CDK2/cyclinA inhibitors: targeting the cyclinA recruitment site with small molecules derived from peptide leads

The syntheses of potent small molecule inhibitors of the CDK2/cyclinA recruitment site are described. Structure-activity trends of nanomolar octapeptides were examined through amino-acid substitution and truncation of the sequence resulting in the identification of a smaller, albeit significantly less potent, tetrapeptide lead. These losses in affinity were recovered by side-chain optimization and by rigidification of the peptide backbone using a combination of solid-phase parallel synthesis and structure-based design. Finally, two guanidine functionalities were replaced to improve drug-like properties, resulting in neutral small molecules equal in activity to that of the peptide lead.     

Genetic typing, based on the 56-kilodalton type-specific antigen gene, of Orientia tsutsugamushi strains isolated from chiggers collected from wild-caught rodents in Taiwan

Orientia tsutsugamushi is the etiological agent of scrub typhus, a mite-borne, febrile illness that occurs in the Asia-Pacific region. We conducted strain characterization of O. tsutsugamushi isolates from chiggers obtained from rodents based the nucleotide sequence of the 56-kDa outer membrane protein gene. With the use of PCR, a total of 68 DNA sequences of 56-kDa antigen genes were amplified. Phylogenetic analysis revealed that there were at least six definable clusters among the 68 isolates: 37% Karp-related strains (25/68), 27% TA763 strains (18/68), 12% JG-related strains (8/68), 19% Kato-related strains (13/68), 4% divergent strains (3/68), and 1% representing a Gilliam prototype strain (1/68). Overall, the O. tsutsugamushi genotypes exhibited a high degree of diversity, similar to that seen in strains from the rest of the areas where scrub typhus is endemic. Moreover, the 56-kDa protein sequence similarity between O. tsutsugamushi isolates from mites and those from human patients (H. Y. Lu et al., Am. J. Trop. Med. Hyg. 83:658-663, 2010) were striking, thus highlighting potential risk factors for this emerging zoonotic disease.     

The SWI/SNF complex acts to constrain distribution of the centromeric histone variant Cse4

In order to gain insight into the function of the Saccharomyces cerevisiae SWI/SNF complex, we have identified DNA sequences to which it is bound genomewide. One surprising observation is that the complex is enriched at the centromeres of each chromosome. Deletion of the gene encoding the Snf2 subunit of the complex was found to cause partial redistribution of the centromeric histone variant Cse4 to sites on chromosome arms. Cultures of snf2Δ yeast were found to progress through mitosis slowly. This was dependent on the mitotic checkpoint protein Mad2. In the absence of Mad2, defects in chromosome segregation were observed. In the absence of Snf2, chromatin organisation at centromeres is less distinct. In particular, hypersensitive sites flanking the Cse4 containing nucleosomes are less pronounced. Furthermore, SWI/SNF complex was found to be especially effective in the dissociation of Cse4 containing chromatin in vitro. This suggests a role for Snf2 in the maintenance of point centromeres involving the removal of Cse4 from ectopic sites.     

Divergent Sp1 protein levels may underlie differential expression of UDP-glucose dehydrogenase by fibroblasts: role in susceptibility to orbital Graves disease

UDP-glucose dehydrogenase (UGDH) catalyzes the formation of UDP-glucuronate. Glucuronate represents an integral component of the glycosaminoglycan, hyaluronan, which accumulates in orbital Graves disease. Here we report that orbital fibroblasts express higher levels of UGDH than do those from skin. This is a consequence of greater UGDH gene promoter activity and more abundant steady-state UGDH mRNA. Six Sp1 sites located in the proximal 550 bp of the UGDH gene promoter appear to determine basal promoter activity, as does a previously unrecognized 49-bp sequence spanning -1436 nucleotides (nt) and -1388 nt that negatively affects activity. Nuclear Sp1 protein is more abundant in orbital fibroblasts, and its binding to specific sites on DNA is greater than that in dermal fibroblasts. Mutating each of these Sp1 sites in a UGDH gene promoter fragment, extending from -1387 to +71 nt and fused to a luciferase reporter, results in divergent activities when transfected in orbital and dermal fibroblasts. Reducing Sp1 attenuated UGDH gene promoter activity, lowered steady-state UGDH mRNA levels, and reduced UGDH enzyme activity. Targeting Sp1 and UGDH with specific siRNAs also lowered hyaluronan synthase-1 (HAS-1) and HAS-2 levels and reduced hyaluronan accumulation in orbital fibroblasts. These findings suggest that orbital fibroblasts express high levels of UGDH in an anatomic-specific manner, apparently the result of greater constitutive Sp1. These high UGDH levels may underlie susceptibility of the orbit to localized overproduction of hyaluronan in Graves disease.     

Comparative genomics of methicillin-resistant Staphylococcus aureus ST239: distinct geographical variants in Beijing and Hong Kong

Background

The ST239 lineage is a globally disseminated, multiply drug-resistant hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA). We performed whole-genome sequencing of representative HA-MRSA isolates of the ST239 lineage from bacteremic patients in hospitals in Hong Kong (HK) and Beijing (BJ) and compared them with three published complete genomes of ST239, namely T0131, TW20 and JKD6008. Orthologous gene group (OGG) analyses of the Hong Kong and Beijing cluster strains were also undertaken.

Results

Homology analysis, based on highest-percentage nucleotide identity, indicated that HK isolates were closely related to TW20, whereas BJ isolates were more closely related to T0131 from Tianjin. Phylogenetic analysis, incorporating a total of 30 isolates from different continents, revealed that strains from HK clustered with TW20 into the ‘Asian clade’, whereas BJ isolates and T0131 clustered closely with strains of the ‘Turkish clade’ from Eastern Europe. HK isolates contained the typical φSPβ-like prophage with the SasX gene similar to TW20. In contrast, BJ isolates contained a unique 15 kb PT1028-like prophage but lacked φSPβ-like and φSA1 prophages. Besides distinct mobile genetic elements (MGE) in the two clusters, OGG analyses and whole-genome alignment of these clusters highlighted differences in genes located in the core genome, including the identification of single nucleotide deletions in several genes, resulting in frameshift mutations and the subsequent predicted truncation of encoded proteins involved in metabolism and antimicrobial resistance.

Conclusions

Comparative genomics, based on de novo assembly and deep sequencing of HK and BJ strains, revealed different origins of the ST239 lineage in northern and southern China and identified differences between the two clades at single nucleotide polymorphism (SNP), core gene and MGE levels. The results suggest that ST239 strains isolated in Hong Kong since the 1990s belong to the Asian clade, present mainly in southern Asia, whereas those that emerged in northern China were of a distinct origin, reflecting the complexity of dissemination and the dynamic evolution of this ST239 lineage.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-529) contains supplementary material, which is available to authorized users.     

Entropic Imaging of Cataract Lens: An In Vitro Study

Phacoemulsification is a common surgical method for treating advanced cataracts. Determining the optimal phacoemulsification energy depends on the hardness of the lens involved. Previous studies have shown that it is possible to evaluate lens hardness via ultrasound parametric imaging based on statistical models that require data to follow a specific distribution. To make the method more system-adaptive, nonmodel-based imaging approach may be necessary in the visualization of lens hardness. This study investigated the feasibility of applying an information theory derived parameter – Shannon entropy from ultrasound backscatter to quantify lens hardness. To determine the physical significance of entropy, we performed computer simulations to investigate the relationship between the signal-to-noise ratio (SNR) based on the Rayleigh distribution and Shannon entropy. Young''s modulus was measured in porcine lenses, in which cataracts had been artificially induced by the immersion in formalin solution in vitro. A 35-MHz ultrasound transducer was used to scan the cataract lenses for entropy imaging. The results showed that the entropy is 4.8 when the backscatter data form a Rayleigh distribution corresponding to an SNR of 1.91. The Young''s modulus of the lens increased from approximately 8 to 100 kPa when we increased the immersion time from 40 to 160 min (correlation coefficient r = 0.99). Furthermore, the results indicated that entropy imaging seemed to facilitate visualizing different degrees of lens hardening. The mean entropy value increased from 2.7 to 4.0 as the Young''s modulus increased from 8 to 100 kPa (r = 0.85), suggesting that entropy imaging may have greater potential than that of conventional statistical parametric imaging in determining the optimal energy to apply during phacoemulsification.     

Chloridzellen der Larven von Caenis diminuta WALKER (Ephemeroptera,Caenidae) bei unterschiedlicher Salinität

The larvae of Caenis diminuta WALKER (Ephemeroptera, Caenidae) have coniform chloride cells which are involved in osmoregulation. The number of chloride cells varies according to salinity conditions of the surrounding waters. The larvae from brackish water have less chloride cells than the larvae from fresh water.