A Simple Construction Procedure for Eesolvable Incomplete Block Designs for any Number of Treatments

A simple, straightforward procedure, which requires no special tables or generators, is presented for constructing resolvable incomplete block designs for v=pk, v=p²k, …, treatments, for k≥p, in incomplete blocks of size k. Also, it is shown, how to obtain incomplete block designs for any v in blocks of size k and k+1. The procedure allows construction of balanced incomplete block designs for p = k a prime number. For p = n not a prime number, incomplete block designs can be obtained by the procedure, but are not balanced. However, for p_s being the smallest prime factor of n, p_s + 1 for v = n², p_s²+ p_s + 1 for v = n³, …, arrangements can be obtained for which the occurrence of any treatment pair in the blocks is either zero or one. This is called a zero-one concurrence design. Procedures are described for obtaining additional zero-one concurrence arrangements. It is shown that the efficiency of these designs is maximum. Both intra-block and inter-block analyses are described.     

Re-engineering Escherichia coli for ethanol production

A lactate producing derivative of Escherichia coli KO11, strain SZ110, was re-engineered for ethanol production by deleting genes encoding all fermentative routes for NADH and randomly inserting a promoterless mini-Tn5 cassette (transpososome) containing the complete Zymomonas mobilis ethanol pathway (pdc, adhA, and adhB) into the chromosome. By selecting for fermentative growth in mineral salts medium containing xylose, a highly productive strain was isolated in which the ethanol cassette had been integrated behind the rrlE promoter, designated strain LY160 (KO11, Δfrd::celY _Ec ΔadhE ΔldhA, ΔackA lacA::casAB _Ko rrlE::(pdc _Zm -adhA _Zm -adhB _Zm -FRT-rrlE) pflB ⁺ ). This strain fermented 9% (w/v) xylose to 4% (w/v) ethanol in 48 h in mineral salts medium, nearly equal to the performance of KO11 with Luria broth.     

Using DMSO for chlorophyll spectroscopy

DMSO (dimethyl sulphoxide, (CH₃)₂SO) is an alternative solvent for spectroscopic assay of chlorophylls (Chls) but has mainly been used on Chl a & b organisms. Here, we develop algorithms for the estimation of Chl a, b & c₁c₂, and d in DMSO solvent. The common unicellular green alga Chlorella sp. (Chl a & b) is used as an example of an oxygenic photo-organism with Chl a as the primary photosynthetic pigment and Chl b as the accessory Chl. The cyanobacterium Synechococcus is used as an example of organisms containing only Chl a. The diatom Chaetoceros sp. is used as the representative Chl a & c₁c₂ organism. The unusual chlorobacterium Acaryochloris marina has Chl d & a. Algorithms for use on mixed phytoplankton and algal mats (Chl a, b & c) were also developed. The algorithms for DMSO solvent are compared to those developed for 90% acetone as the benchmark solvent. DMSO solvent offers the advantages of low volatility, low toxicity, low flammability, biodegradability, and ease of transport and is an effective extractant of chlorophylls. However, there appear to be significant storage problems with DMSO extracts of chlorophylls because of its high freezing point (+18.4°C) and probable breakdown in chlorophylls in DMSO over several days.

     

Simplified formulas for three-way chi-square testing

In order to test against contingency in a 3-way table, simplified X²-formulas are given for (1) the general r xcxl-table, (2) for the rxcx2-table, (3) for the 2x2xl-table, and (4) for the 2×2×2-table. The tests are valid only if a sample of N individuals is observed as to 3 discrete or discretisized (dichotomized) variables which are, under H₀, assumed to be totally independent.     

Kinetic mechanism for the ability of sulfate reducers to out-compete methanogens for acetate

Methanosarcina barkeri and Desulfobacter postgatei are ubiquitous anaerobic bacteria which grow on acetate or acetate plus sulfate, respectively, as sole energy sources. Their apparent K _s values for acetate were determined and found to be approximately 0.2 mM for the sulfate-reducing bacterium and 3 mM for the methanogenic bacterium. In mixed cell suspensions of the two bacteria (adjusted to equal V _max) the rate of acetate consumption by D. postgatei approached 15-fold the rate of M. barkeri at low acetate concentrations. The apparent inhibition of methanogenesis was of the same order as expected from the different K _s value for acetate. Difference in substrate affinities can thus account for the inhibition of methanogenesis from acetate in sulfate-rich environments, where the acetate concentration is well below 1 mM.     

The allometry of safety-factors for plant height

Regression curves for the relation between the critical buckling height H_crit, and the diameter D of columnar support members composed exclusively of different tissues were established based on Greenhill's formula and previously reported mean values for the density-specific stiffness and density-specific strength of parenchyma, primary xylem, sclerenchyma, and wood. These regression curves were used to determine the extent to which the actual heights H of 249 plant species approach or transgress the H_crit for stems relying principally upon different tissue-types for stiffness. Based on empirically determined H and estimated H_crit, the safety-factor H_crit/H (computed on the basis of E/p) against elastic instability resulting from self-loading imposed on stems was determined for dicot and gymnosperm tree species (N = 56), mosses (N = 40), pteridophytes (N = 16), dicot herbs (N = 120), and palms (N = 17). With the exception of tree species, H_crit/H was size-dependent, decreasing with increasing D. This was a consequence of the scaling exponents (i.e., the slopes of the regression curves) for tree H_crit, vs. D and H vs. D which were nearly identical, whereas the scaling exponents for H vs. D for “nonwoody” species were in excess of those for H_crit^, vs. D. With the exception of a few very tall specimens of palm species, however, the majority of nonwoody and woody species did not exceed their estimated H_crit. The upper size-range obtained by the procession of taller plant grades and clades was bounded by the regression curves of H_crit, vs. D established for progressively stiffer plant tissues: parenchyma Å primary xylem Å sclerenchyma Å wood. This appears to be a consequence of the incorporation of progressively stiffer tissues within the stems of taller nonwoody species and the adjustment in the girth of stems, which developmentally occurs for trees.     

Genetic manipulation tools for Dietzia spp.

Aim: To develop an applicable vector system and a transformation method for the manipulation of Dietzia spp. Methods and Results: The pNV18 Nocardia‐E. coli shuttle vector was tested and found to be a replicating plasmid in Dietzia sp. E1. With the use of pNV18, an electroporation method was optimized for the transformation of Dietzia sp. E1, and a transformation efficiency suitable for genetic manipulations was achieved (2·18 × 10⁴ transformants μg^?1 DNA). The method was also applied for the transformation of Dietzia cinnamea, D. maris, D. natronolimnaea and D. psychralcaliphila. Conclusions: The first applicable vectors and a simple electroporation protocol enabling the manipulation of several Dietzia spp. are presented. Significance and Impact of the Study: Dietzia spp. have clinical, industrial and great environmental importance; however, the analysis of the Dietzia genus is currently hampered by the lack of manipulation techniques. The presented basic tools allow the genetic analysis of several Dietzia species, including the human disease‐associated Dietzia maris.     

A robust fed-batch feeding strategy for optimal parameter estimation for baker's yeast production

Parameter identification of structured models is often a problem in biotechnology, because the poor data situation and the number of unknown parameters only allow for inaccurate estimates. But often only a subset of all kinetic parameters of the model are of interest for production purposes, e.g. for fed-batch cultivation. These parameters should be estimated with a given accuracy. In addition, the experiments for information acquisition with respect to these parameters should be as simple as possible and should consider some practical restrictions. In this contribution a fed-batch feeding strategy is proposed to allow for an accurate estimation of yield and of critical growth rate of baker's yeast. The feeding also allows for economic and stereotyped use of staff and equipment and is therefore suitable for routine use in screening of strains and media. The overall pattern is similar to that one, usually used in production scale to minimize errors by limited model validity. After an initial phase for achieving a reproducible state three different growth rates are adjusted to cover the range of possible critical growth rates. From biomass and ethanol measurements yield and critical growth rate can be estimated with an accuracy of about 2.1%. The fermentation pattern ends up with a constant feeding rate to simulate a limited oxygen transfer rate and to allow for an uptake of residual sugar and ethanol before a dough test can be carried out. Beside experimental results simulations and sensitivity analyses are shown.List of Symbols P ethanol concentration - S substrate concentration - S _f substrate concentration in feed - T fermentation time - V fermenter volume - X biomass concentration - C measurement error covariance matrix - F Fisher information matrix - X state variables - Y output variables - X _p state sensitivity functions with respect to parameters - Y _p output sensitivity functions - e eigenvectors - k vector of limitation and inhibition parameters - n number of observations - q _in feeding stream - q _b stream for samples and ammonia feed - r vector of specific turnover rates - y vector of yields - specific weight - eigenvalues - specific growth rate - _set exponent in exponential feeding - standard deviation Dedicated to the 65th birthday of Professor Fritz Wagner.A. O. Ejiofor and B. O. Solomon are grateful to the Alexander von Humboldt Stiftung for granting them fellowships and to GBF for providing all the materials necessary for their successful research stay in Germany.     

Construction of a novel promoter-probe vector and its application for screening strong promoter for Brevibacterium flavum metabolic engineering

Brevibacterium flavum is an important microorganism for the production of amino acids in industrial fermentation. Knowledge of promoters in B. flavum is essential for efficient modulation of gene expression in metabolic engineering. Here we have constructed a novel E. coli-B. flavum promoter-probe vector pDXW-11. The pDXW-11 habors an oriE for replication in E. coli, genes dso and sso for replication in B. flavum, a kan gene used as selected marker, a multiple cloning sites preceded by a rrnBT1T2 terminator and sequentially followed by stop codons, an SD sequence and a cat reporter gene. Using pDXW-11, activities of several promoters were evaluated in B. flavum. A strong promoter, the tac-M promoter, was designed. The tac-M promoter would be very useful for metabolic engineering research in B. flavum.     

Development of nitrilase promoter-derived inducible vectors for Streptomyces

An inducible expression vector, pSH19, which harbors regulatory expression system P_nitA-NitR, for streptomycetes was constructed previously. Here, we have modified pSH19 to obtain shuttle vectors for Streptomyces-E. coli by introducing the replication origin of a plasmid for E. coli (ColE1) and an antibiotic-resistant gene. Six inducible shuttle vectors, pESH19cF, pESH19cR, pESH19kF, pESH19kR, pESH19aF, and pESH19aR, for Streptomyces-E. coli, were successfully developed. The stability of these vectors was examined in five different E. coli strains and Streptomyces lividans TK24. The stability test showed that the pSH19-derived shuttle vectors were stable in E. coli Stbl2 and S. lividans TK24. Heterologous expression experiments involving each of the catechol 2,3-dioxygenase, nitrilase, and N-substituted formamide deformylase genes as a reporter gene showed that pESH19cF, pESH19kF, and pESH19aF possess inducible expression ability in S. lividans TK24. Thus, these vectors were found to be useful expression tools for experiments on both Gram-negative and Gram-positive bacterial genes.     

mRNA extraction of Xanthomonas fragariae in strawberry and validation of reference genes for the RT-qPCR for study of bacterial gene expression

This is the first study which describes a unique procedure of isolating of high-quality, intact RNA from strawberry leaves of Xanthomonas fragariae, three most suitable reference genes, crucial for the normalization of RT-qPCR data for this pathogen and accurate expression analysis of target genes. In our study, various mathematic algorithms: NormFinder geNorm, BestKeeper, the delta CT method, RefFinder were adopted for validation of most stable reference genes from nine candidate genes (ffh, glyA, gyrA, gyrB, proC, pykA, recA, rpoB, rpoD). The analyses allowing to select three most suitable pioneer reference genes, gyrB, ffh, and pykA, that we recommend for the normalization of RT-qPCR data and for the study of the expression of target genes in Xf. Moreover, their combination as references allowed for an accurate expression analysis and computation of the fold change of the flhF and iroN2 genes in Xf. These two genes are important for the success of the colonization of plant tissue and pathogenicity and sequences of primers designed to study these genes, are presented.

     

A simple model for determining affinity from irreversible thermal shifts

Thermal denaturation (Tm) data are easy to obtain; it is a technique that is used by both small labs and large‐scale industrial organizations. The link between ligand affinity (K _D) and ΔTm is understood for reversible denaturation; however, there is a gap in our understanding of how to quantitatively interpret ΔTm for the many proteins that irreversibly denature. To better understand the origin, and extent of applicability, of a K _D to ΔTm correlate, we define equations relating K _D and ΔTm for irreversible protein unfolding, which we test with computational models and experimental data. These results suggest a general relationship exists between K _D and ΔTm for irreversible denaturation.     

Vanadate‐Based Materials for Li‐Ion Batteries: The Search for Anodes for Practical Applications

While the practical application of electrode materials depends intensively on the Li⁺ ion storage mechanisms correlating ultimately with the coulombic efficiency, reversible capacity, and morphology variation of electrode material upon cycling, only intercalation‐type electrode materials have proven viable for commercialization up to now. This paper reviews the promising anode materials of metal vanadates (M_xV_yO_z, M = Co, Cu, Mn, Fe, Zn, Ni, Li) that have high capacity, low cost, and abundant resource, and also discusses the related Li⁺ ion storage mechanism. It is concluded that most of these (M_xV_yO_z, M = Co, Cu, Mn, Fe, Zn, Ni) exhibit irreversible redox reactions upon lithiation/delithiation accompanied by large volume expansion, which is not favorable for industrial applications. In particular, Li₃VO₄ with specific intercalation Li⁺ ion storage mechanism and compatible merits of safety and energy density exhibits great potential for practical application. This review systematically summarizes the latest progress in Li₃VO₄ research, including the representative fabrication approaches for advanced morphology and state‐of‐the‐art technologies to boost performance and the morphology variation associated with Li⁺ ion storage mechanisms. Furthermore, an outlook on where breakthroughs for Li₃VO₄ may be most likely achieved will be provided.     

Bubble-column design for growth of fragile insect cells

A mathematical model for the design of bubble-columns for growth of shear-sensitive insect cells is presented. The model is based on two assumptions. First, the loss of cell viability as a result of aeration is a first-order process. Second, a hypothetical volume X, in which all viable cells are killed, is associated with each air bubble during its lifetime. The model merely consists of an equation for k _d, the first-order death-rate constant, and A _min, the minimum specific surface area of the air bubbles to supply sufficient oxygen. In addition to X, the equation for k _d contains the air flow F, the air-bubble diameter d _b, the diameter D and the height H of the bubble column. This equation has been experimentally validated. Comparison of the equations for k _d and A _min shows that especially H is the key parameter to manipulate in bubble-column design in order to meet the demands set by A _min and k _dg, the first-order growth-rate constant. It is concluded that net growth of cells is enhanced as size and height of the bubble column increase.     

Chemotactic collapse for the Keller-Segel model

 This work is concerned with the system (S) {u _t =Δu − χ∇ (u∇v) for x∈Ω, t>0Γ v _t =Δv+(u−1) for x∈Ω, t>0 where Γ, χ are positive constants and Ω is a bounded and smooth open set in ℝ². On the boundary ∂Ω, we impose no-flux conditions: (N) ∂u∂n =∂v∂n =0 for x∈∂ Ω, t>0 Problem (S), (N) is a classical model to describe chemotaxis corresponding to a species of concentration u(x, t) which tends to aggregate towards high concentrations of a chemical that the species releases. When completed with suitable initial values at t=0 for u(x, t), v(x, t), the problem under consideration is known to be well posed, locally in time. By means of matched asymptotic expansions techniques, we show here that there exist radial solutions exhibiting chemotactic collapse. By this we mean that u(r, t) →Aδ(y) as t→T for some T<∞, where A is the total concentration of the species. Received 9 March 1995; received in revised form 25 December 1995     

Thermal niche for seed germination of Xyris species from Brazilian montane vegetation: Implications for climate change

Understanding how climate change will affect regeneration from seeds is important for developing conservation strategies. We evaluated seed germination requirements for sympatric species of Xyris from montane rupestrian grasslands (campo rupestre) in Brazil to determine their thermal niche and thermal requirements for seed germination. We also assessed whether projected temperature increases would affect seed germination of the species. Seed germination was evaluated at a wide range of constant temperatures (10–40°C) under light (12-hr photoperiod) and dark conditions. Base temperatures (T_b) and thermal times for 50% germination (θ₅₀) were calculated for three species. The effects of projected mean temperature increase on seed germination percentage and timing were evaluated. All species revealed an absolute light requirement for germination. Thermal germination niche breadth was greatest for X. asperula (15 to 35°C) and narrowest for X. seubertii (20 and 25°C). Base temperatures for X. asperula, X. pilosa and X. trachyphylla were 9.0, 12.8 and 11.1°C, respectively. In the scenario with the highest temperature increase (A2), the greatest reductions in seed germination are observed for X. pilosa and X. seubertii. The lowest projected temperature increase (2°C) was sufficient to decrease by 1 day the germination time of X. asperula and X. pilosa. Species of Xyris do not present a pattern for thermal germination niche and thermal requirements values, indicating that the effects of climate warming on the regeneration of these seeds will probably vary among species.     

Marine macroalgae as a source for osmoprotection for Escherichia coli

At elevated osmolarity of the mineral medium M63, marine macroalgae constitute important osmoprotectants and nutrients sources for Escherichia coli. Growth of bacterial population (16 strains) was improved by supplementing M63 salts medium with either aqueous or ethanolic algal extracts obtained from Ascophyllum nodosum, Fucus serratus, Enteromorpha ramulosa, Ulva lactuca, and Palmaria palmata. In their presence, growth was still observed even at 1.02 m NaCl. Furthermore, the E. coli ZB400 growth in presence of whole macroalgae thalli in M63/0.85 m NaCI reached its maximum within 24 h (5 × 10⁷ – 5 × 10⁸ colony-forming units [CFU] per milliliter). In the presence of A. nodosum, bacterial growth was inhibited. In the same experimental conditions, ethanolic extracts improved E. coli growth significantly, because the yield reached 10¹¹ CFU per milliliter. Ulva lactuca and P. palmata allowed the better growth. The Dragendorff-positive compounds extracted from bacterial cells growing on each ethanolic extract exhibited an osmoprotective effect as proved by a disk-diffusion assay. On the other hand, the -onium compounds (quaternary ammonium [betaines] and tertiary sulphonium) and total free amino acid contents of U. lactuca ethanolic extracts were higher than in others. Fucaceae extracts demonstrated especially high protein content. Algal extracts constitute not only an appreciable osmoprotection source for E. coli but also nutrient sources. Correspondence to: J. Minet     

Mapping and marker-assisted selection for a gene for extreme resistance to potato virus Y

The chromosomal location of the major gene Ry _adg controlling extreme resistance to potato virus Y (PVY) in Solanum tuberosum subsp. andigena was identified by RFLP analysis of a diploid potato population. A total of 64 tomato and potato RFLP markers were screened with the bulked segregant analysis (BSA) on segregants extremely resistant, hypersensitive or susceptible to PVY. Four markers TG508, GP125, CD17 and CT168 at the proximal end of chromosome XI showed close linkage with extremely resistant phenotypes. TG508 was identified as the closest marker linked with the Ry _adg locus with the maximum map distance estimated as 2.0 cM. The 4 markers linked with the Ry _adg locus were tested on independent tetraploid and diploid potato clones and were subsequently found useful for marker-assisted selection for plants containing Ry _adg . Received: 5 July 1996 / Accepted: 19 July 1996     

A search for linkage between genes for albinism and parathion resistance inTetranychus Pacificus McGregor

The inheritance of resistance to parathion in the Pacific spider mite was investigated by means of a backcross. A 1:1 ratio of a susceptible and a resistant class indicated the existence of a single dominant gene for resistance.Since the susceptible parental strain was marked with a recessive for albinism, information was obtained of the genetic relationship between the gene for resistance (R _p ) and the gene for albinism (p). The segregation of resistant (R _p r _p ) and susceptible (r _p r_p) classes in pigmented (p+p) and albino (pp) animals revealed that there was no linkage between these genes.     

The Discrete Distribution Used for the Log-rank Test Can Be Inaccurate

When the number of tumors is small, a significance level for the Cox-Mantel (log-rank) test Z is often computed using a discrete approximation to the permutation distribution. For j = 0,…, J let N_j(t) be the number of animals in group j alive and tumor-free at the start of time t. Make a 2 × (1+J) table for each time t of the number of animals R_j(t) with newly palpated tumor out of the total N_j(t) at risk. There are a total of say K tables, one for each distinct time t with observed death or newly palpated tumor. The usual discrete approximation to the permutation distribution of Z is defined by taking tables to be independent with fixed margins N_j(t) and ΣR_j(t) for all t. However, the N_j(t) are random variables for the actual permutation distribution of Z, resulting in dependence among the tables. Calculations for the exact permutation distribution are explained, and examples are given where the exact significance level differs substantially from the usual discrete approximation. The discrepancy arisis primarily because permutations with different Z-scores under the exact distribution can be equal for the discrete approximation, inflating the approximate P-value.