Transition of microbial communities during the adaption to anaerobic digestion of carrot waste

In this study a microbial community suitable for anaerobic digestion of carrot pomace was developed from inocula obtained from natural environmental sources. The changes along the process were monitored using pyrosequencing of the 16S rRNA gene. As the community adapted from a diverse natural community to a community with a definite function, diversity decreased drastically. Major bacterial groups remaining after enrichment were Bacilli (31-45.3%), Porphyromonadaceae (12.1-24.8%) and Spirochaetes (12.5-18.5%). The archaeal population was even less diverse and mainly represented by a single OTU that was 99.7% similar to Methanosarcina mazei. One enrichment which failed to produce large amounts of methane had shifts in the bacterial populations and loss of methanogenic archaea.     

Raman spectroscopy detects phenotypic differences among Escherichia coli enriched for 1‐butanol tolerance using a metagenomic DNA library

Advances in Raman spectroscopy are enabling more comprehensive measurement of microbial cell chemical composition. Advantages include results returned in near real‐time and minimal sample preparation. In this research, Raman spectroscopy is used to analyze E. coli with engineered solvent tolerance, which is a multi‐genic trait associated with complex and uncharacterized phenotypes that are of value to industrial microbiology. To generate solvent tolerant phenotypes, E. coli transformed with DNA libraries are serially enriched in the presence of 0.9% (v/v) and 1.1% (v/v) 1‐butanol. DNA libraries are created using degenerate oligonucleotide primed PCR (DOP‐PCR) from the genomic DNA of E. coli, Clostridium acetobutylicum ATCC 824, and the metagenome of a stream bank soil sample, which contained DNA from 72 different phyla. DOP‐PCR enabled high efficiency library cloning (with no DNA shearing or end‐polishing) and the inclusion un‐culturable organisms. Nine strains with improved tolerance are analyzed by Raman spectroscopy and vastly different solvent‐tolerant phenotypes are characterized. Common among these are improved membrane rigidity from increasing the fraction of unsaturated fatty acids at the expense of cyclopropane fatty acids. Raman spectroscopy offers the ability to monitor cell phenotype changes in near real‐time and is adaptable to high‐throughput screening, making it relevant to metabolic engineering.     

Piperazinyl quinolines as chemosensitizers to increase fluconazole susceptibility of Candida albicans clinical isolates

The effectiveness of the potent antifungal drug fluconazole is being compromised by the rise of drug-resistant fungal pathogens. While inhibition of Hsp90 or calcineurin can reverse drug resistance in Candida, such inhibitors also impair the homologous human host protein and fungal-selective chemosensitizers remain rare. The MLPCN library was screened to identify compounds that selectively reverse fluconazole resistance in a Candida albicans clinical isolate, while having no antifungal activity when administered as a single agent. A piperazinyl quinoline was identified as a new small-molecule probe (ML189) satisfying these criteria.     

Detecting cellulase and esterase enzyme activities encoded by novel genes present in environmental DNA libraries

A genomic DNA library was made from the alkaliphilic cellulase-producing Bacillus agaradhaerans in order to prove our technologies for gene isolation prior to using them with samples of DNA isolated directly from environmental samples. Clones expressing a cellulase activity were identified and sequenced. A new cellulase gene was identified. Genomic DNA libraries were then made from DNA isolated directly from the Kenyan soda lakes, Lake Elmenteita and Crater Lake. Crater Lake clones expressing a cellulase activity and Lake Elmenteita clones expressing a lipase/esterase activity were identified and sequenced. These were encoded by novel genes as judged by DNA sequence comparisons. Genomic DNA libraries were also made from laboratory enrichment cultures of Lake Nakuru and Lake Elmenteita samples. Selective enrichment cultures were grown in the presence of carboxymethylcellulose (CMC) and olive oil. A number of new cellulase and lipase/esterase genes were discovered in these libraries. Cellulase-positive clones from Lake Nakuru were isolated at a frequency of 1 in 15,000 from a library made from a CMC enrichment as compared to 1 in 60,000 from a minimal medium enrichment. Esterase/lipase-positive clones from Lake Elmenteita were isolated with a frequency of 1 in 30,000 from a library made from an olive-oil enrichment as compared to 1 in 100,000 from an environmental library.Communicated by K. Horikoshi     

Isolation of ScFv antibodies of rP27Kip1 from phage display libraries constructed from immunized and non-immunized repertoires

Through mRNA extract, RT and a series of PCR, phage antibody libraries were constructed from rP27^{kip 1}-immunized and non-immunized mice. After only one round of selection with rP27^{kip 1}, clones from each library were chosen randomly and digested byTag I and Hinf I. 11 of 64 clones from the immunized animal had consistent restriction pattern, while none of the 64 clones from the non-immunized animal had, except that one had the same fragments pattern as that of the 11 clones. The 12 fragments were expressed inE. coli BL21(DE3)/pET-28b(+) system. ELISA showed that some of the fragments could bind to rP27^{kip 1} specifically. AU these results implied that specific antibody can be obtained by genetic engineering without hybridoma or many rounds of growth and panning selection.     

A small molecule inhibitor of fungal histone acetyltransferase Rtt109

The histone acetyltransferase Rtt109 is the sole enzyme responsible for acetylation of histone H3 lysine 56 (H3K56) in fungal organisms. Loss of Rtt109 renders fungal cells extremely sensitive to genotoxic agents, and prevents pathogenesis in several clinically important species. Here, via a high throughput chemical screen of >300,000 compounds, we discovered a chemical inhibitor of Rtt109 that does not inhibit other acetyltransferase enzymes. This compound inhibits Rtt109 regardless of which histone chaperone cofactor protein (Asf1 or Vps75) is present, and appears to inhibit Rtt109 via a tight-binding, uncompetitive mechanism.     

Dr. Auzoux's botanical teaching models and medical education at the universities of Glasgow and Aberdeen

In the 1860s, Dr. Louis Thomas Jérôme Auzoux introduced a set of papier-mâché teaching models intended for use in the botanical classroom. These botanical models quickly made their way into the educational curricula of institutions around the world. Within these institutions, Auzoux’s models were principally used to fulfil educational goals, but their incorporation into diverse curricula also suggests they were used to implement agendas beyond botanical instruction. This essay examines the various uses and meanings of Dr. Auzoux’s botanical teaching models at the universities of Glasgow and Aberdeen in the nineteenth century. The two main conclusions of this analysis are: (1) investing in prestigious scientific collections was a way for these universities to attract fee-paying students so that better medical accommodation could be provided and (2) models were used to transmit different kinds of botanical knowledge at both universities. The style of botany at the University of Glasgow was offensive and the department there actively embraced and incorporated ideas of the emerging new botany. At Aberdeen, the style of botany was defensive and there was some hesitancy when confronting new botanical ideas.     

A phylogenetic analysis of Wadi el Natrun soda lake cellulase enrichment cultures and identification of cellulase genes from these cultures

Samples of sediments and surrounding soda soils (SS) from the extremely saline and alkaline lakes of the Wadi el Natrun in the Libyan Desert, Egypt, were obtained in October 2000. Anaerobic enrichment cultures were grown from these samples, DNA isolated, and the bacterial diversity assessed by 16S rRNA gene clone analysis. Clones derived from lake sediments (LS) most closely matched Clostridium spp., Natronoincola histidinovorans, Halocella cellulolytica, Bacillus spp., and the Cytophaga–Flexibacter–Bacteroides group. Similar clones were identified in the SS, but Bacillus spp. predominated. Many of the clones were most closely related to organisms already identified in alkaline or saline environments. Two genomic DNA libraries were made from the pooled LS enrichments and a single SS-enrichment sample. A novel cellulase activity was identified and characterized in each. The lake cellulase ORF encoded a protein of 1,118 amino acids; BLASTP analysis showed it was most closely related to an endoglucanase from Xanthomonas campestris. The soil-cellulase ORF encoded a protein of 634 amino acids that was most closely related to an endoglucanase from Fibrobacter succinogenes.     

Identification of small-molecule inhibitors of Trypansoma cruzi replication

We report the outcome of a high-throughput small-molecule screen to identify novel, nontoxic, inhibitors of Trypansoma cruzi, as potential starting points for therapeutics to treat for both the acute and chronic stages of Chagas disease. Two compounds were identified that displayed nanomolar inhibition of T. cruzi and an absence of activity against host cells at the highest tested dose. These compounds have been registered with NIH Molecular Libraries Program (probes ML157 and ML158).     

Accelerated testing of mold growth on traditional and recycled book paper

The growth of molds on paper containing cellulose is a frequent occurrence when the level of relative air humidity is high or when books become wet due to water leaks in libraries. The aim of this study is to differentiate the bioreceptivity of different types of book paper for different fungi. Laboratory tests were performed with strains of Aspergillus niger, Cladosporium sp., Chaetomium globosum and Trichoderma harzianum isolated from books. Four paper types were evaluated: couché, Pólen (offset), recycled and a reference paper containing only cellulose. The tests were carried out in chambers with relative air humidity of 95% and 100%. Mold growth was greatest in the tests at 100% relative humidity. Results of stereoscopic microscopy observation showed that Cladosporium sp. grew in 74% of these samples, A. niger in 75%, T. harzianum in 72% and C. globosum in 60%. In the chambers with 95% air humidity Cladosporium sp. grew in only 9% of the samples, A. niger in 1%, T. harzianum in 3% and C. globosum did not grow in any sample. The most bioreceptive paper was couché and the least receptive was recycled paper. The composition of the recycled paper, however, varies depending on the types of waste materials used to make it.