Helical peptides with disordered regions for measles viruses provide new generalized insights into fusion inhibitors.

Despite effective vaccines, measles virus (MeV) outbreaks occur sporadically. Therefore, developing anti-MeV agents remains important for suppressing MeV infections. We previously designed peptide-based MeV fusion inhibitors, M1 and M2, that target MeV class I fusion protein (F protein). Here, we developed a novel fusion inhibitor, MEK35, that exerts potent activity against M1/M2-resistant MeV variants. Comparing MEK35 to M1 derivatives revealed that combining disordered and helical elements was essential for overcoming M1/M2 resistance. Moreover, we propose a three-step antiviral process for peptide-based fusion inhibitors: (i) disordered peptides interact with F protein; (ii) the peptides adopt a partial helical conformation and bind to F protein through hydrophobic interactions; and (iii) subsequent interactions involving the disordered region of the peptides afford a peptide-F protein with a high-affinity peptide-F protein interaction. An M1-resistant substitution blocks the second step. These results should aid the development of novel viral fusion inhibitors targeting class I F protein.

Establishing an accurate and sensitive in vitro drug screening system for human adenovirus infection with human corneal cells.

Epidemic keratoconjunctivitis (EKC) is a hazardous and highly contagious disease, with the potential to cause epidemic outbreaks in hospitals and other community settings. There are currently no approved drugs for human adenovirus (HAdV), the causative agent of EKC. To establish a novel drug screening system for ocular HAdV infections, we employed CRL11516, a non-cancerous but immortalized human corneal epithelial cell line. Brincidoforvir and 3'-deoxy-3'-fluorothymidine inhibit replication of HAdV species C type 1 (C1), C2, E4, and C6 to the same extent. This alternative assay system may allow for the evaluation of anti-HAdV activity and cell cytotoxicity of compounds within 2 days and without the need of the rabbit eye infection model.

Serine hydroxymethyltransferase as a potential target of antibacterial agents acting synergistically with one-carbon metabolism-related inhibitors.

Serine hydroxymethyltransferase (SHMT) produces 5,10-methylenetetrahydrofolate (CH2-THF) from tetrahydrofolate with serine to glycine conversion. SHMT is a potential drug target in parasites, viruses and cancer. (+)-SHIN-1 was developed as a human SHMT inhibitor for cancer therapy. However, the potential of SHMT as an antibacterial target is unknown. Here, we show that (+)-SHIN-1 bacteriostatically inhibits the growth of Enterococcus faecium at a 50% effective concentration of 10-11 M and synergistically enhances the antibacterial activities of several nucleoside analogues. Our results, including crystal structure analysis, indicate that (+)-SHIN-1 binds tightly to E. faecium SHMT (efmSHMT). Two variable loops in SHMT are crucial for inhibitor binding, and serine binding to efmSHMT enhances the affinity of (+)-SHIN-1 by stabilising the loop structure of efmSHMT. The findings highlight the potency of SHMT as an antibacterial target and the possibility of developing SHMT inhibitors for treating bacterial, viral and parasitic infections and cancer.

Chances of pregnancy after dropping out from infertility treatments: Evidence from a social survey in Japan.

PURPOSE: We examined a patient's chances of pregnancy after dropping out from infertility treatments, an issue that has been largely understudied. METHOD: Drawing from a nationwide Internet survey in Japan with 1930 respondents, we used data from 199 individuals (109 women and 90 men) who had undergone an infertility treatment. We estimated linear probability models to investigate the extent to which the probability of pregnancy was affected by dropping out after controlling for a couple's attributes. RESULTS: Among the 199 respondents who had experienced an infertility treatment, 91 (45.7% of the entire sample) became pregnant during the treatment, and 108 (54.3%) dropped out. Among these 108 dropouts, 66 (33.2%) eventually became pregnant. After controlling for a couple's attributes, treatment discontinuation reduced the probability of pregnancy by 31.6% (standard error: 5.0%). A relatively limited reduction in the chances of pregnancy was also observed after a patient dropped out of any of the three treatment stages (timed intercourse, intrauterine insemination, and in vitro fertilization). CONCLUSIONS: The results suggest that dropping out from infertility treatments does not preclude any chance of a future pregnancy. More follow-up attention should be provided to dropout patients.

Pyrimidine Analogues as a New Class of Gram-Positive Antibiotics, Mainly Targeting Thymineless-Death Related Proteins.

Multidrug-resistant (MDR) bacteria are widespread throughout the world and pose an increasingly serious threat to human and animal health. Besides implementing strict measures to prevent improper antibiotic use, it remains essential that novel antibiotics must be developed. These antibiotics need to exert their activity via mechanisms different from those employed by currently approved antibiotics. In this study, we used several 5-fluorouracil (5-FU) analogues as chemical probes and investigated the potential of these pyrimidine analogues as antibacterial agents. Several 5-FU derivatives exerted potent activity against strains of Gram-positive cocci (GPC) that are susceptible or resistant toward approved antibiotics, without showing cross-resistance. Furthermore, we have provided evidence that the pyrimidine analogues exerted anti-GPC activity via thymineless death by inhibition of thymidylate synthetase (ThyA) and/or inhibition of RNA synthesis. Interestingly, whole genome resequencing of in vitro-selected, pyrimidine analogue-resistant Staphylococcus aureus mutants indicated that S. aureus strains with pyrimidine-analogue resistance induced an amino acid (AA) substitution, deletion, and/or insertion into thymineless-death related proteins except for ThyA, or enhanced the ThyA transcription level. Thus, S. aureus may avoid altering the ThyA function by introducing an AA substitution, suggesting that the pyrimidine analogues, which directly bind to ThyA without phosphorylation, may be more effective and show a higher genetic barrier than the pyrimidines that depend on phosphorylation for activity. The findings of this study may assist in the future development of a novel class of antibiotics for combating MDR GPC, including methicillin-resistant S. aureus and vancomycin-resistant Enterococci.

1α,25(OH)2D3 inhibits FGF-2 release from oral squamous cell carcinoma cells through down-regulation of HBp17/FGFBP-1.

Heparin-binding protein 17/fibroblast growth factor binding protein-1 (HBp17/FGFBP-1, GenBank accession no. NP-005121) is prominent for its role as the chaperone for fibroblast growth factor-2 (FGF-2), which plays a crucial role in angiogenesis as well as promoting tumor growth. HBp17/FGFBP-1 has been proposed as a candidate biomarker for a number of cancers since it is frequently found to be elevated in many cancer types including in the tissue and cell lines of oral squamous cell carcinomas (OSCC). Previously, we reported that 1α,25(OH)2D3 suppressed the HBp17/FGFBP-1 expression in OSCC by inhibiting nuclear factor-kappaB (NF-κB) expression via vitamin D3 receptor (VDR). In this paper, to further characterize the inhibitory effect of 1α,25(OH)2D3 on HBp17/FGFBP-1, we examined the cellular localization of HBp17/FGFBP-1 protein and FGF-2 protein in the UE OSCC cell line. We found that the treatment of OSCC cells with 40-nM 1α,25(OH)2D3 suppressed HBp17/FGFBP-1 expression both in the nucleus and cytosol and reduced FGF-2 release into the culture medium. The expression of HBp17/FGFBP-1 and FGF-2 was analyzed by immunofluorescence and enzyme-linked immunosorbent assay (ELISA). In summary, the ability of 1α,25(OH)2D3 to suppress the expression of HBp17/FGFBP-1 and FGF-2 strongly suggests a therapeutic potential as a molecular-targeted anticancer drug for FGF-dependent cancers.

Liver X receptors (LXRalpha and LXRbeta) are potent regulators for hepatic Dec1 expression.

DEC1 (BHLHB2/Stra13/Sharp2)-a basic helix-loop-helix transcription factor-is known to be involved in various biological phenomena including clock systems and metabolism. In the clock systems, Dec1 expression is dominantly up-regulated by CLOCK : BMAL1 heterodimer, and it exhibits circadian rhythm in the suprachiasmatic nucleus (SCN)-the central circadian pacemaker-and other peripheral tissues. Recent studies have shown that the strong circadian rhythmicity of Dec1 in the SCN was abolished by Clock mutation, whereas that in the liver was affected, but not abolished, by Clock mutation. Moreover, feeding conditions affected hepatic Dec1 expression, which indicates that Dec1 expression is closely linked with the metabolic functions of the liver. Among ligand-activated nuclear receptors examined, LXRalpha and LXRbeta with T0901317-agonist for LXR-were found to be potent enhancers for Dec1 promoter activity, and a higher expression level of LXRalpha protein was detected in the liver than in the kidney and heart. T0901317 increased the levels of endogenous Dec1 transcript in hepatoma cells. Chromatin immunoprecipitation assay indicated that LXRalpha bound to the Dec1 promoter, and an LXRalpha-binding site was identified. These observations indicate that hepatic DEC1 mediates the ligand-dependent LXR signal to regulate the expression of genes involved in the hepatic clock system and metabolism.

Multiple mechanisms regulate circadian expression of the gene for cholesterol 7alpha-hydroxylase (Cyp7a), a key enzyme in hepatic bile acid biosynthesis.

Cholesterol 7alpha-hydroxylase (CYP7A) and sterol 12alpha-hydroxylase (CYP8B) in bile acid biosynthesis and 3-hydroxyl-3-methylglutaryl CoA reductase (HMGCR) in cholesterol biosynthesis are the key enzymes in hepatic metabolic pathways, and their transcripts exhibit circadian expression profiles in rodent liver. The authors determined transcript levels of these enzymes and the regulatory factors for Cyp7a--including Dbp, Dec2, E4bp4, Hnf4alpha, Pparalpha, Lxralpha, Rev-erbalpha, and Rev-erbbeta--in the liver of wild-type and homozygous Clock mutant mice (Clock/Clock) and examined the effects of these transcription factors on the transcription activities of Cyp7a. The expression profile of the Cyp7a transcript in wild-type mice showed a strong circadian rhythm in both the 12L:12D light-dark cycle and constant darkness, and that in Clock/Clock also exhibited a circadian rhythm at an enhanced level with a lower amplitude, although its protein level became arrhythmic at a high level. The expression profile of Cyp8b mRNA in wild-type mice showed a shifted circadian rhythm from that of Cyp7a, becoming arrhythmic in Clock/Clock at an expression level comparable to that of wild-type mice. The expression profile of Hmgcr mRNA also lost its strong circadian rhythm in Clock/Clock , showing an expression level comparable to that of wild-type mice. The expressions of Dbp, Dec2, Rev-erbalpha, and Rev-erb beta--potent regulators for Cyp7a expression--were abolished or became arrhythmic in Clock/Clock, while other regulators for Cyp7a-Lxralpha, Hnf4alpha, Pparalpha, and E4bp4--had either less affected or enhanced expression in Clock/Clock. In luciferase reporter assays, REV-ERBalpha/beta, DBP, LXRalpha, and HNF4alpha increased the promoter activity of Cyp7a, whereas DEC2 abolished the transcription from the Cyp7a promoter: E4BP4 and PPARalpha were moderate negative regulators. Furthermore, knockdown of REV-ERBalpha/beta with siRNA suppressed Cyp7a transcript levels, and in the electrophoretic mobility shift assay, REV-ERBalpha/beta bound to the promoter of Cyp7a . These observations suggest that (1) active CLOCK is essential for the robust circadian expression of hepatic metabolic enzymes (Cyp7a, Cyp8b, and Hmgcr); (2) clock-controlled genes--DBP, DEC2, and REV-ERBalpha/beta--are direct regulators required for the robust circadian rhythm of Cyp7a; and (3) the circadian rhythm of Cyp7a is regulated by multiple transcription factors, including DBP, REV-ERBalpha/beta, LXRalpha, HNF4alpha DEC2, E4BP4, and PPARalpha.

Effects of fasting and re-feeding on the expression of Dec1, Per1, and other clock-related genes.

To elucidate the food-entrainable oscillatory mechanism of peripheral clock systems, we examined the effect of fasting on circadian expression of clock genes including Dec1 and Dec2 in mice. Withholding of food for 2 days had these effects: the expression level of Dec1 mRNA decreased in all tissues examined, although Per1 mRNA level markedly increased; Per2 expression was reduced in the liver and heart only 42-46 h after the start of fasting; and expression profiles of Dec2 and Bmal1 were altered only in the heart and in the liver, respectively, whereas Rev-erbalpha mRNA levels did not change significantly. Re-feeding after 36-h starvation erased, at least in part, the effect of fasting on Dec1, Dec2, Per1, Per2, and Bmal1 within several hours, and restriction feeding shifted the phase of expression profiles of all examined clock genes including Dec1 and Dec2. These findings indicate that short-term fasting and re-feeding modulate the circadian rhythms of clock genes to different extents in peripheral tissues, and suggest that the expression of Dec1, Per1, and some other clock genes was closely linked with the metabolic activity of these tissues.