Design, synthesis, and studies of small molecule STAT3 inhibitors

A series of small molecule STAT3 inhibitors originally derived from our lead compound STA 21 were synthesized and evaluated. The most potent compound in this series, compound 1, exhibited the same anti-proliferative activities as STA 21 against prostate cancer cell lines that express constitutively active STAT3. Molecular docking showed compound 1 bound to the STAT3beta SH2 domain in a similar manner as STA 21.     

Structure–activity relationship studies of curcumin analogues

Two series of curcumin analogues, a total of twenty-four compounds, were synthesized and evaluated. The most potent compound, compound 23, showed potent growth inhibitory activities on both prostate and breast cancer lines with IC₅₀ values in sub-micromolar range, fifty times more potent than curcumin. Curcumin analogues might be potential anti-tumor agents for breast and prostate cancers.     

Redesign of a protein–peptide interaction: Characterization and applications

The design of protein–peptide interactions has a wide array of practical applications and also reveals insight into the basis for molecular recognition. Here, we present the redesign of a tetratricopeptide repeat (TPR) protein scaffold, along with its corresponding peptide ligand. We show that the binding properties of these protein–peptide pairs can be understood, quantitatively, using straightforward chemical considerations. The recognition pairs we have developed are also practically useful for the specific identification of tagged proteins. We demonstrate the facile replacement of these proteins, which we have termed T‐Mods (TPR‐based recognition module), for antibodies in both detection and purification applications. The new protein–peptide pair has a dissociation constant that is weaker than typical antibody–antigen interactions, yet the recognition pair is highly specific and we have shown that this affinity is sufficient for both Western blotting and affinity purification. Moreover, we demonstrate that this more moderate affinity is actually advantageous for purification applications, because extremely harsh conditions are not required to dissociate the T‐Mod‐peptide interaction. The results we present are important, not only because they represent a successful application of protein design but also because they help define the properties that should be sought in other scaffolds that are being developed as antibody replacements.     

Simultaneous Cellulose Degradation and Electricity Production by Enterobacter cloacae in a Microbial Fuel Cell

Electricity can be directly generated by bacteria in microbial fuel cells (MFCs) from many different biodegradable substrates. When cellulose is used as the substrate, electricity generation requires a microbial community with both cellulolytic and exoelectrogenic activities. Cellulose degradation with electricity production by a pure culture has not been previously demonstrated without addition of an exogenous mediator. Using a specially designed U-tube MFC, we enriched a consortium of exoelectrogenic bacteria capable of using cellulose as the sole electron donor. After 19 dilution-to-extinction serial transfers of the consortium, 16S rRNA gene-based community analysis using denaturing gradient gel electrophoresis and band sequencing revealed that the dominant bacterium was Enterobacter cloacae. An isolate designated E. cloacae FR from the enrichment was found to be 100% identical to E. cloacae ATCC 13047^T based on a partial 16S rRNA sequence. In polarization tests using the U-tube MFC and cellulose as a substrate, strain FR produced 4.9 ± 0.01 mW/m², compared to 5.4 ± 0.3 mW/m² for strain ATCC 13047^T. These results demonstrate for the first time that it is possible to generate electricity from cellulose using a single bacterial strain without exogenous mediators.Exoelectrogenic microorganisms can release electrons to electron acceptors outside the cell, such as iron oxides or carbon anodes in microbial fuel cells (MFCs). Members of many genera, including Rhodoferax (6), Shewanella (13, 14), Pseudomonas (29), Aeromonas (28), Geobacter (2), Geopsychrobacter (10), Desulfuromonas (1), Desulfobulbus (9), Clostridium (27), Geothrix (3), Ochrobactrum (40), and Rhodopseudomonas (38), have been shown to produce electricity in an MFC. These bacteria have been grown on simple soluble substrates, such as glucose or acetate, that can be directly taken into the cell and used for energy production.Cellulose is the most abundant biopolymer in the world, and there is great interest in using this material as a substrate in an MFC. However, use of a particulate substrate in an MFC has not been well investigated. Cellulose must first be hydrolyzed to a soluble substrate that can be taken up by the cell. In previous MFC tests this has required the use of enzymes to hydrolyze the cellulose into sugars or the use of cocultures or mixed cultures (32, 33, 35). For example, Ren et al. (32) used a coculture of the cellulose fermentor Clostridium cellulolyticum and the exoelectrogen Geobacter sulfurreducens to generate electricity in an MFC fed with cellulose. Analysis of the anode microbial communities in other studies of cellulose-fed MFCs showed that Clostridium spp. (in a biofilm) and Comamonadaceae (in suspension) were predominant when rumen contents were used as an inoculum (35), while a rice paddy soil inoculum (12) converged to a Rhizobiales-dominated anode community (more than 30% of the population). To date, it has not been demonstrated that a single microbe can both degrade cellulose and generate current.Conventional methods of isolating exoelectrogenic microorganisms are based primarily on identifying microorganisms that can respire using soluble or insoluble metal oxides in agar plates (20-22). However, not all dissimilatory metal oxide-reducing bacteria are capable of producing electricity in an MFC, and not all bacteria that produce current in an MFC can grow using metal oxides (5, 34). Therefore, these methods may miss important electrochemically active strains of microorganisms. A new method to isolate exoelectrogenic microorganisms was recently developed (40); this method is based on dilution to extinction and a specially designed U-tube MFC that enriches exoelectrogenic bacteria on the anode. Using this method, a bacterium that could produce electricity in an MFC but not respire using iron was isolated (40).The main objective of this study was to isolate a bacterium capable of producing current from particulate cellulose. A cellulose-degrading consortium was diluted and serially transferred into U-tube MFCs using cellulose as the sole electron donor. Community analysis demonstrated the predominance of a single bacterium, which was isolated and compared to a culture collection strain for generation of current in an MFC.     

Carnivora from the Plio-Pleistocene hominin site of Drimolen, Gauteng, South Africa

Drimolen is one of the newest and most productive hominin sites in South Africa, and is dated on faunal grounds between 2.0 Ma to 1.5 Ma. This paper provides the first overview of the Carnivora from Drimolen, updating the previously published preliminary faunal list, and describing all currently prepared craniodental and postcranial material. The Drimolen specimens are described in comparison with other modern and fossil South African carnivore material. The carnivores cover a range of taxa including hyaenids, felids, canids and herpestids. Most notable amongst these are the sabretooth Dinofelis aff. piveteaui craniodental and postcranial remains, which are described in detail, and a Chasmaporthetes nitidula cranium. The genus Chasmaporthetes is found at three other sites in the area - Sterkfontein, Swartkrans and Coopers D. There are two models for the geographic origin of Dinofelis piveteaui, in that it may have arisen in either eastern or southern Africa. These possibilities are discussed in the light of the new South African Dinofelis material, as the Drimolen material appears to represent a more primitive form with affinities with D. piveteaui. Fossil leopard material from Kromdraai B and Drimolen is also discussed, as the metapodia assigned to P. pardus from these two sites are very small, but lie within the variation of modern leopards. Such size differences in fossil postcrania may have implications for the niches that these animals may have occupied in the past.     

Confirmatory analysis of firocoxib in bovine milk by rapid resolution liquid chromatography tandem mass spectrometry

A rapid method has been developed to analyse for firocoxib (FIRO) residue in bovine milk. Milk samples were extracted with acetonitrile and sample extracts were purified on Evolute? ABN solid phase extraction cartridges. Aliquots were analysed by rapid resolution liquid chromatography tandem mass spectrometry (RRLC–MS/MS). The method was validated in bovine milk, according to the criteria defined in Commission Decision 2002/657/EC. The decision limit (CCα) was 1.18 ng/mL and for the detection capability a (CCβ) value of 2.02 ng/mL was obtained. The measurement uncertainty of the method was 27%. Fortifying bovine milk samples (n = 18) in three separate assays, show the accuracy of the method to be between 96 and 105%. The precision of the method, expressed as RSD values for the within-lab reproducibility at the three levels of fortification (5, 7.5 and 10 ng/mL) was less than 11% respectively.     

Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells

Background

Highly pathogenic avian influenza (HPAI) H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease.

Aim

To study influenza A (H5N1) virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease.

Methods

We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces.

Results

We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our data suggests that viremia, secondary to, for example, gastro-intestinal infection, can potentially lead to infection of the lung. HPAI H5N1 virus was a more potent inducer of cytokines (e.g. IP-10, RANTES, IL-6) in comparison to H1N1 virus in alveolar epithelial cells, and these virus-induced chemokines were secreted onto both the apical and basolateral aspects of the polarized alveolar epithelium.

Conclusion

The predilection of viruses for different routes of entry and egress from the infected cell is important in understanding the pathogenesis of influenza H5N1 infection and may help unravel the pathogenesis of human H5N1 disease.     

Natural Selection of Human Embryos: Decidualizing Endometrial Stromal Cells Serve as Sensors of Embryo Quality upon Implantation

Background

Pregnancy is widely viewed as dependent upon an intimate dialogue, mediated by locally secreted factors between a developmentally competent embryo and a receptive endometrium. Reproductive success in humans is however limited, largely because of the high prevalence of chromosomally abnormal preimplantation embryos. Moreover, the transient period of endometrial receptivity in humans uniquely coincides with differentiation of endometrial stromal cells (ESCs) into highly specialized decidual cells, which in the absence of pregnancy invariably triggers menstruation. The role of cyclic decidualization of the endometrium in the implantation process and the nature of the decidual cytokines and growth factors that mediate the crosstalk with the embryo are unknown.

Methodology/Principal Findings

We employed a human co-culture model, consisting of decidualizing ESCs and single hatched blastocysts, to identify the soluble factors involved in implantation. Over the 3-day co-culture period, approximately 75% of embryos arrested whereas the remainder showed normal development. The levels of 14 implantation factors secreted by the stromal cells were determined by multiplex immunoassay. Surprisingly, the presence of a developing embryo had no significant effect on decidual secretions, apart from a modest reduction in IL-5 levels. In contrast, arresting embryos triggered a strong response, characterized by selective inhibition of IL-1β, -6, -10, -17, -18, eotaxin, and HB-EGF secretion. Co-cultures were repeated with undifferentiated ESCs but none of the secreted cytokines were affected by the presence of a developing or arresting embryo.

Conclusions

Human ESCs become biosensors of embryo quality upon differentiation into decidual cells. In view of the high incidence of gross chromosomal errors in human preimplantation embryos, cyclic decidualization followed by menstrual shedding may represent a mechanism of natural embryo selection that limits maternal investment in developmentally impaired pregnancies.     

Natural Selection of Human Embryos: Impaired Decidualization of Endometrium Disables Embryo-Maternal Interactions and Causes Recurrent Pregnancy Loss

Background

Recurrent pregnancy loss (RPL), defined as 3 or more consecutive miscarriages, is widely attributed either to repeated chromosomal instability in the conceptus or to uterine factors that are poorly defined. We tested the hypothesis that abnormal cyclic differentiation of endometrial stromal cells (ESCs) into specialized decidual cells predisposes to RPL, based on the observation that this process may not only be indispensable for placenta formation in pregnancy but also for embryo recognition and selection at time of implantation.

Methodology/Principal Findings

Analysis of mid-secretory endometrial biopsies demonstrated that RPL is associated with decreased expression of the decidual marker prolactin (PRL) but increased levels of prokineticin-1 (PROK1), a cytokine that promotes implantation. These in vivo findings were entirely recapitulated when ESCs were purified from patients with and without a history of RPL and decidualized in culture. In addition to attenuated PRL production and prolonged and enhanced PROK1 expression, RPL was further associated with a complete dysregulation of both markers upon treatment of ESC cultures with human chorionic gonadotropin, a glycoprotein hormone abundantly expressed by the implanting embryo. We postulated that impaired embryo recognition and selection would clinically be associated with increased fecundity, defined by short time-to-pregnancy (TTP) intervals. Woman-based analysis of the mean and mode TTP in a cohort of 560 RPL patients showed that 40% can be considered “superfertile”, defined by a mean TTP of 3 months or less.

Conclusions

Impaired cyclic decidualization of the endometrium facilitates implantation yet predisposes to subsequent pregnancy failure by disabling natural embryo selection and by disrupting the maternal responses to embryonic signals. These findings suggest a novel pathological pathway that unifies maternal and embryonic causes of RPL.     

Accounting for female space sharing in St. Kilda Soay sheep (Ovis aries) results in little change in heritability estimates

When estimating heritability in free‐living populations, it is common practice to account for common environment effects, because of their potential to generate phenotypic covariance among relatives thereby biasing heritability estimates. In quantitative genetic studies of natural populations, however, philopatry, which results in relatives being clustered in space, is rarely accounted for. The two studies that have been carried out so far suggest absolute declines in heritability estimates of up to 43% when accounting for space sharing by relatives. However, due to methodological limitations these estimates may not be representative. We used data from the St. Kilda Soay sheep population to estimate heritabilities with and without accounting for space sharing for five traits for which there is evidence for additive genetic variance (birthweight, birth date, lamb August weight, and female post‐mortem jaw and metacarpal length). We accounted for space sharing by related females by separately incorporating spatial autocorrelation, and a home range similarity matrix. Although these terms accounted for up to 18% of the variance in these traits, heritability estimates were only reduced by up to 7%. Our results suggest that the bias caused by not accounting for space sharing may be lower than previously thought. This suggests that philopatry does not inevitably lead to a large bias if space sharing by relatives is not accounted for. We hope our work stimulates researchers to model shared space when relatives in their study population share space, as doing so will enable us to better understand when bias may be of particular concern.