Comparison of single-phase isotropic elastic and fibril-reinforced poroelastic models for indentation of rabbit articular cartilage

Classically, single-phase isotropic elastic (IE) model has been used for in situ or in vivo indentation analysis of articular cartilage. The model significantly simplifies cartilage structure and properties. In this study, we apply a fibril-reinforced poroelastic (FRPE) model for indentation to extract more detailed information on cartilage properties. Specifically, we compare the information from short-term (instantaneous) and long-term (equilibrium) indentations, as described here by IE and FRPE models. Femoral and tibial cartilage from rabbit (age 0–18 months) knees (n=14) were tested using a plane-ended indenter (diameter=0.544 mm). Stepwise creep tests were conducted to equilibrium. Single-phase IE solution for indentation was used to derive instantaneous modulus and equilibrium (Young's) modulus for the samples. The classical and modified Hayes’ solutions were used to derive values for the indentation moduli. In the FRPE model, the indentation behavior was sample-specifically described with three material parameters, i.e. fibril network modulus, non-fibrillar matrix modulus and permeability. The instantaneous and fibril network modulus, and the equilibrium Young's modulus and non-fibrillar matrix modulus showed significant (p<0.01) linear correlations of R²=0.516 and 0.940, respectively (Hayes’ solution) and R²=0.531 and 0.960, respectively (the modified Hayes’ solution). No significant correlations were found between the non-fibrillar matrix modulus and instantaneous moduli or between the fibril network modulus and the equilibrium moduli. These results indicate that the instantaneous indentation modulus (IE model) provides information on tensile stiffness of collagen fibrils in cartilage while the equilibrium modulus (IE model) is a significant measure for stiffness of PG matrix. Thereby, this study highlights the feasibility of a simple indentation analysis.     

An SIS patch model with variable transmission coefficients

In this paper, an SIS patch model with non-constant transmission coefficients is formulated to investigate the effect of media coverage and human movement on the spread of infectious diseases among patches. The basic reproduction number R₀ is determined. It is shown that the disease-free equilibrium is globally asymptotically stable if R₀?1, and the disease is uniformly persistent and there exists at least one endemic equilibrium if R₀>1. In particular, when the disease is non-fatal and the travel rates of susceptible and infectious individuals in each patch are the same, the endemic equilibrium is unique and is globally asymptotically stable as R₀>1. Numerical calculations are performed to illustrate some results for the case with two patches.     

Electrochemical monitoring of chlorhexidine digluconate effect on polyelectrolyte immobilized bacteria and kinetic cell adhesion

The electrochemical impedance spectroscopy (EIS) technique has been used as a sensitive method to explore the effect of antibacterial molecules on immobilized bacteria and biofilm formation. In this work, we describe the electrochemical spectroscopy as a powerful method to monitor the effect of Chlorhexidine Digluconate (CHX-Dg) on polyelectrolyte immobilized Escherichia coli K12 MG1655 and the kinetics of cell adhesion on gold electrodes. The experimental impedance data were modelised with a Zview program to find the best equivalent electrical circuit and analyse its parameter's properties. Polyelectrolyte multilayer formation on the electrode surface and bacteria immobilization greatly increased the electron-transfer resistance (R_et) and reduced the constant phase element (CPE_dl). The effect of CHX-Dg was studied in a 0.5 × 10⁻⁴ mmol l⁻¹ to 0.5 mmol l⁻¹ range. The relation between the evolution of R_et and CHX-Dg concentration was found to be negatively correlated. When CHX-Dg was added, the electrochemical monitoring of the bacterial kinetic adhesion showed that the electrode's capacity (C_P) variation remained stable, demonstrating that the addition of CHX-Dg in the broth inhibited bacterial adhesion.     

Stereoselective synthesis and characterization of the optically labile chiral Cr(III) complex Δ-(+)589{Cr[(−)bdtp]3}

The diastereoisomeric complex Δ-(+)-tris(cyclicO,O′, 1 (R), 2(R)(−)dimethylethylene dithiophosphato)chromium(III), was synthesized stereoselectively in tetrahydrofuran (THF) solution. The complex proves optically labile, [α]_D=+106, in CHCl₃, changing quickly to [α]_D=+211. The CD spectra in THF enable us to characterize the complex and show a configuration inversion which gives the diastereoisomeric equilibrium Λ⇌Δ with an excess of the Λ-(R,R)(R,R)(R,R) diastereoisomeric form. The equilibrium constant K=0.86 at 25 °C is indicative of a different thermodynamic stability between the two diastereoisomers in THF solution, Λ-(R,R)> Δ-(R,R), δΔH°=1.5 kJ mol⁻¹, δΔG°=0.3 kJ mol⁻¹, δΔS°=4 J mol⁻¹ K⁻¹. The kinetic diastereoisomer Δ-(R,R)(R,R)(R,R) is stabilized in CHCl₃, CH₂Cl₂, EtOH solvents where it is highly soluble and optically stable with a maximum negative chirality factor, g=−5×10⁻³, in CHCl₃.     

Acetylcholine Receptor Channels Activated by a Single Agonist Molecule

The neuromuscular acetylcholine receptor (AChR) is an allosteric protein that alternatively adopts inactive versus active conformations (R↔R^∗). The R^∗ shape has a higher agonist affinity and ionic conductance than R. To understand how agonists trigger this gating isomerization, we examined single-channel currents from adult mouse muscle AChRs that isomerize normally without agonists but have only a single site able to use agonist binding energy to motivate gating. We estimated the monoliganded gating equilibrium constant E₁ and the energy change associated with the R versus R^∗ change in affinity for agonists. AChRs with only one operational binding site gave rise to a single population of currents, indicating that the two transmitter binding sites have approximately the same affinity for the transmitter ACh. The results indicated that E₁ ≈ 4.3 × 10⁻³ with ACh, and ≈1.7 × 10⁻⁴ with the partial-agonist choline. From these values and the diliganded gating equilibrium constants, we estimate that the unliganded AChR gating constant is E₀ ≈ 6.5 × 10⁻⁷. Gating changes the stability of the ligand-protein complex by ∼5.2 kcal/mol for ACh and ∼3.3 kcal/mol for choline.     

Qualitative analysis of models with different treatment protocols to prevent antibiotic resistance

This paper is concerned with the qualitative analysis of two models [S. Bonhoeffer, M. Lipsitch, B.R. Levin, Evaluating treatment protocols to prevent antibiotic resistance, Proc. Natl. Acad. Sci. USA 94 (1997) 12106] for different treatment protocols to prevent antibiotic resistance. Detailed qualitative analysis about the local or global stability of the equilibria of both models is carried out in term of the basic reproduction number R₀. For the model with a single antibiotic therapy, we show that if R₀ < 1, then the disease-free equilibrium is globally asymptotically stable; if R₀ > 1, then the disease-endemic equilibrium is globally asymptotically stable. For the model with multiple antibiotic therapies, stabilities of various equilibria are analyzed and combining treatment is shown better than cycling treatment. Numerical simulations are performed to show that the dynamical properties depend intimately upon the parameters.     

Impact of group mixing on disease dynamics

A general mathematical model is proposed to study the impact of group mixing in a heterogeneous host population on the spread of a disease that confers temporary immunity upon recovery. The model contains general distribution functions that account for the probabilities that individuals remain in the recovered class after recovery. For this model, the basic reproduction number R₀ is identified. It is shown that if R₀<1, then the disease dies out in the sense that the disease free equilibrium is globally asymptotically stable; whereas if R₀>1, this equilibrium becomes unstable. In this latter case, depending on the distribution functions and the group mixing strengths, the disease either persists at a constant endemic level or exhibits sustained oscillatory behavior.     

Repellent activity of catmint, Nepeta cataria, and iridoid nepetalactone isomers against Afro-tropical mosquitoes, ixodid ticks and red poultry mites

The repellent activity of the essential oil of the catmint plant, Nepeta cataria (Lamiaceae), and the main iridoid compounds (4aS,7S,7aR) and (4aS,7S,7aS)-nepetalactone, was assessed against (i) major Afro-tropical pathogen vector mosquitoes, i.e. the malaria mosquito, Anopheles gambiae s.s. and the Southern house mosquito, Culex quinquefasciatus, using a World Health Organisation (WHO)-approved topical application bioassay (ii) the brown ear tick, Rhipicephalus appendiculatus, using a climbing repellency assay, and (iii) the red poultry mite, Dermanyssus gallinae, using field trapping experiments. Gas chromatography (GC) and coupled GC-mass spectrometry (GC-MS) analysis of two N. cataria chemotypes (A and B) used in the repellency assays showed that (4aS,7S,7aR) and (4aS,7S,7aS)-nepetalactone were present in different proportions, with one of the oils (from chemotype A) being dominated by the (4aS,7S,7aR) isomer (91.95% by GC), and the other oil (from chemotype B) containing the two (4aS,7S,7aR) and (4aS,7S,7aS) isomers in 16.98% and 69.83% (by GC), respectively. The sesquiterpene hydrocarbon (E)-(1R,9S)-caryophyllene was identified as the only other major component in the oils (8.05% and 13.19% by GC, respectively). Using the topical application bioassay, the oils showed high repellent activity (chemotype A RD₅₀ = 0.081 mg cm⁻² and chemotype B RD₅₀ = 0.091 mg cm⁻²) for An. gambiae comparable with the synthetic repellent DEET (RD₅₀ = 0.12 mg cm⁻²), whilst for Cx. quinquefasciatus, lower repellent activity was recorded (chemotype A RD₅₀ = 0.34 mg cm⁻² and chemotype B RD₅₀ = 0.074 mg cm⁻²). Further repellency testing against An. gambiae using the purified (4aS,7S,7aR) and (4aS,7S,7aS)-nepetalactone isomers revealed overall lower repellent activity, compared to the chemotype A and B oils. Testing of binary mixtures of the (4aS,7S,7aR) and (4aS,7S,7aS) isomers across a range of ratios, but all at the same overall dose (0.1 mg), revealed not only a synergistic effect between the two, but also a surprising ratio-dependent effect, with lower activity for the pure isomers and equivalent or near-equivalent mixtures, but higher activity for non-equivalent ratios. Furthermore, a binary mixture of (4aS,7S,7aR) and (4aS,7S,7aS) isomers, in a ratio equivalent to that found in chemotype B oil, was less repellent than the oil itself, when tested at two doses equivalent to 0.1 and 0.01 mg chemotype B oil. The three-component blend including (E)-(1R,9S)-caryophyllene at the level found in chemotype B oil had the same activity as chemotype B oil. In a tick climbing repellency assay using R. appendiculatus, the oils showed high repellent activity comparable with data for other repellent essential oils (chemotype A RD₅₀ = 0.005 mg and chemotype B RD₅₀ = 0.0012 mg). In field trapping assays with D. gallinae, addition of the chemotype A and B oils, and a combination of the two, to traps pre-conditioned with D. gallinae, all resulted in a significant reduction of D. gallinae trap capture. In summary, these data suggest that although the nepetalactone isomers have the potential to be used in human and livestock protection against major pathogen vectors, intact, i.e. unfractionated, Nepeta spp. oils offer potentially greater protection, due to the presence of both nepetalactone isomers and other components such as (E)-(1R,9S)-caryophyllene.     

Morphological and physiological adaptive traits of Mediterranean narrow endemic plants: The case of Centaurea gymnocarpa (Capraia Island,Italy)

A case study on Centaurea gymnocarpa Moris & De Not., a narrow endemic species, was carried out by analyzing its morphological, anatomical, and physiological traits in response to natural habitat stress factors under Mediterranean climate conditions. The results underline that the species is particularly adapted to the environment where it naturally grows. At the plant level, the above-ground/below-ground dry mass (1.73 ± 0.60) shows its investment predominately in the above-ground structure with a resulting total leaf area per plant of 1399 ± 94 cm². The senescent attached leaves at the base of the plant contribute to limit leaf transpiration by shading soil around the plant. Moreover, the dense C. gymnocarpa leaf pubescence, leaf rolling, the relatively high leaf mass area (LMA = 12.3 ± 1.3 mg cm⁻²) and leaf tissue density (LTD = 427 ± 44 mg cm⁻³) contribute to limit leaf transpiration, also postponing leaf death under dry conditions. At the physiological level, a relatively low respiration/photosynthesis ratio (R/P_N) in spring results from high R [2.26 ± 0.59 μmol (CO₂) m⁻² s⁻¹] and P_N [12.3 ± 1.5 μmol (CO₂) m⁻² s⁻¹]. The high photosynthetic nitrogen use efficiency [PNUE = 15.5 ± 0.4 μmol (CO₂) g⁻¹ (N) s⁻¹] shows the large amount of nitrogen (N) invested in the photosynthetic machinery of new leaves, associated to a high chlorophyll content (Chl = 35 ± 5 SPAD units). On the contrary, the highest R/P_N ratio (1.75 ± 0.19) in summer is due to a significant P_N decrease and increase of R in response to drought. The low PNUE [1.5 ± 0.2 μmol (CO₂) g⁻¹ (N) s⁻¹] in this season is indicative of a greater N investment in leaf cell walls which may contribute to limit transpiration. On the contrary, the low R/P_N ratio (0.05 ± 0.02) in winter is resulting from the limited enzyme activity of the respiratory apparatus [R = 0.23 ± 0.08 μmol (CO₂) m⁻² s⁻¹] while the low PNUE [3.5 ± 0.2 μmol (CO₂) g⁻¹ (N) s⁻¹] suggests that low temperatures additionally limit plant production. The experiment of the imposed water stress confirms that the C. gymnocarpa growth capability is in conformity with the severe conditions of its natural habitat, likewise as it may be the case with others narrow endemic species that have occupied niches with similar extreme conditions.     

A comprehensive study on the putative δ-opioid receptor (sub)types using the highly selective δ-antagonist, Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH

The goal of our work was a throughout characterization of the pharmacology of the TIPP-analog, Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH and see if putative δ-opioid receptor subtypes can be distinguished. Analgesic latencies were assessed in mouse tail-flick assays after intrathecal administration. In vitro receptor autoradiography, binding and ligand-stimulated [³⁵S]GTPγS functional assays were performed in the presence of putative δ₁-(DPDPE: agonist, BNTX: antagonist), δ₂-(agonist: deltorphin II, Ile^5,6-deltorphin II, antagonist: naltriben) and μ-(DAMGO: agonist) opioid ligands. The examined antagonist inhibited the effect of DPDPE by 60%, but did not antagonize δ₂- and μ-agonist induced analgesia. The radiolabeled form identified binding sites with K_D = 0.18 nM and receptor densities of 102.7 fmol/mg protein in mouse brain membranes. The binding site distribution of the [³H]Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH agreed well with that of [³H]Ile^5,6-deltorphin II as revealed by receptor autoradiography. Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH displayed 2.49 ± 0.06 and 0.30 ± 0.01 nM potency against DPDPE and deltorphin II in the [³⁵S]GTPγS functional assay, respectively. The rank order of potency of putative δ₁- and δ₂-antagonists against DPDPE and deltorphin was similar in brain and CHO cells expressing human δ-opioid receptors. Deletion of the DOR-1 gene resulted in no residual binding of the radioligand and no significant DPDPE effect on G-protein activation. Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH is a highly potent and δ-opioid specific antagonist both in vivo and in vitro. However, the putative δ₁- and δ₂-opioid receptors could not be unequivocally distinguished in vitro.     

A multiplex PCR assay to diagnose and quantify Nosema infections in honey bees (Apis mellifera)

Correct identification of the microsporidia, Nosema apis and Nosema ceranae, is key to the study and control of Nosema disease of honey bees (Apis mellifera). A rapid DNA extraction method combined with multiplex PCR to amplify the 16S rRNA gene with species-specific primers was compared with a previously published assay requiring spore-germination buffer and a DNA extraction kit. When the spore germination-extraction kit method was used, 10 or more bees were required to detect the pathogens, whereas the new extraction method made it possible to detect the pathogens in single bees. Approx. 4-8 times better detection of N. ceranae was found with the new method compared to the spore germination-extraction kit method. In addition, the time and cost required to process samples was lower with the proposed method compared to using a kit. Using the new DNA extraction method, a spore quantification procedure was developed using a triplex PCR involving co-amplifying the N. apis and N. ceranae 16S rRNA gene with the ribosomal protein gene, RpS5, from the honey bee. The accuracy of this semi-quantitative PCR was determined by comparing the relative band intensities to the number of spores per bee determined by microscopy for 23 samples, and a high correlation (R² = 0.95) was observed. This method of Nosema spore quantification revealed that spore numbers as low as 100 spores/bee could be detected by PCR. The new semi-quantitative triplex PCR assay is more sensitive, economical, rapid, simple, and reliable than previously published standard PCR-based methods for detection of Nosema and will be useful in laboratories where real-time PCR is not available.     

In Vitro Characterization of a Recombinant AHL-Lactonase from Bacillus cereus Isolated from a Striped Catfish (Pangasianodon hypophthalmus) Pond

aiiA gene encoding AHL-lactonase was isolated from Bacillus cereus strain N26.2, originating from a striped catfish (Pangasianodon hypophthalmus) pond in Vietnam. This gene, abbreviated as aiiA(N26.2), was cloned and expressed in a competent Escherichia coli strain BL21(DE3)pLysS. The resulting protein, abbreviated as AiiA_N26.2, was highly active in the pH range of 6–8 and could retain 80 % of the maximum activity under storage for 5 days at 4 °C or for 3 days at 20 °C. These properties of AiiA_N26.2 protein confers its future application via feed supplementation, with the purpose of controlling aquaculture pathogens which regulate the virulence via a quorum sensing system.     

The effects of human movement on the persistence of vector-borne diseases

With the recent resurgence of vector-borne diseases due to urbanization and development there is an urgent need to understand the dynamics of vector-borne diseases in rapidly changing urban environments. For example, many empirical studies have produced the disturbing finding that diseases continue to persist in modern city centers with zero or low rates of transmission. We develop spatial models of vector-borne disease dynamics on a network of patches to examine how the movement of humans in heterogeneous environments affects transmission. We show that the movement of humans between patches is sufficient to maintain disease persistence in patches with zero transmission. We construct two classes of models using different approaches: (i) Lagrangian models that mimic human commuting behavior and (ii) Eulerian models that mimic human migration. We determine the basic reproduction number R₀ for both modeling approaches. We show that for both approaches that if the disease-free equilibrium is stable (R₀<1) then it is globally stable and if the disease-free equilibrium is unstable (R₀>1) then there exists a unique positive (endemic) equilibrium that is globally stable among positive solutions. Finally, we prove in general that Lagrangian and Eulerian modeling approaches are not equivalent. The modeling approaches presented provide a framework to explore spatial vector-borne disease dynamics and control in heterogeneous environments. As an example, we consider two patches in which the disease dies out in both patches when there is no movement between them. Numerical simulations demonstrate that the disease becomes endemic in both patches when humans move between the two patches.     

Silver(I) acetylides stabilized by η-alkynes: Synthesis, reaction chemistry and solid state structures

Individual synthetic routes to heterobimetallic Ti(IV)-Ag(I) acetylides of type {[Ti](μ-σ,π-CCR¹)₂}AgCCR² ([Ti] = (η⁵-C₅H₄SiMe₃)₂Ti: R¹ = SiMe₃: 6, R² = SiMe₃; 7, R² = Ph. R¹ = ^tBu: 8, R² = SiMe₃; 9, R² = Ph. [Ti] = (η⁵-C₅H₅)₂Ti): 10, R¹ = ^tBu, R² = SiMe₃) including (i) the reaction of {[Ti](μ-σ, π-CCR¹)₂}AgNO₃ ([Ti] = (η⁵-C₅H₄SiMe₃)₂Ti): 1, R¹ = SiMe₃; 2, R¹ = ^tBu. [Ti] = (η⁵-C₅H₅)₂Ti: 3, R¹ = ^tBu) with LiCCR² (4, R² = SiMe₃; 5, R² = Ph) and (ii) treatment of [Ti](CCSiMe₃)₂ ([Ti] = (η⁵-C₅H₄SiMe₃)₂Ti) (11) with [AgCCR²] (12, R² = SiMe₃; 13, R² = Ph) are described. The reactions of 1-3 with 4 or 5 appeared to be sensitive towards stoichiometry because an excess of 4 or 5 resulted in the formation of [(Ag(CCR²)₂)Li(OEt₂)]_n (14) and [Ti](CCR¹)₂. Coordination polymer 14 is also accessible, when, for example, [AgCCSiMe₃] (12) is treated with 1 eq. of LiCCSiMe₃ (4) in diethyl ether.The titanium(IV)-silver(I) acetylides 6-10 are stable in the dark and at low temperature, while on exposure to light and on heating they decompose to give R²CC-CCR² together with [Ti](CCR¹)₂ and elemental silver.Complexes 6-10 contain a mono-nuclear AgCCR² entity stabilized by the chelate-bonded organometallic π-tweezer molecule [Ti](CCSiMe₃)₂, which was evinced by structure determination of 7 in the solid state. In 14 linear [Me₃SiCC-Ag-CCSiMe₃]⁻ units are connected by [Li(OEt₂)]⁺ building blocks forming a coordination polymer.     

Redox-controlled backbone dynamics of human cytochrome c revealed by N NMR relaxation measurements

Redox-controlled backbone dynamics in cytochrome c (Cyt c) were revealed by 2D ¹⁵N NMR relaxation experiments. ¹⁵N T₁ and T₂ values and ¹H-¹⁵N NOEs of uniformly ¹⁵N-labeled reduced and oxidized Cyt c were measured, and the generalized order parameters (S²), the effective correlation time for internal motion (τ_e), the ¹⁵N exchange broadening contributions (R_ex) for each residue, and the overall correlation time (τ_m) were estimated by model-free dynamics formalism. These dynamic parameters clearly showed that the backbone dynamics of Cyt c are highly restricted due to the covalently bound heme that functions as the stable hydrophobic core. Upon oxidation of the heme iron in Cyt c, the average S² value was increased from 0.88 ± 0.01 to 0.92 ± 0.01, demonstrating that the mobility of the backbone is further restricted in the oxidized form. Such increases in the S² values were more prominent in the loop regions, including amino acid residues near the thioether bonds to the heme moiety and positively charged region around Lys87. Both of the regions are supposed to form the interaction site for cytochrome c oxidase (CcO) and the electron pathway from Cyt c to CcO. The redox-dependent mobility of the backbone in the interaction site for the electron transfer to CcO suggests an electron transfer mechanism regulated by the backbone dynamics in the Cyt c-CcO system.     

Kinetic and Thermodynamic Characterization of Glucoamylase from Colletotrichum sp. KCP1

Extracellular glucoamylase of Colletotrichum sp. KCP1 produced through solid state fermentation was purified by two steps purification process comprising ammonium sulphate precipitation followed by gel permeation chromatography (GPC). The Recovery of glucoamylase after GPC was 50.40 % with 19.3-fold increase in specific activity. The molecular weight of enzyme was found to be 162.18 kDa by native-PAGE and was dimeric protein of two sub-units with molecular weight of 94.62 and 67.60 kDa as determined by SDS-PAGE. Activation energy for starch hydrolysis was 26.45 kJ mol⁻¹ while temperature quotient (Q₁₀) was found to be 1.9. The enzyme was found to be stable over wide pH range and thermally stable at 40–50 °C up to 120 min while exhibited maximum activity at 50 °C with pH 5.0. The pKa₁ and pKa₂ of ionisable groups of active site controlling V_max were 3.5 and 6.8, respectively. V_max, K_m and K_cat for starch hydrolysis were found to be 58.82 U ml⁻¹, 1.17 mg (starch) ml⁻¹ and 449 s⁻¹, respectively. Activation energy for irreversible inactivation (E_a(d)) of glucoamylase was 74.85 kJ mol⁻¹. Thermodynamic parameters of irreversible inactivation of glucoamylase and starch hydrolysis were also determined.     

Construction and evaluation of pMycoFos, a fosmid shuttle vector for Mycobacterium spp. with inducible gene expression and copy number control

Molecular tools for Gram-positive bacteria such as Mycobacterium are less well-developed than those for Gram-negatives such as Escherichiacoli. This has slowed the molecular-genetic characterisation of Mycobacterium spp, which is unfortunate, since this genus has high medical, environmental and industrial significance. Here, we developed a new Mycobacterium shuttle vector (pMycoFos, 12.5 kb, Km^R) which combines desirable features of several previous vectors (controllable copy number in E. coli, inducible gene expression in Mycobacterium) and provides a new multiple cloning site compatible with large inserts of high-GC content DNA. Copy number control in E. coli was confirmed by the increased Km^R of cultures after arabinose induction and the greater DNA yield of vector from arabinose-induced cultures. Measurement of beta-galactosidase activity in pMycoFos clones carrying the lacZ gene showed that in Mycobacterium smegmatis mc²-155, expression was inducible by acetamide, but in E. coli EPI300, the expression level was primarily determined by the vector copy number. Examination of protein profiles on SDS-PAGE gels confirmed the beta-galactosidase assay results. Construction of a fosmid library with the new vector confirmed that it could carry large DNA inserts. The new vector enabled the stable cloning and expression of an ethene monooxygenase gene cluster, which had eluded previous attempts at heterologous expression.     

Prediction of moisture, calorific value, ash and carbon content of two dedicated bioenergy crops using near-infrared spectroscopy

The potential of near infrared spectroscopy in conjunction with partial least squares regression to predict Miscanthus xgiganteus and short rotation coppice willow quality indices was examined. Moisture, calorific value, ash and carbon content were predicted with a root mean square error of cross validation of 0.90% (R² = 0.99), 0.13 MJ/kg (R² = 0.99), 0.42% (R² = 0.58), and 0.57% (R² = 0.88), respectively. The moisture and calorific value prediction models had excellent accuracy while the carbon and ash models were fair and poor, respectively. The results indicate that near infrared spectroscopy has the potential to predict quality indices of dedicated energy crops, however the models must be further validated on a wider range of samples prior to implementation. The utilization of such models would assist in the optimal use of the feedstock based on its biomass properties.     

Application of vibrational spectroscopy in the quality assessment of Buchu oil obtained from two commercially important Agathosma species (Rutaceae)

Quality assessment of natural raw materials and derived consumer products is often done using conventional analytical techniques such as liquid and gas chromatography which are expensive and time consuming. This paper reports on the use of vibrational spectroscopy techniques as possible alternatives for the rapid and inexpensive assessment of the quality of ‘buchu oil’ obtained from two South African species; Agathosma betulina and Agathosma crenulata belonging to the Rutaceae family. Samples of A. betulina (55) and A. crenulata (16) were collected from different natural localities and cultivation sites in South Africa. The essential oil was obtained by hydrodistillation and scanned on Near infrared (NIR), mid infrared (MIR) and Raman spectrometers. The spectral data obtained was processed using chemometric techniques and orthogonal partial least squares discriminant analysis (OPLS-DA) was used to clearly differentiate between A. betulina and A. crenulata. The OPLS-DA technique also proved to be a useful tool to identify wave regions that contain biomarkers (peaks) that contributed to the separation of the two species. The three spectroscopy techniques were also evaluated for their ability to accurately predict the percentage composition of seven major compounds that occur in A. betulina ‘buchu’ oil. Using GC–MS reference data, calibration models were developed for the MIR, NIR and Raman spectral data to predict/profile the major compounds in ‘buchu oil’. A comparison of the three spectroscopy techniques showed that MIR together with PLS algorithms produced the best model (R²X = 0.96; R²Y = 0.88 and Q²Ycum = 0.85) for the quantification of six of the seven major oil constituents. The MIR model showed high predictive power for pseudo-diosphenol (R² = 0.97), isomenthone (R² = 0.97), menthone (R² = 0.90), limonene (R² = 0.91), pulegone (R² = 0.96) and diosphenol (R² = 0.85). These results illustrate the potential of MIR spectroscopy as a rapid and inexpensive alternative to predict the major compounds in buchu oil.     

Estimation of the quality of refined protein crystal structures

Crystallographic R_work and R_free values, which are measures of the ability of the models of macromolecular structures to explain the crystallographic data on which they are based, are often used to assess structure quality. It is widely known, and confirmed here that both are sensitive to the methods used to compute them, and can be manipulated to improve the apparent quality of the model. As an alternative it is proposed here that the quality of crystallographic models should be assessed using a global goodness-of-fit metric R_O2A/R_work where R_O2A is the number of reflections used for refinement divided by the number of nonhydrogen atoms in the structure, and R_work is the working R-factor of the refined structure. Also, analysis of structures in the Protein Data Bank suggests that many data sets have been truncated at high resolution, thereby improving the R-factor statistics. To discourage this practice, it is proposed that the resolution of a dataset be defined as the resolution of the shell of data where <I/σ_I> falls to 1. The proposed goodness-of-fit metric encourages investigators to use all the data available rather than a truncated subset.