High-performance bioreactors: a new generation

Compact loop bioreactors with a total volume of 4 l were equipped with electrically controllable valves and pumps, most of the currently available on-line sensors, direct digital control systems and a process minicomputer; complete automatic operation was thus achieved. These reactors perform excellently: the mixing time is <1 s, oxygen transfer is not a limiting factor and the precision of control of more than 10 process variables is much better than so far reported on other systems. Such high-performance bioreactors were used in investigating the stable synchronous oscillations of Saccharomyces-type yeasts and the reduction of slightly soluble organic compounds with biocatalysts. The inherent advantages of on-line measurement (and control) are discussed.     

Toward a new generation of therapeutics

Scientific evidence in the prevention and treatment of various disorders is accumulating regarding probiotics. The health benefits supported by adequate clinical data include increased resistance to infectious disease, decreased duration of diarrhea, management of inflammatory bowel disease, reduction of serum cholesterol, prevention of allergy, modulation of cytokine gene expression, and suppression of carcinogen production. Recent ventures in metabolic engineering and heterologous protein expression have enhanced the enzymatic and immunomodulatory effects of probiotics and, with time, may allow more active intervention among critical care patients. In addition, a number of approaches are currently being explored, including the physical and chemical protection of cells, to increase probiotic viability and its health benefits. Traditional immobilization of probiotics in gel matrices, most notably calcium alginate and κ-carrageenan, has frequently been employed, with noted improvements in viability during freezing and storage. Conflicting reports exist, however, on the protection offered by immobilization from harsh physiologic environments. An alternative approach, microencapsulation in “artificial cells,” builds on immobilization technologies by combining enhanced mechanical stability of the capsule membrane with improved mass transport, increased cell loading, and greater control of parameters. This review summarizes the current clinical status of probiotics, examines the promises and challenges of current immobilization technologies, and presents the concept of artificial cells for effective delivery of therapeutic bacterial cells.     

HOUDINI: a new approach to computer-based structure generation

A new method of structure generation called convergent structure generation has been developed to address limitations of earlier methods. The features of the program (HOUDINI) based on this method include the following: a single integrated representation of the collective substructural information; the use of parallel atom groups for efficient processing of families of alternative substructural inferences; and a managed structure generation procedure designed to build required structural features early in the process.     

Discotic liquid crystals: a new generation of organic semiconductors

Discotic (disc-like) molecules typically comprising a rigid aromatic core and flexible peripheral chains have been attracting growing interest because of their fundamental importance as model systems for the study of charge and energy transport and due to the possibilities of their application in organic electronic devices. This critical review covers various aspects of recent research on discotic liquid crystals, in particular, molecular design concepts, supramolecular structure, processing into ordered thin films and fabrication of electronic devices. The chemical structure of the conjugated core of discotic molecules governs, to a large extent, their intramolecular electronic properties. Variation of the peripheral flexible chains and of the aromatic core is decisive for the tuning of self-assembly in solution and in bulk. Supramolecular organization of discotic molecules can be effectively controlled by the choice of the processing methods. In particular, approaches to obtain suitable macroscopic orientations of columnar superstructures on surfaces, that is, planar uniaxial or homeotropic alignment, are discussed together with appropriate processing techniques. Finally, an overview of charge transport in discotic materials and their application in optoelectronic devices is given.     

Peripherally metalated secoporphyrazines: a new generation of photoactive pigments

Base-catalyzed cross condensation of dipropylmaleonitrile 1 with bis(dimethylamino)maleonitrile 2 in an equimolar ratio afforded the porphyrazines 3a, 4a, 5a, 6a and 7a. Subsequent demetalation of 5a with TFA followed by remetalation with Zn(OAc)(2) gave ligand 5c in good yield. Compound 5c was, in turn, selectively oxidized and further peripherally functionalized using Pt(PhCN)(2)Cl(2) and PdCl(2) to yield the novel seco solitaire porphyrazines 10a and 10b. The photophysical profiles of the seco solitaire porphyrazines 10a and 10b were evaluated by means of absorption, emission, and transient absorption spectroscopy. The new pigments 10a and 10b were found to be photochemically more stable than the solitaire complexes 3d and 3e and mediated the generation of singlet oxygen with quantum yields of 0.59 and 0.45, respectively.     

Study of the photocatalytic transformation of synephrine: a biogenic amine relevant in anti-doping analysis

The occurrence of some cases of positive results in anti-doping analysis of octopamine requires clarification as to whether its methylated derivative synephrine could be a metabolic precursor of octopamine itself. Synephrine is a natural phenylethylamine derivative present in some food supplements containing Citrus aurantium, permitted in sport regulations. A simulative laboratory study had been done using a photocatalytic process, to identify all possible main and secondary transformation products, in a clean matrix; these were then sought in biological samples obtained from three human volunteers and four rats treated with synephrine; the parent compound and its new potential metabolic products were investigated in human urine and rat plasma samples. The transformation of synephrine and octopamine and the formation of intermediate products were evaluated, adopting titanium dioxide as photocatalyst. Several products were formed and characterized using the HPLC-HRMSⁿ technique. The main intermediates identified in these experimental conditions were compared with the major synephrine metabolites found in in vivo studies on rats and humans. Some more oxidized species, already formed in the photocatalytic process, were also found in urine and plasma samples of treated animals. These new findings could be of interest in further metabolism studies. The main photocatalytic pathway involving synephrine appears to be N-demethylation to give octopamine. On the contrary, we demonstrate the inconsistency of this reaction in both rat and human in vivo determinations, resulting in forensic importance.     

Amorphous fluoropolymers - a new generation of products

Poly(tetrafluoroethylene) (PTFE) already known since 50 years, Is a unique material among plastics due to its chemical inertness, heat resistance, electrical insulation properties and low coefficient of friction. Its high melt viscosity needs special ways of processing. That fact led to the development of melt-processible fluoropolymers such as Perfluoroethylene-propylene (FEP) and Perfluoroalkoxy (PFA). Now we have a third generation which is an amorphous fluorpolymer made by copolymerizing tetrafluoroethylene with 2,2-bis (trifluoromethyl) - 4,5 - difluoro -1,3 - dioxole. The bulky cyclic structure prevents the normal crystallisation as with PTFE polymers. The amorphous fluoropolymers have high clarity and dissolve in selected solvents. Having C-F, C-C and C-O bonds the well known properties as high temperature and chemical resistance are retained. Dielectric constant is in the range 1.83 - 1.93 up to 10.00 MHz the lowest of any plastic material. Optical properties are also very special. Refractive index is very low, in the range 1.29 - 1.32. Transmission is high from UV to IR and the polymer is not photo-degraded. The unique electrical and optical properties, coupled with high chemical and thermal stability, plus the ability to work from solution offers a powerful tool for those working on the frontiers of technology.     

Catalytic antibodies as a new generation of biocatalysts

The present state and perspectives of the creation of a new generation of biocatalysts, catalytic antibodies (CA), and their application to organic synthesis are discussed. The problems to be solved for the development of the practical application of CA are especially noted. This paper was prepared in response to a request from the Editorial board in order to attract the attention of investigators to the creation of artificial enzymes, called catalytic antibodies, which is a new and important direction in science. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 9, pp. 1568–1574, September, 1997     

Boron-nitrogen macrocycles: a new generation of calix[3]arenes

The simple condensation reaction of 3,5-di-tert-butyl salicylaldehyde and 3-aminophenylboronic acid leads to a trimeric macrocyclic compound. The ability of this molecule to include small organic molecules was in a first approximation analyzed by (1)H NMR spectroscopy and X-ray crystallography.     

Structure generation by reduction: a new strategy for computer-assisted structure elucidation

A problem common to computer programs for structure elucidation is the efficient and prospective use of the input information to constrain the structure generation process. The input may consist of potentially overlapping substructure requirements and alternative substructure interpretations of spectral data. Other useful information may be structural features that must not be present in the output structures. All of these may interact in a complex manner that is impossible to determine by use of a bond-by-bond structure assembly algorithm. A new method is described called structure reduction. In contrast to structure assembly, this method begins with a set of all bonds and removes inconsistent bonds as structure generation progresses. This results in a more efficient use of the input information and the ability to use potentially overlapping required substructures. Several examples illustrate the application of our computer program COCOA, which uses this method to solve real-world structure elucidation problems.     

Novel enzyme/DNA/inorganic nanomaterials: a new generation of biocatalysts

The design, synthesis and properties of a new class of enzyme/DNA/inorganic nanobiomaterials are described here. DNA has been used to stabilize the enzymes intercalated in the galleries of the inorganic solid, alpha-Zr(iv) phosphate (alpha-Zr(HPO(4))(2).H(2)O, abbreviated as alpha-ZrP). Interestingly, the presence of DNA improved the activity and stability of the bound enzymes. Key studies leading to the current strategy are presented initially, and these are followed by more recent developments. Several enzymes and proteins, including horseradish peroxidase, lysozyme, glucose oxidase, chymotrypsin, bovine serum albumin, cytochrome c, met-hemoglobin and met-myoglobin are successfully intercalated in the galleries of alpha-ZrP, under benign ambient conditions (aqueous buffered solutions, at room temperature and neutral pH). These novel materials are characterized by XRD, SEM and TEM as well as by biochemical, calorimetric and spectroscopic methods. Spectroscopic studies (circular dichroism, CD), for example, indicated that co-intercalation of DNA improved the retention of bound enzyme structure. The activity was enhanced markedly (five-fold) when DNA is co-intercalated, when compared to the activity in the absence of DNA. Addition of DNA to the sample, after enzyme intercalation, did not make any improvements. Our hypothesis is that enzyme-DNA supramolecular complex binds to the solid and the unfavorable interactions between the enzyme and the solid are minimized. These novel nanobiocomposite materials provide a simple method for packaging DNA and aid in engineering more effective synthetic materials for gene/RNA-delivery and drug delivery applications.     

Immunoaffinity capillary electrophoresis: a new versatile tool for determining protein biomarkers in inflammatory processes

Many diseases caused by inflammatory processes can progress to a chronic state causing deterioration in the quality of life and a poor prognosis for long-term survival. To address inflammatory diseases effectively, early detection and novel therapeutics are required. However, this can be challenging, in part because of the lack of early predictive biomarkers and the limited availability of adequate technologies capable of the identification/characterization of key predictive biomarkers present in biological materials, especially those found at picomolar concentrations and below. This review highlights the need for state-of-the art methodologies, with high-sensitivity and high-throughput capabilities, for determination of multiple biomarkers. Although many new biomarkers have been discovered recently, existing technology has failed to successfully bring this advancement to the patient's bedside. We present an overview of the various advances available today to extend the discovery of predictive biomarkers of inflammatory diseases; in particular, we review the technology of immunoaffinity capillary electrophoresis (IACE), which combines the use of antibodies as highly selective capture agents with the high resolving power of capillary electrophoresis. This two-dimensional hybrid technology permits the quantification and characterization of several protein biomarkers simultaneously, including subtle structural changes such as variants, isoforms, peptide fragments, and post-translational modifications. Furthermore, the results are rapid, sensitive, can be performed at a relatively low cost, without the introduction of false positive or false negative data. The IACE instrumentation can have relevance to medical, pharmaceutical, environmental, military, cultural heritage (authenticity of art work), forensic science, industrial and research fields, and in particular as a point-of-care biomarker analyzer in translational medicine.     

Urine stability and steroid profile: towards a screening index of urine sample degradation for anti-doping purpose

The presence of microorganisms in urine samples, under favourable conditions of storage and transportation, may alter the concentration of steroid hormones, thus altering the correct evaluation of the urinary steroid profile in doping control analysis. According to the rules of the World Anti-Doping Agency (WADA technical document TD2004 EAAS), a testosterone deconjugation higher than 5% and the presence of 5α-androstane-3,17-dione and 5β-androstane-3,17-dione in the deconjugated fraction, are reliable indicators of urine degradation. The determination of these markers would require an additional quantitative analysis since the steroids screening analysis, in anti-doping laboratories, is performed in the total (free + conjugated) fraction. The aim of this work is therefore to establish reliable threshold values for some representative compounds (namely 5α-androstane-3,17-dione and 5β-androstane-3,17-dione) in the total fraction in order to predict directly at the screening stage the potential microbial degradation of the urine samples. Preliminary evidence on the most suitable degradation indexes has been obtained by measuring the urinary concentration of testosterone, epitestosterone, 5α-androstane-3,17-dione and 5β-androstane-3,17-dione by gas chromatography–mass spectrometric every day for 15 days in the deconjugated, glucuronide and total fraction of 10 pools of urines from 60 healthy subjects, stored under different pH and temperature conditions, and isolating the samples with one or more markers of degradation according to the WADA technical document TD2004EAAS. The threshold values for 5α-androstane-3,17-dione and 5β-androstane-3,17-dione were therefore obtained correlating the testosterone deconjugation rate with the urinary concentrations of 5α-androstane-3,17-dione and 5β-androstane-3,17-dione in the total fraction. The threshold values suggested as indexes of urine degradation in the total fraction were: 10 ng mL⁻¹ for 5α-androstane-3,17-dione and 20 ng mL⁻¹ for 5β-androstane-3,17-dione. The validity of this approach was confirmed by the analysis of routine samples for more than five months (i.e. on a total of more than 4000 urine samples): samples with a concentration of total 5α-androstane-3,17-dione and 5β-androstane-3,17-dione higher than the threshold values showed a percentage of free testosterone higher than 5 of its total amount; whereas free testosterone in a percentage higher than 5 of its total amount was not detected in urines with a concentration of total 5α-androstane-3,17-dione and 5β-androstane-3,17-dione lower than the threshold values.     

Mass spectrometry of doping preparations of a new generation: Peroxisome proliferator-activated receptor agonists

The mass spectral properties of the peroxisome proliferator-activated receptor agonists (PPARδ) have been studied by high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) and high-performance liquid chromatography/high resolution mass spectrometry (HPLC/HRMS). Dissociation pathways of the investigated compounds under the conditions of collision activation have been proposed on the basis of the consolidated analysis of the data provided by these methods.     

High-order harmonic generation in rare gases: a new source in photoionization spectroscopy

We demonstrate that we can use the extreme ultraviolet radiation produced by high order harmonic generation to perform photoionization experiments. With harmonics from the 11th to the 69th of a 140 fs Cr:LiSAF laser operating at 825 nm, we measure the relative photoionization cross sections of xenon, krypton, argon and neon over the range 10 to 110 eV. With narrow bandwidth harmonics produced by a tunable, 1 ps dye laser, we observe the autoionizing states between the 4p ⁵ ionization thresholds in krypton.     

Microbubble generation in a co-flow device operated in a new regime

A new regime of operation of PDMS-based flow-focusing microfluidic devices is presented. We show that monodisperse microbubbles with diameters below one-tenth of the channel width (here w = 50 μm) can be produced in low viscosity liquids thanks to a strong pressure gradient in the entrance region of the channel. In this new regime bubbles are generated at the tip of a long and stable gas ligament whose diameter, which can be varied by tuning appropriately the gas and liquid flow rates, is substantially smaller than the channel width. Through this procedure the volume of the bubbles formed at the tip of the gas ligament can be varied by more than two orders of magnitude. The experimental results for the bubble diameter d(b) as function of the control parameters are accounted for by a scaling theory, which predicts d(b)/w ∝ (μ(g)/μ(l))(1/12)(Q(g)/Q(l))(5/12), where μ(g) and μ(l) indicate, respectively, the gas and liquid viscosities and Q(g) and Q(l) are the gas and liquid flow rates. As a particularly important application of our results we produce monodisperse bubbles with the appropriate diameter for therapeutic applications (d(b) ? 5 μm) and a production rate exceeding 10(5) Hz.     

Lewis acid fragmentation of a lithium aryloxide cage: generation of new heterometallic aluminium-lithium species

Heterometallic aluminium-lithium species were prepared by the fragmentation reaction of the hexametallic cage compound [Li{2,6-(MeO)(2)C(6)H(3)O}](6) (1) with alkyl aluminium derivatives. Depending on the aluminium precursor, the species formed present different nuclearities in the solid state as shown by single crystal X-ray analysis. Spectroscopic and computational studies have been performed to study the nuclearity of the synthesized compounds in solution.     

The generation and trapping of organozinc carbenoids from N-diethoxymethyl amides: a new amidocyclopropanation reaction

Amidocyclopropanes are readily prepared by reaction of N-diethoxymethyl amides with alkenes in the presence of zinc amagalm, zinc chloride and chlorotrimethylsilane.