Epitranscriptomic cytidine methylation of the hepatitis B viral RNA is essential for viral reverse transcription and particle production.

Epitranscriptomic RNA modifications have emerged as important regulators of the fate and function of viral RNAs. One prominent modification, the cytidine methylation 5-methylcytidine (m5C), is found on the RNA of HIV-1, where m5C enhances the translation of HIV-1 RNA. However, whether m5C functionally enhances the RNA of other pathogenic viruses remains elusive. Here, we surveyed a panel of commonly found RNA modifications on the RNA of hepatitis B virus (HBV) and found that HBV RNA is enriched with m5C as well as ten other modifications, at stoichiometries much higher than host messenger RNA (mRNA). Intriguingly, m5C is mostly found on the epsilon hairpin, an RNA element required for viral RNA encapsidation and reverse transcription, with these m5C mainly deposited by the cellular methyltransferase NSUN2. Loss of m5C from HBV RNA due to NSUN2 depletion resulted in a partial decrease in viral core protein (HBc) production, accompanied by a near-complete loss of the reverse transcribed viral DNA. Similarly, mutations introduced to remove the methylated cytidines resulted in a loss of HBc production and reverse transcription. Furthermore, pharmacological disruption of m5C deposition led to a significant decrease in HBV replication. Thus, our data indicate m5C methylations as a critical mediator of the epsilon elements' function in HBV virion production and reverse transcription, suggesting the therapeutic potential of targeting the m5C methyltransfer process on HBV epsilon as an antiviral strategy.

Bioactivity Study of Tricyclic and Tetracyclic Genipin Derivatives as Anti-inflammatory Agents.

A series of genipin derivatives included tricyclic cyclopentaimidazopyridine, cyclopentapyridopyrimidine, octahydrocyclopentapyridodiazepine, and tetracyclic decahydrobenzoimidazocyclopentapyridine were synthesized and developed as anti-inflammatory agents. All of them were tested against NO production in LPS-induced RAW264.7 cells. Based on IC50 data and the SAR study, we found that tricyclic cyclopentaimidazopyridines 3d-f and 7-9 presented the better inhibitory activities (≦ 28.1 µM) in comparison with the reference standard Indomethacin (166 µM). On the other hand, all of them showed inactivity for in vitro cyclooxygenase COX-2 inhibition assays and compounds 8 and 9 possessed the cell toxity. To explore the further anti-inflammatory mechanism, Western blot analysis was carried out. Furthermore, compound 3d shown better bioactivity than Indomethacin. The suppression of NF-κB signal pathway by compound 3d was also determined. To sum-up, compound 3d would be the potential anti-inflammatory lead compound.

Synthesis and anti-inflammatory activity evaluation of NO-releasing furoxan/1,2,4-triazole hybrid derivatives.

An efficient synthesis method was developed for furoxan/1,2,4-triazole hybrids 5a-k from methyl 5-(halomethyl)-1-aryl-1H-1,2,4-triazole-3-carboxylates 1 through two-steps reaction including hydrolyzation and esterification. All of the furoxan/1,2,4-triazole hybrid derivatives were characterized by spectroscopy. On the other hand, the influence of newly synthesized multi-substituted 1,2,4-triazoles on the exogenous NO release ability, in vitro and in vivo anti-inflammatory activity, and in silico predictions were experimentally evaluated. Based on the exogenous NO release ability study and SAR studies of in vitro anti-inflammatory activity, all of compounds 5a-k exhibited slightly NO release ability and potential anti-inflammatory activity on LPS-induced RAW264.7 cells (IC50 = 5.74-15.3 µM) compared to Celecoxib (IC50 = 16.5 µM) and Indomethacin (IC50 = 56.8 µM). Furthermore, compounds 5a-k were also subjected to in vitro COX-1/COX-2 inhibition assays. Particularly, compound 5f exhibited extraordinary COX-2 inhibition (IC50 = 0.0455 µM) and selectivity (SI = 209). In addition, compound 5f was also examined in vivo pro-inflammatory cytokine productions and gastric safety and possessed the better inhibition of cytokine and safety compared with Indomethacin at the same concentration. Through the molecular modeling and in silico physicochemical and pharmacokinetic properties prediction, compound 5f was stabilized in COX-2 active binding site and possessed the fundamental strong H-bond interaction with Arg499 to form the significant physicochemical and pharmacological properties as a candidate drug. Following the in vitro, in vivo, and in silico study results, compound 5f demonstrated to be a potential anti-inflammatory agent and had comparable effects with Celecoxib.

Synthesis of ß-Amino Carbonyl 6-(Aminomethyl)- and 6-(Hydroxymethyl)pyrazolopyrimidines for DPP-4 Inhibition Study.

BACKGROUND: Type-2 diabetes is a chronic progressive metabolic disease resulting in severe vascular complications and mortality risk. Recently, DPP-4 inhibitors are conceived as a favorable class of agents for the treatment of type 2 diabetes due to the minimal side effects. METHODS: Sitagliptin is the first medicine approved for DPP-4 inhibitor. Its structure involved three fragments: 2,4,5-triflorophenyl fragment pharmacophore, enantiomerically ß-amino carbonyl linker, and tetrahydrotriazolopyridine. Herein, we are drawn to the possibility of substituting tetrahydrotriazolopyridine motif present in Sitagliptin with a series of new fused pyrazolopyrimidine bicyclic fragment to investigate potency and safety. RESULTS: Two series of fused 6-(aminomethyl)pyrazolopyrimidine and 6-(hydroxymethyl)pyrazolopyrimidine derivatives containing ß-amino ester or amide as linkers were successfully designed for the new DPP-4 inhibitors. Most fused 6-methylpyrazolopyrimidines were evaluated against DPP-4 inhibition and selectivity capacity. Based on research study, ß-amino carbonyl fused 6-(hydroxymethyl)pyrazolopyrimidine possesses the significant DPP-4 inhibition (IC50 ≤ 59.8 nM) and presents similar with Sitagliptin (IC50 = 28 nM). Particularly, they had satisfactory selectivity over DPP-8 and DPP-9, except for QPP. CONCLUSION: ß-Amino esters and amides fused 6-(hydroxymethyl)pyrazolopyrimidine were developed as the new DPP-4 inhibitors. Those compounds with a methyl group or hydrogen in N-1 position and methyl substituted group in C-3 of pyrazolopyrimidine moiety showed better potent DPP-4 inhibition (IC50 = 21.4-59.8 nM). Furthermore, they had satisfactory selectivity over DPP-8 and DPP-9 Finally, the docking results revealed that compound 9n was stabilized at DPP-4 active site and would be a potential lead drug.

A Multisite Study of Family Functioning Impairment in Pediatric Obsessive-Compulsive Disorder.

OBJECTIVE: Familial aspects of pediatric obsessive-compulsive disorder (OCD), including accommodation and treatment, have received notable and warranted attention. However, individual perspectives of its repercussions on family functioning, including emotional and occupational parental burden, have not been closely examined. The present study details this topic using a large multicenter sample. METHOD: Participants included 354 youth affected with OCD and their mothers and fathers ascertained through OCD programs in Boston, Massachusetts (n = 180) and Vancouver, British Columbia (n = 174). The validated OCD Family Functioning Scale and standard OCD measurements were completed. Descriptive, between-site, and cross-perspective comparative analyses were followed by regression model testing to predict family impairment. RESULTS: Family functioning was negatively affected from youth, mother, and father perspectives. Impairment was reportedly more extensive at the time of worst OCD severity and was greater from maternal versus paternal viewpoints. Most frequently affected family tasks and implicated OCD symptoms included morning and bedtime routines and intrusive thoughts. Emotional repercussions in all members included stress and anxiety, followed by frustration or anger in youth and sadness in parents. Nearly half of mothers and one third of fathers reported daily occupational impairment. Compared with youth self-report, parents perceived fewer social and academic effects on their child. Family accommodation most consistently predicted family impairment, especially from parent perspectives. OCD and compulsion severity, contamination and religious obsessions, and comorbidities also predicted various perspectives of family subdomain impairment. CONCLUSION: This study quantitatively details the pervasive burden that pediatric OCD places on families, as reported from complementary relative perspectives. Further attention to this topic is warranted in clinical and research realms.

Development and psychometric properties of the OCD Family Functioning (OFF) Scale.

Obsessive-compulsive disorder (OCD) influences not only patients but also family members. Although the construct of family accommodation has received attention in OCD literature, no measures of overall family functioning are currently available. The OCD Family Functioning (OFF) Scale was developed to explore the context, extent, and perspectives of functional impairment in families affected by OCD. It is a three-part, self-report measure capturing independent perspectives of patients and relatives. A total of 400 subjects were enrolled between 2008 and 2010 from specialized OCD clinics and OCD research studies. Psychometric properties of this scale were examined including internal consistency, test-retest reliability, convergent and divergent validity, and exploratory factor analyses. Both patient and relative versions of the OFF Scale demonstrated excellent internal consistency (Cronbach's alpha coefficient = 0.96). The test-retest reliability was also adequate (ICC = 0.80). Factor analyses determined that the OFF Scale comprises a family functioning impairment factor and four OCD symptom factors that were consistent with previously reported OCD symptom dimension studies. The OFF Scale demonstrated excellent convergent validity with the Family Accommodation Scale and the Work and Social Adjustment Scale. Information gathered regarding emotional impact and family role-specific impairment was novel and not captured by other examined scales. The OFF Scale is a reliable and valid instrument for the clinical and research assessment of family functioning in pediatric and adult OCD. This will facilitate the exploration of family functioning impairment as a potential risk factor, as a moderator and as a treatment outcome measure in OCD.