrate (PDBu) by a factor from human serum was characterized. The serum factor inhibited [3H]PDBu binding in intact monolayer cultures of the rat embryo cell line CREF N and in a subcellular system containing membranes from these cells. Inhibition occurred at both 37 and 4 degrees C and was rapid and reversible. An analysis of [3H]PDBu binding in the presence of the serum factor indicated that inhibition of [3H]PDBu binding by the serum factor was noncompetitive. Using gel filtration to separate the serum factor from free [3H]PDBu, we obtained evidence that the serum factor does not act by binding or trapping the [3H]PDBu. Unlike the phorbol ester tumor promoters, the serum factor alone did not stimulate the release of choline or arachidonic acid from cellular phospholipids, nor did it inhibit the binding of 125I-labeled epidermal growth factor to cellular receptors. The factor did, however, antagonize the inhibition of epidermal growth factor binding induced by PDBu. Sera from pregnant women were, in general, more inhibitory of [3H]PDBu binding than were those from nonpregnant women, which were more inhibitory than those from men. During these studies we found that CREF N cells responded to being grown in the presence of PDBu by partial down regulation of the phorboid receptor. The 50% effective dose for down regulation was 8 nM PDBu, and the maximum effect occurred after 6 h. Taken together, our results indicate that the serum factor inhibits [3H]PDBu binding by a direct physical effect at the level of the phorboid receptors or their associated membranes. It would appear that if this factor acts in vivo, then it might antagonize certain effects of this class of tumor promoters.

DNA microarrays with stem-loop DNA probes: preparation and applications.

We have developed DNA microarrays containing stem-loop DNA probes with short single-stranded overhangs immobilized on a Packard HydroGel chip, a 3-dimensional porous gel substrate. Microarrays were fabricated by immobilizing self-complementary single-stranded oligonucleotides, which adopt a partially duplex structure upon denaturing and re-annealing. Hybridization of single-stranded DNA targets to such arrays is enhanced by contiguous stacking interactions with stem-loop probes and is highly sequence specific. Subsequent enzymatic ligation of the targets to the probes followed by stringent washing further enhances the mismatched base discrimination. We demonstrate here that these microarrays provide excellent specificity with signal-to-background ratios of from 10- to 300-fold. In a comparative study, we demonstrated that HydroGel arrays display 10-30 times higher hybridization signals than some solid surface DNA microarrays. Using Sanger sequencing reactions, we have also developed a method for preparing nested 3'-deletion sets from a target and evaluated the use of stem-loop DNA arrays for detecting p53 mutations in the deletion set. The stem-loop DNA array format is simple, robust and flexible in design, thus it is potentially useful in various DNA diagnostic tests.

The potential impact of the use of homeopathic and herbal remedies on monitoring the safety of prescription products.

The purpose of this paper is to explore the possibility that adverse reactions and drug interactions arising from the use of homeopathic and herbal medicines could lead to confusion when adverse reactions to conventional medicines are reported. An extensive literature review was conducted on the occurrence of adverse reactions and drug interactions following the use of homeopathic or herbal remedies, and the potential for these to confound adverse event reporting to conventional medicines considered. The survey demonstrates the potential for herbal remedies and homeopathic products, to produce adverse drug reactions or drug interactions, and shows the scope for potential for confusion with those arising from conventional medicines. There is a need for greater awareness that adverse reactions apparently due to a conventional medicine, might in reality be due to a herbal medicine or a drug interaction between a herbal medicine and a conventional drug, particularly when a health professional is unaware of the extent of a patient's self-medication with alternative therapies.

X inactivation phenotype in carriers of Pelizaeus-Merzbacher disease: skewed in carriers of a duplication and random in carriers of point mutations.

Pelizaeus-Merzbacher disease (PMD) is an X-linked recessive disease caused by coding sequence mutations in the PLP gene, sub-microscopic duplications of variable sizes including the PLP gene or very rarely deletions of the PLP gene. We analysed the X inactivation pattern in blood of PMD female carriers with duplications and with point mutations. In the majority of duplication carriers (7/11), the X chromosome bearing the duplication was preferentially inactivated, whereas a random pattern of X inactivation was detected in point mutation carriers (3/3), a deletion carrier (1/1), affected females (4/4) who did not have a recognised mutation and normal control females. However 2/5 non-carrier female relatives of patients with a duplication, had skewed X inactivation. The skewed pattern of inactivation observed in most duplication carriers and not in mutation carriers suggests a) that there is selection against those cells in which the duplicated X chromosome is active and b) other expressed sequences within the duplicated region rather than mutant PLP may be responsible. Since the skewed X inactivation did not segregate with the disease in two families and the pattern of X inactivation was variable among the duplication carriers, the pattern X inactivation is an unsuitable diagnostic tool for female carriers of PMD.

Cosmid contigs spanning 9q34 including the candidate region for TSC1.

The tuberous sclerosis disease gene TSC1 has been mapped to 9q34. However, its precise localisation has proved problematic because of conflicting recombination data. Therefore, we have attempted to clone the entire target area into cosmid contigs prior to gene isolation studies. We have used Alu-PCR from irradiation hybrids to produce complex probes from the target region which have identified 1,400 cosmids from a chromosome-specific library. These, along with cosmids obtained by other methods, have been assembled into contigs by a fingerprinting technique. We estimate that we have obtained most of the region in cosmid contigs. These cosmids are a resource for the isolation of expressed genes within the TSC1 interval. In addition, the cosmid contig assembly has demonstrated a number of previously unknown physical connections between genes and markers in 9q34.

The H-reflex to magnetic stimulation of lower-limb nerves.

We elicited H-reflexes by magnetic and electrical stimulation of several different nerves in 10 healthy subjects and two patients with S-1 radiculopathy. The posterior tibial nerve at the popliteal fossa and the femoral nerve at the inguinal ligament were tested with both electrical and magnetic stimulation; the proximal sciatic nerve was tested only with magnetic stimulation. Muscle activity was recorded from the soleus muscle for posterior tibial and sciatic nerve stimulation and from the vastus medialis muscle for femoral nerve stimulation. No significant difference was found between the latency of H-reflexes evoked by magnetic or electrical stimulation. With magnetic stimulation, the mean (+/- SD) Ia sensory fiber conduction velocity in the proximal segment of the sciatic nerve was 72.4 +/- 3.3 m/s, while the motor nerve fiber conduction velocity in the same portion of the nerve was significantly slower, at 60.6 +/- 2.0 m/s. In two patients with unilateral S-1 radiculopathy, the latency of the H-reflex from the soleus muscle to both magnetic and electrical stimulation of the posterior tibial nerve was absent or prolonged on the affected side. Magnetic stimulation can be used to study the H-reflex and Ia fiber conduction velocity and is particularly advantageous when testing deeply located nerve trunks.

3,3-Dialkyl- and 3-alkyl-3-benzyl-substituted 2-pyrrolidinones: a new class of anticonvulsant agents.

A series of 3,3-dialkyl- and 3-alkyl-3-benzyl-substituted 2-pyrrolidinones (lactams) have been prepared and evaluated for their anticonvulsant activities. In the pentylenetetrazole mouse seizure model, 3,3-diethyl lactam 7c and 3-benzyl-3-ethyl lactam 7j are the most effective anticonvulsants (ED50 = 46 and 42 mg/kg, respectively) and have protective index (PI = TD50/ED50) values of 5.65 and 3.00, respectively. These protective index values compare favorably to those of the clinically used antiepileptic drugs ethosuximide (ED50 = 161 mg/kg), phenobarbital (ED50 = 22 mg/kg), and valproic acid (ED50 = 133 mg/kg), which have PI values of 2.35, 4.00, and 2.12, respectively. The benzyl compounds [3-substituents are Bn, H (7h); Bn, Me (7i); and Bn, Et (7j)] are also very effective anticonvulsants against seizures induced by maximal electroshock (ED50 = 41, 55, and 74 mg/kg, respectively) and have PI values of 3.51, 3.04, and 1.70, respectively. The corresponding PI values for phenobarbital and valproic acid are 1.37 and 5.18, respectively. As a class of anticonvulsants, the 3,3-disubstituted 2-pyrrolidinones have a broad spectrum of action and may be useful for the treatment of human epilepsies.

Synthesis and anticonvulsant activities of 3,3-dialkyl- and 3-alkyl-3-benzyl-2-piperidinones (delta-valerolactams) and hexahydro-2H-azepin-2-ones (epsilon-caprolactams).

A series of 3-substituted 2-piperidinone (delta-valerolactam) and hexahydro-2H-azepin-2-one (epsilon-caprolactam) derivatives were prepared and evaluated as anticonvulsants in mice. In the 2-piperidinone series, 3,3-diethyl compound 7b is the most effective anticonvulsant against pentylenetetrazole-induced seizures (ED50, 37 mg/kg; PI (TD50/ED50), 4.46), and 3-benzyl compound 4c (ED50, 41 mg/kg; PI, 7.05) is the most effective anticonvulsant against seizures induced by maximal electroshock. By contrast, none of the epsilon-caprolactams tested had anticonvulsant effects below doses causing rotorod toxicity. log P values were correlated with neurotoxicity and [35S]TBPS displacement, but not with anticonvulsant activity. Electrophysiological evaluations of selected compounds from each series indicated that both the delta-valero-lactams and epsilon-caprolactams potentiated GABA-mediated chloride currents in rat hippocampal neurons.

CNS myelination and PLP gene dosage.

The phenomenon of gene dosage effects demonstrates that the mechanisms of some genetic diseases are best recognised at the genomic level. Classical gene mutation screening approaches utilising PCR are unsuccessful in unravelling the basis of disease because the gene sequence is unaltered and only the copy number is different. Techniques for detecting DNA dosage are required. Examples of haploinsufficiency and gene deletions are well documented, but increased gene dosage is also an important genetic mechanism in disorders involving myelin proteins in the central (CNS) and peripheral nervous system (PNS). Here we review the dosage effects and mutations of the proteolipid protein (PLP) gene that causes Pelizaeus-Merzbacher disease (PMD) and spastic paraplegia Type 2 (SPG2) disorders of CNS myelination. Similarities are drawn with the peripheral neuropathies Charcot-Marie-Tooth disease Type 1 (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) that are also caused by dosage effects and mutations in a single myelin protein gene (peripheral myelin protein 22, PMP-22). We compare the different mutational mechanisms in man and analogous mouse models that suggest a function for PLP beyond its structural role in myelin. We focus on the increased dosage of the PLP gene that is the major cause of PMD and results from a submicroscopic duplication of Xq22. Other clinical phenotypes may arise from gene dosage imbalance with the potential effect of submicroscopic duplications and deletions of the genome being underestimated. Genome sequencing may identify intrinsic structural properties of the DNA with greater susceptibility to these rearrangements and thereby reflect structural changes in the genome.

Microrips of the retinal pigment epithelium.

We describe six patients with exudative age-related macular degeneration who had retinal pigment epithelial detachments with associated overlying neurosensory detachments. During fluorescein angiography, each patient demonstrated a solitary, intense, central serouslike leak at the edge of the retinal pigment epithelial detachment with passage of fluorescein into the subretinal space. In patients in whom the location of associated choroidal neovascularization was evident, the leakage site was remote to the area of neovascularization. Our observations suggest that these leaks result from small retinal epithelial rips, which we termed "microrips," that differ from conventionally described retinal pigment epithelial rips in clinical course and response to laser treatment. We hypothesize that the mechanisms and forces that generate these microrips are different from those producing conventionally described retinal pigment epithelial rips.

Acetylhydrazine hepatotoxicity: the role of covalent binding.

Acetylhydrazine, a human metabolite of isoniazid and a hepatotoxin, binds covalently to rat liver protein and to the proteins of other organs in vivo. The covalent binding to macromolecules in the liver, but not to those of other organs, was increased by pretreatment with phenobarbital. The time course of binding to total hepatic protein and to hepatic subcellular fractions was also altered by phenobarbital pretreatment. There was a concommitant time-dependent decline in cytochrome P-450 levels in the phenobarbital pretreated animals, but not controls. Acetylhydrazine did not produce detectable lesions in organs other than the liver, despite the occurrence of covalent binding to protein in those organs. The results suggest that only a critical fraction of the covalent binding to specific subcellular components is related to the subsequent development of tissue lesions.

Anterior electroencephalographic asymmetry changes in elderly women in response to a pleasant and an unpleasant odor.

Greater left than right frontal EEG activation has been associated with increased positive and/or decreased negative affect, whereas greater right than left frontal activation has been associated with the opposite pattern. Substantial research has documented the trait properties of asymmetry, as well as responses to pleasant and unpleasant stimuli. The present study examined changes in anterior alpha asymmetries in response to pleasant (vanilla), unpleasant (valerian), and neutral (water) odors. As predicted, vanilla produced relative left frontal activation compared to valerian and water. Frontal asymmetry did not differ in response to valerian compared to water. The results are consistent with the hypothesis that the left frontal region of the brain is involved in positive/approach-related emotion, and extend previous results into the olfactory realm.

Postponing sexual intercourse among urban junior high school students-a randomized controlled evaluation.

PURPOSE: To describe a randomized, controlled evaluation of a school-based intervention to delay sexual intercourse among urban junior high school students. METHODS: Six Washington, D.C., junior high schools were randomly assigned to the intervention or nonintervention control condition for an educational program. During the first school year, seventh graders (n = 582) from the six schools obtained written parental consent to participate. Three health professionals (one per intervention school) implemented the program, which consisted of reproductive health classes, the Postponing Sexual Involvement Curriculum, health risk screening, and "booster" educational activities during the following (eighth grade) school year. Cross-sectional surveys were administered at baseline, the end of the seventh grade, and the beginning and end of the eighth grade. Intervention and control group differences in virginity, attitudes toward delayed sex and childbearing, and sexual knowledge and behavior were assessed at all four time points. RESULTS: At baseline, 44% of the seventh grade males and 81% of the seventh grade females reported being virgins. At the end of the seventh grade (first follow-up), after controlling for baseline study group differences, intervention-group females were more likely to report virginity, self-efficacy to refuse sex with a boyfriend, and the intention to avoid sexual involvement during the following 6 months. At the end of the eighth grade, significantly more intervention- than control-group females reported virginity, birth control use at last intercourse (for nonvirgins), and knowledge of adolescent reproductive health and confidentiality rights. No changes in virginity, self-efficacy to refuse sex, or sexual intent for the next 6 months were observed among male participants at any time during the study. However, on all three follow-up surveys, intervention-group males scored significantly higher than their control-group counterparts in knowledge of birth control method efficacy. No change in attitudes toward abstinence was observed for either gender at any follow-up point. CONCLUSIONS: Gender differences in baseline sexual activity rates and in various study outcomes suggest a possible need for separate, gender-specific intervention activities that can adequately address the social and cognitive needs of both sexes.