Molecular basis for lysine specificity in the yeast ubiquitin-conjugating enzyme Cdc34

Ubiquitin (Ub)-conjugating enzymes (E2s) and ubiquitin ligases (E3s) catalyze the attachment of Ub to lysine residues in substrates and Ub during monoubiquitination and polyubiquitination. Lysine selection is important for the generation of diverse substrate-Ub structures, which provides versatility to this pathway in the targeting of proteins to different fates. The mechanisms of lysine selection remain poorly understood, with previous studies suggesting that the ubiquitination site(s) is selected by the E2/E3-mediated positioning of a lysine(s) toward the E2/E3 active site. By studying the polyubiquitination of Sic1 by the E2 protein Cdc34 and the RING E3 Skp1/Cul1/F-box (SCF) protein, we now demonstrate that in addition to E2/E3-mediated positioning, proximal amino acids surrounding the lysine residues in Sic1 and Ub are critical for ubiquitination. This mechanism is linked to key residues composing the catalytic core of Cdc34 and independent of SCF. Changes to these core residues altered the lysine preference of Cdc34 and specified whether this enzyme monoubiquitinated or polyubiquitinated Sic1. These new findings indicate that compatibility between amino acids surrounding acceptor lysine residues and key amino acids in the catalytic core of ubiquitin-conjugating enzymes is an important mechanism for lysine selection during ubiquitination.

Computational model of membrane fission catalyzed by ESCRT-III

ESCRT-III proteins catalyze membrane fission during multi vesicular body biogenesis, budding of some enveloped viruses and cell division. We suggest and analyze a novel mechanism of membrane fission by the mammalian ESCRT-III subunits CHMP2 and CHMP3. We propose that the CHMP2-CHMP3 complexes self-assemble into hemi-spherical dome-like structures within the necks of the initial membrane buds generated by CHMP4 filaments. The dome formation is accompanied by the membrane attachment to the dome surface, which drives narrowing of the membrane neck and accumulation of the elastic stresses leading, ultimately, to the neck fission. Based on the bending elastic model of lipid bilayers, we determine the degree of the membrane attachment to the dome enabling the neck fission and compute the required values of the protein-membrane binding energy. We estimate the feasible values of this energy and predict a high efficiency for the CHMP2-CHMP3 complexes in mediating membrane fission. We support the computational model by electron tomography imaging of CHMP2-CHMP3 assemblies in vitro. We predict a high efficiency for the CHMP2-CHMP3 complexes in mediating membrane fission.

Design and modification of three-component randomly incorporated copolymers for high performance organic photovoltaic applications

In this study we report the molecular design, synthesis, characterization, and photovoltaic properties of a series of diketopyrrolopyrrole (DPP) and dithienothiophene (DTT) based donor-acceptor random copolymers. The six random copolymers are obtained via Stille coupling polymerization using various concentration ratios of donor to acceptor in the conjugated backbone. Bis(trimethylstannyl)thiophene was used as the bridge block to link randomly with the two comonomers 5-(bromothien-2-yl)-2,5-dialkylpyrrolo[3,4-c]pyrrole-1, 4-dione and 2,6-dibromo-3,5-dipentadecyl-dithieno[3,2-b;2′,3′-d] thiophene. The optical properties of these copolymers clearly reveal a change in the absorption band through optimization of the donor-acceptor ratio in the backbone. Additionally, the solution processability of the copolymers is modified through the attachment of different bulky alkyl chains to the lactam N-atoms of the DPP moiety. Applications of the polymers as light-harvesting and electron-donating materials in solar cells, in conjunction with PCBM as acceptor, show power conversion efficiencies (PCEs) of up to 5.02%.

F9 fimbriae of uropathogenic Escherichia coli are expressed at low temperature and recognise Galβ1-3GlcNAc-containing glycans

Uropathogenic Escherichia coli (UPEC) is the leading causative agent of urinary tract infections (UTI) in the developed world. Among the major virulence factors of UPEC, surface expressed adhesins mediate attachment and tissue tropism. UPEC strains typically possess a range of adhesins, with type 1 fimbriae and P fimbriae of the chaperone-usher class the best characterised. We previously identified and characterised F9 as a new chaperone-usher fimbrial type that mediates biofilm formation. However, the regulation and specific role of F9 fimbriae remained to be determined in the context of wild-type clinical UPEC strains. In this study we have assessed the distribution and genetic context of the f9 operon among diverse E. coli lineages and pathotypes and demonstrated that f9 genes are significantly more conserved in a UPEC strain collection in comparison to the well-defined E. coli reference (ECOR) collection. In the prototypic UPEC strain CFT073, the global regulator protein H-NS was identified as a transcriptional repressor of f9 gene expression at 37°C through its ability to bind directly to the f9 promoter region. F9 fimbriae expression was demonstrated at 20°C, representing the first evidence of functional F9 fimbriae expression by wild-type E. coli. Finally, glycan array analysis demonstrated that F9 fimbriae recognise and bind to terminal Galβ1-3GlcNAc structures.

Functional heterogeneity of the UpaH autotransporter protein from Uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) is responsible for the majority of urinary tract infections (UTI). To cause a UTI, UPEC must adhere to the epithelial cells of the urinary tract and overcome the shear flow forces of urine. This function is mediated primarily by fimbrial adhesins, which mediate specific attachment to host cell receptors. Another group of adhesins that contributes to UPEC-mediated UTI is autotransporter (AT) proteins. AT proteins possess a range of virulence properties, such as adherence, aggregation, invasion, and biofilm formation. One recently characterized AT protein of UPEC is UpaH, a large adhesin-involved-in-diffuse-adherence (AIDA-I)-type AT protein that contributes to biofilm formation and bladder colonization. In this study we characterized a series of naturally occurring variants of UpaH. We demonstrate that extensive sequence variation exists within the passenger-encoding domain of UpaH variants from different UPEC strains. This sequence variation is associated with functional heterogeneity with respect to the ability of UpaH to mediate biofilm formation. In contrast, all of the UpaH variants examined retained a conserved ability to mediate binding to extracellular matrix (ECM) proteins. Bioinformatic analysis of the UpaH passenger domain identified a conserved region (UpaHCR) and a hydrophobic region (UpaHHR). Deletion of these domains reduced biofilm formation but not the binding to ECM proteins. Despite variation in the upaH sequence, the transcription of upaH was repressed by a conserved mechanism involving the global regulator H-NS, and mutation of the hns gene relieved this repression. Overall, our findings shed new light on the regulation and functions of the UpaH AT protein.

Molecular characterization of UpaB and UpaC, two new autotransporter proteins of uropathogenic Escherichia coli CFT073

Uropathogenic Escherichia coli (UPEC) is the primary cause of urinary tract infection (UTI) in the developed world. The major factors associated with virulence of UPEC are fimbrial adhesins, which mediate specific attachment to host receptors and trigger innate host responses. Another group of adhesins is represented by the autotransporter (AT) subgroup of proteins. The genome-sequenced prototype UPEC strain CFT073 contains 11 putative AT-encoding genes. In this study, we have performed a detailed molecular characterization of two closely related AT adhesins from CFT073: UpaB (c0426) and UpaC (c0478). PCR screening revealed that the upaB and upaC AT-encoding genes are common in E. coli. The upaB and upaC genes were cloned and characterized in a recombinant E. coli K-12 strain background. This revealed that they encode proteins located at the cell surface but possess different functional properties: UpaB mediates adherence to several ECM proteins, while UpaC expression is associated with increased biofilm formation. In CFT073, upaB is expressed while upaC is transcriptionally repressed by the global regulator H-NS. In competitive colonization experiments employing the mouse UTI model, CFT073 significantly outcompeted its upaB (but not upaC) isogenic mutant strain in the bladder. This attenuated phenotype was also observed in single-challenge experiments, where deletion of the upaB gene in CFT073 significantly reduced early colonization of the bladder.

UpaH Is a newly identified autotransporter protein That contributes to biofilm formation and bladder colonization by Uropathogenic Escherichia coli CFT073

Escherichia coli is the primary cause of urinary tract infection (UTI) in the developed world. The major factors associated with virulence of uropathogenic E. coli (UPEC) are fimbrial adhesins, which mediate specific attachment to host receptors and trigger innate host responses. Another group of adhesins is represented by the autotransporter (AT) subgroup of proteins. In this study, we identified a new AT-encoding gene, termed upaH, present in a 6.5-kb unannotated intergenic region in the genome of the prototypic UPEC strain CFT073. Cloning and sequencing of the upaH gene from CFT073 revealed an intact 8.535-kb coding region, contrary to the published genome sequence. The upaH gene was widely distributed among a large collection of UPEC isolates as well as the E. coli Reference (ECOR) strain collection. Bioinformatic analyses suggest β-helix as the predominant structure in the large N-terminal passenger (α) domain and a 12-strand β-barrel for the C-terminal β-domain of UpaH. We demonstrated that UpaH is expressed at the cell surface of CFT073 and promotes biofilm formation. In the mouse UTI model, deletion of the upaH gene in CFT073 and in two other UPEC strains did not significantly affect colonization of the bladder in single-challenge experiments. However, in competitive colonization experiments, CFT073 significantly outcompeted its upaH isogenic mutant strain in urine and the bladder.

Observing the mother-infant feeding interaction

Early parenting is critical to effective attachment and a range of positive developmental outcomes for children. Feeding is a key task of early parenting and increasing evidence indicates that early feeding practices are important for the development of self-regulation of intake and food preferences which in turn are predictors of later obesity risk. However, relatively little is known about the mother-infant interaction at the transition to solids among typically developing children. This study aimed to describe parenting strategies used by mothers at the transition from milk feeding to solid food. Twenty mother-infant dyads were video-taped during a feeding interaction and data was analysed to describe maternal use of parenting strategies. It was predicted that positive feeding strategies would be correlated with lower levels of Infant Food Refusal (IFR), higher maternal sensitivity, and better overall parenting scores. The opposite was predicted for negative feeding strategies. It was found that positive strategy use and general parenting scores were significantly correlated in the predicted direction, however maternal instruction, aversive contact and ineffective strategy use were significantly correlated with and predictive of IFR. Additionally, it was hypothesised that maternal strategy use would deteriorate towards the end of the interaction, and this hypothesis was partially supported: significantly more negative strategy use was observed in the last third of the interaction, whilst positive strategy use remained consistent through the feeding interaction. The findings have important implications for early feeding parent education and intervention programs.

Evaluation of the potential impact of the global economic crisis on HIV epidemics in Southeast Asia

Economic conditions around the world are likely to deteriorate in the short to medium term. The potential impact of this crisis on the spread of HIV is not clear. Government revenues and aid flows from international donors may face constraints, possibly leading to reductions in funding for HIV programs. Economic conditions (leading to increases in unemployment, for example) may also have an indirect impact on HIV epidemics by affecting the behaviour of individual people. Some behavioural changes may influence the rate of HIV transmission. This report presents findings from a study that investigates the potential impact of the economic crisis on HIV epidemics through the use of mathematical modelling. The potential epidemiological impacts of changes in the economy are explored for two distinctly characterised HIV epidemics: (i) a well-defined, established, and generalised HIV epidemic (specifically Cambodia, where incidence is declining); (ii) an HIV epidemic in its early expansion phase (specifically Papua New Guinea, where incidence has not yet peaked). Country-specific data are used for both settings and the models calibrated to accurately reflect the unique HIV epidemics in each population in terms of both incidence and prevalence. Models calibrated to describe the past and present epidemics are then used to forecast epidemic trajectories over the next few years under assumptions that behavioural or program conditions may change due to economic conditions. It should be noted that there are very limited solid data on how HIV/AIDS program funds may decrease or how social determinants related to HIV risk may change due to the economic crisis. Potential changes in key relevant factors were explored, along with sensitivity ranges around these assumptions, based on extensive discussions with in-country and international experts and stakeholders. As with all mathematical models, assumptions should be reviewed critically and results interpreted cautiously.

Functions and therapeutic roles of exosomes in cancer

The role of exosomes in cancer development has become the focus of much research, due to the many emerging roles possessed by exosomes. These micro-vesicles that are ubiquitously released in to the extracellular milieu, have been found to regulate immune system function, particularly in tumorigenesis, as well as conditioning future metastatic sites for the attachment and growth of tumor tissue. Through an interaction with a range of host tissue, exosomes are able to generate a pro-tumor environment that is essential for carcinogenesis. Herein, we discuss the contents of exosomes and their contribution to tumorigenesis, as well as their role in chemotherapeutic resistance and the development of novel cancer treatments and the identification of cancer biomarkers.

Gold nanoparticles modified electrodes for biosensors

Biomolecules are chemical compounds found in living organisms which are the building blocks of life and perform important functions. Fluctuation from the normal concentration of these biomolecules in living system leads to several disorders. Thus the exact determination of them in human fluids is essential in the clinical point of view. High performance liquid chromatography, flow injection analysis, capillary electrophoresis, fluorimetry, spectrophotometry, electrochemical and chemiluminescence techniques were usually used for the determination of biologically important molecules. Among these techniques, electrochemical determination of biomolecules has several advantages over other methods viz., simplicity, selectivity and sensitivity. In the past two decades, electrodes modified with polymer films, self-assembled monolayers containing different functional groups and carbon paste have been used as electrochemical sensors. But in recent years, nanomaterials based electrochemical sensors play an important role in the improvement of public health because of its rapid detection, high sensitivity and specificity in clinical diagnostics. To date gold nanoparticles (AuNPs) have received arousing attention mainly due to their fascinating electronic and optical properties as a consequence of their reduced dimensions. These unique properties of AuNPs make them as an ideal candidate for the immobilization of enzymes for biosensing. Further, the electrochemical properties of AuNPs reveal that they exhibit interesting properties by enhancing the electrode conductivity, facilitating electron transfer and improving the detection limit of biomolecules. In this chapter, we summarized the different strategies used for the attachment of AuNPs on electrode surfaces and highlighted the electrochemical determination of glucose, ascorbic acid (AA), uric acid (UA) and dopamine derivatives using the AuNPs modified electrodes.

Electrocatalytic oxidation of ascorbic acid using a single layer of gold nanoparticles immobilized on 1,6-hexanedithiol modified gold electrode

This paper describes the electrocatalytic oxidation of ascorbic acid (AA) in phosphate buffer solution by the immobilized citrate capped gold nanoparticles (AuNPs) on 1,6-hexanedithiol (HDT) modified Au electrode. X-ray photoelectron spectrum (XPS) of HDT suggests that it forms a monolayer on Au surface through one of the two single bondSH groups and the other single bondSH group is pointing away from the electrode surface. The free single bondSH groups of HDT were used to covalently attach colloidal AuNPs. The covalent attachment of AuNPs on HDT monolayer was confirmed from the observed characteristic carboxylate ion stretching modes of citrate attached with AuNPs in the infra-red reflection absorption spectrum (IRRAS) in addition to a higher reductive desorption charges obtained for AuNPs immobilized on HDT modified Au (Au/HDT/AuNPs) electrode in 0.1 M KOH when compared to HDT modified Au (Au/HDT) electrode. The electron transfer reaction of [Fe(CN)6]4−/3− was markedly hindered at the HDT modified Au (Au/HDT) electrode while it was restored with a peak separation of 74 mV after the immobilization of AuNPs on Au/HDT (Au/HDT/AuNPs) electrode indicating a good electronic communication between the immobilized AuNPs and the underlying bulk Au electrode through a HDT monolayer. The Cottrell slope obtained from the potential-step chronoamperometric measurements for the reduction of ferricyanide at Au/HDT/AuNPs was higher than that of bare Au electrode indicating the increased effective surface area of AuNPs modified electrode. The Au/HDT/AuNPs electrode exhibits excellent electrocatalytic activity towards the oxidation of ascorbic acid (AA) by enhancing the oxidation peak current to more than two times with a 210 mV negative shift in the oxidation potential when compared to a bare Au electrode. The standard heterogeneous electron transfer rate constant (ks) calculated for AA oxidation at Au/HDT/AuNPs electrode was 5.4 × 10−3 cm s−1. The oxidation peak of AA at Au/HDT/AuNPs electrode was highly stable upon repeated potential cycling. Linear calibration plot was obtained for AA over the concentration range of 1–110 μM with a correlation coefficient of 0.9950. The detection limit of AA was found to be 1 μM. The common physiological interferents such as glucose, oxalate ions and urea do not show any interference within the detection limit of AA. The selectivity of the AuNPs modified electrode was illustrated by the determination of AA in the presence of uric acid.

“This is not a burning issue for me”: How citizens justify their use of wood heaters in a city with a severe air pollution problem

Although wood smoke pollution has been linked to health problems, wood burning remains a popular form of domestic heating in many countries across the world. In this paper, we describe the rhetoric of resistance to wood heater regulation amongst citizens in the regional Australian town of Armidale, where wood smoke levels regularly exceed national health advisory limits. We discuss how this is related to particular sources of resistance, such as affective attachment to wood heating and socio-cultural norms. The research draws on six focus groups with participants from households with and without wood heating. With reference to practice theory, we argue that citizen discourses favouring wood burning draw upon a rich suite of justifications and present this activity as a natural and traditional activity promoting comfort and cohesion. Such discourses also emphasise the identity of the town as a rural community and the supposed gemeinschaft qualities of such places. We show that, in this domain of energy policy, it is not enough to present ‘facts’ which have little emotional association or meaning for the populace. Rather, we need understand how social scripts, often localised, inform identity and practice.

Differential effects of tissue culture coating substrates on prostate cancer cell adherence, morphology and behavior

Weak cell-surface adhesion of cell lines to tissue culture surfaces is a common problem and presents technical limitations to the design of experiments. To overcome this problem, various surface coating protocols have been developed. However, a comparative and precise real-time measurement of their impact on cell behavior has not been conducted. The prostate cancer cell line LNCaP, derived from a patient lymph node metastasis, is a commonly used model system in prostate cancer research. However, the cells’ characteristically weak attachment to the surface of tissue culture vessels and cover slips has impeded their manipulation and analysis and use in high throughput screening. To improve the adherence of LNCaP cells to the culture surface, we compared different coating reagents (poly-L-lysine, poly-L-ornithine, collagen type IV, fibronectin, and laminin) and culturing conditions and analyzed their impact on cell proliferation, adhesion, morphology, mobility and gene expression using real-time technologies. The results showed that fibronectin, poly-L-lysine and poly-L-ornithine improved LNCaP cells adherence and provoked cell morphology alterations, such as increase of nuclear and cellular area. These coating reagents also induced a higher expression of F-actin and reduced cell mobility. In contrast, laminin and collagen type IV did not improve adherence but promoted cell aggregation and affected cell morphology. Cells cultured in the presence of laminin displayed higher mobility than control cells. All the coating conditions significantly affected cell viability; however, they did not affect the expression of androgen receptor-regulated genes. Our comparative findings provide important insight for the selection of the ideal coating reagent and culture conditions for the cancer cell lines with respect to their effect on proliferation rate, attachment, morphology, migration, transcriptional response and cellular cytoskeleton arrangement.

Activation of the canonical Wnt signaling pathway induces cementum regeneration

Canonical Wnt signaling is important in tooth development but it is unclear whether it can induce cementogenesis and promote the regeneration of periodontal tissues lost due to disease. Therefore, the aim of this study is to investigate the influence of canonical Wnt signaling enhancers on human periodontal ligament cell (hPDLCs) cementogenic differentiation in vitro and cementum repair in a rat periodontal defect model. Canonical Wnt signaling was induced by (i) local injection of lithium chloride; (ii) local injection of sclerostin antibody; and (iii) local injection of a lentiviral construct overexpressing β-catenin. The results showed that the local activation of canonical Wnt signaling resulted in significant new cellular cementum deposition and the formation of well-organized periodontal ligament fibers, which was absent in the control group. In vitro experiments using hPDLCs showed that the Wnt signaling pathway activators significantly increased mineralization, alkaline phosphatase (ALP) activity, and gene and protein expression of the bone and cementum markers osteocalcin (OCN), osteopontin (OPN), cementum protein 1 (CEMP1), and cementum attachment protein (CAP). Our results show that the activation of the canonical Wnt signaling pathway can induce in vivo cementum regeneration and in vitro cementogenic differentiation of hPDLCs.