Enhancement of NAD(H) pool for formation of oxidized biochemicals in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The NAD⁺/NADH ratio and the total NAD(H) play important roles for whole-cell biochemical redox transformations. After the carbon source is exhausted, the degradation of NAD(H) could contribute to a decline in the rate of a desired conversion. In this study, methods to slow the native rate of NAD(H) degradation were examined using whole-cell Escherichia coli with two model oxidative NAD⁺-dependent biotransformations. A high phosphate concentration (50 mM) was observed to slow NAD(H) degradation. We also constructed E. coli strains with deletions in genes coding several enzymes involved in NAD⁺ degradation. In shake-flask experiments, the total NAD(H) concentration positively correlated with conversion of xylitol to l-xylulose by xylitol 4-dehydrogenase, and the greatest conversion (80%) was observed using MG1655 nadR nudC mazG/pZE12-xdh/pCS27-nox. Controlled 1-L batch processes comparing E. coli nadR nudC mazG with a wild-type background strain demonstrated a 30% increase in final l-xylulose concentration (5.6 vs. 7.9 g/L) and a 25% increase in conversion (0.53 vs. 0.66 g/g). MG1655 nadR nudC mazG was also examined for the conversion of galactitol to l-tagatose by galactitol 2-dehydrogenase. A batch process using 15 g/L glycerol and 10 g/L galactitol generated over 9.4 g/L l-tagatose, corresponding to 90% conversion and a yield of 0.95 g l-tagatose/g galactitol consumed. The results demonstrate the value of minimizing NAD(H) degradation as a means to improve NAD⁺-dependent biotransformations.     

Ribosome engineering and fermentation optimization leads to overproduction of tiancimycin A,a new enediyne natural product from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CB03234

Tiancimycin (TNM) A, a recently discovered enediyne natural product from Streptomyces sp. CB03234, showed rapid and complete killing of cancer cells and could be used as a payload in antibody drug conjugates. The low yield of TNM A in the wild-type strain promoted us to use ribosome engineering and fermentation optimization for its yield improvement. The Streptomyces sp. CB03234-R-16 mutant strain with a L422P mutation in RpoB, the RNA polymerase β-subunit, was obtained from the rifamycin-resistant screening. After fermentation optimization, the titers of TNM A in Streptomyces sp. CB03234-R-16 reached to 22.5 ± 3.1 mg L^?1 in shaking flasks, and 13 ± 1 mg L^?1 in 15 L fermentors, which were at least 40-fold higher than that in the wild-type strain (~ 0.3 mg L^?1). Quantitative real-time RT-PCR revealed markedly enhanced expression of key genes encoding TNM A biosynthetic enzymes and regulators in Streptomyces sp. CB03234-R-16. Our study should greatly facilitate the future efforts to develop TNM A into a clinical anticancer drug.     

Bottom-up approaches in synthetic biology and biomaterials for tissue engineering applications

Synthetic biologists use engineering principles to design and construct genetic circuits for programming cells with novel functions. A bottom-up approach is commonly used to design and construct genetic circuits by piecing together functional modules that are capable of reprogramming cells with novel behavior. While genetic circuits control cell operations through the tight regulation of gene expression, a diverse array of environmental factors within the extracellular space also has a significant impact on cell behavior. This extracellular space offers an addition route for synthetic biologists to apply their engineering principles to program cell-responsive modules within the extracellular space using biomaterials. In this review, we discuss how taking a bottom-up approach to build genetic circuits using DNA modules can be applied to biomaterials for controlling cell behavior from the extracellular milieu. We suggest that, by collectively controlling intrinsic and extrinsic signals in synthetic biology and biomaterials, tissue engineering outcomes can be improved.     

Proximity and grooming patterns reveal opposite-sex bonding in Rwenzori Angolan colobus monkeys (<Emphasis Type="Italic">Colobus angolensis ruwenzorii</Emphasis>)

Close proximity and social grooming are important bonding mechanisms in primates. These behaviors show the social structure of a species and many studies have found positive correlations between the degree of kinship and grooming and proximity. We used 1 year of data collected via instantaneous scan sampling on a large “supertroop” of Colobus angolensis ruwenzorii at Lake Nabugabo, Uganda, to examine partner preferences for grooming and nearest neighbors in each age-sex class. Little is known about this species, so we based our hypotheses on congeners. Of the five species of black-and-white colobus, data on sex-biased dispersal patterns are available for three (C. guereza, C. vellerosus, and C. polykomos), all of which show male-biased dispersal with occasional female dispersal. We thus predicted that female C. a. ruwenzorii would be more strongly bonded than males, showing greater proximity and grooming. We did not expect bonding between the sexes since congeners do not show this pattern. We found that among adult dyads, males and females were more likely to be found in loose proximity, and to groom, than would be expected given group composition. Conversely, both males and females had relatively weak same-sex relationships. Between the sexes, adult males had higher proximity and grooming indices with adult females without infants than with females with infants. These observations indicate that this subspecies is cross-bonded and that both sexes may disperse. Furthermore, our findings suggest that the social organization and social structure of C. a. ruwenzorii differ greatly from other black-and-white colobus species.     

Depositional facies and cyclic patterns in a subtidal-dominated ramp during the Early-Middle Ordovician in the western Tarim Basin (NW China)

Due to a long-term transgression since the Early Cambrian, an extensive shallow-water carbonate platform was developed in the entire Tarim Basin (NW China). During the deposition of the Yingshan Formation (Early-Middle Ordovician), a carbonate ramp system was formed in the intrashelf basin in the Bachu-Keping area of the western basin. Four well-exposed outcrop sections were selected to investigate their depositional facies, cycles, and sequences, as well as the depositional evolution. Detailed facies analyses permit the recognition of three depositional facies associations, including peritidal, semi-restricted subtidal, and open-marine subtidal facies, and eleven types of lithofacies. These are vertically arranged into meter-scale, shallowing-upward peritidal, semi-restricted subtidal, and open-marine subtidal cycles, in the span of Milankovitch frequency bands, suggesting a dominant control of Earth’s orbital forcing on the cyclic sedimentation on the platform. On the basis of vertical facies (or lithofacies) and cycle stacking patterns, as well as accommodation changes illustrated graphically by Fischer plots at all studied sections, six third-order depositional sequences are recognized and consist of lower transgressive and upper regressive parts. In shallow depositional settings, the transgressive packages are dominated by thicker-than-average, shallow subtidal cycles, whereas the regressive parts are mainly represented by thinner-than-average, relatively shallow subtidal to peritidal cycles. In relatively deep environments, however, the transgressive and regressive successions display the opposite trends of cycle stacking patterns, i.e., thinner-than-average subtidal cycles of transgressive packages. Sequence boundaries are mainly characterized by laterally traceable, transitional zones without apparent subaerial exposure features. Good correlation of the long-term changes in accommodation space inferred from vertical facies and cycle stacking patterns with sea-level fluctuations elsewhere around the world suggests an overriding eustatic control on cycle origination, platform building-up and evolution during the Early-Middle Ordovician, although with localized influences of syndepositional faulting and depositional settings.     

Climate Change Could Increase the Geographic Extent of Hendra Virus Spillover Risk

Disease risk mapping is important for predicting and mitigating impacts of bat-borne viruses, including Hendra virus (Paramyxoviridae:Henipavirus), that can spillover to domestic animals and thence to humans. We produced two models to estimate areas at potential risk of HeV spillover explained by the climatic suitability for its flying fox reservoir hosts, Pteropus alecto and P. conspicillatus. We included additional climatic variables that might affect spillover risk through other biological processes (such as bat or horse behaviour, plant phenology and bat foraging habitat). Models were fit with a Poisson point process model and a log-Gaussian Cox process. In response to climate change, risk expanded southwards due to an expansion of P. alecto suitable habitat, which increased the number of horses at risk by 175–260% (110,000–165,000). In the northern limits of the current distribution, spillover risk was highly uncertain because of model extrapolation to novel climatic conditions. The extent of areas at risk of spillover from P. conspicillatus was predicted shrink. Due to a likely expansion of P. alecto into these areas, it could replace P. conspicillatus as the main HeV reservoir. We recommend: (1) HeV monitoring in bats, (2) enhancing HeV prevention in horses in areas predicted to be at risk, (3) investigate and develop mitigation strategies for areas that could experience reservoir host replacements.     

Introducing DNA-based methods to compare fungal microbiota and concentrations in indoor,outdoor, and personal air

Inhalation of airborne fungi is known to cause respiratory illnesses such as allergies. However, the association between exposure and health outcomes remains largely unclear, in part due to lack of knowledge about fungal exposure in daily life. This study aims to introduce DNA-based methods such as high-throughput sequencing (HTS) and quantitative polymerase chain reaction (qPCR) to compare fungal microbiota and concentrations in indoor, outdoor, and personal air. Five sets of concurrent indoor, outdoor, and personal air samples were collected, each with duration of 4 days. Sequencing analysis revealed greater species richness in personal than indoor air for four out of the five sets, indicating that people are exposed to outdoor species that are not in indoor air. The personal–indoor (P/I) and personal–outdoor (P/O) ratios of total fungi were 1.2 and 0.15, respectively, suggesting that personal exposure to total fungi is better represented by indoor than outdoor concentrations. However, the ratios were taxon dependent, highlighting the complexity of generalizing personal exposure to the diverse kingdom Fungi. These results demonstrate that the HTS/qPCR method is useful for assessing taxon-specific fungal exposure, which might be difficult to achieve effectively using conventional, non-DNA-based techniques.     

α-SNAP Interferes with the Zippering of the SNARE Protein Membrane Fusion Machinery

Neuronal exocytosis is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Before fusion, SNARE proteins form complexes bridging the membrane followed by assembly toward the C-terminal membrane anchors, thus initiating membrane fusion. After fusion, the SNARE complex is disassembled by the AAA-ATPase N-ethylmaleimide-sensitive factor that requires the cofactor α-SNAP to first bind to the assembled SNARE complex. Using chromaffin granules and liposomes we now show that α-SNAP on its own interferes with the zippering of membrane-anchored SNARE complexes midway through the zippering reaction, arresting SNAREs in a partially assembled trans-complex and preventing fusion. Intriguingly, the interference does not result in an inhibitory effect on synaptic vesicles, suggesting that membrane properties also influence the final outcome of α-SNAP interference with SNARE zippering. We suggest that binding of α-SNAP to the SNARE complex affects the ability of the SNARE complex to harness energy or transmit force to the membrane.