Modulated expression of human peripheral blood microRNAs from infancy to adulthood and its role in aging

Accumulating evidence suggests a role for microRNAs (miRNAs) in regulating various processes of mammalian postnatal development and aging. To investigate the changes in blood‐based miRNA expression from preterm infants to adulthood, we compared 365 miRNA expression profiles in a screening set of preterm infants and adults. Approximately one‐third of the miRNAs were constantly expressed from postnatal development to adulthood, another one‐third were differentially expressed between preterm infants and adults, and the remaining one‐third were not detectable in these two groups. Based on their expression in infants and adults, the miRNAs were categorized into five classes, and six of the seven miRNAs chosen from each class except one with age‐constant expression were confirmed in a validation set containing infants, children, and adults. Comparing the chromosomal locations of the different miRNA classes revealed two hot spots: the miRNA cluster on 14q32.31 exhibited age‐constant expression, and the one on 9q22.21 exhibited up‐regulation in adults. Furthermore, six miRNAs detectable in adults were down‐regulated in older adults, and four chosen for individual quantification were verified in the validation set. Analysis of the network functions revealed that differentially regulated miRNAs between infants and adults and miRNAs that decreased during aging shared two network functions: inflammatory disease and inflammatory response. Four expression patterns existed in the 11 miRNAs from infancy to adulthood, with a significant transition in ages 9–20 years. Our results provide an overview on the regulation pattern of blood miRNAs throughout life and the possible biological functions performed by different classes of miRNAs.     

Inducible nitric oxide synthase expression and plasma bilirubin changes in rats under intermittent hypoxia treatment

It has been reported that intermittent hypoxia treatment prevents oxidative injuries to the brain and protects the heart against ischemia-reperfusion injury. Both anti-oxidative defensive systems and prevention of free intracellular calcium overload might be the result of intermittent hypoxia. Thus, the purpose of this study was to explore the effects of intermittent hypoxia (8 h at 12 % O2 per day) for 0, 7 or 14 days on inducible nitric oxide synthase (iNOS) expression in the spleen and on splenic calcium response to the mitogen phytohemagglutinin (PHA). The results demonstrated that administration of intermittent hypoxia for 7 days caused severe hemolysis of erythrocytes in the spleen and the hemolytic condition was ameliorated by intermittent hypoxia for 14 days. However, a significant decline in splenic weight and an increase in plasma total bilirubin levels appeared in rats after hypoxia for 14 days. No calcium response to PHA was observed in splenocytes obtained from rats after intermittent hypoxia for 7 days. After intermittent hypoxia for 14 days, the calcium response to PHA was restored to the level of the controls. Intermittent hypoxia for 7 days was able to induce higher iNOS expression in splenic tissues than hypoxia for 14 days. These results suggested that intermittent hypoxia for 14 days appeared to involve acclimatization that protects the rats from oxidative injury through less hemolysis and iNOS expression in splenic tissues and by the presence of more bilirubin in the plasma. The increase in plasma total bilirubin levels might be the cause of induced adaptation to chronic intermittent hypoxia.     

2E8 Binds to the High Affinity I-domain in a Metal Ion-dependent Manner: A SECOND GENERATION MONOCLONAL ANTIBODY SELECTIVELY TARGETING ACTIVATED LFA-1*

The activation of leukocyte function-associated antigen-1 (LFA-1) plays a critical role in regulating immune responses. The metal ion-dependent adhesion site on the I-domain of LFA-1 α_L subunit is the key recognition site for ligand binding. Upon activation, conformation changes in the I-domain can lead LFA-1 from the low affinity state to the high affinity (HA) state. Using the purified HA I-domain locked by disulfide bonds for immunization, we developed an mAb, 2E8, that specifically binds to cells expressing the HA LFA-1. The surface plasmon resonance analysis has shown that 2E8 only binds to the HA I-domain and that the dissociation constant (K_D) for HA I-domain is 197 nm. The binding of 2E8 to the HA I-domain is metal ion-dependent, and the affinity decreased as Mn²⁺ was replaced sequentially by Mg²⁺ and Ca²⁺. Surface plasmon resonance analysis demonstrates that 2E8 inhibits the interaction of HA I-domain and ICAM-1. Furthermore, we found that 2E8 can detect activated LFA-1 on both JY and Jurkat cells using flow cytometry and parallel plate adhesion assay. In addition, 2E8 inhibits JY cell adhesion to human umbilical vein endothelial cells and homotypic aggregation. 2E8 treatment reduces the proliferation of both human CD4⁺ and CD8⁺ T cells upon OKT3 stimulation without the impairment of their cytolytic function. Taken together, these data demonstrate that 2E8 is specific for the high affinity form of LFA-1 and that 2E8 inhibits LFA-1/ICAM-1 interactions. As a novel activation-specific monoclonal antibody, 2E8 is a potentially useful reagent for blocking high affinity LFA-1 and modulating T cell activation in research and therapeutics.     

Mechanisms underlying the inhibition of murine sperm capacitation by the seminal protein,SPINKL

SPINKL, a serine protease inhibitor kazal‐type‐like protein initially found in mouse seminal vesicle secretions, possesses structurally conserved six‐cysteine residues of the kazal‐type serine protease inhibitor family. However, it has no inhibitory activity against serine proteases. Previously, it was found to have the ability to suppress murine sperm capacitation in vitro. Herein, we investigated the mechanisms underlying the suppressive effect of SPINKL on sperm capacitation. Three in vitro capacitation‐enhancing agents, including bovine serum albumin (BSA), methyl‐beta‐cyclodextrin (MBCD), and dibutyryl cyclic AMP (dbcAMP), coupled with 3‐isobutyl‐1‐methylxanthine (IBMX), were used to evaluate the influence of SPINKL on capacitation signaling. Preincubation of sperm with SPINKL suppressed BSA‐ and MBCD‐induced sperm capacitation by blocking three upstream signals of capacitation that is the cholesterol efflux from sperm plasma membranes, extracellular calcium ion influx into sperm, and increases in intracellular cAMP. Moreover, SPINKL also inhibited downstream signal transduction of capacitation since it suppressed dbcAMP/IBMX and N⁶‐phenyl cAMP (6‐Phe‐cAMP)‐activated cAMP‐dependent protein kinase‐associated protein tyrosine phosphorylation. Such inhibition is probably mediated by attenuation of SRC tyrosine kinase activity. Furthermore, SPINKL could not reverse capacitation once sperm had been capacitated by capacitation‐enhancing agents or capacitated in vivo in the oviduct. SPINKL bound to sperm existed in the uterus but had disappeared from sperm in the oviduct during the sperm's transit through the female reproductive tract. Therefore, SPINKL may serve as an uncapacitation factor in the uterus to prevent sperm from precocious capacitation and the subsequent acrosome reaction and thus preserve the fertilization ability of sperm. J. Cell. Biochem. 114: 888–898, 2013. © 2012 Wiley Periodicals, Inc.     

Synthesis and antiviral evaluation of 9-(S)-[3-alkoxy-2-(phosphonomethoxy)propyl]nucleoside alkoxyalkyl esters: inhibitors of hepatitis C virus and HIV-1 replication

We reported previously that octadecyloxyethyl 9-(S)-[3-hydroxy-2-(phosphonomethoxy)-propyl]adenine (ODE-(S)-HPMPA) was active against genotype 1b and 2a hepatitis C virus (HCV) replicons. This is surprising because acyclic nucleoside phosphonates have been regarded as having antiviral activity only against double stranded DNA viruses, HIV and HBV. We synthesized octadecyloxyethyl 9-(S)-[3-methoxy-2-(phosphonomethoxy)propyl]-adenine and found it to be active in genotype 1b and 2a HCV replicons with EC?? values of 1-2 μM and a CC?? of > 150 μM. Analogs with substitutions at the 3'-hydroxyl larger than methyl or ethyl, or with other purine bases were less active but most compounds had significant antiviral activity against HIV-1 in vitro. The most active anti-HIV compound was octadecyloxyethyl 9-(R)-[3-methoxy-2-(phosphonomethoxy)propyl]guanine with an EC?? < 0.01 nanomolar and a selectivity index of > 4.4 million.     

Autonomic modulation and health-related quality of life among schizophrenic patients treated with non-intensive case management

Background

Schizophrenia is associated with autonomic dysfunction and this may increase cardiovascular mortality. Past studies on autonomic modulation of schizophrenic patients focused on inpatients rather than individuals in a community setting, especially those receiving non-intensive case management (non-ICM). Besides, autonomic modulation and its association with health-related quality of life (HRQoL) in this population remain unexplored.

Methods

A total of 25 schizophrenic patients treated by non-ICM and 40 healthy volunteers were matched by age, gender and body mass index; smokers were excluded. Between the two groups, we compared the individuals'' 5 min resting assessments of heart rate variability and their HRQoL, which was measured using EuroQoL-5D (EQ-5D). Patients with schizophrenia were assessed for psychopathology using the Positive and Negative Syndrome Scale for Schizophrenia (PANSS). We examined the relationship between heart rate variability measurements, HRQoL scores, PANSS scores, and other clinical variables among the schizophrenic patients treated by non-ICM.

Results

Compared to the controls, patients with schizophrenia showed a significant impairment of autonomic modulation and a worse HRQoL. Cardiovagal dysfunction among the schizophrenic patients could be predicted independently based on lower educational level and more negative symptoms. Sympathetic predominance was directly associated with anticholinergics use and EQ-5D using a visual analogue scale (EQ-VAS).

Conclusion

Patients with schizophrenia treated by non-ICM show a significant impairment of their autonomic function and HRQoL compared to the controls. Since the sympathovagal dysfunction is associated with more negative symptoms or higher VAS score, the treatment of the negative symptoms as well as the monitoring of HRQoL might help to manage cardiovascular risk among these individuals. In addition, EQ-VAS scores must be interpreted more cautiously in such a population.     

Genome-wide gene expression analysis implicates the immune response and lymphangiogenesis in the pathogenesis of fetal chylothorax

Fetal chylothorax (FC) is a rare condition characterized by lymphocyte-rich pleural effusion. Although its pathogenesis remains elusive, it may involve inflammation, since there are increased concentrations of proinflammatory mediators in pleural fluids. Only a few hereditary lymphedema-associated gene loci, e.g. VEGFR3, ITGA9 and PTPN11, were detected in human fetuses with this condition; these cases had a poorer prognosis, due to defective lymphangiogenesis. In the present study, genome-wide gene expression analysis was conducted, comparing pleural and ascitic fluids in three hydropic fetuses, one with and two without the ITGA9 mutation. One fetus (the index case), from a dizygotic pregnancy (the cotwin was unaffected), received antenatal OK-432 pleurodesis and survived beyond the neonatal stage, despite having the ITGA9 mutation. Genes and pathways involved in the immune response were universally up-regulated in fetal pleural fluids compared to those in ascitic fluids. Furthermore, genes involved in the lymphangiogenesis pathway were down-regulated in fetal pleural fluids (compared to ascitic fluid), but following OK-432 pleurodesis, they were up-regulated. Expression of ITGA9 was concordant with overall trends of lymphangiogenesis. In conclusion, we inferred that both the immune response and lymphangiogenesis were implicated in the pathogenesis of fetal chylothorax. Furthermore, genome-wide gene expression microarray analysis may facilitate personalized medicine by selecting the most appropriate treatment, according to the specific circumstances of the patient, for this rare, but heterogeneous disease.     

Haplotypes of the D-Amino Acid Oxidase Gene Are Significantly Associated with Schizophrenia and Its Neurocognitive Deficits

D-amino acid oxidase (DAO) has been reported to be associated with schizophrenia. This study aimed to search for genetic variants associated with this gene. The genomic regions of all exons, highly conserved regions of introns, and promoters of this gene were sequenced. Potentially meaningful single-nucleotide polymorphisms (SNPs) obtained from direct sequencing were selected for genotyping in 600 controls and 912 patients with schizophrenia and in a replicated sample consisting of 388 patients with schizophrenia. Genetic associations were examined using single-locus and haplotype association analyses. In single-locus analyses, the frequency of the C allele of a novel SNP rs55944529 located at intron 8 was found to be significantly higher in the original large patient sample (p = 0.016). This allele was associated with a higher level of DAO mRNA expression in the Epstein-Barr virus-transformed lymphocytes. The haplotype distribution of a haplotype block composed of rs11114083-rs2070586-rs2070587-rs55944529 across intron 1 and intron 8 was significantly different between the patients and controls and the haplotype frequencies of AAGC were significantly higher in patients, in both the original (corrected p < 0.0001) and replicated samples (corrected p = 0.0003). The CGTC haplotype was specifically associated with the subgroup with deficits in sustained attention and executive function and the AAGC haplotype was associated with the subgroup without such deficits. The DAO gene was a susceptibility gene for schizophrenia and the genomic region between intron 1 and intron 8 may harbor functional genetic variants, which may influence the mRNA expression of DAO and neurocognitive functions in schizophrenia.