Pig fetal skeletal muscle development is associated with genome-wide DNA hypomethylation and corresponding alterations in transcript and microRNA expression.

Fetal myogenesis represents a critical period of porcine skeletal muscle development and requires coordinated expression of thousands of genes. Epigenetic mechanisms, including DNA methylation, drive transcriptional regulation during development; however, these processes are understudied in developing porcine tissues. We performed bisulfite sequencing to assess DNA methylation in pig longissimus dorsi muscle at 41- and 70-days gestation (dg), as well as RNA- and small RNA-sequencing to identify coordinated changes in methylation and expression between myogenic stages. We identified 45 739 differentially methylated regions (DMRs) between stages, and the majority (N = 34 232) were hypomethylated at 70 versus 41 dg. Integration of methylation and transcriptomic data revealed strong associations between differential gene methylation and expression. Differential miRNA methylation was significantly negatively correlated with abundance, and dynamic expression of assayed miRNAs persisted postnatally. Motif analysis revealed significant enrichment of myogenic regulatory factor motifs among hypomethylated regions, suggesting that DNA hypomethylation may function to increase accessibility of muscle-specific transcription factors. We show that developmental DMRs are enriched for GWAS SNPs for muscle- and meat-related traits, demonstrating the potential for epigenetic processes to influence phenotypic diversity. Our results enhance understanding of DNA methylation dynamics of porcine myogenesis and reveal putative cis-regulatory elements governed by epigenetic processes.

Bilateral internal carotid artery occlusion with compensatory perfusion in a HIV-infected patient.

SUMMARY: HIV infection occlusive arteriopathies may result in neurological symptoms. We report a case of bilateral complete occlusion of the extracranial portions of the internal carotid arteries in a HIV+ve patient who presented with a syncopal episode due to intraventricular haemorrhage. Compensatory blood flow from the posterior cerebral circulation via the circle of Willis resulted in small telangiectatic vessels arising from the posterior cerebral circulation which probably accounted for this rare haemorrhagic complication of an occlusive arteriopathy.

Differential loss of neurochemical markers following quinolinic acid-induced lesions of rat striatum.

Twenty-one days following bilateral striatal injections of the excitotoxin quinolinic acid (QA) (75, 100, or 150 nmol) or vehicle there were differential losses of various neurochemical markers. Choline acetyltransferase was relatively resistant to QA-induced lesions while glutamate decarboxylase activity was more sensitive. The binding of [3H]glutamate to the N-methyl-D-aspartate receptor was very sensitive to QA-induced lesions while the loss of [3H]MK801 binding was less sensitive. The differential loss of [3H]glutamate and [3H]MK801 binding indicated that these sites may represent distinct molecules which are differentially located or differentially regulated. The binding of [3H]SCH23390 to the D1 dopamine receptor was also very sensitive to QA-induced lesions. [3H]SCH23390 binding may represent a relatively simple and sensitive neurochemical assay of QA-induced neurotoxicity.

Selection of Streptomyces ambofaciens mutants that produce large quantities of spiramycin and determination of optimal conditions for spiramycin production.

The aim of this work was to develop a strategy to isolate a morphologically stable mutant of Streptomyces ambofaciens ATCC 15154 which produced high titers of spiramycin. The rationale was to grow a nitrosoguanidine-mutated population for many generations under nonselective conditions followed by two cycles of protoplast formation and regeneration. A total of 2,400 surviving colonies were then screened for spiramycin production and subsequently checked for stability. From this experiment, strain 6-37 was isolated that produced 181 mg of spiramycin per liter and only one morphological type. The parent strain (ATCC 15154) produced 107 mg of spiramycin per liter and four morphological types. Strain 6-37 was then mutated with nitrosoguanidine, and 14,000 colonies were screened for spiramycin production. From this experiment, five strains were isolated that produced titers ranging from 187 to 373 mg of spiramycin per liter. Subsequent media and time studies with these strains resulted in a fermentation that produced 1,728 mg of spiramycin per liter.

Results of N-methyl-D-aspartate antagonists in perinatal cerebral asphyxia therapy.

Perinatal cerebral asphyxia, which results in significant neurologic and cognitive disabilities in infants and children, remains a major health problem. Potential neurologic sequelae include cerebral palsy, mental retardation, and epilepsy. Over the next few years, neuroprotective agents that prevent asphyxial neuronal injury and death are likely to be developed. These agents may also be effective in prophylaxis and treatment of chronic neurologic disorders, including epilepsy and neurodegenerative disorders, such as Huntington disease.

Neural grafts and pharmacological intervention in a model of Huntington's disease.

Using the excitotoxic animal model of Huntington's disease, two experimental treatments were evaluated. The first experiment explored the effect of MK801 (a systemically active anticonvulsant, and noncompetitive NMDA antagonist) pretreatment on quinolinic acid (QA)-induced striatal degeneration and behavioral deficits. MK801 prevented QA-induced neuropathological changes in the striatum and the anatomical protection was correlated with the absence of deficits in the cataleptic response to haloperidol. The second experiment tested the ability of three types of fetal grafts to reverse behavioral deficits induced by kainic acid (KA) lesion. Fetal (E15-16) striatal, cortical and tectal grafts were delivered into the KA-lesioned striatum one week or one month after lesion. Animals in this experiment were evaluated on a motor coordination task, haloperidol-induced catalepsy and amphetamine-induced locomotor activity. Striatal grafts attenuated the deficits induced by KA in all of the tasks observed, and no effect of time of grafting was detected. Tectal grafts had a partial beneficial effect, attenuating the decrease in the cataleptic response to haloperidol observed after KA lesions. No effect of time of grafting was detected for these grafts. In contrast, a clear effect of time of grafting was detected for the cortical grafts. Early cortical grafts reversed the exaggerated response to amphetamine observed after KA lesions whereas late cortical grafts resulted in sham-like scores on the catalepsy test. Histochemical analysis showed that most of the grafts survived, had acetylcholinesterase (AChE) positive fibers and cell bodies, and were metabolically active as indicated by cytochrome oxidase (CO) positive staining. It is suggested that striatal grafts may have restored to some extent the striatal GABAergic control over output structures, and that trophic factors play a role in behavioral recovery as is evident from the beneficial effects of the tectal grafts. Although the mechanisms underlying the differential effects observed after early or late cortical grafts are unknown, the interaction between the cellular components and trophic factors present in the cortical grafts and the condition of the lesioned host at the time of grafting may yield a host-graft complex with a unique profile.

MK801 prevents quinolinic acid-induced behavioral deficits and neurotoxicity in the striatum.

Bilateral intrastriatal injections of quinolinic acid (QA) (180 nmoles) induced weight loss and neurologic and behavioral deficits including convulsions, decreased catalepsy response to haloperidol, increased nocturnal locomotor activity, and abnormal feeding behavior in adult male Sprague-Dawley rats. Pretreatment with the noncompetitive N-methyl-D-aspartate (NMDA) antagonist, MK801 (4 mg/kg IP) 30 min prior to stereotaxic surgery prevented the appearance of all QA-induced behavioral abnormalities and prevented weight loss. Twelve weeks after surgery the QA-lesioned animals recovered to sham levels on feeding behavior and nocturnal locomotor activity, but showed persistent reductions in haloperidol-induced catalepsy. Histological examination of the QA-lesioned brains showed extensive lesions of the dorsolateral striatum and frontoparietal cortex. MK801 pretreatment protected against these lesions. These results confirm that MK801 treatment prevents the appearance of neuropathological damage after QA neurotoxicity, and further show that neuronal protection with MK801 is correlated with the absence of QA-induced behavioral deficits.

Chronic treatment with MK-801 increases the quinolinic acid-induced loss of D-1 dopamine receptors in rat striatum.

Following withdrawal from short-term treatment with the non-competitive N-methyl-d-aspartate receptor antagonist MK-801 (1 mg/kg per day) there was no significant change in the Bmax or KD of [3H]SCH23390 binding to dopamine D-1 receptors in rat striatum. Intrastriatal injection of the excitotoxin quinolinic acid (100 nmol) produced a significant decrease in [3H]SCH23390 binding. In rats withdrawn from chronic MK-801 treatment quinolinic acid produced a significantly greater loss of [3H]SCH23390 binding sites than in rats not treated with MK-801. These data indicated that striatal neurons are hypersensitive to the neurotoxic actions of quinolinic acid following withdrawal from chronic MK-801 treatment.

The topography of MK-801-induced locomotor patterns in rats.

Locomotor patterns in rats given systemic injections of the novel, noncompetitive N-methyl-D-aspartate (NMDA) antagonist, MK-801 were characterized using Digiscan Animal Activity Monitoring System. At low systemic doses, MK-801 produced an activity pattern most similar to patterns previously described for less potent noncompetitive NMDA antagonists; this was typified by hyperactive locomotor behavior, with increases in distance travelled, speed, and clockwise/anticlockwise locomotion, and a marked decrease in rearing behavior. Although MK-801 elicited some motor patterns similar to those previously described for sympathomimetic agents, including hyperactivity and increased stereotypy, it did not produce increased rearing behavior, the most prominent sympathomimetic effect. These results demonstrated that the topography of locomotion elicited by low systemic doses of MK-801 is most similar to locomotor patterns previously described for noncompetitive NMDA receptor antagonists.

MK-801 prevents hippocampal neurodegeneration in neonatal hypoxic-ischemic rats.

In cerebral asphyxia, enhanced postsynaptic stimulation of N-methyl-D-aspartate (NMDA) receptor by excessive glutamate may mediate neuronal injury and death. The neuroprotective potential of the novel, potent NMDA receptor antagonist MK-801 was assessed by evaluating hippocampal behavioral and histologic outcomes in an experimental rat model of neonatal hypoxia/ischemia. Seven-day-old rats with and without MK-801 pretreatment were subjected to unilateral carotid ligation followed by 2 hours of hypoxia. At age 30 days, spontaneous alternation behavior was measured using a conventional wooden T maze. Hypoxic-ischemic animals pretreated with saline demonstrated a significant impairment in spontaneous alternation behavior compared with that of normal control rats and the hypoxic-ischemic rats pretreated with MK-801. Hippocampal neuronal damage in the CA1 and CA3 regions was prevented in animals pretreated with MK-801 vs saline-treated controls. Therefore, while saline-treated rats with hippocampal lesions showed defective memory and hippocampal neuronal destruction, pretreatment with MK-801 protected rats. Thus, MK-801 appears to protect hippocampal neurons from hypoxia/ischemia and may be potentially beneficial in preventing neonatal asphyxial brain damage.

Neurologic status and intracranial hemorrhage in very-low-birth-weight preterm infants. Outcome at 1 year and 5 years.

Twenty-six very-low-birth-weight preterm infants with and without intracranial hemorrhage (ICH) were followed up prospectively from birth to school age to determine the relationship between ICH and subsequent neurologic and cognitive outcomes. All children had sequential cranial ultrasound examinations at birth and neurologic assessments at 3-month intervals during the first year, at 1 year of age, and at 5 to 6 years; psychometric assessments were done at 5 to 6 years. Seventeen children had no ICH, 3 had grade 1 ICH, 1 had grade 3 ICH, and 5 had grade 4 ICH. The 1-year Amiel-Tison neurologic assessment in 25 infants demonstrated that 14 were normal, 3 were suspect, and 8 were abnormal. By 5 to 6 years of age, 5 of 8 children neurologically abnormal at 1 year remained abnormal, 2 of 3 children neurologically suspect at 1 year remained suspect; while 9 of 15 children neurologically normal at 1 year remained normal, the remaining 6 had become suspect. The predominant neurologic abnormality at 5 to 6 years was subtle neurologic dysfunctioning. The Wechsler Preschool and Primary Scale of Intelligence at 5 to 6 years revealed a mean group IQ score of 92.1. The Beery Visual Motor Integration Test results demonstrated that 18 of 26 children had mild to severe visual motor perceptual difficulties. Severe ICH (grades 3 and 4) correlated with abnormal neurologic performances at 1 and 5 to 6 years. Mild ICH (grade 1) and no ICH did not correlate with any one of the 1-year neurologic classifications. The 1-year status correlated with the 5- to 6-year neurologic outcome best for children who were either neurologically suspect or abnormal at age 1 year. The 1-year neurologic score did not correlate with 5- to 6-year IQ and Beery Visual Motor Integration Test scores.

Very-low-birth-weight, preterm infants with or without intracranial hemorrhage. Neurologic, cognitive and cranial MRI correlations at 4-8-year follow-up.

Fourteen very-low-birth-weight (VLBW) preterm infants with and without intracranial hemorrhage (ICH) were prospectively followed from birth to 4 to 8 years for the purpose of determining neurologic and cognitive sequelae associated with ICH severity and to correlate outcomes with brain morphology as determined by Magnetic Resonance Imaging (MRI). Intracranial hemorrhage was documented by cranial ultrasonography performed in early life. Follow-up assessments included neurologic and psychometric examinations and cranial MRI scans. Of six children with no ICH, five had normal results on all three follow-up measures. Three children with Grade I-II ICH had mild to moderate neurologic and cognitive sequelae with focal white matter MRI abnormalities. Five children with Grade III-IV ICH had severe neurologic, cognitive, and MRI deficits, including MRI regional and diffuse white matter abnormalities and/or cortical atrophy. Focal and diffuse neurologic deficits correlated with the extent of MRI morphologic abnormalities. Results of this study indicate that ICH severity correlated with outcomes in children at follow-up; the more severe the ICH, the more adverse the neurologic, cognitive, and MRI results. MRI white matter abnormalities were present in all children with any degree ICH, while ventriculomegaly was seen only in severe ICH (Grade III-IV ICH). Neurologic deficits correlated with MRI structural abnormalities.