Thin-Layer Chromatography with Ultraviolet and Mass Spectrometric Detection: From Preparative-Layer to Miniaturized Ultra-Thin-Layer Technique

The present challenge in drug discovery is to synthesize new compounds efficiently in minimal time. The trend is towards carefully designed and well-characterized compound libraries because fast and effective synthesis methods easily produce thousands of new compounds. The need for rapid and reliable analysis methods is increased at the same time. Quality assessment, including the identification and purity tests, is highly important since false (negative or positive) results, for instance in tests of biological activity or determination of early-ADME parameters in vitro (the pharmacokinetic study of drug absorption, distribution, metabolism, and excretion), must be avoided. This thesis summarizes the principles of classical planar chromatographic separation combined with ultraviolet (UV) and mass spectrometric (MS) detection, and introduces powerful, rapid, easy, low-cost, and alternative tools and techniques for qualitative and quantitative analysis of small drug or drug-like molecules. High performance thin-layer chromatography (HPTLC) was introduced and evaluated for fast semi-quantitative assessment of the purity of synthesis target compounds. HPTLC methods were compared with the liquid chromatography (LC) methods. Electrospray ionization mass spectrometry (ESI MS) and atmospheric pressure matrix-assisted laser desorption/ionization MS (AP MALDI MS) were used to identify and confirm the product zones on the plate. AP MALDI MS was rapid, and easy to carry out directly on the plate without scraping. The PLC method was used to isolate target compounds from crude synthesized products and purify them for bioactivity and preliminary ADME tests. Ultra-thin-layer chromatography (UTLC) with AP MALDI MS and desorption electrospray ionization mass spectrometry (DESI MS) was introduced and studied for the first time. Because of the thinner adsorbent layer, the monolithic UTLC plate provided 10 100 times better sensitivity in MALDI analysis than did HPTLC plates. The limits of detection (LODs) down to low picomole range were demonstrated for UTLC AP MALDI and UTLC DESI MS. In a comparison of AP and vacuum MALDI MS detection for UTLC plates, desorption from the irregular surface of the plates with the combination of an external AP MALDI ion source and an ion trap instrument provided clearly less variation in mass accuracy than the vacuum MALDI time-of-flight (TOF) instrument. The performance of the two-dimensional (2D) UTLC separation with AP MALDI MS method was studied for the first time. The influence of the urine matrix on the separation and the repeatability was evaluated with benzodiazepines as model substances in human urine. The applicability of 2D UTLC AP MALDI MS was demonstrated in the detection of metabolites in an authentic urine sample.

Lantibiotic Nisin and Its Detection Methods

The type A lantibiotic nisin produced by several Lactococcus lactis strains, and one Streptococcus uberis strainis a small antimicrobial peptide that inhibits the growth of a wide range of gram-positive bacteria, such as Bacillus, Clostridium, Listeria and Staphylococcus species. It is nontoxic to humans and used as a food preservative (E234) in more than 50 countries including the EU, the USA, and China. National legislations concerning maximum addition levels of nisin in different foods vary greatly. Therefore, there is a demand for non-laborious and sensitive methods to identify and quantify nisin reliably from different food matrices. The horizontal inhibition assay, based on the inhibitory effect of nisin to Micrococcus luteus is the base for most quantification methods developed so far. However, the sensitivity and accuracy of the agar diffusion method is affected by several parameters. Immunological tests have also been described. Taken into account the sensitivity of immunological methods to interfering substances within sample matrices, and possible cross-reactivities with lantibiotics structurally close to nisin, their usefulness for nisin detection from food samples remains limited. The proteins responsible for nisin biosynthesis, and producer self-immunity are encoded by genes arranged into two inducible operons, nisA/Z/QBTCIPRK and nisFEG, which also contain internal, constitutive promoters PnisI and PnisR. The transmembrane histidine kinase NisK and the response regulator NisR form a two-component signal transduction system, in which NisK autophosphorylates after exposure to extra cellular nisin, and subsequently transfers the phosphate to NisR. The phosphorylated NisR then relays the signal downstream by binding to two regulated promoters in the nisin gene cluster, i.e the nisA/Z/Qand the nisF promoters, thus activating transcription of the structural gene nisA/Z/Q and the downstream genes nisBTCIPRK from the nisA/Z/Q promoter, and the genes nisFEG from the nisF promoter. In this work two novel and highly sensitive nisin bioassays were developed. Both of these quantification methods were based on NisRK mediated, nisin induced Green Fluorescent Protein (GFP) fluorescence. The suitabilities of these assays for quantifica¬tion of nisin from food samples were evaluated in several food matrices. These bioassays had nisin sensitivities in the nanogram or picogram levels. In addition, shelf life of nisin in cooked sausages and retainment of the induction activity of nisin in intestinal chyme (intestinal content) was assessed.

Modern techniques in detection, identification and quantification of bacteria and peptides from foods

Standards have been placed to regulate the microbial and preservative contents to assure that foods are safe to the consumer. In a case of a food-related disease outbreak, it is crucial to be able to detect and identify quickly and accurately the cause of the disease. In addition, for every day control of food microbial and preservative contents, the detection methods must be easily performed for numerous food samples. In this present study, quicker alternative methods were studied for identification of bacteria by DNA fingerprinting. A flow cytometry method was developed as an alternative to pulsed-field gel electrophoresis, the golden method . DNA fragment sizing by an ultrasensitive flow cytometer was able to discriminate species and strains in a reproducible and comparable manner to pulsed-field gel electrophoresis. This new method was hundreds times faster and 200,000 times more sensitive. Additionally, another DNA fingerprinting identification method was developed based on single-enzyme amplified fragment length polymorphism (SE-AFLP). This method allowed the differentiation of genera, species, and strains of pathogenic bacteria of Bacilli, Staphylococci, Yersinia, and Escherichia coli. These fingerprinting patterns obtained by SE-AFLP were simpler and easier to analyze than those by the traditional amplified fragment length polymorphism by double enzyme digestion. Nisin (E234) is added as a preservative to different types of foods, especially dairy products, around the world. Various detection methods exist for nisin, but they lack in sensitivity, speed or specificity. In this present study, a sensitive nisin-induced green fluorescent protein (GFPuv) bioassay was developed using the Lactococcus lactis two-component signal system NisRK and the nisin-inducible nisA promoter. The bioassay was extremely sensitive with detection limit of 10 pg/ml in culture supernatant. In addition, it was compatible for quantification from various food matrices, such as milk, salad dressings, processed cheese, liquid eggs, and canned tomatoes. Wine has good antimicrobial properties due to its alcohol concentration, low pH, and organic content and therefore often assumed to be microbially safe to consume. Another aim of this thesis was to study the microbiota of wines returned by customers complaining of food-poisoning symptoms. By partial 16S rRNA gene sequence analysis, ribotyping, and boar spermatozoa motility assay, it was identified that one of the wines contained a Bacillus simplex BAC91, which produced a heat-stable substance toxic to the mitochondria of sperm cells. The antibacterial activity of wine was tested on the vegetative cells and spores of B. simplex BAC91, B. cereus type strain ATCC 14579 and cereulide-producing B. cereus F4810/72. Although the vegetative cells and spores of B. simplex BAC91 were sensitive to the antimicrobial effects of wine, the spores of B. cereus strains ATCC 14579 and F4810/72 stayed viable for at least 4 months. According to these results, Bacillus spp., more specifically spores, can be a possible risk to the wine consumer.

Lactic acid bacteria and their antimicrobial peptides : Induction,detection,partial characterization, and potential applications

Bacteriocin-producing lactic acid bacteria and their isolated peptide bacteriocins are of value to control pathogens and spoiling microorganisms in foods and feed. Nisin is the only bacteriocin that is commonly accepted as a food preservative and has a broad spectrum of activity against Gram-positive organisms including spore forming bacteria. In this study nisin induction was studied from two perspectives, induction from inside of the cell and selection of nisin inducible strains with increased nisin induction sensitivity. The results showed that a mutation in the nisin precursor transporter NisT rendered L. lactis incapable of nisin secretion and lead to nisin accumulation inside the cells. Intracellular proteolytic activity could cleave the N-terminal leader peptide of nisin precursor, resulting in active nisin in the cells. Using a nisin sensitive GFP bioassay it could be shown, that the active intracellular nisin could function as an inducer without any detectable release from the cells. The results suggested that nisin can be inserted into the cytoplasmic membrane from inside the cell and activate NisK. This model of two-component regulation may be a general mechanism of how amphiphilic signals activate the histidine kinase sensor and would represent a novel way for a signal transduction pathway to recognize its signal. In addition, nisin induction was studied through the isolation of natural mutants of the GFPuv nisin bioassay strain L. lactis LAC275 using fl uorescence-activated cell sorting (FACS). The isolated mutant strains represent second generation of GFPuv bioassay strains which can allow the detection of nisin at lower levels. The applied aspect of this thesis was focused on the potential of bacteriocins in chicken farming. One aim was to study nisin as a potential growth promoter in chicken feed. Therefore, the lactic acid bacteria of chicken crop and the nisin sensitivity of the isolated strains were tested. It was found that in the crop Lactobacillus reuteri, L. salivarius and L. crispatus were the dominating bacteria and variation in nisin resistance level of these strains was found. This suggested that nisin may be used as growth promoter without wiping out the dominating bacterial species in the crop. As the isolated lactobacilli may serve as bacteria promoting chicken health or reducing zoonoosis and bacteriocin production is one property associated with probiotics, the isolated strains were screened for bacteriocin activity against the pathogen Campylobacter jejuni. The results showed that many of the isolated L. salivarius strains could inhibit the growth of C. jejuni. The bacteriocin of the L. salivarius LAB47 strain, with the strongest activity, was further characterized. Salivaricin 47 is heat-stable and active in pH range 3 to 8, and the molecular mass was estimated to be approximately 3.2 kDa based on tricine SDS-PAGE analysis.

Rhizoctonia -Scots pine interactions: detection, impact on seedling performance and host defence gene response

Rhizoctonia spp. are ubiquitous soil inhabiting fungi that enter into pathogenic or symbiotic associations with plants. In general Rhizoctonia spp. are regarded as plant pathogenic fungi and many cause root rot and other plant diseases which results in considerable economic losses both in agriculture and forestry. Many Rhizoctonia strains enter into symbiotic mycorrhizal associations with orchids and some hypovirulent strains are promising biocontrol candidates in preventing host plant infection by pathogenic Rhizoctonia strains. This work focuses on uni- and binucleate Rhizoctonia (respectively UNR and BNR) strains belonging to the teleomorphic genus Ceratobasidium, but multinucleate Rhizoctonia (MNR) belonging to teleomorphic genus Thanatephorus and ectomycorrhizal fungal species, such as Suillus bovinus, were also included in DNA probe development work. Strain specific probes were developed to target rDNA ITS (internal transcribed spacer) sequences (ITS1, 5.8S and ITS2) and applied in Southern dot blot and liquid hybridization assays. Liquid hybridization was more sensitive and the size of the hybridized PCR products could be detected simultaneously, but the advantage in Southern hybridization was that sample DNA could be used without additional PCR amplification. The impacts of four Finnish BNR Ceratorhiza sp. strains 251, 266, 268 and 269 were investigated on Scot pine (Pinus sylvestris) seedling growth, and the infection biology and infection levels were microscopically examined following tryphan blue staining of infected roots. All BNR strains enhanced early seedling growth and affected the root architecture, while the infection levels remained low. The fungal infection was restricted to the outer cortical regions of long roots and typical monilioid cells detected with strain 268. The interactions of pathogenic UNR Ceratobasidium bicorne strain 1983-111/1N, and endophytic BNR Ceratorhiza sp. strain 268 were studied in single or dual inoculated Scots pine roots. The fungal infection levels and host defence-gene activity of nine transcripts [phenylalanine ammonia lyase (pal1), silbene synthase (STS), chalcone synthase (CHS), short-root specific peroxidase (Psyp1), antimicrobial peptide gene (Sp-AMP), rapidly elicited defence-related gene (PsACRE), germin-like protein (PsGER1), CuZn- superoxide dismutase (SOD), and dehydrin-like protein (dhy-like)] were measured from differentially treated and un-treated control roots by quantitative real time PCR (qRT-PCR). The infection level of pathogenic UNR was restricted in BNR- pre-inoculated Scots pine roots, while UNR was more competitive in simultaneous dual infection. The STS transcript was highly up-regulated in all treated roots, while CHS, pal1, and Psyp1 transcripts were more moderately activated. No significant activity of Sp-AMP, PsACRE, PsGER1, SOD, or dhy-like transcripts were detected compared to control roots. The integrated experiments presented, provide tools to assist in the future detection of these fungi in the environment and to understand the host infection biology and defence, and relationships between these interacting fungi in roots and soils. This study further confirms the complexity of the Rhizoctonia group both phylogenetically and in their infection biology and plant host specificity. The knowledge obtained could be applied in integrated forestry nursery management programmes.

Detection of clinical mastitis with the help of a thermal camera

Increasing dairy farm size and increase in automation in livestock production require that new methods are used to monitor animal health. In this study, a thermal camera was tested for its capacity to detect clinical mastitis. Mastitis was experimentally induced in 6 cows with 10 mu g of Escherichia coli lipopolysaccharide (LPS). The LPS was infused into the left forequarter of each cow, and the right forequarters served as controls. Clinical examination for systemic and local signs and sampling for indicators of inflammation in milk were carried out before morning and evening milking throughout the 5-d experimental period and more frequently on the challenge day. Thermal images of experimental and control quarters were taken at each sampling time from lateral and medial angles. The first signs of clinical mastitis were noted in all cows 2 h postchallenge and included changes in general appearance of the cows and local clinical signs in the affected udder quarter. Rectal temperature, milk somatic cell count, and electrical conductivity were increased 4 h postchallenge and milk N-acetyl-beta-D-glucosaminidase activity 8 h postchallenge. The thermal camera was successful in detecting the 1 to 1.5 degrees C temperature change on udder skin associated with clinical mastitis in all cows because temperature of the udder skin of the experimental and control quarters increased in line with the rectal temperature. Yet, local signs on the udder were seen before the rise in udder skin and body temperature. The udder represents a sensitive site for detection of any febrile disease using a noninvasive method. A thermal camera mounted in a milking or feeding parlor could detect temperature changes associated with clinical mastitis or other diseases in a dairy herd.

Early Growth and Later Health, Focus on Metabolic Syndrome, Obesity and Physical Activity

The Developmental Origins of Health and Disease Hypothesis proposes that adverse health outcomes in adult life are in part programmed during fetal life and infancy. This means that e.g. restricted nutrition during pregnancy programmes the offspring to store fat more effectively, to develop faster and to reach puberty earlier. These adaptations are beneficial in terms of short term survival. However, in developed countries these adaptations often lead to an increased risk of obesity and metabolic disturbances in later life, due to a mismatch between the prenatal and postnatal environment. This thesis aimed to study the role of early growth in people who are obese as adults, but metabolically healthy as well as in those who are normal in weight but metabolically obese. Other study aims were to assess whether physical activity and cardiorespiratory fitness are programmed early in life. The role of socioeconomic status in the development of obesity from a life course setting was also studied. These studies included 2003 men and women born in Helsinki between 1934 and 1944 with detailed information of their prenatal and childhood growth as well as living conditions. They participated in the detailed clinical examination during the years 2001-2004. A sub-group of the subjects participated in the UKK Institute 2-kilometre walk test. Metabolic syndrome was defined according to the 2005 criteria of the International Diabetes Federation. Among the obese men and women 20 % were metabolically healthy. Those with metabolic syndrome did not differ in birth size compared to the healthy ones, but by two years of age, they were lighter and thinner, and remained so up to 11 years. The period when changes in BMIs were predictive of the metabolic syndrome was from birth to 7 years. Of the normal weight individuals 17 % were metabolically obese. Again, there were no differences in birth size. However, by the age 7 years, those men who later developed metabolic syndrome were thinner. Gains in BMI during the first two years of life were protective of the syndrome. Children who were heavier, and especially taller, were more physically active, exercised with higher intensity and had higher cardiorespiratory fitness in their adult life than those who were shorter and thinner as children. Lower educational attainment and lower adult social class were associated with obesity in both men and women. Childhood social class was inversely associated with body mass index only in men while lower household income was associated with higher BMI in women. These results support the role of early life factors in the development of metabolic syndrome and adult life style. Early detection of risk factors predisposing to these conditions is highly relevant from a public health point of view.