Synthesis and in vitro anti-HSV-1 activity of a novel Hsp90 inhibitor BJ-B11

In this study, a novel Hsp90 inhibitor BJ-B11, was synthesized and evaluated for in vitro antiviral activity against several viruses. Possible anti-HSV-1 mechanisms were also investigated. BJ-B11 displayed no antiviral activity against coxsackievirus B₃ (CVB₃), human respiratory syncytial virus (RSV) and influenza virus (H1N1), but exhibited potent anti-HSV-1 and HSV-2 activity with EC₅₀ values of 0.42 ± 0.18 μM and 0.60 ± 0.21 μM, respectively. Additionally, the inhibitory effects of BJ-B11 against HSV-1 were likely to be introduced at early stage of infection. Our results indicate that BJ-B11 with alternative mechanisms of action is potent as an anti-HSV clinical trial candidate.     

Labeling and Localization of the Herpes Simplex Virus Capsid Protein UL25 and Its Interaction with the Two Triplexes Closest to the Penton

The herpes simplex virus type 1 UL25 protein is one of seven viral proteins that are required for DNA cleavage and packaging. Together with UL17, UL25 forms part of an elongated molecule referred to as the C-capsid-specific component (CCSC). Five copies of the CCSC are located at each of the capsid vertices on DNA-containing capsids. To study the conformation of UL25 as it is folded on the capsid surface, we identified the sequence recognized by a UL25-specific monoclonal antibody and localized the epitope on the capsid surface by immunogold electron microscopy. The epitope mapped to amino acids 99-111 adjacent to the region of the protein (amino acids 1-50) that is required for capsid binding. In addition, cryo-EM reconstructions of C-capsids in which the green fluorescent protein (GFP) was fused within the N-terminus of UL25 localized the point of contact between UL25 and GFP. The result confirmed the modeled location of the UL25 protein in the CCSC density as the region that is distal to the penton with the N-terminus of UL25 making contact with the triplex one removed from the penton. Immunofluorescence experiments at early times during infection demonstrated that UL25-GFP was present on capsids located within the cytoplasm and adjacent to the nucleus. These results support the view that UL25 is present on incoming capsids with the capsid-binding domain of UL25 located on the surface of the mature DNA-containing capsid.     

Exacting predictions by cybernetic model confirmed experimentally: Steady state multiplicity in the chemostat

We demonstrate strong experimental support for the cybernetic model based on maximizing carbon uptake rate in describing the microorganism's regulatory behavior by verifying exacting predictions of steady state multiplicity in a chemostat. Experiments with a feed mixture of glucose and pyruvate show multiple steady state behavior as predicted by the cybernetic model. When multiplicity occurs at a dilution (growth) rate, it results in hysteretic behavior following switches in dilution rate from above and below. This phenomenon is caused by transient paths leading to different steady states through dynamic maximization of the carbon uptake rate. Thus steady state multiplicity is a manifestation of the nonlinearity arising from cybernetic mechanisms rather than of the nonlinear kinetics. The predicted metabolic multiplicity would extend to intracellular states such as enzyme levels and fluxes to be verified in future experiments. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Dark recovery of UV-irradiated phage T1 I. A minor recovery effect whose exclusion permits the study of survival kinetics under presumably repairless conditions

The survival of UV-irradiated phage T1 is much lower in excision repair-deficient than in excision repair-proficient E. coli cells, due to lack of “host cell reactivation” (HCR). An additional decrease in phage survival occurs when repair-deficient (HCR⁻) host cells have been exposed to UV doses from 3000–10 000 erg mm⁻² of 254 nm UV-radiation prior to infection. The observed effect is attributed to loss of a minor phage recovery process, which requires neither the bacterial excision repair nor the bacterial REC repair system. This type of recovery is little affected by caffeine or acriflavine at concentrations that preclude HCR completely. Its full inhibition by UV-irradiation of the cells requires an approximately 8 times larger dose than complete inhibition of HCR.

In heavily preirradiated cells, the T1 burst size is extremely small and multiplicity reactivation is considerably less extensive than in unirradiated cells. Presumably the survival of singly infecting T1 in these cells reflects absence of any type of repair. The observed phage sensitivity and shape of the curve are compatible with the expectation for completely repairless conditions. The mechanism underlying the minor recovery is not known; theoretical considerations make a phage REC repair mechanism seem likely.     

Bayesian latent variable models for mixed discrete outcomes

In studies of complex health conditions, mixtures of discrete outcomes (event time, count, binary, ordered categorical) are commonly collected. For example, studies of skin tumorigenesis record latency time prior to the first tumor, increases in the number of tumors at each week, and the occurrence of internal tumors at the time of death. Motivated by this application, we propose a general underlying Poisson variable framework for mixed discrete outcomes, accommodating dependency through an additive gamma frailty model for the Poisson means. The model has log-linear, complementary log-log, and proportional hazards forms for count, binary and discrete event time outcomes, respectively. Simple closed form expressions can be derived for the marginal expectations, variances, and correlations. Following a Bayesian approach to inference, conditionally-conjugate prior distributions are chosen that facilitate posterior computation via an MCMC algorithm. The methods are illustrated using data from a Tg.AC mouse bioassay study.     

粘质沙雷菌裂解性噬菌体ФSM9-3Y的分离和鉴定

目的从医院污水中分离粘质沙雷菌噬菌体,并分析其生物学特性,为进一步研究针对耐药性粘质沙雷菌的噬菌体制剂提供依据。方法采用双层琼脂平板法分离纯化针对粘质沙雷菌的裂解性噬菌体,观察噬菌体对宿主菌的裂解特异性,通过负染法电镜观察噬菌体的形态结构,提取噬菌体核酸进行酶切电泳,测定噬菌体的最佳感染复数和一步生长曲线,SDS-PAGE电泳初步分析噬菌体的结构蛋白和非结构蛋白。结果从医院污水分离出7株可裂解粘质沙雷菌的噬菌体,对其中一株噬菌体(命名为ФSM9-3Y)的生物学特征进行了初步研究。电镜显示噬菌体呈蝌蚪状,头部为20面体立体对称、直径约70 nm;尾部长约50 nm。ФSM9-3Y的最佳感染复数为1。一步生长曲线表明;ФSM9-3Y的潜伏期约30 min,暴发时间70 min,暴发量为629 PFU/cell。凝胶电泳显示噬菌体基因组为双链DNA、大小约54 kb。SDS-PAGE呈现至少包括13种蛋白,相对分子质量范围在25～130 kD,其中主要蛋白的相对分子质量约为48 kD。结论此次分离的噬菌体ФSM9-3Y为裂解性噬菌体,根据形态和结构特征,粘质沙雷菌噬菌体ФSM9-3Y属于有尾病毒目,肌尾噬菌体科。     

Molecular cloning of cDNA from foot-and-mouth disease virus C1-Santa Pau (C-S8). Sequence of protein-VP1-coding segment

cDNA segments copied from the RNA of foot-and-mouth disease virus (FMDV) C₁-Santa Pau (isolate C-S8) have been cloned in plasmid pBR322. A 998-bp DNA fragment, that includes the region coding for capsid protein VP1, the carboxy terminus of VP3, and the amino terminus of precursor protein p52 has been sequenced. Comparison of the nucleotide sequence with those from FMDV O₁K, A₁₀61, a₁₂ and C₃ Indaial (Kurz et al., Nucl. Acids Res. 9 (1981) 1919–1931; Kleid et al., Science 214 (1981) 1125–1129; Boothroyd et al., Gene 17 (1982) 153–161; Makoff et al., Nucl. Acids Res. 10 (1982) 8285–8295) indicates extensive variability between the corresponding gene segments, including short insertions and deletions. Base transversions are more frequent than transitions within the VP1 coding segment, but not in the sequence coding for the amino-terminal end of p52. The nucleotide sequence divergence is reflected in variability in both the primary and the predicted higher-order structures of the encoded VP1s.     

Effects of p-fluorophenylalanine on the induction of mutations in bacteriophage T4: I. 5-Bromouracil mutagenesis

The amino acid analogue p-fluorophenylalanine (PFPA) was found to have no mutagenic activity in the r system of bacteriophage T4. However, under standard conditions for 5-bromouracil (5-BU) mutagenesis, PFPA depressed the induced frequencies for both forward and reverse mutations. When the folate antagonist sulphanilamide (SU) was omitted from the mutagenic treatment medium or when it was replaced by Trimethoprim (TM), another folate antagonist, this depressive effect was abolished. It was proposed that PFPA alleviated the inhibitory action of SU.