Glucosyltransferase Mutants of Leuconostoc mesenteroides NRRL B-1355

Leuconostoc mesenteroides NRRL B-1355 produces dextrans and alternan from sucrose. Alternan is an unusual dextran-like polymer containing alternating α(1→6)/α(1→3) glucosidic bonds. Cultures were mutagenized with UV and ethyl methanesulfonate, and colony morphology mutants were selected on 10% sucrose plates. Colony morphology variants exhibited changes from parent cultures in the production of one or more glucosyltransferases (GTFs) and glucans. Mutants were characterized by measuring resistance of glucan products to dextranase digestion, by electrophoresis, and by high-pressure liquid chromatography of maltose acceptor products generated from sucrose-maltose mixtures. Some mutants produced almost pure fraction L dextran, and cultures exhibited a single principal GTF band on sodium dodecyl sulfate-acrylamide gels. Other mutants produced glucans enriched for alternan. Colony morphology characteristics (size, smoothness, and opacity) and liquid culture properties (clumpiness, color, and viscosity in 10% sucrose medium) were explained on the basis of GTF production. Three principal GTF bands were detected.     

Understanding the Reduced Efficiencies of Organic Solar Cells Employing Fullerene Multiadducts as Acceptors

The use of fullerenes with two or more adducts as acceptors has been recently shown to enhance the performance of bulk‐heterojunction solar cells using poly(3‐hexylthiophene) (P3HT) as the donor. The enhancement is caused by a substantial increase in the open‐circuit voltage due to a rise in the fullerene lowest unoccupied molecular orbital (LUMO) level when going from monoadducts to multiadducts. While the increase in the open‐circuit voltage is obtained with many different polymers, most polymers other than P3HT show a substantially reduced photocurrent when blended with fullerene multiadducts like bis‐PCBM (bis adduct of Phenyl‐C₆₁‐butyric acid methyl ester) or the indene C₆₀ bis‐adduct ICBA. Here we investigate the reasons for this decrease in photocurrent. We find that it can be attributed partly to a loss in charge generation efficiency that may be related to the LUMO‐LUMO and HOMO‐HOMO (highest occupied molecular orbital) offsets at the donor‐acceptor heterojunction, and partly to reduced charge carrier collection efficiencies. We show that the P3HT exhibits efficient collection due to high hole and electron mobilities with mono‐ and multiadduct fullerenes. In contrast the less crystalline polymer Poly[[9‐(1‐octylnonyl)‐9H‐carbazole‐2,7‐diyl]‐2,5‐thiophenediyl‐2,1,3‐benzothiadiazole‐4,7‐diyl‐2,5‐thiophenediyl (PCDTBT) shows inefficient charge carrier collection, assigned to low hole mobility in the polymer and low electron mobility when blended with multiadduct fullerenes.     

Cellulosomics, a gene-centric approach to investigating the intraspecific diversity and adaptation of Ruminococcus flavefaciens within the rumen

Background

The bovine rumen maintains a diverse microbial community that serves to break down indigestible plant substrates. However, those bacteria specifically adapted to degrade cellulose, the major structural component of plant biomass, represent a fraction of the rumen microbiome. Previously, we proposed scaC as a candidate for phylotyping Ruminococcus flavefaciens, one of three major cellulolytic bacterial species isolated from the rumen. In the present report we examine the dynamics and diversity of scaC-types both within and between cattle temporally, following a dietary switch from corn-silage to grass-legume hay. These results were placed in the context of the overall bacterial population dynamics measured using the 16S rRNA.

Principal Findings

As many as 117 scaC-types were estimated, although just nineteen were detected in each of three rumens tested, and these collectively accounted for the majority of all types present. Variation in scaC populations was observed between cattle, between planktonic and fiber-associated fractions and temporally over the six-week survey, and appeared related to scaC phylogeny. However, by the sixth week no significant separation of scaC populations was seen between animals, suggesting enrichment of a constrained set of scaC-types. Comparing the amino-acid translation of each scaC-type revealed sequence variation within part of the predicted dockerin module but strong conservation in the N-terminus, where the cohesin module is located.

Conclusions

The R. flavefaciens species comprises a multiplicity of scaC-types in-vivo. Enrichment of particular scaC-types temporally, following a dietary switch, and between fractions along with the phylogenetic congruence suggests that functional differences exist between types. Observed differences in dockerin modules suggest at least part of the functional heterogeneity may be conferred by scaC. The polymorphic nature of scaC enables the relative distribution of R. flavefaciens strains to be examined and represents a gene-centric approach to investigating the intraspecific adaptation of an important specialist population.     

Unique properties of muscularis mucosae smooth muscle in guinea pig urinary bladder

The muscularis mucosae, a type of smooth muscle located between the urothelium and the urinary bladder detrusor, has been described, although its properties and role in bladder function have not been characterized. Here, using mucosal tissue strips isolated from guinea pig urinary bladders, we identified spontaneous phasic contractions (SPCs) that appear to originate in the muscularis mucosae. This smooth muscle layer exhibited Ca(2+) waves and flashes, but localized Ca(2+) events (Ca(2+) sparks, purinergic receptor-mediated transients) were not detected. Ca(2+) flashes, often in bursts, occurred with a frequency (～5.7/min) similar to that of SPCs (～4/min), suggesting that SPCs are triggered by bursts of Ca(2+) flashes. The force generated by a single mucosal SPC represented the maximal force of the strip, whereas a single detrusor SPC was ～3% of maximal force of the detrusor strip. Electrical field stimulation (0.5-50 Hz) evoked force transients in isolated detrusor and mucosal strips. Inhibition of cholinergic receptors significantly decreased force in detrusor and mucosal strips (at higher frequencies). Concurrent inhibition of purinergic and cholinergic receptors nearly abolished evoked responses in detrusor and mucosae. Mucosal SPCs were unaffected by blocking small-conductance Ca(2+)-activated K(+) (SK) channels with apamin and were unchanged by blocking large-conductance Ca(2+)-activated K(+) (BK) channels with iberiotoxin (IbTX), indicating that SK and BK channels play a much smaller role in regulating muscularis mucosae SPCs than they do in regulating detrusor SPCs. Consistent with this, BK channel current density in myocytes from muscularis mucosae was ～20% of that in detrusor myocytes. These findings indicate that the muscularis mucosae in guinea pig represents a second smooth muscle compartment that is physiologically and pharmacologically distinct from the detrusor and may contribute to the overall contractile properties of the urinary bladder.     

Synthesis of novel steroidal agonists,partial agonists,and antagonists for the glucocorticoid receptor

Adverse effects of glucocorticoids could be limited by developing new compounds that selectively modulate anti-inflammatory activity of the glucocorticoid receptor (GR). We have synthesized a novel series of steroidal GR ligands, including potent agonists, partial agonists and antagonists with a wide range of effects on inhibiting secretion of interleukin-6. Some of these new ligands were designed to directly impact conformational stability of helix-12, in the GR ligand-binding domain (LBD). These compounds modulated GR activity and glucocorticoid-induced gene expression in a manner that was inversely correlated to the degree of inflammatory response. In contrast, compounds designed to directly modulate LBD epitopes outside helix-12, led to dissociated levels of GR-mediated gene expression and inflammatory response. Therefore, these new series of compounds and their derivatives will be useful to dissect the ligand-dependent features of GR signaling specificity.     

Predictors of Condom Use among Peer Social Networks of Men Who Have Sex with Men in Ghana,West Africa

Ghanaian men who have sex with men (MSM) have high rates of HIV infection. A first step in designing culturally relevant prevention interventions for MSM in Ghana is to understand the influence that peer social networks have on their attitudes and behaviors. We aimed to examine whether, in a sample of Ghanaian MSM, mean scores on psychosocial variables theorized to influence HIV/STI risk differed between peer social networks and to examine whether these variables were associated with condom use. We conducted a formative, cross-sectional survey with 22 peer social networks of MSM (n = 137) in Ghana. We assessed basic psychological-needs satisfaction, HIV/STI knowledge, sense of community, HIV and gender non-conformity stigmas, gender equitable norms, sexual behavior and condom use. Data were analyzed using analysis of variance, generalized estimating equations, and Wilcoxon two sample tests. All models were adjusted for age and income, ethnicity, education, housing and community of residence. Mean scores for all psychosocial variables differed significantly by social network. Men who reported experiencing more autonomy support by their healthcare providers had higher odds of condom use for anal (AOR = 3.29, p<0.01), oral (AOR = 5.06, p<0.01) and vaginal (AOR = 1.8, p<0.05) sex. Those with a stronger sense of community also had higher odds of condom use for anal sex (AOR = 1.26, p<0.001). Compared to networks with low prevalence of consistent condom users, networks with higher prevalence of consistent condom users had higher STD and HIV knowledge, had norms that were more supportive of gender equity, and experienced more autonomy support in their healthcare encounters. Healthcare providers and peer social networks can have an important influence on safer-sex behaviors in Ghanaian MSM. More research with Ghanaian MSM is needed that considers knowledge, attitudes, and norms of their social networks in the development and implementation of culturally relevant HIV/STI prevention intervention strategies.     

Kinase-mediated trapping of bi-functional conjugates of paclitaxel or vinblastine with thymidine in cancer cells

In the present work, we explore the possibility of introducing selectivity to existing chemotherapeutics via the design of non-pro-drug, bi-functional molecules comprising a microtubule-binding agent and a substrate for a disease-associated kinase. The design, synthesis, and in vitro biological evaluation of paclitaxel-thymidine and vinblastine-thymidine bi-functional conjugates are reported here. This work provides the first account of 'kinase-mediated trapping' of cancer therapeutics.     

Calcium measurement in living filamentous fungi expressing codon-optimized aequorin

Calcium signalling is little understood in filamentous fungi largely because easy and routine methods for calcium measurement in living hyphae have previously been unavailable. We have developed the recombinant aequorin method for this purpose. High levels of aequorin expression were obtained in Neurospora crassa, Aspergillus niger and Aspergillus awamori by codon optimization of the aequorin gene. Three external stimuli (mechanical perturbation, hypo-osmotic shock and high external calcium) were found transiently to increase [Ca(2+)](c). Each of the calcium signatures associated with these physico-chemical treatments was unique, suggesting the involvement of three distinct calcium-mediated signal transduction pathways. The fungal calcium channel blocker KP4 inhibited the [Ca(2+)](c) responses to hypo-osmotic shock and high external calcium, but not to mechanical perturbation. The divalent cation chelator BAPTA inhibited [Ca(2+)](c) responses to mechanical perturbation and hypo-osmotic shock. The calcium agonists A23187 and cyclopiazonic acid increased [Ca(2+)](c) levels.     

Electrostatic and non-electrostatic contributions of proteoglycans to the compressive equilibrium modulus of bovine articular cartilage

This study presents direct experimental evidence for assessing the electrostatic and non-electrostatic contributions of proteoglycans to the compressive equilibrium modulus of bovine articular cartilage. Immature and mature bovine cartilage samples were tested in unconfined compression and their depth-dependent equilibrium compressive modulus was determined using strain measurements with digital image correlation analysis. The electrostatic contribution was assessed by testing samples in isotonic and hypertonic saline; the combined contribution was assessed by testing untreated and proteoglycan-depleted samples.Though it is well recognized that proteoglycans contribute significantly to the compressive stiffness of cartilage, results demonstrate that the combined electrostatic and non-electrostatic contributions may add up to more than 98% of the modulus, a magnitude not previously appreciated. Of this contribution, about two thirds arises from electrostatic effects. The compressive modulus of the proteoglycan-depleted cartilage matrix may be as low as 3 kPa, representing less than 2% of the normal tissue modulus; experimental evidence also confirms that the collagen matrix in digested cartilage may buckle under compressive strains, resulting in crimping patterns. Thus, it is reasonable to model the collagen as a fibrillar matrix that can sustain only tension. This study also demonstrates that residual stresses in cartilage do not arise exclusively from proteoglycans, since cartilage remains curled relative to its in situ geometry even after proteoglycan depletion. These increased insights on the structure–function relationships of cartilage can lead to improved constitutive models and a better understanding of the response of cartilage to physiological loading conditions.