Permethylation as a strategy for high-molecular-weight polysaccharide structure analysis by nuclear magnetic resonance-Case study of Xylella fastidiosa extracellular polysaccharide.

Current practices for structural analysis of extremely large-molecular-weight polysaccharides via solution-state nuclear magnetic resonance (NMR) spectroscopy incorporate partial depolymerization protocols that enable polysaccharide solubilization in suitable solvents. Non-specific depolymerization techniques utilized for glycosidic bond cleavage, such as chemical degradation or ultrasonication, potentially generate structural fragments that can complicate complete and accurate characterization of polysaccharide structures. Utilization of appropriate enzymes for polysaccharide degradation, on the other hand, requires prior structural knowledge and optimal enzyme activity conditions that are not available to an analyst working with novel or unknown compounds. Herein, we describe an application of a permethylation strategy that allows the complete dissolution of intact polysaccharides for NMR structural characterization. This approach is utilized for NMR analysis of Xylella fastidiosa extracellular polysaccharide (EPS), which is essential for the virulence of the plant pathogen that affects multiple commercial crops and is responsible for multibillion dollar losses each year.

Acetylation in Ionic Liquids Dramatically Increases Yield in the Glycosyl Composition and Linkage Analysis of Insoluble and Acidic Polysaccharides.

Glycosyl composition and linkage analyses are important first steps toward understanding the structural diversity and biological importance of polysaccharides. Failure to fully solubilize samples prior to analysis results in the generation of incomplete and poor-quality composition and linkage data by gas chromatography-mass spectrometry (GC-MS). Acidic polysaccharides also do not give accurate linkage results, because they are poorly soluble in DMSO and tend to undergo ß-elimination during permethylation. Ionic liquids can solubilize polysaccharides, improving their derivatization and extraction for analysis. We show that water-insoluble polysaccharides become much more amenable to chemical analysis by first acetylating them in an ionic liquid. Once acetylated, these polysaccharides, having been deprived of their intermolecular hydrogen bonds, are hydrolyzed more readily for glycosyl composition analysis or methylated more efficiently for glycosyl linkage analysis. Acetylation in an ionic liquid greatly improves composition analysis of insoluble polysaccharides when compared to analysis without acetylation, enabling complete composition determination of normally recalcitrant polysaccharides. We also present a protocol for uronic acid linkage analysis that incorporates this preacetylation step. This protocol produces partially methylated alditol acetate derivatives in high yield with minimal ß-elimination and gives sensitive linkage results for acidic polysaccharides that more accurately reflect the structures being analyzed. We use important plant polysaccharides to show that the preacetylation step leads to superior results compared to traditional methodologies.

Prediction of anisotropic NMR data without knowledge of alignment medium structure by surface decomposition.

Prediction of anisotropic NMR data directly from solute-medium interaction is of significant theoretical and practical interest, particularly for structure elucidation, configurational analysis and conformational studies of complex organic molecules and natural products. Current prediction methods require an explicit structural model of the alignment medium: a requirement either impossible or impractical on a scale necessary for small organic molecules. Here we formulate a comprehensive mathematical framework for a parametrization protocol that deconvolutes an arbitrary surface of the medium into several simple local landscapes that are distributed over the medium's surface by specific orientational order parameters. The shapes and order parameters of these local landscapes are determined via fitting that maximizes the congruence between experimentally determined anisotropic NMR measurables and their predicted counterparts, thus avoiding the need for an a priori knowledge of the global medium morphology. This method achieves substantial improvements in the accuracy of predicted anisotropic NMR values compared to current methods, as demonstrated herein with sixteen natural products. Furthermore, because this formalism extracts structural commonalities of the medium by combining anisotropic NMR data from different compounds, its robustness and accuracy are expected to improve as more experimental data become available for further re-optimization of fitting parameters.

Evaluating the Utility of Permethylated Polysaccharide Solution NMR Data for Characterization of Insoluble Plant Cell Wall Polysaccharides.

Plant cell wall polysaccharide analysis encompasses the utilization of a variety of analytical tools, including gas and liquid chromatography, mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectroscopy. These methods provide complementary data, which enable confident structural proposals of the many complex polysaccharide structures that exist in the complex matrices of plant cell walls. However, cell walls contain fractions of varying solubilities, and a few techniques are available that can analyze all fractions simultaneously. We have discovered that permethylation affords the complete dissolution of both soluble and insoluble polysaccharide fractions of plant cell walls in organic solvents such as chloroform or acetonitrile, which can then be analyzed by a number of analytical techniques including MS and NMR. In this work, NMR structure analysis of 10 permethylated polysaccharide standards was undertaken to generate chemical shift data providing insights into spectral changes that result from permethylation of polysaccharide residues. This information is of especial relevance to the structure analysis of insoluble polysaccharide materials that otherwise are not easily investigated by solution-state NMR methodologies. The preassigned NMR chemical shift data is shown to be vital for NMR structure analysis of minor polysaccharide components of plant cell walls that are particularly difficult to assign by NMR correlation data alone. With the assigned chemical shift data, we analyzed the permethylated samples of destarched, alcohol-insoluble residues of switchgrass and poplar by two-dimensional NMR spectral profiling. Thus, we identified, in addition to the major polysaccharide components, two minor polysaccharides, namely, <5% 3-linked arabinoxylan (switchgrass) and <2% glucomannan (poplar). In particular, the position of the arabinose residue in the arabinoxylan of the switchgrass sample was confidently assigned based on chemical shift values, which are highly sensitive to local chemical environments. Furthermore, the high resolution afforded by the 1H NMR spectra of the permethylated switchgrass and poplar samples allowed facile relative quantitative analysis of their polysaccharide composition, utilizing only a few milligrams of the cell wall material. The concepts herein developed will thus facilitate NMR structure analysis of insoluble plant cell wall polysaccharides, more so of minor cell wall components that are especially challenging to analyze with current methods.

Developing nonuniform sampling strategies to improve sensitivity and resolution in 1,1-ADEQUATE experiments.

Nonuniform sampling (NUS) strategies are developed for acquiring highly resolved 1,1-ADEQUATE spectra, in both conventional and homodecoupled (HD) variants with improved sensitivity. Specifically, the quantile-directed and Poisson gap methods were critically compared for distributing the samples nonuniformly, and the quantile schedules were further optimized for weighting. Both maximum entropy and iterative soft thresholding spectral estimation algorithms were evaluated. All NUS approaches were robust when the degree of data reduction is moderate, on the order of a 50% reduction of sampling points. Further sampling reduction by NUS is facilitated by using weighted schedules designed by the quantile method, which also suppresses sampling noise well. Seed independence and the ability to specify the sample weighting in quantile scheduling are important in optimizing NUS for 1,1-ADEQUATE data acquisition. Using NUS yields an improvement in sensitivity, while also making longer evolution times accessible that would be difficult or impractical to attain by uniform sampling. Theoretical predictions for the sensitivity enhancements in these experiments are in the range of 5-20%; NUS is shown to disambiguate weak signals, reveal some n JCC correlations obscured by noise, and improve signal strength relative to uniform sampling in the same experimental time. This work presents sample schedule development for applying NUS to challenging experiments. The schedules developed here are made available for general use and should facilitate the broader utilization of ADEQUATE experiments (including 1,1-, 1,n-, and HD- variants) for challenging structure elucidation problems.

Determination of Complex Small-Molecule Structures Using Molecular Alignment Simulation.

Correct structural assignment of small molecules and natural products is critical for drug discovery and organic chemistry. Anisotropy-based NMR spectroscopy is a powerful tool for the structural assignment of organic molecules, but it relies on the utilization of a medium that disrupts the isotropic motion of molecules in organic solvents. Here, we establish a quantitative correlation between the atomic structure of the alignment medium, the molecular structure of the small molecule, and molecule-specific anisotropic NMR parameters. The quantitative correlation uses an accurate three-dimensional molecular alignment model that predicts residual dipolar couplings of small molecules aligned by poly(γ-benzyl-l-glutamate). The technique facilitates reliable determination of the correct stereoisomer and enables unequivocal, rapid determination of complex molecular structures from extremely sparse NMR data.

pKa measurements for the SAMPL6 prediction challenge for a set of kinase inhibitor-like fragments.

Determining the net charge and protonation states populated by a small molecule in an environment of interest or the cost of altering those protonation states upon transfer to another environment is a prerequisite for predicting its physicochemical and pharmaceutical properties. The environment of interest can be aqueous, an organic solvent, a protein binding site, or a lipid bilayer. Predicting the protonation state of a small molecule is essential to predicting its interactions with biological macromolecules using computational models. Incorrectly modeling the dominant protonation state, shifts in dominant protonation state, or the population of significant mixtures of protonation states can lead to large modeling errors that degrade the accuracy of physical modeling. Low accuracy hinders the use of physical modeling approaches for molecular design. For small molecules, the acid dissociation constant (pKa) is the primary quantity needed to determine the ionic states populated by a molecule in an aqueous solution at a given pH. As a part of SAMPL6 community challenge, we organized a blind pKa prediction component to assess the accuracy with which contemporary pKa prediction methods can predict this quantity, with the ultimate aim of assessing the expected impact on modeling errors this would induce. While a multitude of approaches for predicting pKa values currently exist, predicting the pKas of drug-like molecules can be difficult due to challenging properties such as multiple titratable sites, heterocycles, and tautomerization. For this challenge, we focused on set of 24 small molecules selected to resemble selective kinase inhibitors-an important class of therapeutics replete with titratable moieties. Using a Sirius T3 instrument that performs automated acid-base titrations, we used UV absorbance-based pKa measurements to construct a high-quality experimental reference dataset of macroscopic pKas for the evaluation of computational pKa prediction methodologies that was utilized in the SAMPL6 pKa challenge. For several compounds in which the microscopic protonation states associated with macroscopic pKas were ambiguous, we performed follow-up NMR experiments to disambiguate the microstates involved in the transition. This dataset provides a useful standard benchmark dataset for the evaluation of pKa prediction methodologies on kinase inhibitor-like compounds.

Enabling Fast Pseudo-2D NMR Spectral Acquisition for Broadband Homonuclear Decoupling: The EXACT NMR Approach.

Pseudo-2D NMR spectroscopy provides a means of acquiring broadband homonuclear decoupled spectra useful for structural characterization of complex molecules. However, data points concatenated in the direct dimension in these experiments are acquired over incremented time periods-leading to long acquisition times with no sensitivity benefits due to the absence of signal averaging between scans. Herein, the concept of EXACT NMR spectroscopy ("burst" non-uniform sampling of data points) is explored in pseudo-2D experiments with results revealing little or no loss in spectral quality or signal intensity despite the acceleration of acquisition-up to 400 % in some cases.

EXtended ACquisition Time (EXACT) NMR-A Case for 'Burst' Non-Uniform Sampling.

A strong case exists for the introduction of burst non-uniform sampling (NUS) in the direct dimension of NMR spectroscopy experiments. The resulting gaps in the NMR free induction decay can reduce the power demands of long experiments (by switching off broadband decoupling for example) and/or be used to introduce additional pulses (to refocus homonuclear coupling, for example). The final EXtended ACquisition Time (EXACT) spectra are accessed by algorithmic reconstruction of the missing data points and can provide higher resolution in the direct dimension than is achievable with existing non-NUS methods.

Permethylation as a Strategy for High Molecular Weight Polysaccharide Structure Analysis by NMR - Case Study of Xylella fastidiosa EPS.

Current practices for structure analysis of extremely large molecular weight polysaccharides via solution-state NMR spectroscopy incorporate partial depolymerization protocols that enable polysaccharide solubilization in suitable solvents. Non-specific depolymerization techniques utilized for glycosidic bond cleavage, such as chemical degradation or ultrasonication, potentially generate structure fragments that can complicate the complete characterization of polysaccharide structures. Utilization of appropriate enzymes for polysaccharide degradation, on the other hand, requires prior structure information and optimal enzyme activity conditions that are not available to the analyst working with novel or unknown compounds. Herein, we describe the application of a permethylation strategy that allows the complete dissolution of the intact polysaccharides for NMR structure characterization. This approach is utilized for NMR analysis of Xylella fastidiosa EPS, which is essential for the virulence the plant pathogen that affects multiple commercial crops and is responsible for multibillion dollar losses each year.

Indiscriminate slaughter of pregnant goats for meat in Enugu, Nigeria: Causes, prevalence, implications and ways-out.

BACKGROUND: The indiscriminate slaughter of pregnant goats (SPGs) undermines meat production and food security especially in developing countries. It also connotes animal cruelty, depletion of goat population and may enhance the spread of zoonotic pathogens inhabiting the female reproductive tract during carcass processing. Consequently, this study determined the causes and prevalence of slaughtering pregnant goats for meat in Enugu, Nigeria. The study also estimated the economic losses associated with SPGs, discussed the negative public health consequences and suggested the ways-out. METHODS: Structured, validated and pilot-tested questionnaire was used to ascertain the reasons for SPGs for meat among 78 willing and randomly selected respondents. The questionnaire survey was conducted in the form of interview. Pregnancy statuses of the goats slaughtered were ascertained by visual inspection and palpation of the eviscerated and longitudinally incised uteri and the horns for macroscopic evidence of pregnancy. Ages of the dams were estimated by dentition method. Estimation of the gestational age was performed by crown-rump length method. The study lasted for six months, comprised of three months (December to March) during the dry/hot season and another three months (May to August) during the wet/rainy season. Economic loss estimation was based on the current monetary values of a matured (30 kilogram) goat and one kilogram of chevon in Enugu, Nigeria; which was determined through market survey. Pearson's Chi-square test was used to determine whether there were significant (P<0.05) statistical associations between SPGs and age and season. RESULTS: Major reasons adduced for SPGs were: economic hardship (41%), ignorance of the goat's pregnancy status (21%), increased demand for chevon (13%) and feed scarcity during drought (11%). Of the 1,658 does examined during the six months study, 589 (35.5%) were pregnant. The majority (876/1658, 52.8%) of the female goats slaughtered were in their active reproductive age of ≤ 4 years, while 782 (47.2%) were aged > 4 years. Similarly, majority (1007/1658, 60.7%) of the does/nannies were slaughtered during the dry/hot season. A total of 907 foetuses at first (n = 332, 36.6%), second (n = 486, 53.6%) and third (n = 89, 9.8%) trimesters of gestation were recovered from the 589 PGs. Singleton, twin and triplet pregnancies were observed in 312 (53%), 236 (40%) and 41 (7%) PGs, respectively. About ₦34.44 million ($83,390) would have been earned if the foetuses were born alive and raised to maturity. Additionally, 19,136 kg of chevon, valued at ₦47,841, 000 ($115,838), which would have accrued from the wasted foetuses was also lost. CONCLUSION: Considering the economic, zoonotic and livestock production implications of this work, frantic efforts to reduce SPGs in Enugu, Nigeria is imperative. This could be achieved through advocacy, goat farmers' enlightenment, ante-mortem pregnancy diagnosis, provision of subsidized feed materials during the dry season and strict enforcement of the Nigerian Meat Edict law, which proscribes unapproved slaughter of gravid animals. These measures may improve food safety and security, improve goat reproduction and production, reduce protein malnutrition, limit dissemination of zoonotic pathogens during carcass processing and hence protect public health in Nigeria.

Comparative performance of bio-based coatings formulated with cellulose, chitin, and chitosan nanomaterials suitable for fruit preservation.

Sustainable nanomaterials (SNMs) from wood, sugarcane and crab shell were prepared and used to coat selected fruits. The properties of SNMs and selected fruits were characterized and strawberry was used as an example to test antifungal activity and freshness preservation of the SNMs. The SNMs with their nano-structured morphology form strong shear-thinning dispersions for easy spraying on fruit surfaces. The fruit surface free energy was influenced by its surface morphology, predominant surface wax components, and cutin monomers. The antifungal activity of SNMs was influenced by their surface functional groups and particle size (crystals vs fibers). The coblend of wood nanocrystals (WCNCs) and chitosan nanofiber (CSNFs) exhibited the best antifungal property, which was comparable with the performance of the fungicide thiabendazole (80 mg L-1). The weight loss and color change of the WCNC/CSNF coated strawberries decreased by nearly half compared with the control samples, showing coating effectiveness on preserving fruit freshness.

Epidemiology of gastrointestinal worm infections in pigs reared in Enugu State, Nigeria.

Gastrointestinal worm infections (GWI) constrain pig production and zoonotic pig parasites make pork unsafe for human consumption. This study determined the distributions, determinants and dynamics of GWI and also the effect of the infection on production parameters in pigs reared in Enugu State, Nigeria. The GWI were determined by faecal egg counts following standard procedure. Sixty piggeries and 564 pigs were randomly selected for the study. Questionnaire survey was conducted to obtain data on some production parameters and risk practices aiding GWI in the piggeries. An overall prevalence of 88.3% (53/60) and 68.1% (384/564) at farm and individual pig levels respectively were recorded. High egg counts of single and mixed infections involving Strongyle, Ascaris and Trichuris species were found. The infection predominated in young (74.1%, 240/324) and female (72.3%, 272/376) pigs during the rainy/wet season (74.5%, 204/274). Rearing pigs of different ages together, feeding pigs with untreated abattoir/poultry waste, and unhygienic on-farm feed compounding were the major risk practices underpinning acquisition and spread of GWI. Infected piggeries had less litter weight and reduced mean weight at weaning and maturity. Pre-weaning piglet mortality was 15.5%. The seasonality and preponderance of the infection in young and female pigs are useful epidemiological findings which could be exploited for development of an effective control strategy against the parasitic infections. An overhaul of parasitic disease control measures in piggeries in Enugu State is imperative for greater productivity and profitability in swine production, and to boost availability of safe and wholesome pork for human consumption. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12639-021-01377-y.

Unequivocal structure confirmation of a breitfussin analog by anisotropic NMR measurements.

Structural features of proton-deficient heteroaromatic natural products, such as the breitfussins, can severely complicate their characterization by NMR spectroscopy. For the breitfussins in particular, the constitution of the five-membered oxazole central ring cannot be unequivocally established via conventional NMR methods when the 4'-position is halogenated. The level of difficulty is exacerbated by 4'-iodination, as the accuracy with which theoretical NMR parameters are determined relies extensively on computational treatment of the relativistic effects of the iodine atom. It is demonstrated in the present study, that the structure of a 4'-iodo breitfussin analog can be unequivocally established by anisotropic NMR methods, by adopting a reduced singular value decomposition (SVD) protocol that leverages the planar structures exhibited by its conformers.

Glucuronidation of Methylated Quercetin Derivatives: Chemical and Biochemical Approaches.

Botanical supplements derived from grapes are functional in animal model systems for the amelioration of neurological conditions, including cognitive impairment. Rats fed with grape extracts accumulate 3'-O-methyl-quercetin-3-O-ß-d-glucuronide (3) in their brains, suggesting 3 as a potential therapeutic agent. To develop methods for the synthesis of 3 and the related 4'-O-methyl-quercetin-7-O-ß-d-glucuronide (4), 3-O-methyl-quercetin-3'-O-ß-d-glucuronide (5), and 4'-O-methyl-quercetin-3'-O-ß-d-glucuronide (6), which are not found in the brain, we have evaluated both enzymatic semisynthesis and full chemical synthetic approaches. Biocatalysis by mammalian UDP-glucuronosyltransferases generated multiple glucuronidated products from 4'-O-methylquercetin, and is not cost-effective. Chemical synthetic methods, on the other hand, provided good results; 3, 5, and 6 were obtained in six steps at 12, 18, and 30% overall yield, respectively, while 4 was synthesized in five steps at 34% overall yield. A mechanistic study on the unexpected regioselectivity observed in the quercetin glucuronide synthetic steps is also presented.

Synergism of anisotropic and computational NMR methods reveals the likely configuration of phormidolide A.

Characterization of the complex molecular scaffold of the marine polyketide natural product phormidolide A represents a challenge that has persisted for nearly two decades. In light of discordant results arising from recent synthetic and biosynthetic reports, a rigorous study of the configuration of phormidolide A was necessary. This report outlines a synergistic effort employing computational and anisotropic NMR investigation, that provided orthogonal confirmation of the reassigned side chain, as well as supporting a further correction of the C7 stereocenter.

PBLG as a versatile liquid crystalline medium for anisotropic NMR data acquisition.

Anisotropic NMR of small molecules has thus far utilized different solvent systems and alignment media to analyze molecules of different polarities. The value of utilizing PBLG liquid crystalline phase in a co-solvent system, expanding its range as a versatile alignment medium, is shown with three molecules of different polarities: strychnine, parthenolide, and sucrose.

13C NMR-Based Approaches for Solving Challenging Stereochemical Problems.

Determining the configuration of proton-deficient molecules is challenging using conventional NMR methods including nuclear Overhauser effect (NOE) and the proton-dependent J-based configuration analysis (JBCA). The problem is exacerbated when only one stereoisomer is available. Alternative methods based on the utilization of 13C NMR chemical shifts, 13C-13C homonuclear couplings measured at natural abundance, and residual chemical shift anisotropy measurements in conjunction with density functional theory calculations are illustrated with a proton-deficient model compound.

Ileosigmoid knotting--a case for primary anastomosis.

BACKGROUND: Resection of gangrenous/non-viable bowel followed by primary anastomoses is known to be a viable option for treatment of ileosigmoid knotting. We here report a case of ileosigmoid knot and examined the options for treatment. METHOD: A case report of a patient with ileosigmoid knotting and discussion of relevant literature for surgical treatment. RESULT: A 26 year old man presented to the accident and emergency unit with two weeks' history of fever and alternating diarrhea and constipation followed by features of intestinal obstruction and later, generalized peritonitis. Plain abdominal X-rays showed dilated loops of bowel and multiple air-fluid levels. The diagnosis of ileosigmoid knotting was missed preoperatively. At exploratory laparotomy, ileosigmoid knot was encountered with non-viable segments of the sigmoid colon and ileum. Resection of the non-viable bowel was carried out with primary anastomoses with good postoperative outcome. CONCLUSION: Primary anastomoses after resection of gangrenous bowel is a safe surgical treatment option in ileosigmoid knotting.

Traumatic Intracranial Aerocele With Progressive Blindness - A case report.

BACKGROUND: Traumatic intracranial aerocele, also known as pneumocephalus, is an uncommon condition that may be asymptomatic or may present with progressive neurological deficits and life threatening conditions that demand urgent decompressive craniotomy to reduce the acute rise in intracranial pressure and the sequelae. AIMS & OBJECTIVES: A high degree of suspicion and continuous neurological monitoring are essential for the early detection and the prompt neurosurgical intervention demanded for the achievement of a good outcome in patients following traumatic acute severe head injury with life threatening neurological complications. METHOD: Presentation of a young motorcyclist who was not wearing a crash helmet and was involved in a road traffic accident in which he sustained a compound cranio-facial injury with loss of consciousness and symptomatic intracranial aerocele. RESULTS: The case of a 28-year old motorcyclist without a helmet, following a road traffic accident, sustained compound skull fracture with CSF rhinorrhea, ventricular aerocele and progressive blindness who recovered his vision fully following bitemporal decompressive craniotomy. CONCLUSION: A high index of suspicion enabled early detection and prompt decompressive craniotomy that stemmed the progressive loss of vision in this patient with an uncommon but symptomatic intracranial aerocele and cranio-facial compound head injury.