Automated Projection Spectroscopy (APSY) for the Assignment of NMR Resonances of Biological Macromolecules

High resolution nuclear magnetic resonance (NMR) spectroscopy in solution is an established technique in structural biology. Detailed functional and structural studies of biological macromolecules by NMR require the assignment of the chemical shifts to specific nuclei. In biological applications, the necessary data is usually obtained from a number of two- and three-dimensional (2D and 3D) NMR experiments. Often, these data cannot be fully analyzed by automated computer programs due to insufficient separation and resolution of the signals in the available spectra. Then, complete resonance assignment requires manual interaction and can become a long and labor-intensive task. Automated projection spectroscopy (APSY) allows the substantial improvement of the resolution by providing spectral information from four and higher dimensional experiments without measuring the full spectrum, which would by far exceed any acceptable measuring time. APSY only measures a series of projections of the high-dimensional spectrum which can be obtained in a much shorter time. Peak picking of the projection spectra provides the basis for the calculation of the high-dimensional chemical shift correlation space by the algorithm GAPRO. The resulting high-dimensional peak lists are commonly artifact-free and of exceptional precision. Along with their high number of correlated nuclei they provide an ideal basis for reliable automated assignment. We will introduce the basic concepts of APSY, illustrate them with an application of a 6D APSY-seq-HNCOCANH experiment, and discuss some practical aspects

4D APSY-HBCB(CG)CDHD experiment for automated assignment of aromatic amino acid side chains in proteins

A four-dimensional (4D) APSY (automated projection spectroscopy)-HBCB(CG)CDHD experiment is presented. This 4D experiment correlates aromatic with aliphatic carbon and proton resonances from the same amino acid side chain of proteins in aqueous solution. It thus allows unambiguous sequence-specific assignment of aromatic amino acid ring signals based on backbone assignments. Compared to conventional 2D approaches, the inclusion of evolution periods on 1Hβ and 13Cδ efficiently removes overlaps, and provides two additional frequencies for consequent automated or manual matching. The experiment was successfully applied to three proteins with molecular weights from 6 to 13kDa. For the complementation of the assignment of the aromatic resonances, TOCSY- or COSY-based versions of a 4D APSY-HCCHaro sequence are propose

4D experiments measured with APSY for automated backbone resonance assignments of large proteins

Detailed structural and functional characterization of proteins by solution NMR requires sequence-specific resonance assignment. We present a set of transverse relaxation optimization (TROSY) based four-dimensional automated projection spectroscopy (APSY) experiments which are designed for resonance assignments of proteins with a size up to 40kDa, namely HNCACO, HNCOCA, HNCACB and HN(CO)CACB. These higher-dimensional experiments include several sensitivity-optimizing features such as multiple quantum parallel evolution in a ‘just-in-time' manner, aliased off-resonance evolution, evolution-time optimized APSY acquisition, selective water-handling and TROSY. The experiments were acquired within the concept of APSY, but they can also be used within the framework of sparsely sampled experiments. The multidimensional peak lists derived with APSY provided chemical shifts with an approximately 20 times higher precision than conventional methods usually do, and allowed the assignment of 90% of the backbone resonances of the perdeuterated primase-polymerase ORF904, which contains 331 amino acid residues and has a molecular weight of 38.4kD

Sugar-to-base correlation in nucleic acids with a 5D APSY-HCNCH or two 3D APSY-HCN experiments

A five-dimensional (5D) APSY (automated projection spectroscopy) HCNCH experiment is presented, which allows unambiguous correlation of sugar to base nuclei in nucleic acids. The pulse sequence uses multiple quantum (MQ) evolution which enables long constant-time evolution periods in all dimensions, an improvement that can also benefit non-APSY applications. Applied with an RNA with 23 nucleotides the 5D APSY-HCNCH experiment produced a complete and highly precise 5D chemical shift list within 1.5h. Alternatively, and for molecules where the out-and-stay 5D experiment sensitivity is not sufficient, a set of out-and-back 3D APSY-HCN experiments is proposed: an intra-base (3D APSY-b-HCN) experiment in an MQ or in a TROSY version, and an MQ sugar-to-base (3D APSY-s-HCN) experiment. The two 3D peak lists require subsequent matching via the N1/9 chemical shift values to one 5D peak list. Optimization of the 3D APSY experiments for maximal precision in the N1/9 dimension allowed matching of all 15N chemical shift values contained in both 3D peak lists. The precise 5D chemical shift correlation lists resulting from the 5D experiment or a pair of 3D experiments also provide a valuable basis for subsequent connection to chemical shifts derived with other experiment

Strategy for automated NMR resonance assignment of RNA: application to 48-nucleotide K10

A procedure is presented for automated sequence-specific assignment of NMR resonances of uniformly [13C, 15N]-labeled RNA. The method is based on a suite of four through-bond and two through-space high-dimensional automated projection spectroscopy (APSY) experiments. The approach is exemplified with a 0.3mM sample of an RNA stem-loop with 48 nucleotides, K10, which is responsible for dynein-mediated localization of Drosophila fs(1)K10 mRNA transcripts. The automated analysis of the APSY data led to highly accurate and precise 3- to 4-dimensional peak lists. They provided a reliable basis for the subsequent sequence-specific resonance assignment with the algorithm FLYA and resulted in the fully automated resonance assignment of more than 80% of the resonances of the 13C-1H moieties at the 1′, 2′, 5, 6, and 8 positions in the nucleotides. The procedure was robust with respect to numerous impurity peaks, low concentration of this for NMR comparably large RNA, and structural features such as a loop, single-nucleotide bulges and a non-Watson-Crick wobble base pairs. Currently, there is no precise chemical shift statistics (as used by FLYA) for RNA regions which deviate from the regular A-form helical structure. Reliable and precise peak lists are thus required for automated sequence-specific assignment, as provided by APSY

The Use of Garcinia Extract (Hydroxycitric Acid) as a Weight loss Supplement: A Systematic Review and Meta-Analysis of Randomised Clinical Trials

The aim of this systematic review is to examine the efficacy of Garcinia extract, hydroxycitric acid (HCA) as a weight reduction agent, using data from randomised clinical trials (RCTs). Electronic and nonelectronic searches were conducted to identify relevant articles, with no restrictions in language or time. Two independent reviewers extracted the data and assessed the methodological quality of included studies. Twenty-three eligible trials were identified and twelve were included. Nine trials provided data suitable for statistical pooling. The meta-analysis revealed a small, statistically significant difference in weight loss favouring HCA over placebo (MD: −0.88 kg; 95% CI: −1.75, −0.00). Gastrointestinal adverse events were twice as common in the HCA group compared with placebo in one included study. It is concluded that the RCTs suggest that Garcinia extracts/HCA can cause short-term weight loss. The magnitude of the effect is small, and the clinical relevance is uncertain. Future trials should be more rigorous and better reported

Use of Complementary and Alternative Medicine in the context of cancer; prevalence, reasons for use, disclosure, information received, risks and benefits reported by people with cancer in Norway

Background Research exploring the use of specific Complementary and Alternative Medicine (CAM) modalities by Norwegian cancer patients is sparse. The aims of this study were therefor to map the different CAM modalities cancer patients use and further investigate their rationale for use, communication about use, self-reported benefits and harms, and their sources of information about the different modalities. Methods In cooperation with the Norwegian Cancer Society (NCS), we conducted an online cross-sectional study among members of their user panel with present or previously cancer (n = 706). The study was carried out in September/October 2021 using a modified cancer-specific version of the International Questionnaire to Measure Use of Complementary and Alternative Medicine (I-CAM-Q). In total, 468 members, 315 women and 153 men, agreed to participate resulting in a response rate of 67.2%. The study was reported in accordance with the National Research Center in Complementary and Alternative Medicine’s (NAFKAM) model of reporting CAM use. Results A large proportion of the participants (79%, n = 346) had used some form of CAM with a mean of 3.8 modalities each (range 1-17); 33% (n = 143) had seen a CAM provider, 52% (n = 230) had used natural remedies, while 58% (n = 253) had used self-help practices. Most of the participants used CAM to increase their quality of life, cope with the cancer disease or for relaxation/well-being (64%-94%), mostly with high satisfaction and low rates of adverse effects. Few used CAM to treat cancer or prevent it from spreading (16%, n = 55). The main information sources were health care providers (47%), the internet (47%), and family and friends (39%). More than half (59%) of the cancer patients discussed their use of at least one CAM modality with a physician. Conclusions The results of this survey will provide health professionals with more in-depth insight into the patterns of CAM use by cancer patients and facilitate better-informed discussions with their patients. Considering the high use of CAM, reliable information provision supporting cancer care providers’ knowledge and health literacy among patients as well as good communication are crucial. The cooperation between the NCS and NAFKAM provides an example of how to address these issues

The use of Garcinia extract (Hydroxycitric Acid) as a weight loss supplement: a systematic review and meta-analysis of randomized clinical trials,”

The aim of this systematic review is to examine the efficacy of Garcinia extract, hydroxycitric acid (HCA) as a weight reduction agent, using data from randomised clinical trials (RCTs). Electronic and nonelectronic searches were conducted to identify relevant articles, with no restrictions in language or time. Two independent reviewers extracted the data and assessed the methodological quality of included studies. Twenty-three eligible trials were identified and twelve were included. Nine trials provided data suitable for statistical pooling. The meta-analysis revealed a small, statistically significant difference in weight loss favouring HCA over placebo (MD: −0.88 kg; 95% CI: −1.75, −0.00). Gastrointestinal adverse events were twice as common in the HCA group compared with placebo in one included study. It is concluded that the RCTs suggest that Garcinia extracts/HCA can cause shortterm weight loss. The magnitude of the effect is small, and the clinical relevance is uncertain. Future trials should be more rigorous and better reported

Automated NMR resonance assignment strategy for RNA via the phosphodiester backbone based on high-dimensional through-bond APSY experiments

A fast, robust and reliable strategy for automated sequential resonance assignment for uniformly [13C, 15N]-labeled RNA via its phosphodiester backbone is presented. It is based on a series of high-dimensional through-bond APSY experiments: a 5D HCP-CCH COSY, a 4D H1′C1′CH TOCSY for ribose resonances, a 5D HCNCH for ribose-to-base connection, a 4D H6C6C5H5 TOCSY for pyrimidine resonances, and a 4D H8C8(C)C2H2 TOCSY for adenine resonances. The utilized pulse sequences are partially novel, and optimized to enable long evolution times in all dimensions. The highly precise APSY peak lists derived with these experiments could be used directly for reliable automated resonance assignment with the FLYA algorithm. This approach resulted in 98% assignment completeness for all 13C-1H, 15N1/9 and 31P resonances of a stem-loop with 14 nucleotides

Prevalence of late and long-term effects of cancer (treatment) and use of complementary and alternative medicine in Norway

Background The increasing number of patients surviving cancer leads to more people experiencing late and long term-effects from the disease and its treatment. Fatigue, sleep disorders, early menopause, pain, and nerve damage are commonly reported. Methods helping people to recover after cancer treatment are therefore essential. The aims of this study were threefold; (1) to determine the level of cancer patients suffering from late and long-term effects of cancer diagnosis and treatment in Norway, (2) explore complementary and alternative medicine (CAM) modalities used for managing these adversities, and (3) describe self-perceived benefits and harms of the CAM interventions. Methods The study was conducted in cooperation with the Norwegian Cancer Society (NCS) and consisted of an online cross-sectional study among members of the NCS user panel with present or previous cancer (n = 706). The study was carried out in September/October 2021 using a modified cancer-specific version of the International Questionnaire to Measure Use of Complementary and Alternative Medicine (I-CAM-Q). A total of 315 women and 153 men agreed to participate, resulting in a response rate of 67%. Results Most of the participants (83%) suffered from late and long-term effects of cancer treatment; mostly fatigue (59.2%), sleep disorder (41.5%), hot flashes (39.2%), nerve damage (polyneuropathy, 38.0%), and pain (36.6%) with a mean number of 5.1 different late and long-term effects. Late and long-term effects were positively associated with younger age and college/university education. Nearly half of the participants experiencing late and long-term effects (43%) reported having used CAM to treat these complaints. Most frequently used were self-help practices (26%) such as relaxation therapy (19%), yoga (14%) and meditation (13%), but also visits to CAM providers were reported by 22%. Herbal- and other natural remedies to treat late and long-term effects were used by 13%. A high percentage of CAM users reported self-perceived improvements of their symptoms (86% for self-help practices, 90% for visits to CAM providers). Few experienced adverse effects of the CAM treatment. Conclusion A large proportion of cancer patients suffered from a wide range of late and long-term effects of cancer diagnosis and treatment, and they use CAM to treat these complaints to a rather high degree. Relaxation therapy, yoga, meditation, massage, and acupuncture were the most frequently used therapies regardless of complaint. The therapies used are generally considered to be both safe and beneficial for the respective complaint, indicating that the participants seem to be well informed about the choices they make