High-Throughput Metabolic Fingerprinting of Legume Silage Fermentations via Fourier Transform Infrared Spectroscopy and Chemometrics

Silage quality is typically assessed by the measurement of several individual parameters, including pH, lactic acid, acetic acid, bacterial numbers, and protein content. The objective of this study was to use a holistic metabolic fingerprinting approach, combining a high-throughput microtiter plate-based fermentation system with Fourier transform infrared (FT-IR) spectroscopy, to obtain a snapshot of the sample metabolome (typically low-molecular-weight compounds) at a given time. The aim was to study the dynamics of red clover or grass silage fermentations in response to various inoculants incorporating lactic acid bacteria (LAB). The hyperspectral multivariate datasets generated by FT-IR spectroscopy are difficult to interpret visually, so chemometrics methods were used to deconvolute the data. Two-phase principal component-discriminant function analysis allowed discrimination between herbage types and different LAB inoculants and modeling of fermentation dynamics over time. Further analysis of FT-IR spectra by the use of genetic algorithms to identify the underlying biochemical differences between treatments revealed that the amide I and amide II regions (wavenumbers of 1,550 to 1,750 cm⁻¹) of the spectra were most frequently selected (reflecting changes in proteins and free amino acids) in comparisons between control and inoculant-treated fermentations. This corresponds to the known importance of rapid fermentation for the efficient conservation of forage proteins.     

傅立叶变换红外光谱在生命科学中的应用

主要综述了傅立叶变换红外光谱在生命科学中的广泛应用.阐述了傅立叶变换红外光谱在生物大分子中的吸收峰位置、振动方式以及谱带归属,介绍了傅立叶变换红外光谱目前在肿瘤方面的研究,为从分子的角度研究癌变机理,提供了重要依据.     

Identification of Trueperella pyogenes Isolated from Bovine Mastitis by Fourier Transform Infrared Spectroscopy

The present study was designed to investigate the potential of Fourier transform infrared (FT-IR) spectroscopy to identify Trueperella (T.) pyogenes isolated from bovine clinical mastitis. FT-IR spectroscopy was applied to 57 isolates obtained from 55 cows in a period from 2009 to 2012. Prior to FT-IR spectroscopy these isolates were identified by phenotypic and genotypic properties, also including the determination of seven potential virulence factor encoding genes. The FT-IR analysis revealed a reliable identification of all 57 isolates as T. pyogenes and a clear separation of this species from the other species of genus Trueperella and from species of genus Arcanobacterium and Actinomyces. The results showed that all 57 isolates were assigned to the correct species indicating that FT-IR spectroscopy could also be efficiently used for identification of this bacterial pathogen.     

Fourier Transform Infrared Emission Spectroscopy in the Study of Biological Molecules

Biophysics - Abstract—Infrared spectroscopy is a powerful analytical method that is not completely developed in relation to biological systems as yet. Previously, this method has been...     

Interaction between Soluble and Membrane-Embedded Potassium Channel Peptides Monitored by Fourier Transform Infrared Spectroscopy

Recent studies have explored the utility of Fourier transform infrared spectroscopy (FTIR) in dynamic monitoring of soluble protein-protein interactions. Here, we investigated the applicability of FTIR to detect interaction between synthetic soluble and phospholipid-embedded peptides corresponding to, respectively, a voltage-gated potassium (Kv) channel inactivation domain (ID) and S4–S6 of the Shaker Kv channel (KV1; including the S4–S5 linker “pre-inactivation” ID binding site). KV1 was predominantly α-helical at 30°C when incorporated into dimyristoyl-l-α-phosphatidylcholine (DMPC) bilayers. Cooling to induce a shift in DMPC from liquid crystalline to gel phase reversibly decreased KV1 helicity, and was previously shown to partially extrude a synthetic S4 peptide. While no interaction was detected in liquid crystalline DMPC, upon cooling to induce the DMPC gel phase a reversible amide I peak (1633 cm⁻¹) consistent with novel hydrogen bond formation was detected. This spectral shift was not observed for KV1 in the absence of ID (or vice versa), nor when the non-inactivating mutant V7E ID was applied to KV1 under similar conditions. Alteration of salt or redox conditions affected KV1-ID hydrogen bonding in a manner suggesting electrostatic KV1-ID interaction favored by a hairpin conformation for the ID and requiring extrusion of one or more KV1 domains from DMPC, consistent with ID binding to S4–S5. These findings support the utility of FTIR in detecting reversible interactions between soluble and membrane-embedded proteins, with lipid state-sensitivity of the conformation of the latter facilitating control of the interaction.     

Identification of Yersinia enterocolitica at the Species and Subspecies Levels by Fourier Transform Infrared Spectroscopy

Yersinia enterocolitica and other Yersinia species, such as Y. pseudotuberculosis, Y. bercovieri, and Y. intermedia, were differentiated using Fourier transform infrared spectroscopy (FT-IR) combined with artificial neural network analysis. A set of well defined Yersinia strains from Switzerland and Germany was used to create a method for FT-IR-based differentiation of Yersinia isolates at the species level. The isolates of Y. enterocolitica were also differentiated by FT-IR into the main biotypes (biotypes 1A, 2, and 4) and serotypes (serotypes O:3, O:5, O:9, and “non-O:3, O:5, and O:9”). For external validation of the constructed methods, independently obtained isolates of different Yersinia species were used. A total of 79.9% of Y. enterocolitica sensu stricto isolates were identified correctly at the species level. The FT-IR analysis allowed the separation of all Y. bercovieri, Y. intermedia, and Y. rohdei strains from Y. enterocolitica, which could not be differentiated by the API 20E test system. The probability for correct biotype identification of Y. enterocolitica isolates was 98.3% (41 externally validated strains). For correct serotype identification, the probability was 92.5% (42 externally validated strains). In addition, the presence or absence of the ail gene, one of the main pathogenicity markers, was demonstrated using FT-IR. The probability for correct identification of isolates concerning the ail gene was 98.5% (51 externally validated strains). This indicates that it is possible to obtain information about genus, species, and in the case of Y. enterocolitica also subspecies type with a single measurement. Furthermore, this is the first example of the identification of specific pathogenicity using FT-IR.The genus Yersinia belongs to the bacterial family Enterobacteriaceae and encompasses three well-known human pathogens: Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica. Pathogenic strains of Y. enterocolitica cause yersiniosis, an acute enteric disease. In Germany and Switzerland, strains of Y. enterocolitica belong to the most frequently isolated pathogens connected with bacterial gastroenteritis (27, 31). Y. enterocolitica also causes other clinical syndromes, such as enterocolitis, acute mesenteric lymphadenitis, mimicking appendicitis, postinfectious arthritis, and systemic infections (7, 21). It is assumed that the main contamination source is food of animal origin, especially pork meat or raw milk (8, 21, 27). Therefore, the focus of diagnosis for these bacteria as food-borne pathogens includes the examination of food samples in food inspection and veterinary controls of livestock.The species Y. enterocolitica sensu lato as described by Frederiksen (9) was recently subdivided into several species: Y. enterocolitica sensu stricto, Y. intermedia, Y. frederiksenii, Y. kristensenii, Y. aldovae, Y. mollaretii, Y. rohdei, and Y. bercovieri (20). The identification of Y. enterocolitica sensu stricto by traditional agar plate techniques (ISO standard 10273:2003) is complicated by the fact that on the commonly used selective agar plates, especially the cefsulodin-irgasan-novobiocin (CIN) agar, several unrelated bacteria also grow (1, 20). In addition, some Yersinia strains are inhibited by CIN agar (10). The differentiation of putative Yersinia strains isolated from the CIN agar is additionally impeded because the commonly used commercial identification systems (for example, API 20E or API Rapid 32IDE) do not include all Yersinia strains in their databases and usually misidentify them as Y. enterocolitica (12). Nevertheless, the biochemical test system API 20E is still used as an affordable tool for the identification of Y. enterocolitica. This probably results in a constant misidentification of certain Yersinia species, particularly Y. bercovieri, Y. rohdei, and Y. intermedia, as Y. enterocolitica (1, 12, 15).Y. enterocolitica sensu stricto comprises pathogenic and nonpathogenic members. The species can be grouped into various biotypes by biochemical tests and independently into different serotypes by immunological tests. Both types are connected with different pathogenic potential. The most common biotype-serotype combinations associated with human diseases were biotype 1B/serotype O:8, 2/O:5,27, 2/O:9, 3/O:3, and 4/O:3 (7). Biotype 1A is deemed to be non- or less pathogenic for humans. Biotype 1B is widespread in the United States and only rarely detected in Europe and Japan (11, 14, 26, 28). Based on different DNA-DNA hybridization values and 16S rRNA gene sequences, it was proposed to name the “American” strains Y. enterocolitica subsp. enterocolitica (19). Biotypes 2 and 4 are often isolated from yersiniosis patients, and biotype 3 seems to be pathogenic but rare (6, 21).Pathogenic strains of Y. enterocolitica harbor certain virulence factors, such as the plasmid-encoded yadA gene and the chromosomally encoded ail gene (17, 32). In contrast, apathogenic strains of Y. enterocolitica do not contain these two genes. However, the plasmid harboring the yadA gene can be lost under certain cultivation conditions in the laboratory (4). This may lead to false-negative results in any test system based on the presence of this plasmid. Therefore, the ail gene appears to be the best-suited marker for the detection of pathogenic Y. enterocolitica strains. The product of the ail gene is an adhesion and invasion factor (17). Therefore, the detection of the ail gene by PCR is used as an indication of the presence of pathogenic strains of Y. enterocolitica in selective enrichments or isolated pure cultures (33).Recently, Fourier transform infrared spectroscopy (FT-IR) has been established as a new method for identification of bacteria, yeasts, and other microorganisms (3, 16, 22, 24, 38). This method analyzes the total composition of all components of the cell using infrared spectroscopy (13, 18). The FT-IR method is rapid and reliable and therefore can be easily adapted to routine analysis. Furthermore, there accrue almost no costs for consumables during sample preparation and measurements. The technique offers a wide range of applications for differentiation at the species and subspecies levels. It has already been used for the differentiation of several food-borne pathogens, like Listeria monocytogenes (25), Escherichia coli (13), and Bacillus cereus (23, 29). Recently, promising results were obtained by combination of FT-IR and multivariate methods for data processing, in particular artificial neural networks (ANN) (25, 35).In the present work, FT-IR combined with ANN analysis was applied for classification of Yersinia strains at the species level and of Y. enterocolitica at the subspecies level. Furthermore, differentiation between pathogenic and apathogenic strains of Y. enterocolitica by FT-IR was attempted.     

Microwave-Induced Structural Changes in Bacteriorhodopsin: Studies by Optical and Fourier Transform Infrared Difference Spectroscopy

Biophysics - Abstract—Using optical and Fourier transform infrared (FTIR) difference spectroscopy, microwave radiation was found to affect the bacteriorhodopsin (BR) structure in films at a...     

Characterisation of Authentic Lignin Biorefinery Samples by Fourier Transform Infrared Spectroscopy and Determination of the Chemical Formula for Lignin

Efficient methods for lignin characterisation are increasingly important as the field of lignin valorisation is growing with the increasing use of lignocellulosic feedstocks, such as wheat straw and corn stover, in biorefineries. In this study, we characterised a set of authentic lignin biorefinery samples in situ with no prior purification and minimal sample preparation. Lignin chemical formulas and lignin Fourier transform infrared (FTIR) spectra were extracted from mixed spectra by filtering out signals from residual carbohydrates and minerals. From estimations of C, H and O and adjustment for cellulose and hemicelluloses contents, the average chemical formula of lignin was found to be C₉H_10.2O_3.4 with slight variations depending on the biomass feedstock and processing conditions (between C₉H_9.5O_2.8 and C₉H_11.1O_3.6). Extracted FTIR lignin spectra showed many of the same characteristic peaks as organosolv and kraft lignin used as benchmark samples. Some variations in the lignin spectra of biorefinery lignin residue samples were found depending on biomass feedstock (wheat straw, corn stover or poplar) and on pretreatment severity, especially in the absorbance of bands at 1267 and 1032 cm^?1 relative to the strong band at ~1120 cm^?1. The suggested method of FTIR spectral analysis with adjustment for cellulose and hemicellulose is proposed to provide a fast and efficient way of analysing lignin in genuine lignin samples resulting from biorefineries.     

油菜籽含油量傅里叶变换近红外模型的修正

为了提高近红外模型的精确度与准确度,需要定期地对原模型进行修正。常用的方法是在原模型中添加一些包含新信息的新样品,因此,样品的选择成为模型维护过程中的关键因素之一。以利用近红外光谱分析法测定油菜籽含油量为例,向原模型中添加不同偏差的样品建立独立的近红外模型,并设计相应的验证集对各模型的预测性能进行全面评价。结果表明:不同偏差的样品对模型预测性能的改善效果有差异,只有当新样品的偏差与原模型的预测偏差相匹配时,添加的新样品才能更有效地对原模型进行修正。依据偏差选择样品的新思路为近红外模型的维护提供了一条有效地途径。     

Application of Fourier Transform Infrared Spectroscopy and Chemometrics for Differentiation of Salmonella enterica Serovar Enteritidis Phage Types

Fourier transform infrared (FT-IR) spectroscopy and chemometric techniques were used to discriminate five closely related Salmonella enterica serotype Enteritidis phage types, phage type 1 (PT1), PT1b, PT4b, PT6, and PT6a. Intact cells and outer membrane protein (OMP) extracts from bacterial cell membranes were subjected to FT-IR analysis in transmittance mode. Spectra were collected over a wavenumber range from 4,000 to 600 cm⁻¹. Partial least-squares discriminant analysis (PLS-DA) was used to develop calibration models based on preprocessed FT-IR spectra. The analysis based on OMP extracts provided greater separation between the Salmonella Enteritidis PT1-PT1b, PT4b, and PT6-PT6a groups than the intact cell analysis. When these three phage type groups were considered, the method based on OMP extract FT-IR spectra was 100% accurate. Moreover, complementary local models that considered only the PT1-PT1b and PT6-PT6a groups were developed, and the level of discrimination increased. PT1 and PT1b isolates were differentiated successfully with the local model using the entire OMP extract spectrum (98.3% correct predictions), whereas the accuracy of discrimination between PT6 and PT6a isolates was 86.0%. Isolates belonging to different phage types (PT19, PT20, and PT21) were used with the model to test its robustness. For the first time it was demonstrated that FT-IR analysis of OMP extracts can be used for construction of robust models that allow fast and accurate discrimination of different Salmonella Enteritidis phage types.Over the past 10 years there has been an increase in the incidence of gastrointestinal infections caused by Salmonella enterica serovar Enteritidis, which is now one of the leading S. enterica serotypes worldwide (21, 27). Poultry, poultry products, cattle, and dairy products are the predominant sources of Salmonella-contaminated food products that cause human salmonellosis (28). Large-scale infections continue to occur in developed countries (8). Unrestricted international movement of commercially prepared food and food ingredients and dissimilarities in government and industry food safety controls during the processing, distribution, and marketing of products have surely contributed to the increase in food-borne outbreaks. Salmonella is a tremendous challenge for the agricultural and food processing industries because of its ability to survive under adverse conditions, such as low levels of nutrients and suboptimal temperatures (4, 13).Salmonella Enteritidis isolates can be categorized for epidemiological purposes by using a variety of typing tools (13). These tools include typing techniques such as serological and phage typing (29) and antibiotic resistance patterns (25). These methods are now supplemented by molecular genetics techniques, such as DNA fingerprinting (23), plasmid profiling (16), and pulsed-field gel electrophoresis (26). Phage typing has been used to diagnose Salmonella outbreaks, including S. enterica serovar Typhi and S. enterica serovar Typhimurium outbreaks (29). It is useful to evaluate whether isolates obtained from different sources at different times are similar or distinct in terms of their reactions with a specific collection of bacteriophages used for typing. The correlation between phage type and the source of an epidemic is high (22). Although very effective, existing classification methods are time-consuming, laborious, and expensive, and they often require special training of personnel and expertise, which can prevent a rapid response to the presence of pathogenic bacterial species.Fourier transform infrared (FT-IR) spectroscopy has been successfully used for differentiation and classification of microorganisms at the species and subspecies levels (7, 9, 12, 15, 18, 19, 20). This technique has been shown to have high discriminatory power and allows identification of bacteria at distinct taxonomic levels based on differences in the infrared absorption patterns of microbial cells. FT-IR spectroscopy has been used to differentiate and characterize intact microbial cells based on outer membrane cell components, including lipopolysaccharides (LPS), lipoproteins, and phospholipids (24). Several studies in which S. enterica serotypes have been discriminated using multivariate data analysis and FT-IR spectroscopy have been performed (1, 2, 10, 11). Kim et al. (11) compared the FT-IR spectra of intact cells and the FT-IR spectra of outer membrane protein (OMP) extracts from S. enterica serotypes to discriminate serotypes. Analysis of spectra of OMP extracts in the 1,800- to 1,500-cm⁻¹ region resulted in 100% correct classification of the serotypes investigated.Previously, there have been no reports of differentiation of Salmonella Enteritidis phage types by FT-IR spectroscopy and chemometric methods. To discriminate closely related phage types of Salmonella Enteritidis in this study, intact cells and OMP extracts of bacterial cell membranes were subjected to FT-IR analysis. The isolates analyzed included isolates belonging to five of the phage types of Salmonella Enteritidis found most frequently in Portuguese hospitals in the period from 2004 to 2006, phage type 1 (PT1), PT1b, PT4b, PT6, and PT6a (5, 14). Chemometric models were used to discriminate between phage types based on infrared spectra.     

Molecular View by Fourier Transform Infrared Spectroscopy of the Relationship between Lactocin 705 and Membranes: Speculations on Antimicrobial Mechanism

Lactocin 705 is a bacteriocin whose activity depends upon the complementation of two peptides, termed Lac705α and Lac705β. Neither Lac705α nor Lac705β displayed bacteriocin activity by itself when the growth of sensitive cells was monitored. To obtain molecular insights into the lactocin 705 mechanism of action, Fourier transform infrared spectroscopy was used to investigate the interactions of each peptide (Lac705α and Lac705β) with dipalmitoylphosphatidylcholine liposomal membranes. Both peptides show the ability to interact with the zwitterionic membrane but at different bilayer levels. While Lac705α interacts with the interfacial region inducing dehydration, Lac705β peptide interacts with only the hydrophobic core. This paper presents the first experimental evidence that supports the hypothesis that Lac705α and Lac705β peptides could form a transmembrane oligomer. From the obtained results, a mechanism of action of lactocin 705 on membrane systems is proposed. The component Lac705α could induce the dehydration of the bilayer interfacial region, and the Lac705β peptide could insert in the hydrophobic region of the membrane where the peptide has adequate conditions to achieve the oligomerization.     

Identification of Early Biomarkers during Acetaminophen-Induced Hepatotoxicity by Fourier Transform Infrared Microspectroscopy

Acetaminophen is a widely prescribed drug used to relieve pain and fever; however, it is a leading cause of drug-induced liver injury and a burden on public healthcare. In this study, hepatotoxicity in mice post oral dosing of acetaminophen was investigated using liver and sera samples with Fourier Transform Infrared microspectroscopy. The infrared spectra of acetaminophen treated livers in BALB/c mice show decrease in glycogen, increase in amounts of cholesteryl esters and DNA respectively. Rescue experiments using L-methionine demonstrate that depletion in glycogen and increase in DNA are abrogated with pre-treatment, but not post-treatment, with L-methionine. This indicates that changes in glycogen and DNA are more sensitive to the rapid depletion of glutathione. Importantly, analysis of sera identified lowering of glycogen and increase in DNA and chlolesteryl esters earlier than increase in alanine aminotransferase, which is routinely used to diagnose liver damage. In addition, these changes are also observed in C57BL/6 and Nos2^−/− mice. There is no difference in the kinetics of expression of these three molecules in both strains of mice, the extent of damage is similar and corroborated with ALT and histological analysis. Quantification of cytokines in sera showed increase upon APAP treatment. Although the levels of Tnfα and Ifnγ in sera are not significantly affected, Nos2^−/− mice display lower Il6 but higher Il10 levels during this acute model of hepatotoxicity. Overall, this study reinforces the growing potential of Fourier Transform Infrared microspectroscopy as a fast, highly sensitive and label-free technique for non-invasive diagnosis of liver damage. The combination of Fourier Transform Infrared microspectroscopy and cytokine analysis is a powerful tool to identify multiple biomarkers, understand differential host responses and evaluate therapeutic regimens during liver damage and, possibly, other diseases.     

An Evaluation of High-Throughput Approaches to QTL Mapping in Saccharomyces cerevisiae

Dissecting the molecular basis of quantitative traits is a significant challenge and is essential for understanding complex diseases. Even in model organisms, precisely determining causative genes and their interactions has remained elusive, due in part to difficulty in narrowing intervals to single genes and in detecting epistasis or linked quantitative trait loci. These difficulties are exacerbated by limitations in experimental design, such as low numbers of analyzed individuals or of polymorphisms between parental genomes. We address these challenges by applying three independent high-throughput approaches for QTL mapping to map the genetic variants underlying 11 phenotypes in two genetically distant Saccharomyces cerevisiae strains, namely (1) individual analysis of >700 meiotic segregants, (2) bulk segregant analysis, and (3) reciprocal hemizygosity scanning, a new genome-wide method that we developed. We reveal differences in the performance of each approach and, by combining them, identify eight polymorphic genes that affect eight different phenotypes: colony shape, flocculation, growth on two nonfermentable carbon sources, and resistance to two drugs, salt, and high temperature. Our results demonstrate the power of individual segregant analysis to dissect QTL and address the underestimated contribution of interactions between variants. We also reveal confounding factors like mutations and aneuploidy in pooled approaches, providing valuable lessons for future designs of complex trait mapping studies.     

Rapid Quantification of Methamphetamine: Using Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) and Chemometrics

In Australia and increasingly worldwide, methamphetamine is one of the most commonly seized drugs analysed by forensic chemists. The current well-established GC/MS methods used to identify and quantify methamphetamine are lengthy, expensive processes, but often rapid analysis is requested by undercover police leading to an interest in developing this new analytical technique. Ninety six illicit drug seizures containing methamphetamine (0.1%–78.6%) were analysed using Fourier Transform Infrared Spectroscopy with an Attenuated Total Reflectance attachment and Chemometrics. Two Partial Least Squares models were developed, one using the principal Infrared Spectroscopy peaks of methamphetamine and the other a Hierarchical Partial Least Squares model. Both of these models were refined to choose the variables that were most closely associated with the methamphetamine % vector. Both of the models were excellent, with the principal peaks in the Partial Least Squares model having Root Mean Square Error of Prediction 3.8, R² 0.9779 and lower limit of quantification 7% methamphetamine. The Hierarchical Partial Least Squares model had lower limit of quantification 0.3% methamphetamine, Root Mean Square Error of Prediction 5.2 and R² 0.9637. Such models offer rapid and effective methods for screening illicit drug samples to determine the percentage of methamphetamine they contain.     

Differentiation ofFusarium spp. by Fourier Transform Infrared (FT-IR) spectroscopy

Fourier Transform Infrared (FT-IR) spectroscopy, in combination with chemometrics, was investigated as a simple method to differentiateFusarium spp. Strains were grown on solid culture plates for sporulation. Spectral data of spores ofFusarium spp. were recorded within the range from 4000 to 400 cm^?1. Hierarchical cluster analysis (HCA) and principal components analysis (PCA) were used to study the clusters in the data of spectral regions of 3000–2800 and 1800–1480 cm^?1. The virtual rate of correct differentiation (100%) was achieved for both the HCA and PCA. FT-IR technique shows high potential as a novel, accurate and easy-to-use differentiation method eagerly sought for the routine differentiation ofFusarium isolates from a wide range of environmental sources.     

Noninvasive High-Throughput Single-Cell Analysis of the Intracellular pH of Saccharomyces cerevisiae by Ratiometric Flow Cytometry

The ability of cells to maintain pH homeostasis in response to environmental changes has elicited interest in basic and applied research and has prompted the development of methods for intracellular pH measurements. Many traditional methods provide information at population level and thus the average values of the studied cell physiological phenomena, excluding the fact that cell cultures are very heterogeneous. Single-cell analysis, on the other hand, offers more detailed insight into population variability, thereby facilitating a considerably deeper understanding of cell physiology. Although microscopy methods can address this issue, they suffer from limitations in terms of the small number of individual cells that can be studied and complicated image processing. We developed a noninvasive high-throughput method that employs flow cytometry to analyze large populations of cells that express pHluorin, a genetically encoded ratiometric fluorescent probe that is sensitive to pH. The method described here enables measurement of the intracellular pH of single cells with high sensitivity and speed, which is a clear improvement compared to previously published methods that either require pretreatment of the cells, measure cell populations, or require complex data analysis. The ratios of fluorescence intensities, which correlate to the intracellular pH, are independent of the expression levels of the pH probe, making the use of transiently or extrachromosomally expressed probes possible. We conducted an experiment on the kinetics of the pH homeostasis of Saccharomyces cerevisiae cultures grown to a stationary phase after ethanol or glucose addition and after exposure to weak acid stress and glucose pulse. Minor populations with pH homeostasis behaving differently upon treatments were identified.     

Biochemical properties of Saccharomyces cerevisiae DNA polymerase IV

Although mammals encode multiple family X DNA polymerases implicated in DNA repair, Saccharomyces cerevisiae has only one, DNA polymerase IV (pol IV). To better understand the repair functions of pol IV, here we characterize its biochemical properties. Like mammalian pol beta and pol lambda, but not pol mu, pol IV has intrinsic 5'-2-deoxyribose-5-phosphate lyase activity. Pol IV has low processivity and can fill short gaps in DNA. Unlike the case with pol beta and pol lambda, the gap-filling activity of pol IV is not enhanced by a 5'-phosphate on the downstream primer but is stimulated by a 5'-terminal synthetic abasic site. Pol IV incorporates rNTPs into DNA with an unusually high efficiency relative to dNTPs, a property in common with pol mu but not pol beta or pol lambda. Finally, pol IV is highly inaccurate, with an unusual error specificity indicating the ability to extend primer termini with limited homology. These properties are consistent with a possible role for pol IV in base excision repair and with its known role in non-homologous end joining of double strand breaks, perhaps including those with damaged ends.     

Gypsophile Chemistry Unveiled: Fourier Transform Infrared (FTIR) Spectroscopy Provides New Insight into Plant Adaptations to Gypsum Soils

Gypsum soils are among the most restrictive and widespread substrates for plant life. Plants living on gypsum are classified as gypsophiles (exclusive to gypsum) and gypsovags (non-exclusive to gypsum). The former have been separated into wide and narrow gypsophiles, each with a putative different ecological strategy. Mechanisms displayed by gypsum plants to compete and survive on gypsum are still not fully understood. The aim of this study was to compare the main chemical groups in the leaves of plants with different specificity to gypsum soils and to explore the ability of Fourier transform infrared (FTIR) spectra analyzed with neural network (NN) modelling to discriminate groups of gypsum plants. Leaf samples of 14 species with different specificity to gypsum soils were analysed with FTIR spectroscopy coupled to neural network (NN) modelling. Spectral data were further related to the N, C, S, P, K, Na, Ca, Mg and ash concentrations of samples. The FTIR spectra of the three groups analyzed showed distinct features that enabled their discrimination through NN models. Wide gypsophiles stood out for the strong presence of inorganic compounds in their leaves, particularly gypsum and, in some species, also calcium oxalate crystals. The spectra of gypsovags had less inorganic chemical species, while those of narrow gypsum endemisms had low inorganics but shared with wide gypsophiles the presence of oxalate. Gypsum and calcium oxalate crystals seem to be widespread amongst gypsum specialist plants, possibly as a way to tolerate excess Ca and sulphate. However, other mechanisms such as the accumulation of sulphates in organic molecules are also compatible with plant specialization to gypsum. While gypsovags seem to be stress tolerant plants that tightly regulate the uptake of S and Ca, the ability of narrow gypsum endemisms to accumulate excess Ca as oxalate may indicate their incipient specialization to gypsum.