The Fantastic Four: A plug 'n' play set of optimal control pulses for enhancing nmr spectroscopy

We present highly robust, optimal control-based shaped pulses designed to replace all 90{\deg} and 180{\deg} hard pulses in a given pulse sequence for improved performance. Special attention was devoted to ensuring that the pulses can be simply substituted in a one-to-one fashion for the original hard pulses without any additional modification of the existing sequence. The set of four pulses for each nucleus therefore consists of 90{\deg} and 180{\deg} point-to-point (PP) and universal rotation (UR) pulses of identical duration. These 1 ms pulses provide uniform performance over resonance offsets of 20 kHz (1H) and 35 kHz (13C) and tolerate reasonably large radio frequency (RF) inhomogeneity/miscalibration of (+/-)15% (1H) and (+/-)10% (13C), making them especially suitable for NMR of small-to-medium-sized molecules (for which relaxation effects during the pulse are negligible) at an accessible and widely utilized spectrometer field strength of 600 MHz. The experimental performance of conventional hard-pulse sequences is shown to be greatly improved by incorporating the new pulses, each set referred to as the Fantastic Four (Fanta4).Comment: 28 pages, 19 figure

Time Optimal Control in Spin Systems

In this paper, we study the design of pulse sequences for NMR spectroscopy as a problem of time optimal control of the unitary propagator. Radio frequency pulses are used in coherent spectroscopy to implement a unitary transfer of state. Pulse sequences that accomplish a desired transfer should be as short as possible in order to minimize the effects of relaxation and to optimize the sensitivity of the experiments. Here, we give an analytical characterization of such time optimal pulse sequences applicable to coherence transfer experiments in multiple-spin systems. We have adopted a general mathematical formulation, and present many of our results in this setting, mindful of the fact that new structures in optimal pulse design are constantly arising. Moreover, the general proofs are no more difficult than the specific problems of current interest. From a general control theory perspective, the problems we want to study have the following character. Suppose we are given a controllable right invariant system on a compact Lie group, what is the minimum time required to steer the system from some initial point to a specified final point? In NMR spectroscopy and quantum computing, this translates to, what is the minimum time required to produce a unitary propagator? We also give an analytical characterization of maximum achievable transfer in a given time for the two spin system.Comment: 20 Pages, 3 figure

Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli strain disabled in the TCA cycle

Escherichia coli (E. coli) is an ideal organism to tailor-make labeled nucleotides for biophysical studies of RNA. Recently, we showed that adding labeled formate enhanced the isotopic enrichment at protonated carbon sites in nucleotides. In this paper, we show that growth of a mutant E. coli strain DL323 (lacking succinate and malate dehydrogenases) on 13C-2-glycerol and 13C-1,3-glycerol enables selective labeling at many useful sites for RNA NMR spectroscopy. For DL323 E. coli grown in 13C-2-glycerol without labeled formate, all the ribose carbon atoms are labeled except the C3′ and C5′ carbon positions. Consequently the C1′, C2′ and C4′ positions remain singlet. In addition, only the pyrimidine base C6 atoms are substantially labeled to ~96% whereas the C2 and C8 atoms of purine are labeled to ~5%. Supplementing the growth media with 13C-formate increases the labeling at C8 to ~88%, but not C2. Not unexpectedly, addition of exogenous formate is unnecessary for attaining the high enrichment levels of ~88% for the C2 and C8 purine positions in a 13C-1,3-glycerol based growth. Furthermore, the ribose ring is labeled in all but the C4′ carbon position, such that the C2′ and C3′ positions suffer from multiplet splitting but the C5′ position remains singlet and the C1′ position shows a small amount of residual C1′–C2′ coupling. As expected, all the protonated base atoms, except C6, are labeled to ~90%. In addition, labeling with 13C-1,3-glycerol affords an isolated methylene ribose with high enrichment at the C5′ position (~90%) that makes it particularly attractive for NMR applications involving CH2-TROSY modules without the need for decoupling the C4′ carbon. To simulate the tumbling of large RNA molecules, perdeuterated glycerol was added to a mixture of the four nucleotides, and the methylene TROSY experiment recorded at various temperatures. Even under conditions of slow tumbling, all the expected carbon correlations were observed, which indicates this approach of using nucleotides obtained from DL323 E. coli will be applicable to high molecular weight RNA systems

Asymmetry of 13C labeled 3-pyruvate affords improved site specific labeling of RNA for NMR spectroscopy

Selective isotopic labeling provides an unparalleled window within which to study the structure and dynamics of RNAs by high resolution NMR spectroscopy. Unlike commonly used carbon sources, the asymmetry of 13C-labeled pyruvate provides selective labeling in both the ribose and base moieties of nucleotides using E. coli variants, that until now were not feasible. Here we show that an E. coli mutant strain that lacks succinate and malate dehydrogenases (DL323) and grown on [3-13C]-pyruvate affords ribonucleotides with site specific labeling at C5′ (~95%) and C1′ (~42%) and minimal enrichment elsewhere in the ribose ring. Enrichment is also achieved at purine C2 and C8 (~95%) and pyrimidine C5 (~100%) positions with minimal labeling at pyrimidine C6 and purine C5 positions. These labeling patterns contrast with those obtained with DL323 E. coli grown on [1, 3-13C]-glycerol for which the ribose ring is labeled in all but the C4′ carbon position, leading to multiplet splitting of the C1′, C2′ and C3′ carbon atoms. The usefulness of these labeling patterns is demonstrated with a 27-nt RNA fragment derived from the 30S ribosomal subunit. Removal of the strong magnetic coupling within the ribose and base leads to increased sensitivity, substantial simplification of NMR spectra, and more precise and accurate dynamic parameters derived from NMR relaxation measurements. Thus these new labels offer valuable probes for characterizing the structure and dynamics of RNA that were previously limited by the constraint of uniformly labeled nucleotides

Mucosal Leishmaniasis Caused by Leishmania (Viannia) braziliensis and Leishmania (Viannia) guyanensis in the Brazilian Amazon

Background: Leishmania (Viannia) braziliensis is a parasite recognized as the most important etiologic agent of mucosal leishmaniasis (ML) in the New World. In Amazonia, seven different species of Leishmania, etiologic agents of human Cutaneous Leishmaniasis, have been described. Isolated cases of ML have been described for several different species of Leishmania: L. (V.) panamensis, L. (V.) guyanensis and L. (L.) amazonensis. Methodology: Leishmania species were characterized by polymerase chain reaction (PCR) of tissues taken from mucosal biopsies of Amazonian patients who were diagnosed with ML and treated at the Tropical Medicine Foundation of Amazonas (FMTAM) in Manaus, Amazonas state, Brazil. Samples were obtained retrospectively from the pathology laboratory and prospectively from patients attending the aforementioned tertiary care unit. Results: This study reports 46 cases of ML along with their geographical origin, 30 cases caused by L. (V.) braziliensis and 16 cases by L. (V.) guyanensis. This is the first record of ML cases in 16 different municipalities in the state of Amazonas and of simultaneous detection of both species in 4 municipalities of this state. It is also the first record of ML caused by L. (V.) guyanensis in the states of Para, Acre, and Rondonia and cases of ML caused by L. (V.) braziliensis in the state of Rondonia. Conclusions/Significance: L. (V.) braziliensis is the predominant species that causes ML in the Amazon region. However, contrary to previous studies, L. (V.) guyanensis is also a significant causative agent of ML within the region. The clinical and epidemiological expression of ML in the Manaus region is similar to the rest of the country, although the majority of ML cases are found south of the Amazon River.SUFRAMA[016/2004

The “minimal boundary curve for endothermy” as a predictor of heterothermy in mammals and birds: a review

According to the concept of the “minimal boundary curve for endothermy”, mammals and birds with a basal metabolic rate (BMR) that falls below the curve are obligate heterotherms and must enter torpor. We examined the reliability of the boundary curve (on a double log plot transformed to a line) for predicting torpor as a function of body mass and BMR for birds and several groups of mammals. The boundary line correctly predicted heterothermy in 87.5% of marsupials (n = 64), 94% of bats (n = 85) and 82.3% of rodents (n = 157). Our analysis shows that the boundary line is not a reliable predictor for use of torpor. A discriminate analysis using body mass and BMR had a similar predictive power as the boundary line. However, there are sufficient exceptions to both methods of analysis to suggest that the relationship between body mass, BMR and heterothermy is not a causal one. Some homeothermic birds (e.g. silvereyes) and rodents (e.g. hopping mice) fall below the boundary line, and there are many examples of heterothermic species that fall above the boundary line. For marsupials and bats, but not for rodents, there was a highly significant phylogenetic pattern for heterothermy, suggesting that taxonomic affiliation is the biggest determinant of heterothermy for these mammalian groups. For rodents, heterothermic species had lower BMRs than homeothermic species. Low BMR and use of torpor both contribute to reducing energy expenditure and both physiological traits appear to be a response to the same selective pressure of fluctuating food supply, increasing fitness in endothermic species that are constrained by limited energy availability. Both the minimal boundary line and discriminate analysis were of little value for predicting the use of daily torpor or hibernation in heterotherms, presumably as both daily torpor and hibernation are precisely controlled processes, not an inability to thermoregulate

Altered Metabolism of Growth Hormone Receptor Mutant Mice: A Combined NMR Metabonomics and Microarray Study

Growth hormone is an important regulator of post-natal growth and metabolism. We have investigated the metabolic consequences of altered growth hormone signaling in mutant mice that have truncations at position 569 and 391 of the intracellular domain of the growth hormone receptor, and thus exhibit either low (around 30% maximum) or no growth hormone-dependent STATS signaling respectively. These mutants result in altered liver metabolism, obesity and insulin resistance

A Global Metabolic Shift Is Linked to Salmonella Multicellular Development

Bacteria can elaborate complex patterns of development that are dictated by temporally ordered patterns of gene expression, typically under the control of a master regulatory pathway. For some processes, such as biofilm development, regulators that initiate the process have been identified but subsequent phenotypic changes such as stress tolerance do not seem to be under the control of these same regulators. A hallmark feature of biofilms is growth within a self-produced extracellular matrix. In this study we used metabolomics to compare Salmonella cells in rdar colony biofilms to isogenic csgD deletion mutants that do not produce an extracellular matrix. The two populations show distinct metabolite profiles. Even though CsgD controls only extracellular matrix production, metabolite signatures associated with cellular adaptations associated with stress tolerances were present in the wild type but not the mutant cells. To further explore these differences we examine the temporal gene expression of genes implicated in biofilm development and stress adaptations. In wild type cells, genes involved in a metabolic shift to gluconeogenesis and various stress-resistance pathways exhibited an ordered expression profile timed with multicellular development even though they are not CsgD regulated. In csgD mutant cells, the ordered expression was lost. We conclude that the induction of these pathways results from production of, and growth within, a self produced matrix rather than elaboration of a defined genetic program. These results predict that common physiological properties of biofilms are induced independently of regulatory pathways that initiate biofilm formation

Reversal of MDR1-associated resistance to topotecan by PAK-200S, a new dihydropyridine analogue, in human cancer cell lines

Recent data suggest that expression of the membrane P170-glycoprotein (P-gp) may confer resistance to the topoisomerase- I-interactive agent topotecan. The present study describes the cellular effects of a new dihydropyridine analogue, PAK-200S, on P-gp-mediated resistance to topotecan in human breast and ovarian tumour cells. PAK-200S at a non-cytotoxic concentration of 2.0 μM completely reversed resistance to topotecan in P-gp-expressing MCF-7/adr (breast) and A2780/Dx5 (ovarian) tumour cells, respectively, with no effects on parental cells. Cellular pharmacokinetic studies by reversed-phase high-performance liquid chromatography analysis showed significantly lower cellular drug concentrations of the pharmacologically active closed-ring lactone of topotecan in multidrug-resistant cells than in parental cells. PAK-200S was effective in restoring the cellular lactone concentrations of topotecan in resistant MCF-7/adr cells to levels comparable to those obtained in parental cells. Furthermore, exposure of MCF-7/adr cells to topotecan in the presence of PAK-200S significantly increased the induction of protein-linked DNA breaks. PAK-200S did not alter nuclear topoisomerase I-mediated ex vivo pBR322 DNA plasmid unwinding activity and topoisomerase-I protein expression. These results suggest that reversal of P-gp-mediated resistance to topotecan by PAK-200S was related to the restoration of cellular drug concentrations of the active lactone form of topotecan rather than a direct effect on topoisomerase-I function. © 1999 Cancer Research Campaig