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Drainage and conversion of natural peatlands, which increases fire frequency, haze air pollution and carbon emissions, also affects the physical and chemical properties of peat soils. Although there has been continued interest in research on tropical peat soil properties, no attempt has yet been made to synthesise these results. We conducted a systematic literature review and meta-analysis of sixty-six papers published in English language academic literature to explore the current state of knowledge of peat soil properties of Southeast Asia and to compare physical and chemical peat properties (e.g. bulk density, carbon content, pH) under different land uses and depths. Most of these studies were undertaken in Indonesia (56.1%) and Malaysia (28.8%), where substantial tracts of peat soils occur. We extracted data from these papers to calculate the mean of each peat property and compare results between land uses and depths. Linear mixed-effects models were used to test the significance of land use and depth on each peat property. We found that bulk density (44 papers), carbon (C) content (43 papers), pH (42 papers) and nitrogen (N) content (39 papers) were the most widely reported, while other properties remain less studied. Bulk density, pH, phosphorus (P) and calcium (Ca) showed significant differences between land uses and depths. Fibre fraction, potassium (K), iron (Fe) and zinc (Zn) levels showed a significant difference between land uses only, while N differed significantly only between soil depths. Other physical properties such as hydraulic conductivity, porosity, woody fraction, amorphic fraction and chemical properties such as electrical conductivity (EC), C, ammonium (NH4+), nitrate (NO3−), available nitrogen (available N), magnesium (Mg), aluminium (Al), copper (Cu), manganese (Mn), sulphur (S) and silicon (Si) showed no significant differences between land uses or depths. This review identifies key research gaps, including underrepresented geographic areas and peat properties and highlights the need for standardised methodologies for measuring peat soil properties.

Antimicrobial peptides (AMPs), which typically disrupt the bacterial wall prompting leakage or lysis of the cell, form a growing contingent in the arsenal against antibiotic resistant bacteria. The effectiveness of AMPs is, however, hampered by their low solubility, general chemical and physical instability, and short half-life in vivo. Lipid nanocarriers such as cubosomes are effective at encapsulating and protecting proteins while simultaneously showing promise in delivery applications. Here, the efficacy of cubosome mediated delivery of AMPs is evaluated by the in-situ surface characterization of model membranes with varying composition. The cubosomes were observed to initially fuse with the membranes, with subsequent membrane disruption observed after approximately 20 - 60 min. The time for the disruption was sensitive to the charge of the cubosome as well as the composition of the bilayer. More physiologically relevant bilayers including lipids with phospho-(1'-rac-glycerol) (PG) or phosphoethanolamine (PE) headgroups were more vulnerable than those of neat phosphocholine (PC). Notably, disruption to the bilayer occurred an order of magnitude faster for encapsulated AMP compared to free AMP.

A novel chemoselective [3 + 2] annulation reaction of easily accessible ketoxime acetate with 2-aryl-3-ethoxycarbonyl pyrroline-4,5-dione has been developed for the synthesis of unknown pyrrolo[2,3-b]pyrrole frameworks. This method involves copper-mediated N–O bond cleavage followed by the formation of carbon–carbon and carbon–nitrogen bonds. This operationally simple protocol provides broader functional group compatibility and good yields.

The resistance of pathogenic bacteria toward traditional biocidal treatment methods is a growing concern in various settings, including that of water treatment and in the medical industry. As such, advanced antibacterial technologies are needed to prevent infections, against which current antibiotics are failing. This study introduces copper oxide nanoparticles (CuONPs) doped in graphene oxide (GO) as a potential pathogenic bacterial treatment. The aim of the study was to evaluate the antibacterial properties of the GO-CuONP hybridized material against pathogenic Escherichia coli ATCC 8739 (E. coli) and Salmonella typhimurium ATCC 14028 (S. typhimurium). GO was synthesized using a modified Hummer's method and doped with 40% w/w CuONPs using a series of thermal chemical reactions. The resulting hybrids were then characterized using scanning electron microscopic (SEM) and spectroscopic studies. These studies revealed that the hybrid material was considerably altered by the inclusion of CuONPs. The live and dead bacteria attached to the GO-CuONP material were detected using confocal laser scanning microscopy (CLSM). The antibacterial activity assay of the GO-CuONP material was conducted using a standard plate count method. Importantly, the GO-CuONP nanocomposite was determined to be an effective antibacterial nanomaterial, significantly inhibiting the growth of both E. coli and S. typhimurium bacteria compared to that observed on the pristine GO material. This study suggests that GO-CuONP composites are a promising high-efficacy antibacterial nanomaterial.

Here, we report a microwave-assisted, one-pot, three-component, 1,3-dipolar cycloaddition reaction to produce highly regioselective and stereoselective bis-spirooxindoles as potential inhibitors against amyloid-β fibrillation. Ease of synthesis, promising anti-amyloidogenic activity, low toxicity, and in vitro blood brain barrier permeability makes these compounds attractive therapeutic leads to treat Alzheimer's disease.

A novel chemoselective [3 + 2] annulation reaction of easily accessible ketoxime acetate with 2-aryl-3-ethoxycarbonyl pyrroline-4,5-dione has been developed for the synthesis of unknown pyrrolo[2,3

The increase in resistant bacterial strains necessitates the identification of new antimicrobial molecules. Antimicrobial peptides (AMPs) are an attractive option because of evidence that bacteria cannot easily develop resistance to AMPs. The peptaibols, a class of naturally occurring AMPs, have shown particular promise as antimicrobial drugs, but their development has been hindered by their mechanism of action not being clearly understood. To explore how peptaibols might interact with membranes, circular dichroism, vibrational circular dichroism, linear dichroism, Raman spectroscopy, Raman optical activity, neutron reflectivity and molecular dynamics simulations have been used to study a small library of peptaibol mimics, the Aib‐rich peptides. All the peptides studied quickly partitioned and oriented in membranes, and we found evidence of chiral interactions between the phospholipids and membrane‐embedded peptides. The protocols presented in this paper open new ground by showing how chiro‐optical spectroscopies can throw light on the mechanism of action of AMPs., Sit up or lie down: Linear dichroism, circular dichroism and Raman spectroscopy, neutron reflectivity and molecular dynamics simulations have been used to explore how mimics of peptaibol antibiotics (Aib foldamers) interact with membranes. These studies revealed all peptides partitioned well into the bilayers, with orientation dependent upon peptide length, as well as chiral interactions between peptides and the phospholipids.

Keratin was extracted from different segments of disposable waste chicken feathers (CF) including the whole feathers, calamus/rachis (β-sheet) and barbs/barbules (α-helix), using sodium sulfide and l -cysteine. The yield of extracted keratin from sodium sulfide and l -cysteine was ˜88% and ˜66% respectively. The mass ratio of feathers to reducing agent was 1:20 and the reaction temperature was 40 °C for 6 h. Concentration of keratin extracted by each method was measured using the Bradford assay. The protein extracted from each feather section was characterised using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, vibrational spectroscopy including FTIR and Raman, nuclear magnetic resonance, and thermogravimetry. These results confirmed the keratin structures after each extraction methods. The study showed that α-helix and s-sheet based keratin could be extracted from CF using sodium sulfide and l -cysteine with high yields. This is the first report of CF keratin extraction using l -cysteine.

Aquaculture represents a major part of the world&rsquo, s food supply. This area of food production is developing rapidly, and as such the tools and analytical techniques used to monitor and assess the quality of fish need to also develop and improve. The use of spatially off-set Raman spectroscopy (SORS) is particularly well-suited for these applications, given the ability of this technique to take subsurface measurements as well as being rapid, non-destructive and label-free compared to classical chemical analysis techniques. To explore this technique for analysing fish, SORS measurements were taken on commercially significant whole fish through the skin in different locations. The resulting spectra were of high quality with subsurface components such as lipids, carotenoids, proteins and guanine from iridophore cells clearly visible in the spectra. These spectral features were characterised and major bands identified. Chemometric analysis additionally showed that clear differences are present in spectra not only from different sections of a fish but also between different species. These results highlight the potential application for SORS analysis for rapid quality assessment and species identification in the aquaculture industry by taking through-skin measurements.

Helical α-aminoisobutyric acid (Aib) foldamers show great potential as devices for the communication of conformational information across phospholipid bilayers, but determining their conformation in bilayers remains a challenge. In the present study, Raman, Raman optical activity (ROA), infrared (IR) and vibrational circular dichroism (VCD) spectroscopies have been used to analyze the conformational preferences of Aib foldamers in solution and when interacting with bilayers. A 310 -helix marker band at 1665-1668 cm-1 in Raman spectra was used to show that net helical content increased strongly with oligomer length. ROA and VCD spectra of chiral Aib foldamers provided the chiroptical signature for both left- and right-handed 310 -helices in organic solvents, with VCD establishing that foldamer screw-sense was preserved when the foldamers became embedded within bilayers. However, the population distribution between different secondary structures was perturbed by the chiral phospholipid. These studies indicate that ROA and VCD spectroscopies are valuable tools for the study of biomimetic structures, such as artificial signal transduction molecules, in phospholipid bilayers.

A polyurethane-based polymer, and a sustainable, natural resource in the form of chicken feather (keratin) fibers were combined to form a bio-composite via solvent-casting-evaporation at 0, 10, 20, 30, 40, 50, 60, and 70%·w/w. The thermo-mechanical properties of the composites were assessed using thermogravimetry, dynamic mechanical analysis, and stress-strain measurements with hysteresis loops. The uniformity of the dispersion of the feather fiber in the polyurethane matrix was investigated via macro-photography. Scanning electron microscopy of fracture surfaces was used to verify that the adhesion between fiber and polymer was effective. A molecular modeling visualization predicted the existence of hydrogen bonding between fibers and polyurethane molecules and this result was supported by Fourier transform infrared analysis of the final composites. Addition of chicken feather fibers to the polyurethane matrix was found to decrease the glass transition temperature, recovery strain and mass loss of the composites, but increase the elastic modulus, storage modulus and char level. The results demonstrate that keratin derived from what is currently a waste product from the poultry industry (with significant economic and environmental disposal costs) can effectively and cheaply improve the thermo-mechanical properties of composite materials, with potentially large environmental benefits.

Resveratrol is a stilbenoid phytoalexin with promising myriad health benefits predominantly contributed by the trans ( E) diastereomeric form. A recent study has implicated the cis ( Z) diastereomer in human health. This stereoisomer binds with high affinity to human tyrosyl-tRNA synthetase, initiating a downstream cascade that promotes the expression of genes associated with the cellular stress response. We discovered that the nonplanar structure of the cis-resveratrol conformer possesses certain chiral signals in its simulated vibrational circular dichroism (VCD) and Raman optical activity (ROA) spectra. These features may be used for the optical detection of the binding event and in understanding the more diversified biological roles of trans-resveratrol over cis-resveratrol. We use a density functional theory model, which is validated against the known results for the E diastereomer. The Z diastereomer is significantly nonplanar and can exist in two helical atropisomeric forms. These forms exchange rapidly in solution, but only one is observed to bind with the synthetase. This suggests that the binding may generate an enantiomeric excess, leading to detectable changes in the vibrational optical activity spectra. We identify candidate features at 998, 1649, and 1677 cm

The early stages of fibril formation are difficult to capture in solution. We use cold-ion spectroscopy to examine an 11-residue peptide derived from the protein transthyretin and clusters of this fibre-forming peptide containing up to five units in the gas phase. For each oligomer, the UV spectra exhibit distinct changes in the electronic environment of aromatic residues in this peptide compared to that of the monomer and in the bulk solution. The UV spectra of the tetra- and pentamer are superimposable but differ significantly from the spectra of the monomer and trimer. Such a spectral evolution suggests that a common structural motif is formed as early as the tetramer. The presence of this stable motif is further supported by the low conformational heterogeneity of the tetra- and pentamer, revealed from their IR spectra. From comparison of the IR-spectra in the gas and condensed phases, we propose putative assignments for the dominant motif in the oligomers.

Complex coacervates of bovine lactoferrin (LF) and sodium alginate (NaAlg) were prepared aiming to protect the structure and associated function of LF in gastric stage of digestion. The parameters affecting the formation of complex coacervates (pH, LF-to-NaAlg ratio) were optimized using electrostatic charge density, turbidity and yield of the complex coacervates. The breaking down of LF structure into smaller peptides, antioxidant capacity and iron release/binding properties of LF and LF-NaAlg were determined in oral, gastric and intestinal stages of in-vitro digestion. The highest yield of LF-NaAlg complex coacervates was achieved at pH 4.5 and the LF-to-NaAlg ratio of 8:1. UP to 30% LF was delivered to the intestinal stage in complex coacervate form while the entire uncomplexed LF was degraded at the gastric stage. The antioxidant capacity of LF in complex coacervate was increased by 12%. The iron binding/release property of LF was not affected in LF-NaAlg complex coacervates.

A library of Sox-pyrrolizidines was rapidly prepared by microwave-assisted, one-pot, three-component, 1,3-dipolar cycloaddition of azomethine ylides from l-proline and isatin, with various β-nitrostyrenes. Nitro-Sox compounds, 4b, 4d and 4e inhibit HEWL amyloid fibril formation by ThT studies with percentages of fluorescence intensity of 55.4, 42.9 and 40.3%, respectively. Further studies with MTT assay, Raman spectroscopy, TEM and molecular docking supported these promising candidates for activity against amyloid misfolding, a phenomenon leading to Alzheimer's disease pathology.

The U rhynchophylla, U tomentosa, Isatis indigotica Fortune, Voacanga Africana, herbal constituents, fungal extracts from Aspergillus duricaulis culture media, include spirooxindoles, polyphenols or bridged spirocyclic alkaloids. Their constituents exhibit specific and synergistic multiple neuroprotective properties including inhibiting of Aβ fibril induced cytotoxicity, NMDA receptor inhibition in mice models of Alzheimer’s disease (AD). The pioneering research from Woodward to Waldmann has advanced the synthesis of spirocyclic alkaloids. Furthermore, the elucidation of the genetic analysis, biochemical pathways that links strictosidine to the alkaloids akuammicine, stemmadenine, tabersonine, catharanthine, will now enable the biotechnological generation, also stimulate synthesis of related bridged spirocyclic alkaloids for medicinal investigations. From the value of spirocyclic structures as multi target dementia leads, we hypothesise that simpler Lipinski-like natural/synthetic alkaloid analogues may likewise be discovered that provide neurocognitive enhancing activities against dementia and AD.

Here we reveal details of the interaction between human lysozyme proteins, both native and fibrils, and their water environment by intense terahertz time domain spectroscopy. With the aid of a rigorous dielectric model, we determine the amplitude and phase of the oscillating dipole induced by the THz field in the volume containing the protein and its hydration water. At low concentrations, the amplitude of this induced dipolar response decreases with increasing concentration. Beyond a certain threshold, marking the onset of the interactions between the extended hydration shells, the amplitude remains fixed but the phase of the induced dipolar response, which is initially in phase with the applied THz field, begins to change. The changes observed in the THz response reveal protein-protein interactions me-diated by extended hydration layers, which may control fibril formation and may have an important role in chemical recognition phenomena.

In-vitro digestion behaviour of iron saturated (holo)-, native- and iron depleted (apo)- bovine lactoferrin (LF) was studied in simulated oral, gastric and intestinal digestion conditions. All LFs were hydrolysed to

Raman spectroscopy has attracted considerable interest during the past two decades as a vibrational technique used for the molecular characterisation of different molecules. Whilst the Raman effect is known to be generally weak, it is also known that this can be greatly improved using surface-enhanced Raman scattering (SERS). Indeed, in recent years, the power of SERS for rapid identification and quantification of target analytes in a wide range of applications has been repeatedly demonstrated in multiple studies. Moreover, the application of SERS in combination with an isotopically labelled compound (ILC), as an internal standard, has also very recently shown promising results for quantitative SERS measurements, by improving both its accuracy and precision. This is due to the 12C and 13C or 1H and 2H (D) having similar physicochemical properties. The use of these internal standards results in the reduction of any influences due to the number of nanoparticles within the analysis zone and fluctuations in laser fluence. Thus, in this study we have employed SERS for quantitative detection of tryptophan (Trp) and caffeine. These have been chosen because Trp is readily available as the deuterated form and caffeine is available in both 12C and 13C. Quantum chemical calculations based on density functional theory (DFT) have been utilized to determine the vibrational characteristics of the target analytes. For SERS analysis incorporating isotopologues of tryptophan three independent experiments were conducted with three different batches of nanoparticles over a 12 month period; our results show that the use of this internal standard improves quantification of this target molecule. In particular for the independent test sets (i.e., samples not used in quantitative partial least squares regression (PLSR) model construction) we observed improvements in the linearity for test set predictions, as well as lower errors in test set predictions, when isotope internal standards were used during SERS for both deuterated tryptophan as well as 13C caffeine. This work is an extension of and a natural progression from our earlier studies. By exploring additional analytes of interest, allowing for the assessment of the different types of stable isotopes as internal standards, and demonstrating the transfer/robustness of isotopologues for use with SERS, we believe this approach could be readily extended to other biologically-relevant compounds.

Spatially off-set Raman spectroscopy (SORS) offers non-invasive chemical characterisation of the sub-surface of various biological tissues as it permits the assessment of diffusely scattering samples at depths of several orders of magnitude deeper than conventional Raman spectroscopy. Chemicals such as glycogen, glucose, lactate and cortisol are predictors of meat quality, however detection of these chemicals is limited to the surface of meat using conventional Raman spectroscopy as their concentration is higher within the tissue. Here, we have used SORS to detect spectral bands for glycogen, lactate, glucose and cortisol beneath the surface of meat tissue by spiking. To our knowledge, this is the first report on this method for potential application in meat quality analysis. We further validate our SORS spectral results using chemometric analysis to determine chemical-specific spectral characteristics suitable for chemical identification. The chemometric analysis clearly shows distinction of spiked metabolites into four distinct groups, even for such chemically similar compounds as glucose, glycogen and lactate.

Structural analysis of carbohydrates is a complicated endeavour, due to the complexity and diversity of the samples at hand. Herein, we apply a combined computational and experimental approach, employing molecular dynamics (MD) and density functional theory (DFT) calculations together with NMR and Raman optical activity (ROA) measurements, in the structural study of three mannobiose disaccharides, consisting of two mannoses with varying glycosidic linkages. The disaccharide structures make up the scaffold of high mannose glycans and are therefore important targets for structural analysis. Based on the MD population analysis and NMR, the major conformers of each mannobiose were identified and used as input for DFT analysis. By systematically varying the solvent models used to describe water interacting with the molecules and applying overlap integral analysis to the resulting calculational ROA spectra, we found that a full quantum mechanical/molecular mechanical approach is required for an optimal calculation of the ROA parameters. Subsequent normal mode analysis of the predicted vibrational modes was attempted in order to identify possible marker bands for glycosidic linkages. However, the normal mode vibrations of the mannobioses are completely delocalised, presumably due to conformational flexibility in these compounds, rendering the identification of isolated marker bands unfeasible.

Vegetable gardens in cities provide communities with fresh vegetables but also may contribute towards public exposure to metals present in soil from historical pollution. Contamination of some Melbourne garden soils with Pb (range 12.9-773 mg kg-1 in soil) was found with some soils exceeding the Australian human health screening criteria for residential land use of 300 mg kg-1. Cadmium concentrations (0.12-1.04 mg kg-1) were above the ambient background soil concentrations of

Preharvest sprouting of grains causes significant losses to growers and buyers. The problem occurs throughout the world, and economic losses can be millions of dollars. Visual assessment of germination is ineffective because even grains showing no external signs (e.g., having a visible shoot) may still have germinated sufficiently so that it is no longer possible for high-quality products to be made from them. Current procedures to determine whether a grain has germinated are based around measuring enzyme or starch levels. However, despite protracted efforts to develop enhanced ways to measure germination over the last 50 years, there are currently no sufficiently accurate or reliable approaches available. The present work assesses the potential for infrared spectroscopy-based metabolomic profiling to assess the germination of barley and wheat. The results indicate that mid- and near-infrared spectroscopy are able to determine if the grain has germinated and give an indication of how long the germination ...

The drying and denaturation characteristics of native, iron-depleted (apo-), and iron-saturated (holo-)lactoferrin (LF) were studied in convective air-drying (CD) process using single droplet drying. The extent of loss of LF due to denaturation and changes in the secondary structures during CD and isothermal water heating (IHT) were measured. The holo-LF was found to be most stable among these three forms both at 70 and 95°C. The apo-LF was the least stable compared to other two. The secondary structural features of LF were altered to significantly higher extent when subjected to IHT than to CD both at 70 and 95°C. Holo-LF should be preferred when producing LF powder through a convective drying process.

Environmental pollution is usually monitored via mass spectrometry-based approaches. Such techniques are extremely sensitive but have several disadvantages. The instruments themselves are expensive, require specialized training to use and usually cannot be taken into the field. Samples also usually require extensive pre-treatment prior to analysis which can affect the final result. The development of analytical methods that matched the sensitively of mass spectrometry but that could be deployed in the field and require minimal sample processing would be highly advantageous for environmental monitoring. One method that may meet these criteria is Surface Enhanced Raman Spectroscopy (SERS). This is a surface-sensitive technique that enhances Raman scattering by molecules adsorbed on rough nanostructure surfaces such as gold or silver nanoparticles. SERS gives selective spectral enhancement such that increases in sensitivity of 1010 to 1014 have been reported. While this means SERS is, theoretically at least, capable of single molecule detection such a signal enhancement is hard to achieve in practice. In this review the background of SERS is introduced for the environmental scientist and the recent literature on the detection of several classes of environmental pollutants using this technique is discussed. For heavy metals the lowest limit of detection reported was 0.45 μg/L for Mercury; for pharmaceuticals, 2.4 μg/L for propranolol; for endocrine disruptors, 0.35 μg/L for 17β-estradiol; for perfluorinated compounds, 500 μg/L for perfluorooctanoic acid and for inorganic pollutants, 37g/L for general pesticide markers. The signal enhancements achieved in each case show great promise for the detection of pollutants at environmentally relevant concentrations and, although it does not yet routinely match the sensitivity of mass spectrometry. Further work to develop SERS methods and apply them for the detection of contaminants could be of wide benefit for environmental science.

A basic environmental risk assessment was carried out for the top 10 dispensed pharmaceuticals in Melbourne, Australia, in contrast to the more commonly assessed measure of the most used drugs by physical mass. This allowed for the evaluation of compounds that had not previously been the subject of risk assessment. Estimations of the possible fate and behaviour of the target pharmaceuticals in sewage treatment plants were also made. The predicted removal rates of most drugs within standard sewage treatment were expected to be low, with the exception of the statins, which had high removal rates. Each pharmaceutical was predicted to be present in Melbourne wastewater at the nanogram per litre range or lower. All compounds were predicted to be of low toxicity risk, although it was not possible to model mixture effects. Atorvastatin and Irbesartan were also found to possess the potential to possibly bioaccumulate in the aquatic food chain but not to the extent that would require regulation or labelling.

A new orbitide named ribifolin was isolated and characterized from Jatropha ribifolia using mass spectrometry, NMR spectroscopy, quantitative amino acid analysis, molecular dynamics/simulated annealing, and Raman optical activity measurements and calculations. Ribifolin (1) and its linear form (1a) were synthesized by solid-phase peptide synthesis, followed by evaluation of its antiplasmodial and cytotoxicity activities. Compound 1 was moderately effective (IC50 = 42 μM) against the Plasmodium falciparum strain 3D7.

Lactoferrin (LF) is a multifunctional protein occurring in many biological secretions including milk. It possesses iron binding/transferring, antibacterial, antiviral, antifungal, anti-inflammatory and anti-carcinogenic properties. These functional properties intimately depend on the structural integrity of LF especially its higher order conformation. LF is primarily extracted from bovine milk and it is subsequently added into many commercial products such as nutritional supplements, infant formula, cosmetics and toothpaste. LF is sensitive to denaturation induced by temperature and other physicochemical stresses. Hence, the extraction, powder formation processes of LF and processing parameters of LF-containing products have to be optimized to minimise its undesired denaturation. This review documents the advances made on structure-function relationships and discusses the effectiveness of methods used to preserve the structure of LF during thermal processing. Oral delivery, as the most convenient way for administering LF, is also discussed focusing on digestion of LF in oral, gastric and intestinal stages. The effectiveness of methods used to deliver LF to intestinal digestion stage in structurally intact form is also compared. Altogether, this work comprehensively reviews the fate of LF during thermal processing and digestion, and suggests suitable means to preserve its structural integrity and functional properties. Scope of review The manuscript aims at providing a comprehensive review of the latest publications on four aspects of LF: structural features, functional properties, nature and extent of denaturation and gastrointestinal digestion. It also analyses how these publications benefit food and pharmaceutical industries.

Made available in DSpace on 2018-12-11T17:29:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-07-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The combination of experimental and theoretical Raman optical activity data for the flavone C-diglycoside isoswertisin-4′-methyl-ether-2′′α-L-rhamnoside provided detailed information about its conformational preferences in aqueous solution as well as the absolute configuration of the glycosidic linkage. This work also demonstrated the importance of explicit solvation for accurate predictions of the conformational ensemble and vibrational optical activity properties of natural products in water, as opposed to gas-phase or polarizable continuum model calculations. Department of Chemistry Federal University of São Carlos—UFSCar, Rod. Washington Luís km 235 Institute of Chemistry São Paulo State University—UNESP, Rua Prof. Francisco Degni, 55 School of Applied Sciences RMIT University, GPO Box 2476 Institute of Chemistry São Paulo State University—UNESP, Rua Prof. Francisco Degni, 55 FAPESP: 2014/25222-9 FAPESP: 2015/07089-2

In this study surface enhanced Raman scattering (SERS) combined with the isotopic labelling (IL) principle has been used for the quantification of codeine spiked into both water and human plasma. Multivariate statistical approaches were employed for the analysis of these SERS spectral data, particularly partial least squares regression (PLSR) which was used to generate models using the full SERS spectral data for quantification of codeine with, and without, an internal isotopic labelled standard. The PLSR models pro-vided accurate codeine quantification in water and human plasma with high prediction accuracy (Q2). In addition, the employment of codeine-d6 as the internal standard further improved the accuracy of the model, by increasing the Q2 from 0.89 to 0.94 and decreasing the low root-mean-square error of predic-tions (RMSEP) from 11.36 to 8.44. Using the peak area at 1281 cm−1 assigned to C–N stretching, C–H wagging and ring breathing, the limit of detection was calculated in both water and human plasma to be 0.7 μM (209.55 ng mL−1) and 1.39 μM (416.12 ng mL−1), respectively. Due to a lack of definitive codeine vibrational assignments, density functional theory (DFT) calculations have also been used to assign the spectral bands with their corresponding vibrational modes, which were in excellent agreement with our experimental Raman and SERS findings. Thus, we have successfully demonstrated the application of SERS with isotope labelling for the absolute quantification of codeine in human plasma for the first time with a high degree of accuracy and reproducibility. The use of the IL principle which employs an isotopolog (that is to say, a molecule which is only different by the substitution of atoms by isotopes) improves quantifi-cation and reproducibility because the competition of the codeine and codeine-d6 for the metal surface used for SERS is equal and this will offset any difference in the number of particles under analysis or any\ud fluctuations in laser fluence. It is our belief that this may open up new exciting opportunities for testing SERS in real-world samples and applications which would be an area of potential future studies.

The poly(L-proline) II (PPII) helix is considered to be a major conformation in disordered polypeptides and unfolded proteins in aqueous solution. The PPII conformation can be identified by using Raman optical activity (ROA), which measures the different intensities of right- and left-circularly polarized Raman scattered light from chiral molecules and provides information on stereochemistry associated with vibrational motions. In the present study, we used tetra-alanine (Ala4) as a model system, since its central amide bond adopts the PPII conformation. The predominance of the PPII conformation was supported by 11 ns molecular dynamics (MD) simulations at 300 K. The MD snapshots were used for subsequent quantum mechanical/molecular mechanical (QM/MM) calculations to compute the Raman and ROA spectra. The present MD + QM/MM analysis leads to a good agreement between the observed and simulated spectra, allowing us to assign most of the spectral features including the ROA band near 1320 cm−1, which has been used as a marker for the PPII conformation. This positive ROA band has three components. The lower frequency component near 1310 cm−1 arises from an internal peptide bond, whereas the higher frequency components around 1320–1335 cm−1 appear due to N- and C-terminal peptide groups. The MD + QM/MM calculations also reproduced the electronic circular dichroism spectra of Ala4. The present results provide a satisfactory framework for future investigations of unfolded/disordered proteins as well as peptides in solutions by chiral spectroscopic methods.

Raman optical activity (ROA) provides exquisite sensitivity to molecular stereochemistry and its dynamics, but at the cost of relatively weak signal intensity. Increasing ROA signals through the use of plasmon resonance enhancements has long been seen as a desirable strategy, but unfortunately has not proven to be an easy solution to implement. This review discusses recent developments that can now provide spectroscopists with the ability to combine the structural selectivity of ROA with the quantitative sensitivity of SERS.

Recent Raman and Raman optical activity (ROA) results have demonstrated that dimethyl sulfoxide (DMSO) induces the selective conversion of α-helix motifs into the poly(L-proline) II (PPII) helix conformation in an array of proteins, while β-sheets remain mostly unaffected. Human serum albumin (HSA), a highly α-helical protein, underwent the most dramatic changes and, therefore, was selected as a model for further investigations into the mechanism of this conformational change. Herein we report the use of two-dimensional ROA correlation analysis applying synchronous, autocorrelation, and moving windows approaches in order to understand the conformational transitions in HSA as a function of DMSO concentration. Our results indicate that the destabilization of native α-helix starts at DMSO concentrations as little as 20% in water (v/v), with the transition to PPII helix being complete at ~80% DMSO. These results clearly indicate that any protein preparation containing relatively low concentrations of DMSO should consider possible disruptions in α-helical domains.

We illustrate the great potential of Raman and ROA spectroscopies for investigating the structure and organisation of glycoproteins and the complex matrices they can form. In combination these spectroscopic techniques are sensitive to changes in conformation revealing details of secondary and tertiary structures, probing hydrogen bonding interactions, as well as resolving side chain orientation and the absolute configuration of chiral substructures. To demonstrate this potential we have characterised the structural changes in a complex glycoprotein, mucin. Spectral changes were observed during the entanglement transition as the mucin concentration was increased. By applying two-dimensional correlation analysis (2DCos) to the ROA and Raman concentration-dependent spectral sets delicate transitions in mucin conformation could also be determined. From ~20-40 mg/ml conformational transitions assigned mainly to the sugar N-acetyl-d-galactosamine (GalNAc), which is the linking saccharide unit to the protein backbone, were monitored. Further changes in local oligosaccharide conformation above 40 mg/ml were also monitored, together with other structural transitions observed in the protein core, particularly β-structure formation. Consequently, these spectral techniques were shown to monitor the formation of transient entanglements formed by brush-brush interactions between oligosaccharide combs of mucin molecules identifying changes in both carbohydrate and protein moieties. This work clearly shows how these methods can be used to elucidate fresh insights into the complex behaviour of these large complex molecules.

Bovine lactoferrin (LFb) powders were produced using spray drying and freeze drying. Industrially obtained fresh liquid-LFb was used as starting material. The antioxidant capacity, solubility in water, moisture sorption behaviour, the extent of denaturation and changes in the secondary structural features of spray-dried (SDLFb) and freeze-dried bovine lactoferrin (FDLFb) powders were determined. The residual moisture content, water activity, particle size and amorphous/crystalline nature of the SDLFb and FDLFb were also measured. Results showed that both SDLFb and FDLFb powders had negligible denaturation and conformation changes compared to the liquid-LFb. Both SDLFb and FDLFb showed type II sorption behaviour with almost identical monolayer moisture content. The SDLFb powders were amorphous in nature with >98% solubility in water. The antioxidant activity of SDLFb was similar to that of the liquid-LFb while it was ∼6% less in FDLFb. Based on the residual moisture content, water activity, solubility and preservation of secondary structure of LFb in resultant powders, a spray drying process with 180 °C inlet and 95 °C outlet temperature was found to produce similar or better quality LFb powders compared to the ones produced through a freeze drying process.

Infrared (IR) spectra contain substantial information aboutprotein structure. This has previously most often been exploited by usingknown band assignments. Here, we convert spectral intensities in bins withinAmide I and II regions to vectors and apply machine learning methods todetermine protein secondary structure. Partial least squares was performedon spectra of 90 proteins in H2O. After preprocessing and removal ofoutliers, 84 proteins were used for this work. Standard normal variate andsecond-derivative preprocessing methods on the combined Amide I and IIdata generally gave the best performance, with root-mean-square values forprediction of ∼12% for α-helix, ∼7% for β-sheet, 7% for antiparallel β-sheet,and ∼8% for other conformations. Analysis of Fourier transform infrared(FTIR) spectra of 16 proteins in D2O showed that secondary structuredetermination was slightly poorer than in H2O. Interval partial least squareswas used to identify the critical regions within spectra for secondarystructure prediction and showed that the sides of bands were most valuable, rather than their peak maxima. In conclusion, wehave shown that multivariate analysis of protein FTIR spectra can give α-helix, β-sheet, other, and antiparallel β-sheet contentswith good accuracy, comparable to that of circular dichroism, which is widely used for this purpose.

The Raman optical activity spectra of the epimers β-D-glucose and β-D-galactose, two monosaccharides of biological importance, have been calculated using molecular dynamics combined with a quantum mechanics/molecular mechanics approach. Good agreement between theoretical and experimental spectra is observed for both monosaccharides. Full band assignments have been carried out, which has not previously been possible for carbohydrate epimers. For the regions where the spectral features are opposite in sign, the differences in the vibrational modes have been noted and ascribed to the band sign changes.

Colloidal-based surface-enhanced Raman scattering (SERS) is a complex technique, where interaction between multiple parameters, such as colloid type, its concentration, and aggregating agent, is poorly understood. As a result SERS has so far achieved limited reproducibility. Therefore the aim of this study was to improve enhancement and reproducibility in SERS, and to achieve this, we have developed a multiobjective evolutionary algorithm (MOEA) based on Pareto optimality. In this MOEA approach, we tested a combination of five different colloids with six different aggregating agents, and a wide range of concentrations for both were explored; in addition we included in the optimization process three laser excitation wavelengths. For this optimization of experimental conditions for SERS, we chose the β-adrenergic blocker drug propranolol as the target analyte. The objective functions chosen suitable for this multiobjective problem were the ratio between the full width at half-maximum and the half-maximum intensity for enhancement and correlation coefficient for reproducibility. To analyze a full search of all the experimental conditions, 7785 experiments would have to be performed empirically; however, we demonstrated the search for acceptable experimental conditions of SERS can be achieved using only 4% of these possible experiments. The MOEA identified several experimental conditions for each objective which allowed a limit of detection of 2.36 ng/mL (7.97 nM) propranolol, and this is significantly lower (>25 times) than previous SERS studies aimed at detecting this β-blocker.

Raman and Raman optical activity (ROA) spectra are presented for adenosine and seven of its derivative ribonucleotides. Both of these spectroscopic techniques are shown to be sensitive to the site and degree of phosphorylation, with a considerable number of marker bands being identified for these ribonucleotides. ROA spectra are shown to provide the most sensitive diagnostic tool for phosphorylation characterization and quantification.