Phylogeography of Francisella tularensis: global expansion of a highly fit clone.

Francisella tularensis contains several highly pathogenic subspecies, including Francisella tularensis subsp. holarctica, whose distribution is circumpolar in the northern hemisphere. The phylogeography of these subspecies and their subclades was examined using whole-genome single nucleotide polymorphism (SNP) analysis, high-density microarray SNP genotyping, and real-time-PCR-based canonical SNP (canSNP) assays.

Developmental validation of a single-tube amplification of the 13 CODIS STR loci, D2S1338, D19S433, and amelogenin: the AmpFlSTR Identifiler PCR Amplification Kit.

Analysis of length polymorphism at short tandem repeat (STR) loci utilizing the polymerase chain reaction (PCR) process has proven to be an ideal assay for human identification purposes. The short length of STR loci coupled with the amplification of target sequence through PCR allows for a robust, sensitive, and specific assay for highly polymorphic markers. A multiplex containing fifteen STR loci plus the gender-determining locus Amelogenin was developed to provide a single amplification/detection of all CODIS (Combined DNA Index System) STR loci (CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, TH01, TPOX, and vWA) as well as two internationally-accepted STRs (D2S1338 and D19S433).

Development of the AmpFISTR SEfiler PCR amplification kit: a new multiplex containing the highly discriminating ACTBP2 (SE33) locus.

The AmpFISTR SEfiler kit co-amplifies 11 short tandem repeat loci including SE33 in a single multiplex. After establishing the optimum in primer titration studies, the primer concentrations of all loci in the multiplex were chosen such that the heterozygote peak height ratios of each of the loci were balanced. The combined primer set was then tested to determine the robustness of the multiplex under various conditions.

Variation in urine composition in the human urinary tract: evidence of urothelial function in situ?

Purpose: An increased awareness of the concept that the urothelium has a significant transport function led us to question whether urine composition changes as it passes along the human lower urinary tract.

Materials And Methods: Urine samples from the bladder and renal pelvis were collected from 30 adults who underwent percutaneous nephrolithotomy (27) or ureteral stent insertion before lithotripsy (3). Urine was obtained from the 2 renal pelves (operative and contralateral sides) in 6 patients (24%).

The comparison of plasma deproteinization methods for the detection of low-molecular-weight metabolites by (1)H nuclear magnetic resonance spectroscopy.

Blood plasma is the major vehicle by which metabolites are transported around the body in mammalian species, and chemical analysis of plasma can provide a wealth of information relating to the biochemical status of an individual and is important for diagnostic purposes. However, plasma is very complex in physicochemical terms because it is composed of a range of organic and inorganic constituents with a wide range of molecular weights and chemical classes and this makes analysis non-trivial. It is now well established that high-resolution (1)H NMR spectroscopy of blood plasma provides useful qualitative and quantitative biochemical information relating to metabolic disorders.

Distinction between normal and renal cell carcinoma kidney cortical biopsy samples using pattern recognition of (1)H magic angle spinning (MAS) NMR spectra.

The technique of magic angle spinning (MAS) high resolution (1)H NMR spectroscopy applied to intact tissues provides excellent peak resolution and thus much biochemical information. The use of computer-based pattern recognition techniques to classify human renal cortex tissue samples as normal or tumour based on their (1)H MAS NMR spectra has been investigated. In this preliminary study of 22 paired control and tumour samples, exploratory data analysis using principal components based on NMR spectral intensities showed clear separation of the two classes.

Nuclear magnetic resonance spectroscopy in the diagnosis of modifications of the oxidative stress in red blood cell during pre-eclampsia.

Unlabelled: Our study uses the technique of nuclear magnetic resonance (NMR) spectroscopy to determine the changes of antioxidant (AO) system in red blood cells during pre-eclampsia (PEC) as compared with normal pregnancies 28 and 34 weeks of gestation.

Material And Method: The study was carried out in 30 women divided into 3 groups: Group I included 10 women with pre-eclampsia, Group II A included 10 women with normal pregnancy, 28 weeks of gestation and Group II B included 10 women with normal pregnancy, 34 weeks of gestation. The balance was determined by measuring the glutathione (GSH) levels.

Urinary proton magnetic resonance studies of early ifosfamide-induced nephrotoxicity and encephalopathy.

Ifosfamide is an oxazophosphorine widely used in the treatment of cancer in children and adults. Nephrotoxicity and neurotoxicity are major side effects. The aim of this study was to use high-resolution proton nuclear magnetic resonance (1H NMR) spectroscopy of urine to identify novel biochemical markers of ifosfamide-induced toxicity.

Nuclear magnetic resonance spectroscopy: a non-invasive probe of kidney metabolism and function.

Major advances in nuclear magnetic resonance (NMR) spectroscopic methods and technology have led to the increased use of this technique to study kidney metabolism and function. These studies include: (1) the identification of organic osmolytes in the renal medulla and their role as potential markers of medullary development and damage; (2) changes in renal epithelial cell organic solute transport, such as autosomal dominant polycystic kidney disease, and (3) the biochemical heterogeneity of the nephron and identification of markers of site-specific renal damage in experimental animals and man. The present review summarises these data with the aim of demonstrating how NMR can be used as an indirect, and non-invasive probe of homeostatic mechanisms in vivo and in vitro.

Biochemical classification of kidney carcinoma biopsy samples using magic-angle-spinning 1H nuclear magnetic resonance spectroscopy.

High resolution 1H nuclear magnetic resonance (NMR) spectra using spinning at the magic angle (1H MAS NMR) have been obtained on intact normal and pathological kidney tissue samples from patients undergoing surgery for renal cell carcinoma (RCC). The spectra were measured on ca. 80 mg samples and provided high resolution 1H NMR spectra in which effects of dipolar couplings, chemical shift anisotropy and magnetic susceptibility differences are minimised thus yielding high spectral resolution.

Short-term urinary flow impairment deregulates PAX2 and PCNA expression and cell survival in fetal sheep kidneys.

Renal malformations account for most children with chronic renal failure and are often associated with urinary tract anatomical obstruction. We examined cellular and molecular events after experimental urinary flow impairment in fetal sheep. Ovine gestation lasts 144 to 150 days with the metanephros appearing at 27 to 30 days.

Nuclear magnetic resonance spectroscopic and principal components analysis investigations into biochemical effects of three model hepatotoxins.

1H NMR spectroscopy of urine combined with pattern recognition (PR) methods of data analysis has been used to investigate the time-related biochemical changes induced in Sprague-Dawley rats by three model hepatotoxins: alpha-naphthyl isothiocyanate (ANIT), d-(+)-galactosamine (GalN), and butylated hydroxytoluene (BHT). The development of hepatic lesions was monitored by conventional plasma analysis and liver histopathology. Urine was collected continuously postdosing up to 144 h and analyzed by 600-MHz 1H NMR spectroscopy.

High resolution 1H NMR spectroscopic studies on dynamic biochemical processes in incubated human seminal fluid samples.

High resolution 600 MHz 1H NMR spectroscopy was used to investigate the changes in biochemical composition of whole human seminal fluid (SF) and an artificial mixture of prostatic (PF) and seminal vesicle fluid (SVF). A variety of time-related biochemical changes were monitored simultaneously and non-invasively in SF, including enzymatic hydrolysis of phosphorylcholine to choline and polypeptides to amino acids. The fastest NMR-observable reactions in SF were the conversion of phosphorylcholine to choline (t1/2 approximately equal to 9 min) and uridine-5'-monophosphate (UMP) to uridine (t1/2 < 2 min).

750 MHz 1H NMR spectroscopy characterisation of the complex metabolic pattern of urine from patients with inborn errors of metabolism: 2-hydroxyglutaric aciduria and maple syrup urine disease.

750 MHz 1H NMR spectroscopy has been used to characterise in detail the abnormal low molecular weight metabolites of urine from two patients with inborn errors of metabolism. One case of the rare condition 2-hydroxyglutaric aciduria has been examined. There is at present no rapid routine method to detect this genetic defect, although NMR spectroscopy of urine is shown to provide a distinctive pattern of resonances.

HPLC fractions of human uremic plasma inhibit the RBC membrane calcium pump.

We have reported that uremic plasma filtrates (UF) inhibit the red blood cell (RBC) membrane calcium pump. The inhibitor was dialyzable, smaller than 3,000 molecular weight, heat-stable, and protease-resistant. In the present study, we used reverse-phase preparative HPLC, analytical HPLC, and Sephadex G-25 elution to identify inhibitory fractions.

Uroscopy in the 21st century: high-field NMR spectroscopy.

From the experiments described, it can be seen that there are different research approaches that can be taken and these are summarized in Table 1. Whereas much scientific research is principally hypothesis led, there remains, nevertheless, an important place for exploratory research. High resolution NMR can measure, directly and simultaneously, a wide range of endogenous metabolites in biological fluids and has the unique capability of providing structural information on the metabolites detected.

Differentiation of H. pylori Strains Using PCR RFLP.

Helicobacter pylori strains have been shown to display considerable heterogeneity with respect to DNA sequence. Diverse restriction fragment length polymorphism (RFLP) patterns are generated among strains by restriction endonuclease digestion of whole chromosomal DNA (1-3), digestion of specific polymerase chain reaction (PCR) products (4), or arbitrary primer-PCR and random amplified polymorphic DNA-PCR (5-7). These techniques demonstrate numerous distinct and reproducible patterns that can be used to differentiate strains.

Combined HPLC, NMR spectroscopy, and ion-trap mass spectrometry with application to the detection and characterization of xenobiotic and endogenous metabolites in human urine.

The direct coupling of HPLC with NMR spectroscopy has been extended by splitting the HPLC eluent after conventional UV detection and sending part to a NMR spectrometer and part to an ion-trap mass spectrometer in a "triplehyphenated" HPLC-NMR-MS system. Combined UV, 1H NMR, and positive-ion electrospray MS detection was achieved in the continuous-flow mode using whole human urine from a subject dosed with acetaminophen. By means of HPLC-NMR-MS, the structural information available from the complementary spectroscopic techniques provided rapid confirmation of the identity of the acetaminophen glucuronide and sulfate metabolites, together with a number of endogenous metabolites.

Nuclear magnetic resonance and high-performance liquid chromatography-nuclear magnetic resonance studies on the toxicity and metabolism of ifosfamide.

A combination of high-resolution nuclear magnetic resonance (NMR) and high-performance liquid chromatography (HPLC)-NMR spectroscopic methods has been used to analyse urine from humans and rats treated with the anticancer drug ifosfamide. It was possible to detect a range of abnormal endogenous metabolites in urine after ifosfamide administration to human subjects undergoing cancer therapy and to relate the metabolic perturbations to the nephrotoxic effects of the drug. Changes observed by 1H NMR included increases in levels of urinary glucose, glycine, alanine, histidine, lactate, acetate, succinate, and trimethylamine-N-oxide and decreases in the levels of hippurate and citrate.

Analysis of fetal and neonatal urine using proton nuclear magnetic resonance spectroscopy.

Aim: To use high field proton nuclear magnetic resonance spectroscopy (1H NMR) to characterise the low molecular weight metabolite composition of neonatal and fetal urine in relation to gestational age and perinatal outcome.

Methods: The first urine passed by two neonatal groups, six full term and five preterm infants with normal renal function, was analysed by 1H NMR and compared with fetal urine from 14 cases with obstructive uropathy.

Results: The mean ratios of taurine, myo-inositol, and trimethylamine-N-oxide (TMAO) to creatinine were 4.

750 MHz 1H and 1H-13C NMR spectroscopy of human blood plasma.

High-resolution 750 MHz 1H NMR spectra of control human blood plasma have been measured and assigned by the concerted use of a range of spin-echo, two-dimensional J-resolved, and homonuclear and heteronuclear (1H-13C) correlation methods. The increased spectral dispersion and sensitivity at 750 MHz enable the assignment of numerous 1H and 13C resonances from many molecular species that cannot be detected at lower frequencies. This work presents the most comprehensive assignment of the 1H NMR spectra of blood plasma yet achieved and includes the assignment of signals from 43 low M(r) metabolites, including many with complex or strongly coupled spin systems.

Automatic data reduction and pattern recognition methods for analysis of 1H nuclear magnetic resonance spectra of human urine from normal and pathological states.

Multivariate data analysis techniques have been used to compare 600-MHz 1H nuclear magnetic resonance (NMR) spectra of urine obtained from patients with inborn errors of metabolism (IEM) and urine obtained from healthy subjects. These spectra are very complex; each contains many thousands of resonances with a high dynamic range. A consistent method of reducing this wealth of data to manageable proportions is presented as a two-stage process.

Ultra high field NMR spectroscopic studies on human seminal fluid, seminal vesicle and prostatic secretions.

Ultra high field 1H-NMR spectroscopic methods have been used to analyse the composition of seminal fluid and its component secretions, prostatic and seminal vesicle fluids from normal human subjects and those with vasal aplasia and non-obstructive infertility. The 1H-NMR spectrum of whole seminal fluid is extremely complex and many resonances are extensively overlapped in single pulse spectra even when measured at 600 or 750 MHz 1H resonance frequency. A combination of 2-D 1H-NMR methods (including J-Resolved and various 1H homonuclear correlation and 1H-13C heteronuclear correlation techniques) were applied at 600 or 750 MHz in order to extensively assign the signals from the organic components of seminal fluid.

Urinary protein excretion and the diagnosis of graft rejection or renal dysfunction in renal transplant patients.

Urinary proteins have been found to be a sensitive marker of renal damage caused by nephrotoxic agents. An electrophoretic method was used to investigate the potential value of the pattern of urinary protein excretion in 14 cyclosporin-treated renal transplant patients, to differentiate between graft rejection episodes and other causes of renal dysfunction. Urinary protein excretion consistent with renal damage was observed in all of the patients studied, with no marked differences between those with signs of graft rejection, those with renal dysfunction, or those with stable renal function.