Efficient production of a recombinant ι-carrageenase in Brevibacillus choshinensis using a new integrative vector for the preparation of ι-carrageenan oligosaccharides

In this study, a new integrative vector (pBCGA) was developed and used for the high-level expression of an optimized ι-carrageenase gene (CGIOP) in Brevibacillus choshinensis. The pBCGA vector allowed multiple copies of the gene CGIOP to be stably integrated into the genomic DNA of B. choshinensis. The recombinant strain I24 could produce an extracellular ι-carrageenase activity of 38.9 U/mL within 72 h, which remained stable after five sequential inoculations and cultivations under the antibiotic-free culture conditions. Furthermore, the strain I24 was applied to 10-L fermentation under the antibiotic-free culture condition, resulting in the highest observed ι-carrageenase activity of 182.4 U/mL within 24 h. Subsequently, recombinant ι-carrageenase (rCgiA) was purified and characterized, exhibiting an optimal pH and temperature of 8.0 and 45 °C, respectively. Notably, rCgiA showed considerable stability below 45 °C and over a relatively broad pH range of 6.0–11.0. In addtion, the activity of rCgiA was significantly stimulated by NaCl, Mg²⁺, and Ca²⁺. HILIC LC-LTQ-Orbitrap-FTMS analysis revealed that rCgiA hydrolyzed ι-carrageenan via a processive mechanism with the major product of ι-carrageenan tetrasaccharide. Thus, the strain B. choshinensis I24 had broad potential for use in the environment-friendly and large-scale production of ι-carrageenase and ι-carrageenan oligosaccharides.     

Uniform magnetic fields and double-wrapped coil systems: improved techniques for the design of bioelectromagnetic experiments.

A common mistake in biomagnetic experimentation is the assumption that Helmholtz coils provide uniform magnetic fields; this is true only for a limited volume at their center. Substantial improvements on this design have been made during the past 140 years with systems of three, four, and five coils. Numerical comparisons of the field uniformity generated by these designs are made here, along with a table of construction details and recommendations for their use in experiments in which large volumes of uniform intensity magnetic exposures are needed. Double-wrapping, or systems of bifilar windings, can also help control for the non-magnetic effects of the electric coils used in many experiments. In this design, each coil is wrapped in parallel with two separate, adjacent strands of copper wire, rather than the single strand used normally. If currents are flowing in antiparallel directions, the magnetic fields generated by each strand will cancel and yield virtually no external magnetic field, whereas parallel currents will yield an external field. Both cases will produce similar non-magnetic effects of ohmic heating, and simple measures can reduce the small vibration and electric field differences. Control experiments can then be designed such that the only major difference between treated and untreated groups is the presence or absence of the magnetic field. Double-wrapped coils also facilitate the use of truly double-blind protocol, as the same apparatus can be used either for experimental or control groups.     

Fasciola hepatica: stereological analysis of effects of certain metabolic inhibitors on synthesis of secretory bodies by the tegument of tissue slices.

The influence of several well-known inhibitors on the production and distribution of Type 1 secretory bodies in the tegument of Fasciola hepatica slices has been analyzed stereologically.Cycloheximide was found to inhibit the production of these bodies by the Golgi complex of the tegumental cells, but apparently had little effect on intracellular transport and discharge. The metabolic inhibitors, DNP and iodoacetate, seemed to block all three processes.The results provide physiological evidence that the GER-Golgi system in the tegumental cells of this fluke carry out protein or glycoprotein synthesis, that a functional relationship exists between the tegumental cells and the surface syncytium in the transport and discharge of the secretory bodies, and that synthesis, transport, and discharge of the bodies are all energy-dependent processes.     

Climatic controls of vegetation vigor in four contrasting forest types of India—evaluation from National Oceanic and Atmospheric Administration’s Advanced Very High Resolution Radiometer datasets (1990–2000)

Ten-day advanced very high resolution radiometer images from 1990 to 2000 were used to examine spatial patterns in the normalized difference vegetation index (NDVI) and their relationships with climatic variables for four contrasting forest types in India. The NDVI signal has been extracted from homogeneous vegetation patches and has been found to be distinct for deciduous and evergreen forest types, although the mixed-deciduous signal was close to the deciduous ones. To examine the decadal response of the satellite-measured vegetation phenology to climate variability, seven different NDVI metrics were calculated using the 11-year NDVI data. Results suggested strong spatial variability in forest NDVI metrics. Among the forest types studied, wet evergreen forests of north-east India had highest mean NDVI (0.692) followed by evergreen forests of the Western Ghats (0.529), mixed deciduous forests (0.519) and finally dry deciduous forests (0.421). The sum of NDVI (SNDVI) and the time-integrated NDVI followed a similar pattern, although the values for mixed deciduous forests were closer to those for evergreen forests of the Western Ghats. Dry deciduous forests had higher values of inter-annual range (RNDVI) and low mean NDVI, also coinciding with a high SD and thus a high coefficient of variation (CV) in NDVI (CVNDVI). SNDVI has been found to be high for wet evergreen forests of north-east India, followed by evergreen forests of the Western Ghats, mixed deciduous forests and dry deciduous forests. Further, the maximum NDVI values of wet evergreen forests of north-east India (0.624) coincided with relatively high annual total precipitation (2,238.9 mm). The time lags had a strong influence in the correlation coefficients between annual total rainfall and NDVI. The correlation coefficients were found to be comparatively high (R²=0.635) for dry deciduous forests than for evergreen forests and mixed deciduous forests, when the precipitation data with a lag of 30 days was correlated against NDVI. Using multiple regression approach models were developed for individual forest types using 16 different climatic indices. A high proportion of the temporal variance (>90%) has been accounted for by three of the precipitation parameters (maximum precipitation, precipitation of the wettest quarter and driest quarter) and two of the temperature parameters (annual mean temperature and temperature of the coldest quarter) for mixed deciduous forests. Similarly, in the case of deciduous forests, four precipitation parameters and three temperature parameters explained nearly 83.6% of the variance. These results suggest differences in the relationship between NDVI and climatic variables based upon the time of growing season, time interval and climatic indices over which they were summed. These results have implications for forest cover mapping and monitoring in tropical regions of India.     

Measurement of concurrent selection episodes

Current methods for measuring selection with longitudinal data have been developed with the assumption that episodes of selection are sequential. However, a number of empirical examinations have demonstrated that natural and sexual selection may act concurrently and in opposing directions. Other recent work has highlighted the difficulty of assigning fitness values for survival when reproduction and mortality within a population temporally overlap. I treat these as facets of a single problem; how to analyze selection where mortality and reproduction are concurrent. To address this problem, I formalize a method to estimate total fitness of individuals over a period of time utilizing longitudinal data. I then show how the fitness may be partitioned to provide two separate estimates of fitness for reproductive opportunity and reproductive success. In addition, another total fitness estimate for the period can be obtained from the two partitioned estimates. This procedure will allow calculation of total fitness where there are some missing datapoints for reproductive success of an individual. A simulation indicates that bias is generally low for the various fitness estimates. These methods should expand our ability to understand the interaction of different selection episodes.     

Characterizing functional consequences of DNA copy number alterations in breast and ovarian tumors by spaceMap

We propose a novel conditional graphical model—spaceMap—to construct gene regulatory networks from multiple types of high dimensional omic profiles. A motivating application is to characterize the perturbation of DNA copy number alterations(CNAs) on downstream protein levels in tumors. Through a penalized multivariate regression framework, spaceMap jointly models high dimensional protein levels as responses and high dimensional CNAs as predictors. In this setup, spaceMap infers an undirected network among proteins together with a directed network encoding how CNAs perturb the protein network. spaceMap can be applied to learn other types of regulatory relationships from high dimensional molecular profiles, especially those exhibiting hub structures. Simulation studies show spaceMap has greater power in detecting regulatory relationships over competing methods. Additionally, spaceMap includes a network analysis toolkit for biological interpretation of inferred networks. We applies spaceMap to the CNAs, gene expression and proteomics data sets from CPTAC-TCGA breast(n=77) and ovarian(n=174) cancer studies. Each cancer exhibits disruption of ‘ion transmembrane transport' and‘regulation from RNA polymerase Ⅱ promoter' by CNA events unique to each cancer. Moreover, using protein levels as a response yields a more functionally-enriched network than using RNA expressions in both cancer types. The network results also help to pinpoint crucial cancer genes and provide insights on the functional consequences of important CNA in breast and ovarian cancers. The R package spaceMap—including vignettes and documentation—is hosted on https://topherconley.github.io/spacemap.     

Observations on integrative transformation in Schizosaccharomyces pombe

Summary Three different Schizosaccharomyces pombe strains have been transformed with a circular or linearized non-ars plasmid carrying the ura4 ⁺ gene as a selectable marker. The first strain shows full homology between the genomic ura4-294 gene (point mutation) and the marker gene on the plasmid. The second strain carries a 600 bp deletion (ura4-D6) that decreases homology between plasmid and chromosome. No homology remains in the third strain which has a complete deletion of the ura4 gene on the chromosome (ura4-D18). When sequence homology exists between transforming DNA and the chromosomal ura4 region, gene conversion is strongly preferred over integration of the circular plasmid. Reduction of the length of homology leads to a decrease of transformation frequencies, and homology dependent as well as a minority of homology independent integrations are observed. In the complete absence of homology two rate types of transformants are encountered: either the circular plasmid replicates autonomously, although it is devoid of an ars sequence, or alternatively the plasmid integrates into the genome at various positions. Transformation with plasmid cut within the coding region of ura4 can lead to tandemly arranged multiple integrations, when no homology exists between the free ends and the chromosome. The integrations occur at the ura4 locus, when homology is retained between plasmid and chromosome, and at various sites in the genome of the strain with a complete deletion of the ura4 gene. The results suggest that homology dependent events (conversion, integration) are strongly preferred in transformation of S. pombe with non-ars plasmids. In addition low frequency integration by illegitimate recombination is observed. Linearized plasmid can be ligated in vivo to form monomers or multimers in the absence of homology between the free plasmid ends and the chromosomal genome.     

The INDICARE-model – measuring and caring about participation in higher education's sustainability assessment

The implementation of sustainability in higher education has been advanced over at least the last two decades and brought sustainability assessment on the research agenda of Education for Sustainable Development (ESD) and sustainability science. Participatory approaches have gained increasing attention in these endeavours, but remain often vague and less addressed in sustainability assessment procedures. To fill in this gap, an indicator-based model, INDICARE, was developed that can assist in assessing participatory processes within higher education's sustainability initiatives. The objective of this paper is to introduce and discuss the model's theoretical background, its structure, applicability, and how it can broaden the perspectives on participation and sustainability assessment in the university context.Embedded in a cross-sectional qualitative research design, the model was developed in iterative stages and was presented and adjusted along six feedback loops, having been presented to 98 persons during conferences, workshops and university meetings. Inspired by biophilic ideas, transformative learning theories and participatory evaluation, INDICARE follows an ecocentric and integrative perspective that places the earth and its community at the centre of attention. A preliminary set of thirty indicators and practices, grouped in three categories of context, process, and transformation, is proposed. The assessment process itself is considered as a thought-provoking exercise rather than as a control tool and emphasizes the interplay of personal reflection and action-oriented outreach. INDICARE intends to invigorate the sustainability debate in higher education, in particular by proposing a more holistic approach to assessment that underlines experiencing the interconnectedness of human–nature relationships, combined with reflective exercises that can respond better to the call for transformation on individual and institutional level.     

Augmenting podocyte injury promotes advanced diabetic kidney disease in Akita mice

To determine if augmenting podocyte injury promotes the development of advanced diabetic nephropathy (DN), we created mice that expressed the enzyme cytosine deaminase (CD) specifically in podocytes of diabetic Akita mice (Akita-CD mice). In these mice, treatment with the prodrug 5-flucytosine (5-FC) causes podocyte injury as a result of conversion to the toxic metabolite 5-fluorouracil (5-FU). We found that treatment of 4–5 week old Akita mice with 5-FC for 5 days caused robust albuminuria at 16 and 20 weeks of age compared to 5-FC treated Akita controls, which do not express CD (Akita CTLs). By 20 weeks of age, there was a significant increase in mesangial expansion in Akita-CD mice compared to Akita CTLs, which was associated with a variable increase in glomerular basement membrane (GBM) width and interstitial fibrosis. At 20 weeks of age, podocyte number was similarly reduced in both groups of Akita mice, and was inversely correlated with the albuminuria and mesangial expansion. Thus, enhancing podocyte injury early in the disease process promotes the development of prominent mesangial expansion, interstitial fibrosis, increased GBM thickness and robust albuminuria. These data suggest that podocytes play a key role in the development of advanced features of diabetic kidney disease.