Morphological, structural and thermal properties of developed CNC and electrospun nanofibers were characterized. X-ray diffraction and thermal analysis revealed that the crystallinity of PV was reduced by the electrospinning process, and the incorporation of CNC increased the thermal stability of biodegradable nanofibers. Interactions between CNC and PV polymer also enhanced the thermal stability of CNC and improved the dispersion of CNC within the PLA matrix. PLA materials with CNC lyophilized were also casted in order to compare the properties with materials based on CNC containing nanofibers. Nanofibers and CNC were incorporated into PLA at three concentrations: 0.5%, 1% and 3% (CNC respect to polymer weight) and nanocomposites were fully characterized. Overall, nanofibers containing CNC positively modified the physical properties of PLA materials, such as the crystallinity degree of PLA which was greatly enhanced. Specifically, materials with 1% nanofiber 1PLA(PV/CNC)n presented highest improvements related to mechanical and barrier properties; elongation at break was enhanced almost four times and the permeation of oxygen was reduced by approximately 30%.Electrospun nanofibers of poly  (PV) were obtained to improve dispersion of cellulose nanocrystals (CNC) within hydrophobic biopolymeric matrices, such as poly(lactic acid) (PLA). Electrospun nanofibers (PV/CNC) Facing the need to reduce the negative effects on the environment caused by the accumulation of conventional petroleum-based polymer waste, the efforts of material researchers have focused on the development of new materials based on biopolymers; these are degraded under natural conditions by microorganisms without leaving toxic or harmful waste in the environment. These biodegradable materials present a number of excellent and promising properties in several applications, including in packaging, and the automotive and biomedical sectors. Some of their properties need to be improved, such as excessive brittleness and insufficient barrier properties; however, the development of nanocomposites has been considered a promising solution to these disadvantages. A nanocomposite is a multiphase composite where at least one of the phases is present in the nanoscale dimension.Over the last years, nanocellulose and particularly cellulose nanocrystals (CNCs) have attracted much interest for the production of fully-renewable and biodegradable nanocomposites. CNCs are natural nanofillers obtained from cellulose, a fibrous, hard, and water-insoluble substance that plays an essential role in maintaining the structure of plant cell walls. The multiple connections between cellulose chains through hydrogen bonding constitute cellulosic fibrils which have highly ordered  and unordered (amorphous) regions. Amorphous regions can be selectively hydrolyzed through acid hydrolysis, to obtain nanosized crystalline regions called cellulose nanocrystals . Many stDespite the lack of apparent compatibility between these two polymers, the results were interesting. The morphology, thermal, mechanical and barrier properties of PLA nanocomposites were studied and compared to PLA nanocomposites with freeze-dried CNCs. Moreover, the study of the effect of the electrospinning process and the incorporation of CNC in the PV properties were also investigated.® Co. . Gohsenol type AH-17 polyvinyl alcohol (PV) (saponification degree 97–98.5% and viscosity 25–30 mPa·s) was obtained from The Nippon Synthetic Chemical Co. . Cellulose fibers (CF) (powder 80–145 µm), and polyethylene glycol (PEG) were supplied by Sigma Aldrich. Chloroform and sulfuric acid 95–97% were supplied by Merck. Low-flow PES 170 dialysis membranes  were purchased from Nipro Medical Corporation .Poly(lactic acid) (PLA), 2003D ) was purchased from NatureworksCellulose nanocrystals were prepared following the procedure of Bondenson et al. (2006) with some modifications . Ten graw/v) owing to achieve a high incorporation degree of CNC into the electrospun PV nanofibers in order to incorporate the minimum concentration of PV in the final PLA nanocomposite. 1.2 g poly (PV) was added to 20 mL of CNC solution and stirred at 90 °C until polymer was dissolved. PV solution at the same concentration without CNC was also electrospun. Solutions were transferred to 5 mL plastic syringes and connected through a PTFE tube to a 18-gauge blunt stainless steel needle charged by a high voltage power supply with a range of 0–30 kV. The collector plate was fixed at a working distance of 10 cm below the needle tip and connected to the grounded counter electrode of the power supply. A voltage of approximately 10 kV and the flow rate of 0.25 mL/h were used. CNC containing PV nanofibers were named “(PV/CNC)n”, while neat PV nanofibers were named “(PV)n”.CNC solution obtained from dialysis was concentrated through evaporation until a final concentration of 1.8% n, 1PLA(PV/CNC)n and 3PLA(PV/CNC)n”, respectively. Thus, it was necessary the incorporation of the electrospun nanofibers (PV/CNC)n at 2.2% for 0.5PLA(PV/CNC)n, 4.7% for 1PLA(PV/CNC)n, and 13% for 3PLA(PV/CNC)n n, 1PLA(PV)n and 3PLA(PV)n. It is crucial to elucidate the effect of each component on the final PLA properties changes. PLA blank was also casted and named “PLA”. PEG was added at 5% w/w polymer for all formulations to facilitate the casting process. PLA based films were obtained by solution-extension-evaporation process (“casting”). Electrospun (PV/CNC)shown in . ControlThe morphology of the electrospun nanofibers and the nanocomposites were studied using a scanning electron microscope (SEM) JSM-5410 Jeol  with accelerating voltage at 10 kV. Films were fracturated using Tensile Tester because it was not possible to obtain the samples through cryo-fracture. Then, samples were coated with gold palladium using a Sputtering System Hummer 6.2., and SEM micrographs of the surface and the cross-section of the materials were taken. Nanofibers were also analyzed through transmission electron microscopy (TEM). Nanofibers were placed on a cupper grid and examined under a Phillips Tecnai 12 Bio Twin TEM at 80 kV. Images were recorded using a CCD camera Olympus Megaview G2 at different magnifications.Structures of CNC, nanofibers and PLA nanocomposites were evaluated with X-ray diffraction (XRD). XRD patterns were measured using a Siemens Diffractometer D5000  with 30 mA and 40 kV using CuKa (λ = 1.54 Å) radiation at room temperature. All scans were performed in a 2θ range 2–12° at 0.02°/s.. Samples (ca. 9 mg) were heated from 20 to 600 °C at 10 °C min−1 under nitrogen atmosphere (flow rate 50 mL·min−1).Thermogravimetric analyses (TGA) were carried out using a Mettler Toledo Gas Controller GC20 Stare System TGA/DCS −1 heating rate. Sample weight was about 8–10 mg. The degree of crystallinity (cX) of the PLA materials was deduced using the equation :mH is the specific melting enthalpy of the sample (J g−1); ΔccH is the specific cold crystallization enthalpy of the sample (J g−1) and ΔH0m is the specific melting enthalpy of a wholly crystalline PLA (93.1 J g−1) [Differential Scanning Calorimetry (DSC) analyses were also performed with a Mettler Toledo DSC-822e calorimeter . Thermograms were obtained from −20° to 220°, cooling to −20 °C, and a second heating process to 220 °C with 10 °C min−1. Results are the average of 8 specimens for each film.Tensile testing of each material was measured using a Zwick Roell model BDOFB 0.5 TH Tensile Tester , according to ASTM D-882. Strips (10 cm × 2.5 cm) of films were cut using a die cutter and kept at 25 °C and 50% RH for 48 h before the test. Analyses were carried out with a 1 kN load cell. The initial grip separation was 10 cm and the crosshead speed used was 50 mm·minThe oxygen permeation rates of the materials were determined at 0%, 35%, 50% and 75% relative humidity (RH), and 23 °C using an Oxtran model 2/21 ML Mocon . Films were previously purged with nitrogen for a minimum of 16 h in the RH desired, prior to exposure to an oxygen flow of 10 mL/min. Permeation values were determined every 45 min until constant.p < 0.05.A randomized experimental design was considered for the experiments. Data analysis was carried out using Statgraphics Plus 5.1 . This software was used to implement variance analysis and Fisher’s LSD test. Differences were considered significant at n were successfully obtained with a final relation of 23/77 (w/w) CNC/PV. TEM and SEM microscopies were useful tools to observe the morphology of CNCs and the electrospun nanofibers (PV)n and (PV/CNC)n ((PV/CNC)PV/CNC)n A–E. As FPV/CNC)n D,E revean at lowest concentration (film 0.5PLA(PV/CNC)n) had a smooth surface, but, at higher concentrations (films 1PLA(PV/CNC)n and 3PLA(PV/CNC)n), it was possible to distinguish two different surfaces due to the process used: ‘casting’. The surface, which was in contact with the glass during casting, presented also a smooth surface while the other side presented some roughness  and nanocomposites at the highest concentration of these nanocomponents, as an example, are plotted in n, presented only the main crystallographic peak at 2θ = 19.5°, confirming that the electrospinning process changed PV crystallinity. Certainly, the crystallization process during electrospinning implied the generation of different spherulite sizes, although crystallinity degree remained the same (as shown later in DSC results). On the other hand, the (PV/CNC)n diffraction pattern exhibited two PV characteristic peaks (2θ = 19.5° and 22.4°), probably because the presence of CNC favored the polymer crystallization during the electrospinning process. In addition, a new peak at 2θ = 15.5° appeared, which was attributed to the presence of CNC.Evaluations with X-ray diffraction (XRD) of cellulose nanocrystals and nanofibers (CNC and (PV/CNC)n enhanced the degree of crystallinity of PLA, with new diffraction peaks appearing at 2θ = 14.9°, 19.1°, and 22.5°, which agreed with data obtained by Pagés et al. [The XRD analyses of the developed materials were performed to obtain information about material crystallinity C. The PLs et al. . With res et al. .n, presented an earlier degradation with a maximum at approximately 305 °C attributed to the electrospinning process causing a change in the polymer nanoscale structure, which meant that it had more surface and, hence, the heat penetrated faster. On the other hand, nanofibers containing CNC, (PV/CNC)n, presented better thermal stability. As onsetT and the temperature of maximum degradation of PV nanofibers containing CNC due to the formation of hydrogen bonds between PV side chains and CNCs [and CNCs . In addin to the PLA matrix slightly decreased the thermal stability of nanocomposites, probably due to the earlier degradation of the PV polymer matrix. As concentration of (PV/CNC)n increased, temperature of maximum degradation shifted to lower temperatures, although these decreases were not significantly different. On the other hand, nanocomposites with CNC did not present significant changes on the maximum degradation temperature but, as Regarding the nanocomposites, they all presented one main degradation process which indicated good compatibility between the constituents of the films. In general, the addition of nanofibers (PV/CNC)In addition, DSC thermograms were performed to deduce, firstly, the effect of the incorporation of CNC and the electrospinning process on PV crystallinity, and, secondly, the effect of the incorporation of cellulose nanocrystals and electrospun nanofibers on PLA thermal properties. In the case of electrospun nanofibers, both the first and second heating processes were analyzed. As gT value, while crystallinity was not significantly affected. Surely, the decrease on gT value was due to the presence of residual water, confirmed by the mass loss on TGA analysis, that resulted in a plasticizing effect. Therefore, after removing the thermal history of the materials, gT values of the PV control and electrospun nanofibers were not significantly different. Nevertheless, melting enthalpies, which were initially similar, during second heating process, melting enthalpy of (PV)n nanofibers was greatly higher than PV control, probably due to the lower degradation suffered during the first heating process.The electrospinning process also influenced PV thermal properties since the first heating process showed a decrease on glass transition temperature, n presented an enhancement of gT value, indicating that the mobility of the chains of the polymer was reduced by the presence of the CNCs due to the interactions between PV and the CNCs. In addition, CNC incorporation affected the crystallinity conformation because any enthalpic process was observed during the cooling and second heating processes, suggesting the first heating process completely degraded the crystal structure. As n started suffering a degradation process that was confirmed by running a DSC of this sample until 300 °C.CNC incorporation into PV nanofibers also had an interesting effect. Electrospun (PV/CNC)ccH and ΔmH) were corrected for PLA content. Nanocomposites showed a slight decrease on glass transition temperatures, cold crystallization and melting temperatures associated with the plasticizing effect of (PV/CNC)n nanofibers [n were well dispersed and probably intermingled between polymer chains enhancing their mobility. However, when nanofiller concentration was 3%, this trend changed and the rigidity of materials increased. Nanofibers (PV/CNC)n facilitated the crystallization process of PLA biopolymer. This fact was already observed with the XRD results. Other works have found that cellulose fibers induced crystal nucleation at the fiber surface, and this effect was called the transcrystallinity effect.nofibers . As SEM This result is very positive from a barrier perspective, since crystals are typically impermeable systems but, mechanically, could be negative because crystallization can promote additional rigidity and, hence, fragility for the biopolymer mechanical performance ,36,37. RThe mechanical properties of the films were characterized by Young’s modulus (YM), tensile strength (TS), and elongation at break (EB) values and the results are summarized in n increased YM, although statistical analysis revealed the results were not significantly different. As already observed on DSC analysis, the presence of (PV/CNC)n induced an extra polymer crystallization that could result in this enhancement of Young’s modulus because the material also increased their rigidity. Moreover, the presence of these nanofibers increased considerably the elongation at break values of these nanocomposites. Although this enhancement on EB values was due to the presence of PV electrospun fibers since composites with (PV)n also presented some increase, this effect was greatly higher in the nanocomposites with CNC-containing electrospun nanofibers. EB value of 1PLA(PV/CNC)n material was approximately three times higher than those values published in different literature studies [In general, the incorporation of electrospun nanofibers (PV/CNC) studies ,13,15,39n, were also performed in order to study the influence of PV on the modification of these parameters. As n presented similar effects than nanofibers with embedded CNCs, excepting for elongation at break values, where the presence of CNCs considerably increased this parameter. Arrieta et al. already observed a similar effect when the modification of CNCs improved their dispersion into PLA-PHB materials and generated better interaction between PLA and PHB [Mechanical properties of control films based on PV nanofibers without CNC, (PV) and PHB .Ultimately, the TS value showed a slight decrease with respect to PLA. The reduction of TS have occurred in previous works and the values are within the range reported by other authors, which are between 30 and 60 MPa ,15,19,20−19 and 3 × 10−18 m3 m/s m2 Pa, which are in agreement with those reported in the literature for PLA [ for PLA ,20,41. TIn general, transport properties are known to be strongly influenced by tortuous path altering factors, including shape and aspect ratio of the filler, degree of dispersion, filler loading, orientation, adhesion to the matrix, moisture activity, filler-induced crystallinity, polymer chain immobilization, and filler induced solvent retention ,8,12. Thn was the nanocomposite that exhibited the highest oxygen barrier, mainly at high RH. Indeed, this material showed a reduction in the oxygen permeation approximately of 30% at 35%, 50% and 75% RH. In general, permeation values of PLA nanocomposites with (PV/CNC)n were very similar to results obtained by Martinez-Sanz et col. with oxygen permeability values ranging from 1.5 × 10−18 to 2.2 × 10−18 m3 m/s m2 Pa [As /s m2 Pa . Permeatn (grey bars) also presented a decrease on oxygen permeability values. The presence of PV nanofibers contributed to the improvement of barrier properties but, mainly in the best performance material, the presence of CNCs also played an important role. Probably, the strong interactions between CNCs and PV based on hydrogen bonding  decreased the effect of humidity on PV polymer due to the reduction of available PV hydroxyl groups to interact with water.The effect of the incorporation of the PV polymeric matrix in the formulation of these nanocomposites is worthy of highlighting. As shown in On the other hand, CNCs-containing nanocomposites presented higher values of oxygen permeation than when compared to the PLA control. Contrary to other works, nanocomposites with freeze-dried CNCs did not present improvement on barrier properties ,15. AlthAnother interesting fact to highlight was that oxygen permeation was not affected by water activity. In some cases, gas permeability was lower when relative humidity was very high, and no significant differences were observed between RH. This effect has already been reported ,41. The n. Results were not so striking due to the presence of PEG as the plasticizer and, principally, the effect of the obtaining process. On the other hand, the characterization of PLA nanocomposites revealed that the incorporation of electrospun nanofibers with and without CNC considerably affected the principal properties. Electronic microscopy revealed it was possible to obtain a high dispersion of CNCs when they were incorporated into PLA matrix by means of PV electrospun nanofibers. There were some improvements in the properties of the nanocomposite materials compared to pure PLA. Results indicated that the incorporation of (PV/CNC)n modified the structural properties of PLA, and the performance of CNCs on the film for reinforcement is better through electrospun nanofibers. It was clearly evidenced that the incorporation of PV also influenced PLA properties. The films with electrospun nanofibers containing CNCs presented an improvement on the mechanical behavior, resulting in a more flexible and tough film. The ductility of the nanocomposite with electrospun nanofibers containing CNCs was clearly improved. Although it was not observed for all nanocomposites, barrier properties were significantly enhanced with the incorporation of CNCs through electrospun PV nanofibers at 1% when compared to CNCs-containing nanocomposite films. Undoubtedly, the material 1PLA(PV/CNC)n presented the best performance on mechanical and oxygen barrier properties. The performance of these novel nanocomposites was a sum of the effect of the incorporation of PV nanofibers and the presence of CNCs.The main goal of this study was to improve the dispersion of cellulose nanowhiskers (CNCs) within PLA matrices in order to improve its physical properties. The strategy used was the incorporation of CNCs into electrospun nanofibers of polyvinyl alcohol, (PV/CNC)


Antimicrobials represent one of humanity’s medical revolutions enabling us to treat both human and veterinary bacterial infections. It is, therefore, of utmost importance to preserve their effectiveness. However, during the last decades, the continuing rapid development of antimicrobial resistance (AMR) has emerged as a major global public health concern . ResistaEnterobacteriaceae and the recently detected plasmid-mediated colistin resistance (mcr-1 gene) in livestock, food, and humans in China  associated with AMU in livestock.Surveys and expert opinions are well-accepted approaches for exploring the behavioral basis of AMU and AMR. These methods could provide informative data on the attitudes, motivation, and knowledge of veterinarians and farmers toward AMU and AMR . On the Bacteria can be naturally resistant against specific antimicrobial classes (intrinsic resistance) , howeverThe complex epidemiology of AMR together with the socio-economical drivers make this topic the quintessential One-Health issue. Transectoral and transdisciplinary approaches are a “must-do” to tackle AMR appropriately. A reduction in AMU was not always followed by a decline in AMR, as demonstrated in the case of VRE . ReducinStaphylococcus aureus (LA-MRSA). Here, animal trade has been identified to be a major driver of LA-MRSA dissemination (Enterobacteriaceae and in particular Escherichia (E.) coli, fecal shedding represents the main route of dissemination, thus not only host, but also environmental reservoirs may exist which constitute multiple, complex ways of resistance introduction and transmission. So far, experimental studies have demonstrated animal-to-animal transmission of resistant E. coli under controlled conditions within confined compartments  for dissemination to other species, including humans .It is, therefore, important to improve our knowledge on how animal contacts and trade (direct transmission), farm management, and the wider farm environment (indirect transmission) drive the dissemination of AMR and to identify potential interventions to counteract this phenomenon. Farm management studies could include all those practices that potentially facilitate spread of resistant bacteria within and between farms and from farms to the environment, such as farm hygiene and biosecurity, animal waste management, structure  of holdings as well as animal production intensity.vice versa. Source attribution can be of help to shed light on the contribution of AMR originating from livestock to the public health resistance burden. Moreover, it can also be an important piece of evidence when developing targeted interventions against AMR. Genomic data might also provide some additional information on potential evolutionary processes in bacteria during transmission within the studied populations. Furthermore, molecular epidemiology data can shed some light on how much of the resistance reservoir is attributed to the spread of resistant bacteria or de novo emergence due to AMU selection pressure in the studied farms.Holistic, One-Health approaches should always be backed with molecular epidemiological data, which can provide information about links between resistance genes observed in different samples, such as from animals of different origin. Resistance genes should be studied not only in animal samples but also in the wider farm environment, such as farmers, other livestock species, farm pets, wildlife, manure, and water. These ecological data can provide the molecular link to characterize reservoirs of resistant bacteria and could support studies on transmission pathways between animal populations but also from animals to humans and AMR is a complex phenomenon and is driven by biological processes and socio-economical factors. Understanding the attitude and knowledge of farmers and veterinarians toward AMU and AMR is a crucial step for the design of strategies to combat this public health threat. The lack of detailed AMU data impacts our ability to interpret surveillance data on AMR and to design efficient interventions. Therefore, monitoring systems to fill this knowledge gap should be prioritized. Finally, the ecology of AMR should be addressed with a holistic, One-Health approach combining expertise from different disciplines, such as veterinary clinicians, public health scientists, microbiologists, wildlife veterinarians, environmental scientists (ecologists), agricultural/forestry scientists, and epidemiologists.IM wrote the manuscript. LPC provided valuable expertise on monitoring systems for antimicrobial usage and social sciences. KS provided valuable expertise on the topics of One-Health and social sciences. GS-R provided valuable expertise and feedback in all topics included in this opinion manuscript and assisted IM in the conceptualization of the manuscript. All the authors have read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Randomized clinical trials have shown the efficacy and safety of short-acting exenatide in patients with type 2 diabetes mellitus (T2DM). The aim of this observational study was to investigate the effectiveness and safety of exenatide twice a day in Korean patients with T2DM who are suboptimally controlled with oral hypoglycemic agents.This study was a post hoc analysis of multi-center (71 centers), prospective, observational, single-arm, post-marketing study of short-acting exenatide 5 to 10 μg twice a day from March 2008 to March 2014 and analyzed those who finished the follow-up over 20 weeks of medication. Changes of hemoglobin A1c (HbA1c), fasting plasma glucose (FPG), and body weight values before and after exenatide treatment were analyzed. Adverse events and adverse drug reactions were estimated in patients who were treated with exenatide at least once and for whom follow-up for safety has been completed.p < 0.001). Subjects with higher baseline glucose and HbA1c levels showed an independent association with a greater reduction in glucose level. In addition, short duration of diabetes less than 5 years was an independent predictor for the improvement in glucose level. The majority of study subjects showed a reduction in both body weight and glucose level (63.3%) after exenatide treatment. In terms of safety profile, exenatide treatment was generally well-tolerated and the incidence of severe adverse event was rare (0.8%). The gastrointestinal side effects were most common and hypoglycemia was reported in 1.7% of subjects.After 20 weeks treatment with exenatide, mean HbA1c and body weight were significantly reduced from 8.4% to 7.7% and from 83.4 kg to 80.2 kg, respectively  contains supplementary material, which is available to authorized users. The prevalence of obesity and type 2 diabetes mellitus (T2DM) is now increasing rapidly in Korea and Asian countries –3. HowevExenatide is an incretin mimetic that activates glucagon-like-peptide-1 (GLP-1) receptors. It mainly decreases postprandial plasma glucose level by stimulating insulin secretion, suppressing glucagon secretion and delaying gastric emptying in a glucose-dependent manner , 8. Numeβ-cell dysfunction is a main pathogenic mechanism for the development of T2DM in Asian population but, a lesser degree of insulin resistance compared with Caucasian [However, the pathophysiology of T2DM could be different according to ethnicity and it was reported that aucasian , 14. In aucasian . Furtheraucasian .Therefore, the aim of this study was to investigate the effects of exenatide twice a day injection in a real clinical practice on blood glucose level, body weight, and safety profiles in obese Korean patients with T2DM who are suboptimally controlled with oral hypoglycemic agents.ClinicalTrials.gov, number NCT02090673). The investigators’ decisions regarding the proper treatment and care of the patient were made in the course of the normal clinical practice; without blinding or randomization to particular comparator arms or therapies. In this study, patients were enrolled for the collection of their data on observations made during normal clinical practice such as serum HbA1c, fasting plasma glucose (FPG), body weight, medical history, and adverse events.This is a post hoc analysis of multi-center (71 centers), prospective, observational, single-arm, post-marketing surveillance (PMS) study to evaluate the safety and effectiveness of short-acting exenatide (Byetta®), conducted from March 2008 to March 2014, in patients with T2DM  . The basPatients who were participating the study could reject or withdraw the continuation of treatment or the study with the reason of follow-up lost, adverse events, patient’s decision, or physician’s decision. If a patient discontinued the treatment during the study, the patients were contacted to collect all adverse events information and reasons why they want to stop the treatment. All relevant information was captured by data form. Patients who were discontinued from study were included to safety population if they had taken at least one dose of exenatide. We calculated the discontinuation rate due to any adverse events. Adverse events and serious adverse events which were recorded in electronic case report form were recoded and reanalyzed according to the current routine classification. For the serious adverse events, we reviewed the patient record and revealed the diagnosis of them.All statistical analysis was performed with SAS  for the numerics, and NCSS  for the graphics. For effectiveness analysis, missing values was imputed using the last observation carried forward methods. Baseline characteristics were shown in descriptive statistics such as mean and standard deviation for continuous data, or frequency and percentage for categorical data. Safety analysis was also summarized descriptively.p-value is less than 0.05, it was considered statistically significant.Clinical factors which could affect the HbA1c reduction between responder and non-responder groups were analyzed using chi-square test in 3 different strata. We performed multivariate logistic regression test to adjust the potential variables for the HbA1c reduction in each stratum. To confirm the correlation between changes of HbA1c and body weight, scattered graph was rendered and correlation coefficient was calculated. If Of the total 1269 patients who received more than one-time injection of exenatide twice a day, data retrieved from 754 patients who completed 20 weeks treatment with exenatide were analyzed for the safety profiles . Among the 754 participants, 619 patients who measured HbA1c level both at baseline and 20-week endpoint were analyzed for the effectiveness .n = 754), the mean age and BMI of study subjects were 48.8 years and 31.2 kg/m2, respectively. 34.0% of the subjects were men. Approximately one third of the study subjects had a family history of diabetes and duration of diabetes was 7.7 years. At baseline, mean HbA1c and FPG level were 8.4% and 164.3 mg/dL, respectively  and HbA1c (−0.75%) levels from baseline were observed over 20-week of exenatide treatment. To determine glucose-lowering effect of exenatide, we used three different criteria to define responder to exenatide treatment. The characteristics of responders by three definitions as stratum are shown in Table  mg/dL anp < 0.001, Fig. We next determined the weight change with exenatide treatment. After 20 weeks treatment with exenatide twice a day, body weight was significantly reduced from 83.4 kg to 80.2 kg . Consecutively, the discontinuation rate due to adverse events between 8 and 20 weeks of medication showed 4.5% (49 patients out of 1088 patients)  who completed 20 weeks treatment with exenatide, severe adverse event was very rare (0.8%) but 173 cases (22.9%) of adverse events were reported. The gastrointestinal side effects, mostly nausea, were the most common side effect and hypoglycemia was reported in 1.7% of subjects  such as hypoglycemia and weight gain etc., incretin-based drugs including GLP-1 analogues and dipeptidyl peptidase-4 (DPP4) inhibitors have been progressively developed and been regarded as a recommended drug for glycemic control in patients with T2DM . FurtherFrom this prospective, 20-week observational, phase 4 PMS study with short-acting exenatide twice a day in Korean patients with T2DM, we demonstrated 3 main findings. First, exenatide twice a day significantly induced HbA1c and weight reduction in real clinical practice. Second, higher baseline glucose level and short duration of diabetes were independent predictor for good glycemic control after exenatide treatment. Third, adverse events of exenatide in real-life setting are similar to those of RCTs .With respect to study design, this analysis retrieved from the prospective, observational, PMS study. RCTs and OSs have both benefits and drawbacks reciprocally, which should be interpreted with balanced stance. Briefly, the inclusion and exclusion criteria of RCTs tend to be defined narrowly to conduct either the regulatory requirements in drug trials or the specific aims targeting selected population. The characteristics of patients who participate in the clinical trials are to be more concerned on their health status and care by health providers, and compliant with the drugs. In these regards, they might provide limited usefulness on the effectiveness and safety of a drug to generalize to the real world practice. In contrast, the design of PMS OSs in everyday practice setting is able to cover a broader population without stringent inclusion/exclusion criteria, and characteristics of patients. Furthermore, it also provides valuable information on the physician’s preference and ideas . These un = 369) experienced the both benefits of GLP-1 analogues on HbA1c reduction and weight loss. Those who got none benefits of exenatide were just 3.77%. In the study, BMI did not affect glucose-lowering effect of exenatide. In one study which was aiming to investigate metabolic outcomes in patients with type 2 diabetes treated with exenatide, the response in HbA1c reduction was independent of baseline weight, and BMI [With respect to the glycemic effectiveness of exenatide twice a day in real-life setting, exenatide improved glycemic control through up-regulating β-cells and down-regulating α-cells residing in pancreatic islets. In recent meta-analysis, short-acting exenatide twice a day was effective in reducing HbA1c  compared with placebo in patients with T2DM . Further and BMI . Also, mn = 754) at 20 weeks in this PMS OS, 22.9% subjects reported any adverse events. Based on this finding, caution should be paid to the patients’ tolerability.With respect to the adverse events of exenatide, of 181 participants who discontinued exenatide injection, 37% ones failed to continue on exenatide within 8 weeks. Of 334 participants who discontinued exenatide injection between 8 weeks and 20 weeks, 14.7% of subjects discontinued on exenatide injection. Taken together, of 1269 subjects retrieved from safety analysis set, 161 (9.1%) reported to stop the exenatide injection. Those who were analyzed for effectiveness analysis set (β-cell functions and peripheral insulin sensitivity which are the key pathophysiologic factors in the development and progress of T2DM. The clinical implications of duration of diabetes, baseline FPG and HbA1c, and numbers of ADDs which were different between the responders and non-responders might be explained in these assessments. Second, in the balanced stance of both studies design of RCTs and OS, this study might remain observer and selection bias which are reflected by not scientific milieu but socio-economic one. Underreporting also occurred especially in drug adverse events. A final major limitation is the drop out data from missing data and adherence to physicians’ orders.The strengths of this study are the large sample number and the real-world clinical practice setting with patients of only Asian ethnic background. It, however, also has important weaknesses. This study did not provide the laboratory data to evaluate the pancreatic In conclusion, the results of the study of real-life clinical practice in Korea demonstrate that, in Korean subjects with T2DM, exenatide twice a day resulted significant reduction in glycemic control and relative tolerance in Korean patients with T2DM in real-world clinical setting.


Kaposi sarcoma is an angioproliferative disorder that requires infection with human herpesvirus 8 (HHV-8) for its development. The majority of cases are associated with HIV infection or other immunocompromising conditions. Thymomas are occasionally associated to cytopenia, which may alter the patients' immune responses.  Case report using clinical records.  Case report of a 46-year-old male patient diagnosed with thymoma and myasthenia gravis. The patient was referred to an otolaryngology consultation with complaints of facial pain in the right malar region, interpreted as an acute sinusitis. Following examination, an expansive maxillary sinus mass was found, and endoscopic surgery was undertaken. After careful investigation, it was diagnosed as a Kaposi sarcoma.  It is thought to be the first described case of a maxillary sinus Kaposi sarcoma in an HIV-negative patient. Thus, this entity has to be considered in the differential diagnosis of sinus masses, even in non-HIV patients. Thymic neoplasms are responsible for 0.2 to 1.5% of total tumor incidence and are the most common neoplasms arising in the anterior mediastinum in adults [There are no known risk factors, and there is a strong association with paraneoplastic syndromes, the most common being myasthenia gravis (MG). MG has been diagnosed in 30 to 40 percent of thymic neoplasms; other paraneoplastic syndromes are rare . MG is aKaposi sarcoma (KS) is an angioproliferative disorder that requires infection with human herpesvirus 8 (HHV-8) for its development. As many as 4–15% of the healthy population may be seropositive for HHV-8; however, only under certain circumstances does KS develop, namely, in immunocompromised patients. KS is classified into four types based on the clinical ground in which it develops: (1) classic ; (2) endemic ; 3) iatrogenic ; and (4) AIDS-associated (epidemic KS) [ iatrogenIn recent literature, some cases of association between KS and thymoma have been reported, most of them with cytopenia secondary to the thymic neoplasm . Some reported cases include KS in HIV-negative patients, some in unique presentation sites .We report the case of a male patient aged 46. The patient had been diagnosed with MG at age 26, when symptoms of facial and limb extremities weakness were noticed for the first time. Following investigation, a thymoma was found as the cause of MG, and at age 32, he was submitted to a thymectomy and treated with acetylcholinesterase inhibitors and immunosuppressants, namely, corticosteroids.At the age of 45, the patient was referred for the first time with complaints of facial pain in the right malar region and purulent nasal discharge from the same side. He was clinically diagnosed with acute sinusitis and underwent medical treatment, with slight relief of symptoms. As symptoms persisted after medical management, a CT scan was ordered, revealing an expansive maxillary sinus opacification with erosion of the right maxillary sinus medial wall . The magAn endoscopy for biopsy purposes was performed, but it was inconclusive due to clinically relevant hemorrhage from a vascularized lesion protruding in the medial wall of the maxillary sinus. An angiography and preoperative embolization  of the rThe patient remained stable with only symptomatic treatment until the age of 50 when, following worsening of MG symptoms , metastatic lung deposits of the thymoma were diagnosed. Surgical removal of the metastasis was undertaken.The thymoma was classified as a B3 , and therefore, the patient was also treated with adjuvant radiotherapy. During radiotherapy and due to worsening of MG symptoms, human immunoglobulin and corticosteroids were administered.Three months after these events, the patient had a new relapse from his MG and started corticotherapy. Two weeks into the treatment, he referred to newly skin pigmentation on the left wrist and legs. He also stated that these skin lesions were present 3 months earlier but smaller in size, growing with the recently added immunosuppressant therapy. These lesions were biopsied, and the histologic result was Kaposi sarcoma (KS). The patient was found to be HHV-8 IgG positive and HIV 1/2 and HTLV I/II negative.It was the diagnosis of KS in the biopsy of the limbs' lesions that led us to rethink the previous finding of the maxillary sinus kaposiform hemangioendothelioma, since the latter is usually not associated with HHV-8 IgG positivity nor is seen in adults. An histological review of the operatory sample was performed, and with immunohistochemistry technique , it was rediagnosed as a KS of the maxillary sinus.KS in paranasal sinuses has been reported extensively in the literature, however, always associated with HIV infection or with immunosuppressant therapy. KS associated with hematologic, lymphoid, and thymic neoplasia has also been described, but no cases have been reported in the paranasal sinuses . The KS Extensive research on the characteristics of the paranasal sinus histology was undertaken, in order to explain the propensity of the maxillary sinus to develop KS (in both HIV-positive and HIV-negative patients) compared to the other paranasal cavities. No differences between maxillary sinus and the other paranasal sinuses were found to be significant in the literature to explain this propensity, and therefore it is still an open subject for further investigation.Using the words “kaposi,” “sarcoma,” “maxillary sinus,” “HIV,” and “HIV negative” in different combinations in the databases PubMed/MEDLINE and Cochrane Database of Systematic Reviews , there were no findings of cases in the recent literature of a maxillary sinus KS in an HIV-negative patient. Therefore, the authors here present what is thought to be the first described case of a maxillary sinus KS in an HIV-negative patient. We alert for care in the differential diagnosis of paranasal sinus lesions if concomitant with a thymoma, because the immunosuppressive treatment of the second, or the disease itself, can cause the development of the KS, regardless of an associated HIV infection.There is still the need to keep investigating the specific features of the maxillary sinus that lead it to be more susceptible to these lesions.


Prionace glauca) cartilage and prepared KS oligosaccharides (KSO) through keratanase II-catalyzed hydrolysis. The structures of KS and KSO were characterized using multi-dimensional nuclear magnetic resonance (NMR) spectra and liquid chromatography-mass spectrometry (LC-MS). Shark cartilage KS was highly sulfated and modified with ~2.69% N-acetylneuraminic acid (NeuAc) through α-linked to galactose. Additionally, KS exhibited binding affinity to Ricinus communis agglutinin I (RCA120) in a concentration-dependent manner, a highly toxic lectin from beans of the castor plant. Furthermore, KSO from dp2 to dp8 bound to RCA120 in the increasing trend while the binding affinity of dp8 was superior to polysaccharide. These results define novel structural features for KS from Prionace glauca cartilage and demonstrate the potential application on ricin-antidote exploitation.As an important glycosaminoglycan, keratan sulfate (KS) mainly exists in corneal and cartilage, possessing various biological activities. In this study, we purified KS from blue shark ( GAGs are categorized into four classes on the basis of variations in monosaccharide compositions, linkage types, and the degree and pattern of sulfated substitution: chondroitin sulfate (CS)/dermatan sulfate (DS), heparin (HP)/heparan sulfate (HS), hyaluronan (HA), and keratan sulfate (KS) [Glycosaminoglycans (GAGs) are anionic, linear polysaccharides consisting of repeating disaccharide units of hexuronic acid (ate (KS) ,2.N-acetylglucosamine (GlcNAc) with sulfation occurring at the C6 of both saccharide units [N-acetylneuraminic acid (NeuAc) residues can be either α - or α -linked to Gal and typically occupy non-reducing terminal positions, while the fucose residues are α -linked to GlcNAc [KS is the only type of GAGs without uronic acid, and mainly composed of alternating repeating disaccharide unit of β 1,3)-linked galactose  and β 1,4)-linked -linked g-linked -de units . In genede units , and brade units . In addide units ,10,11,12o GlcNAc .O-sulfated-GlcNAc and non-sulfated Gal, is required in a critical period of visual cortex plasticity [Additionally, KS participates in diversely important biological processes in vivo. The high abundance of KS in cornea and cartilage is crucial in maintaining the proper hydration levels and keeps the cornea transparent ,14. In aasticity . KS in masticity ,18. KS casticity . Furtherasticity . Shiratoasticity . KS disaasticity .Ricinus communis). It is composed of chain A and chain B, linked by a disulfide bond. Ricin A chain (32 kDa) is a ribosome-inactivating enzyme, while ricin B chain (34 kDa) is a galactose/N-acetylgalactosamine binding lectin [Ricinus communis agglutinin I (RCA120) recognizes carbohydrate chains with non-reducing terminal β-d-galactose and Galβ1→4GlcNAc sequence [Ricin is a potent cytotoxic glycoprotein derived from the beans of castor plant  and liquid chromatography-mass spectrometry (LC-MS) are all powerful techniques for structural elucidation. Multi-dimensional NMR spectra have been applied to characterizing the capping segments, main chain repeating unit and linkage region of KS derived from bovine tracheal cartilage . Due to Prionace glauca cartilage. The structure of KS and KSO generated through enzymatic hydrolysis were elucidated by NMR and hydrophilic interaction liquid chromatography-Fourier transform mass spectrometry (HILIC-FTMS) analysis. The interaction of KS and KSO with RCA120 was also determined using SPR and MOE docking methods, providing a foundation for further pharmaceutical exploitation of Prionace glauca cartilage KS.In the present study, a highly sulfated KS was isolated from Prionace glauca cartilage was generated through proteolysis as previously reported [Mw: 45,980) was much bigger than that from bovine corneal and chicken egg white , which might be related to the unique marine environment, such as low temperature; high pressure and salt; and lack of oxygen, light, and nutrition [The crude polysaccharide from reported . The polutrition ,30.1H-NMR, DEPTQ NMR, and 2D 1H-1H COSY, 1H-13C HSQC, as well as 1H-13C HMBC. Major 1H- and 13C-chemical shifts identified from these spectra were assigned as The structural features of KS were properly characterized through a combination of 1D 1H-NMR spectrum, KS had a crowded region between 3.4 and 4.8 ppm, resulting in a severe signal overlap for the majority of the resonances. Two notable anomeric proton signals at 4.66 and 4.48 ppm were identical with H-1 of GlcNAc and Gal, respectively -linked fucose  and Gal (denoted G1) residues, respectively , assigned to 6-O-sulfo GlcNAc and 6-O-sulfo Gal residues, respectively -glucosaminidic linked to galactose in KS . On cleae GlcNAc . The KSOe GlcNAc . A total1H-NMR spectrum was 1:22, facilitating the calculation of NeuAc content as 2.69%, which closely agreed with the results calculated by GlycResoft. Based on the determined molecular weight and NeuAc content, we deduced that four NeuAc residues were present in each KS chain. This suggested that KS might have at least four branches since NeuAc was only believed to occupy the non-reducing ends of chains.Moreover, the integration ratio between H3ax of NeuAc and H1 of Gal in KS sample was partly digested by keratanase II, and then fractionated on a Bio-Gel P6 column. The elution curve illuminated five prominent oligosaccharide fractions . Based o1H-13C HMBC, C2, C4, and C5 of NeuAc were assigned by the cross-peaks with H3ax and H3eq. Then the proton in Gal (H-X) correlating with the C2 of NeuAc was identified -linked to Gal  had been described previously through SPR, the interaction between RCA120 and KS polysaccharide in vitro was scarce [120 in a concentration-dependent manner with DK values of 1.22 × 10−7 M and 1.37 × 10−7 M, respectively . The chondroitin ABC lyase and heparin lyases I, II, III were performed in our laboratory following the reported method [Escherichia coli was prepared as previously described [d method . XB-SAX escribed . AcetoniPrionace glauca cartilage was prepared by methods reported previously with minor modification [KS from fication . The crufication . The fra2-Gelatin method [Mw) were determined by high performance gel filtration chromatography system and detected by refractive index detector (RID) and multi-angle laser scattering system (MALLS)  [Uronic acid content was determined by modified carbazole method . Sulfaten method . Purity ra, USA) . Monosacra, USA) .The KS sample was completely degraded by enzymatic hydrolysis. KS (100 µg) was dissolved in 100 μL of digestion buffer containing 50 mM ammonium acetate (pH 7.0). Excess keratanase II (50 mU) was added to KS sample and incubated at 37 °C overnight with gentle agitation. Enzymatic digestion was terminated by heating in a 100 °C water bath, and then spun down at 12,000 rpm for 5 min; supernatant was used directly for HILIC-FTMS analysis.4OAc/98% acetonitrile (solvent A) and 5 mM NH4OAc/H2O (solvent B) at a flow rate of 150 μL/min. The gradient was programmed as 92% A initially and then linearly changed to 60% A over 58 min. The analysis was performed in the negative ion mode using a capillary temperature of 275 °C. The spray voltage was 4.2 kV and Nitrogen dry gas flowed at 40 L/min. Data acquisition and analysis were performed using Xcalibur 2.0 software  and GlycReSoft 1.0 software .HILIC-FTMS analysis was performed on an Agilent 1290 LC ultra-performance liquid chromatography (UPLC) system  equipped with a LTQ ORBITRAP XL mass spectrometer . The KSO were separated by a Luna HILIC column  at 25 °C. The mobile phase was a mixture of 5 mM NH4HCO3 at a flow rate of 0.2 mL/min. Next, each fraction was analyzed by ESI-MS at the negative-ion mode. For collision induced dissociation (CID)-MSn scanning, helium was used as collision gas with collision energy of 20–25 eV [KS sample (200 mg) was partly digested by keratanase II (5 IU) at 37 °C for 5 h with gentle agitation. The reaction was terminated in 100 °C water bath, and the precipitant was removed by centrifugation at 12,000 r/min. The supernatant was loaded on Bio-Gel P6 column connected to an ÄKTA-fast protein liquid chromatography (FPLC) system . The column was then eluted by 0.2 mol/L NH20–25 eV .2O) respectively and freeze-dried three times to replace all exchangeable protons with deuterium, then redissolved in 500 μL D2O. One-dimensional (1D) 1H-NMR, DEPTQ NMR, and two-dimensional (2D) 1H-1H COSY, 1H-13C HSQC, 1H-13C HMBC were performed at 298K on Bruker BioSpin GmbH 600 MHz  with Topspin 2.1.6 software . Chemical shifts were displayed relative to internal deuterated acetone for 1H and 13C.Samples of KS and KSO were dissolved in 500 μL 99.9% deuterium oxide  with the free amino groups and the residue with the reducing end in the polysaccharide chain following a published procedure [120 were injected over the sensor chip at a flow-rate of 30 µL/min for a period of 3 min followed by 3 min dissociation period. For the oligosaccharides binding, RCA120 was immobilized to CM5 chip using amine coupling based on the manufacturer’s protocol. The successful immobilization of RCA120 was confirmed by the observation of a ~2000 RU increase in the sensor chip. KSO from dp2 to dp8 and polysaccharide (1 mg/mL) in HBS-EP buffer ) were injected at a flow rate of 30 µL/min for 3 min. At the end of the sample injection, the same buffer was flowed over the sensor surface to facilitate dissociation. The sensor chip was regenerated by injecting with 30 µL of 2 M NaCl and 30 µL of running buffer to get a fully regenerated surface. SPR experiments were performed in triplicate at each concentration, confirming reproducibility. The binding sensor grams (RU versus time) were pooled, trimmed, double referenced, and experimentally fit to different kinetic models using BIAevaluation software v4.0.1 .SPR measurements were performed using a Biacore 3000 SPR instrument . For the polysaccharides binding, biotinylated shark cartilage KS and chicken egg white KS (From lab of Robert J. Linhardt) [rocedure . Two-folMolecular docking was performed using MOE with the AMBER12: EHT force field. Ricin B-like lectin crystal structures used for the ADT calculations above were utilized. The induced fit docking approach was applied with consideration of the side chain flexibility of KSO at the binding site. The ligand binding site was defined using the bound ligands in the crystal structures. Ten docking conformations of the ligands were produced, and the best scored conformation with minimum binding energy was selected for analysis.Prionace glauca cartilage. A series of KSO were also obtained by KS digestion with keratanase II. The structure of KS and KSO were confirmed by multi-dimensional NMR spectra and HILIC-FTMS. The results showed that shark cartilage KS was highly sulfated and the major disaccharide repeating unit was -3Gal6Sβ1→4GlcNAc6Sβ1-. Moreover, this KS was modified by NeuAc capped non-reducing ends of chains, and the NeuAc was α -linked to galactose.Herein, we reported a purified KS from 120 in a concentration-dependent manner with the DK value of 1.22 × 10−7 M. Furthermore, KS oligosaccharides from dp2 to dp8 bound to RCA120 in the increasing trend, while the binding affinity of dp8 was superior to polysaccharide. MOE docking assays also verified the results. In conclusion, these results define novel structural features for KS from Prionace glauca cartilage and demonstrate the potential application in ricin-antidote exploitation.SPR showed that KS bound to RCA


Finally, we have validated the accuracy of cuRRBS predictions for single and double enzyme digestions using two independent experimental datasets.DNA methylation is an important epigenetic modification in many species that is critical for development, and implicated in ageing and many complex diseases, such as cancer. Many cost-effective genome-wide analyses of DNA modifications rely on restriction enzymes capable of digesting genomic DNA at defined sequence motifs. There are hundreds of restriction enzyme families but few are used to date, because no tool is available for the systematic evaluation of restriction enzyme combinations that can enrich for certain sites of interest in a genome. Herein, we present customised Reduced Representation Bisulfite Sequencing (cuRRBS), a novel and easy-to-use computational method that solves this problem. By computing the optimal enzymatic digestions and size selection steps required, cuRRBS generalises the traditional MspI-based Reduced Representation Bisulfite Sequencing (RRBS) protocol to all restriction enzyme combinations. In addition, cuRRBS estimates the fold-reduction in sequencing costs and provides a robustness value for the personalised RRBS protocol, allowing users to tailor the protocol to their experimental needs. Moreover, we show  With the advent of next-generation sequencing (NGS) scientists are studying the biology of life at unprecedented resolution . UnfortuTo address these cost inefficiencies, many methods have been developed to reduce the number of fragments that need to be sequenced for a given biological system or WGBS validation experiment –25. ThesαI have also been used , Mus musculus (mm10) and Arabidopsis thaliana (TAIR10). Scaffolds not assembled into the main chromosomes were discarded. Genomic annotation for the human genome (hg38) was obtained from GENCODE  (CpG islands (CGIs), which were extracted from the UCSC Genome Browser  relative to the TSS of all protein-coding transcripts in GENCODE, similar to the strategy used in Taher et al.  , with th Browser . GC contrections . Promoter et al. . Genomicr et al. . All ann library ,49.Restriction package from Biopython v1.68 to digest the different genomes with the appropriate restriction enzymes in silico (in silico digestions was stored (pre-computed files) and subsequently read by cuRRBS when needed to reduce the computational time . When assessing enzyme combinations, the information from the appropriate individual pre-computed files  were combined by the software to compute all the necessary variables.We used the n silico . Only thTo ensure the user has full control over the enzymes that cuRRBS will use to derive the desired enrichments, one of the inputs given to cuRRBS is an enzyme annotation file. This file contains the desired isoschizomer families that the user wishes to be tested by cuRRBS. In the GitHub repository we have already defined enzyme annotation files for enzymes that are methylation-insensitive in a CG context and in CG, CHG and CHH contexts . HoweverIn addition, the output file from cuRRBS contains, by default, 30 cuRRBS protocols that would enrich for the user's sites of interest. Therefore, the user can determine which enzyme combination and size range would be the simplest and most appropriate for the given application. This provides the user with the opportunity to consider experimental factors that may complicate the protocol, such as buffer compatibility and whether consecutive digestions would be required.Enrichment Value (EV) can be calculated as:NF is the number of genomic fragments that will theoretically be sequenced (i.e. those whose lengths after the in silico digestion are within the size range); n is the total number of sites of interest; max_Score is the Score obtained if all the sites of interest were sequenced and:iw is the weight of the ith site of interest and iγ is 1 if the ith site would be theoretically sequenced (i.e. present in a size selected fragment and ≤ read length base pairs away from one of the ends of the fragment) and 0 otherwise. Since the objective of cuRRBS is to maximize the Score while minimizing the NF, the best results will be obtained when EV is minimized.cuRRBS makes use of several variables in order to find the best enzyme combination and size range which enriches for a certain set of genomic sites defined by the user (see ‘cuRRBS overview’). For a given enzyme (combination) and size range, the Cost Reduction Factor (CRF) for a given cuRRBS protocol can be calculated as:refNF is the estimated number of fragments that would be sequenced in a Whole Genome Bisulfite Sequencing (WGBS) experiment, that can be roughly calculated as the genome size (g) divided by the read length (r).cuRRBS output also contains other variables that may be of interest to the user (see ‘cuRRBS overview’). The robustness (R) of a given enzyme (combination) is calculated as:a,bEV is the EV for the optimal size range  and δ is the experimental error (in bp) that is assumed during the size selection step. The robustness will take values in the interval . This could further reduce the computational time, especially if combinations of more than two enzymes were being evaluated.The CPU time required by cuRRBS depends on several parameters, including the number of enzymes checked, the experimental error, the number of sites of interest or the genome size . The RAMin silico the ability of cuRRBS to enrich for the sites of interest in a selection of different biological systems where DNA methylation has an important functional role. In some of these systems, described below, previous analysis was performed in order to obtain the genomic coordinates for the sites:Exon-intron boundaries in human. Exons and introns were obtained from protein-coding genes using GENCODE annotation data. Those CpG sites that were found within ± 5 bp of a canonical splice site  were selected.Epigenetic clock in human. These sites were obtained from Horvath . Brieflyet al., 2016  were kindly provided by Maurano  or more than 50% gain in methylation relative to the genome average loss  were used in this analysis.Induced pluripotent stem cell (iPSC) demethylated and maintained sites in mouse. These were obtained as described previously , with anin vivo NRF1 binding sites in mouse. ChIP-seq data was processed as described in the original publication , with the exception of the human epigenetic clock system, where the sites were assigned the absolute value of the weights in the linear model , for a read length (r) of 75 bp and a Score threshold (c) of 0.25. In the mouse and human examples we considered 128 isoschizomer families that contained enzymes that were not sensitive to CpG methylation. In the case of Arabidopsis thaliana we used 28 isoschizomer families that contained enzymes that were not sensitive to 5mC in any context .cuRRBS was run in the different systems described above using the default parameters  and had base pairs removed from the 3′ end to avoid including filled-in nucleotides with artificial methylation states . The data was then mapped to the human genome  or the mouse genome  using Bismark (0.18.0) (XmaI-RRBS data generated on the Ion Torrent platform  and MspIplatform  were qua(0.18.0) . In eachTP): number of CGI-CpGs that cuRRBS predicted to be sequenced and were indeed found in the RRBS data.True positives (TN): number of CGI-CpGs that cuRRBS predicted to be absent and were not found in the RRBS data.True negatives (FP): number of CGI-CpGs that cuRRBS predicted to be sequenced but were not found in the RRBS data.False positives (FN): number of CGI-CpGs that cuRRBS predicted to be absent but were found in the RRBS data.False negatives  in RRBS (Reduced Representation Bisulfite Sequencing), which is used to enrich for CG dinucleotides (CpGs) contained in promoters and CpG islands  Figure . HoweverPrevious studies have tested the potential of other restriction enzymes and enzyme combinations to expand the range of CpG sites that can be targeted in a genome ,30,56,57We have developed a novel computational method (cuRRBS) that determines the optimal combination of restriction enzymes and size range to enrich for any given set of sites of interest in any genome. In other words, by modifying two of the steps in the original RRBS protocol Figure , cuRRBS in silico the potential of all single enzymes and double-enzyme combinations to enrich for the sites of interest using the following two key variables :NF, which reflects the theoretical number of fragments that will be sequenced after the size selection step. Assuming that the sequencing cost is proportional to NF, cuRRBS attempts to minimise this value.Score, which reflects the theoretical number of sites of interest that will be sequenced after the size selection step. cuRRBS attempts to maximise this value.The software takes as input the genomic coordinates that the user wants to target Figure . AfterwaNF and Score variables are positively correlated with one another, such that the more genomic fragments sequenced, the more sites of interest are likely to be contained within the reduced representation  and minimum Score that would be acceptable for a given application , a variable that combines both NF and Score into a single number . cuRRBS repeats this procedure for every single enzyme and enzyme combination and reports those with the best hits (i.e. those with the lowest EVs)  .Cost Reduction Factor (CRF). This estimates the theoretical fold-reduction in sequencing costs for the cuRRBS protocol when compared to Whole Genome Bisulfite Sequencing (WGBS).Robustness (R). This assesses how much the cuRRBS prediction varies if a slightly different size range is used :cuRRBS provides a way to effectively interrogate DNA methylation in any biological system for which the reference genome is available. Besides reducing the cost for organisms currently under intensive study , cuRRBS opens the door to the cost-effective study of DNA methylation in species with large genomes or where DNA methylation in non-CpG contexts is common, such as plants , which cin silico include genomic regions whose methylation status is important during cellular reprogramming   . In addiFurthermore, we have also observed that many of the top hits reported by cuRRBS are digestions of two restriction enzymes , highligαI)  and the other from a combination of two restriction enzymes (MspI and TaqαI) ,31. By eTo test the accuracy of cuRRBS predictions in the context of a single enzyme digestion, we utilised the non-canonical RRBS dataset generated from human DNA using the restriction enzyme XmaI . This darobustness variable as part of the cuRRBS output in order to judge the consequences of these experimental errors.Interestingly, the original theoretical size range that the study was aiming for (110–200 bp) was slightly different to the one achieved in the actual experiments (90–185 bp) . We ran αI  to NF = 331 058 (for MspI and TaqαI). This represents approximately a 30-fold difference in the number of recoverable fragments and demonstrates that cuRRBS predictions, even for low NF values, are experimentally feasible. Importantly, in the nine theoretical examples that we report .Lastly, the number of fragments that were theoretically recoverable in each of our experimental systems ranged from e report , the numcuRRBS provides a new framework that allows the user to optimise RRBS for the biological system of interest by using novel combinations of restriction enzymes. Therefore, cuRRBS makes the study of DNA methylation more affordable across all species for which genomic sequences are available. Furthermore, it can open the door to the design of future studies in a clinical context , which rCurrently, cuRRBS only considers combinations of up to two restriction enzymes. However, in the future, it would be possible to adapt the software to explore combinations that contain higher numbers of enzymes, which could theoretically allow targeting the sites of interest even more efficiently . MoreoveFinally, the potential of restriction enzymes to target different genomic coordinates is not limited to DNA methylation. As such, it would be conceivable for cuRRBS to be adapted to enrich for SNPs of interest ,62 or toαI-RRBS) respectively.cuRRBS and its documentation are freely distributed under GNU General Public License v3.0 and can be accessed via GitHub . The pubSupplementary DataClick here for additional data file.


Therefore, we hypothesized that MATE mediates dopamine secretion, a cationic catecholamine, into the tubule lumen, thereby regulating natriuresis. Here, we show that [3H]dopamine uptake in human (h) MATE1-, hMATE-2K- and mouse (m) MATE-expressing cells exhibited saturable kinetics. Fluid retention and decreased urinary excretion of dopamine and Na+ were observed in Mate1-knockout mice compared to that in wild-type mice. Imatinib, a MATE inhibitor, inhibited [3H]dopamine uptake by hMATE1-, hMATE2-K- and mMATE1-expressing cells in a concentration-dependent manner. At clinically-relevant concentrations, imatinib inhibited [3H]dopamine uptake by hMATE1- and hMATE2-K-expressing cells. The urinary excretion of dopamine and Na+ decreased and fluid retention occurred in imatinib-treated mice. In conclusion, MATE transporters secrete renally-synthesized dopamine, and therefore, urinary dopamine has the potential to be an index of the MATE transporter activity.Dopamine is a cationic natriuretic catecholamine synthesized in proximal tubular cells (PTCs) of the kidney before secretion into the lumen, a key site of its action. However, the molecular mechanisms underlying dopamine secretion into the lumen remain unclear. Multidrug and toxin extrusion (MATE) is a H The transport characteristics of MATE transporters were examined at 1 min in subsequent experiments because of technical limitations and reproducibility. An “overshoot” was observed because the driving force (an outward H+ gradient for MATE) was depleted during the uptake experiments, and substrate backflow occurred [3H]dopamine uptake by hMATE1, hMATE2-K and mMATE1, concentration-dependent uptake was examined, and the dopamine uptake by all three transporters exhibited saturable kinetics, following the Michaelis–Menten equation (Vmax), Michaelis–Menten constant (Km) and Vmax/Km values are summarized in 3H]dopamine transport activity (Vmax/Km) was mMATE1 > hMATE1, hMATE2-K.To examine whether dopamine is a substrate of MATE transporters, we carried out uptake experiments, which are frequently used to evaluate MATE transport properties ,23,24,25h) MATE1 a, hMATE2h) MATE1 a and mouh) MATE1 b compareoccurred ,27. To eequation c,d,e. Th+ excretion in vivo, we carried out acute saline volume expansion experiments in wild-type and Mate1-knockout mice. This is because intravenous saline infusion is known to accelerate dopamine synthesis in the kidney and promotes urinary Na+ excretion dopamine transport activity of hMATE1 and hMATE2-K was similar  . Further similar . Functio+ excretion effect remains unclear. The urinary Na+ level in the volume expansion period was 38.9 and 9.8 µ equivalent (Eq)/h for the wild-type and Mate1-knockout mice, respectively  are taken up from the circulation by the membrane potential-dependent OCT2, a basolateral transporter, and secreted into the tubular lumen by MATE1 and MATE2-K, which are both apical transporters ,41,42. DCtrough) of imatinib must be >1000 ng/mL (2 µM) to achieve clinical efficacy in patients with chronic myelocytic leukemia (CML) or gastrointestinal stromal tumors dopamine  were added. In the cis-inhibition experiments, various concentrations of imatinib, dasatinib and nilotinib were added in addition to [3H]dopamine. Following the indicated incubation times at 37 °C, the medium containing [3H]dopamine was aspirated, and the monolayers were rapidly rinsed twice with 1 mL of ice-cold incubation medium without 10 µM U0521. The cells were dissolved in 0.5 mL of 0.5 normality (N) sodium hydroxide (NaOH), and then, the radioactivity in 200 µL was determined using liquid scintillation counting following addition to 2 mL of Ultima Gold (PerkinElmer). The protein content of each NaOH solution was determined using a Bio-Rad Protein Assay Kit  by using bovine γ-globulin as a standard. The Km and Vmax values were calculated from the saturation curve using the Michaelis–Menten equation, V = Vmax[S]/(Km + [S]), with Kaleidagraph Version 4.00  after the nonspecific uptake values were subtracted. The nonspecific uptake values were calculated from the linear model obtained from 1 and 5 mM [3H]dopamine uptake values in the presence of 5 mM tetraethylammonium. The IC50 values were calculated from the inhibition plots using the equation, V = V0/[1 + ([I]/IC50)n], with Kaleidagraph Version 4.00, where V and V0 are the uptake amounts of [3H]dopamine in the presence and absence of the inhibitor, respectively, [I] is the concentration of the inhibitor and n is the Hill coefficient.The MATE-expressing HEK293 cells were preincubated with 0.2 mL of incubation medium . The fixed tissues were embedded in OCT compound , frozen rapidly in liquid nitrogen; 6 µm-thick sections were cut and then incubated with 10 mM of citrate buffer pH 6 (D1 and D5) or 1 mM of EDTA-2Na buffer pH 9 (NHE3) at 95 °C for 40 min for antigen retrieval. After washing with PBS, the sections were incubated with 3% skim milk in PBS at room temperature for 15 min. Following another PBS wash, the sections were incubated at 4 °C overnight with specific antiserum specific for D1  (1:4500 dilution), D5  (1:1600 dilution) or anti-NHE3 antibody  (1:1000 dilution). Following three PBS washes (5 min each), the sections were incubated with hydrogen peroxide (0.3% in methanol) at room temperature for 30 min. After three additional PBS washes (5 min each), the sections were incubated with EnVision Horseradish Peroxidase  at room temperature for 30 min. After washing with distilled water, the nuclei were stained with hematoxylin, and the images were captured using an Aperio .Mate1-knockout mice were anesthetized with isoflurane. Blood was collected from the inferior vena cava, and the chemical parameters were measured using a CHEM8+ cartridge and the i-STAT analyzer . Then, the mouse carcasses were heated in an oven at 60 °C over 14 days or until there was no further change in weight loss on 2 consecutive days. The difference between the live mouse weight and dried carcass weight was used to determine the fluid content of each mouse [The imatinib-  and vehicle-treated (lactated Ringer’s solution) wild-type, intact wild-type and intact  Mate1-knch mouse . The tot4 was added to kidney collection tube to prevent dopamine degradation. In the imatinib inhibition study, 500 mg/kg imatinib dissolved in lactated Ringer’s solution were administered orally to wild-type mice 3 h or immediately before saline infusion.For the saline infusion, a catheter was inserted into the femoral vein with polyethylene tubing  in mice anesthetized with 50 mg/kg of pentobarbital. To collect the urine, the urinary bladder was catheterized with SP-31 tubing . The saline was infused using an automatic infusion pump  at 0.24 mL/h for 30 min in the stabilization period, 0.12 mL/h for 60 min in the control period and 3 mL/h for 30 min in the volume expansion period. Following the latter period, the infusion pump was stopped, and the mice were then left for 30 min before the kidney samples were collected. The urinary electrolyte levels of the mice were measured using an ion-selective electrode . For dopamine detection, 1 N HCl was added to the urinary collection tube, and 0.2 N HClOThe catecholamines in the urine reagent kit  were used for dopamine sample preparation according to the manufacturer’s instructions with some modifications. For urinary dopamine detection, the urine was first diluted to a total volume of 300 µL with 1% 6 N HCl, and then, 50 µL of the internal standard (DHBA) were then added, followed by 6 mL of neutralization buffer. We applied 600 µL elution buffer to the column in a final step and added formic acid to the collected eluate. Forty microliters of sample was injected into the LC-MS/MS system after being filtrated with a filter . The kidney homogenate was diluted 5- or 6-fold with 1% 6 N HCl. After centrifugation, 300 µL of the supernatant were subjected to the same procedure used for the urine sample above.For imatinib detection, the mouse plasma and kidney homogenate samples were diluted 40× and 400× with saline, respectively, and 10 µL of roscovitine solution (1 µg/mL) were added to 100 µL of the diluted samples. Then, the samples were deproteinized with 200 µL of acetonitrile for 10 min with agitation. After centrifugation  using the Centrifuge 5424 , the supernatant was diluted with 200 µL of 0.2% formic acid, and 1 µL of the sample was injected into the LC-MS/MS system after filtration using a filter .m/z 154.1 → 91.2 for dopamine and m/z 140.1 → 77.1 for DHBA. For imatinib detection, gradient elution was carried out at a flow rate of 0.2 mL/min. Roscovitine was used as an internal standard. Detection was carried out in the multiple reaction-monitoring mode by monitoring ion transitions of m/z 494.171 → 393.900 for imatinib and m/z 355.047 → 233.100 for roscovitine.The dopamine and imatinib were analyzed by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A liquid chromatography system consisting of a Prominence series chromatograph  coupled to an API4000 triple-quadrupole tandem mass spectrometer  was used for dopamine detection, and an Eksigent ultra-performance liquid chromatography (AB SCIEX) coupled to a QTRAP4500 triple-quadrupole tandem mass spectrometer (AB SCIEX) was used for imatinib detection. An Inertsil ODS-3  was used for chromatographic separation. The mobile phase was composed of 0.1% formic acid and acetonitrile containing 0.1% formic acid. For dopamine detection, gradient elution was carried out at a flow rate of 0.25 mL/min. 3,4-dihydroxybenzylamine (DHBA) was used as an internal standard. The detection was carried out in the multiple reaction monitoring mode by monitoring ion transitions of t-test.All data are expressed as the mean ± standard error (S.E.) and were analyzed statistically using the unpaired


The recently theoretically described nuclear spin-induced circular dichroism (NSCD) is a promising method for the optical detection of nuclear magnetization. NSCD involves both optical excitations of the molecule and hyperfine interactions and, thus, it offers a means to realize a spectroscopy with spatially localized, high-resolution information. To survey the factors relating the molecular and electronic structure to the NSCD signal, we theoretically investigate NSCD of twenty structures of the four most common nucleic acid bases . The NSCD signal correlates with the spatial distribution of the excited states and couplings between them, reflecting changes in molecular structure and conformation. This constitutes a marked difference to the nuclear magnetic resonance (NMR) chemical shift, which only reflects the local molecular structure in the ground electronic state. The calculated NSCD spectra are rationalized by means of changes in the electronic density and by a sum-over-states approach, which allows to identify the contributions of the individual excited states. Two separate contributions to NSCD are identified and their physical origins and relative magnitudes are discussed. The results underline NSCD spectroscopy as a plausible tool with a power for the identification of not only different molecules, but their specific structures as well. Recent years have seen both experimental progress12346789101112131415196913122122The NMOS effects offer possibilities to develop new optical spectroscopy approaches with a high, nucleus-specific and spatial resolution, due to the fact that hyperfine interactions localized at magnetic nuclei are involved, in analogy to nuclear magnetic resonance (NMR) spectroscopyi.e., the rotation of linearly polarized light by an ensemble of spin-polarized nuclei, was described in 2006 by Savukov, Lee and Romalis6256912131415The first observed NMO phenomenon, NSOR, NSCD manifests itself as the difference in the absorption of left- and right-circularly polarized light or, equivalently, as ellipticity induced into the incident, linearly polarized light, in samples with macroscopic nuclear spin polarization in the direction parallel with the light beam. It should be stressed that the NSCD effect is not a perturbation of natural or magnetic circular dichroism, but has a different physical origin. NSCD arises from the real part of the nuclear spin-inflicted antisymmetric polarizability, which gains non-vanishing values only at frequencies in the vicinity of optical transitionsTheoretical studies of NSCD were so far mainly concerned with small model organic moleculesBd and Ba terms based on their band-shapes resembling those characteristic for dispersion and absorption, respectively. We describe how the spectra exhibit the local nature of NSCD via the mutual orientation of the transition moments involving the orbital hyperfine operator Bd-term dominated spectra.This paper thus dives deeper into the effects underlying NSCD spectra in order to further the understanding of this phenomenon. We show that NSCD is an effect with pronounced sensitivity to the electronic structure and that even changing the structure to a different conformer may significantly influence the spectra. This sensitivity can be linked to the changes in the electron density upon excitation. Conversely, even molecules with rather different structures can give rise to similar spectra if the spatial distribution of their excited state densities is comparable. By using a sum-over-states approach we also show that the NSCD signal can be decomposed into two contributions, which we call The studied structures of all four nucleic acid bases are presented in cyt2/cyt3 shown in cyt2/cyt3 conformers have remarkably similar NSCD signal as well as very similar difference densities, as could be expected since the hydroxyl group does not participate extensively in the excitations. The spectra are simple and all atoms give rise to a similar couplet feature with the two lobes at the energies of the first two excitations. The signals corresponding to the C1, C2 and C4 nuclei have the positive sign on the low energy side of the couplet, whereas the opposite situation pertains for the C3 nucleus. The reason for this behaviour lies in the different orientation of the 3, as compared to others, as will be discussed later.We begin with the discussion of the NSCD spectra of the pair cyt2/cyt3, the pair cyt4/cyt5  is shown in The second group of thymine structures . As a result, nucleus in one structure may not have the corresponding, chemically similar partner in the other one, making the comparison qualitatively different from the case of 13C. For this reason, the discussion of 1H nuclei in the present work is limited only to comparison within pairs, which consist of conformers with identical bonding arrangements. The results are included in the i.e., that similarities in the difference densities correspond to similar NSCD.There is, however, another set of nuclei suitable for experiments, protons. The analysis in the To rationalize the NSCD signals of different nuclei, we performed an analysis in terms of the individual excited states. As the complex polarization propagator  calculations used to produce the spectra of the previous figures do not allow to simply distinguish such individual contributions, we used the sum-over-states (SOS) approach, instead.K, as16The NSCD signal is computed through quadratic response functions and can be expressed, for nucleus ω, μ0, c0, NA and 〈IK〉 refer to the angular frequency, permittivity of vacuum, speed of the light in vacuum, Avogadro constant and nuclear spin polarization, respectively. For a particular transition from the ground electronic state |0〉 to the excited state |m〉, NSCD is proportional to the residues of the quadratic response function29where k〉 is a member of a set of further excited states with the corresponding energies of the states E0, Em and Ek, m〉, ωb is the frequency of the external perturbing field, α, β, γ denote Cartesian coordinates.where |Bd-term , both electric dipole transition moments are between the ground state and an excited state, while the matrix element of Bd-term contains the difference of the energies of the two excited states.The two contributions in eq.  are qualBa-term (since its bandshape resembles an absorption band), involves the ground state in one matrix element of the electric dipole moment, as well as in that of the m〉 and |k〉 involves now the operator The second term in eq. , which wBd and Ba contributions. The denominator in Ba-type terms corresponds to excitation energies, hence it can never be smaller than the relative energy of the first excited state. In addition, the Ba-type terms can be expected to contribute on account of the denominators progressively less to the NSCD spectrum as excited state energies rise. On the other hand, the energy difference of the two excited states in the Bd-type terms can in principle be very small, allowing a very large contribution from this type of terms. For this reason, it may be reasonably expected that the Bd-terms are dominant in cases of excited states packed closely together.The energy denominators play a significant role in the overall NSCD intensity and the relative significance of the Bd and Ba terms of individual transitions from the ground state contribute to the overall spectrum in Bd contributions to the NSCD signal of a pair of excited states |k〉 and |m〉 are identical in magnitude but of opposite signs when the states are interchanged. Where the electric dipole operators appear “symmetrically” in the first term of Bd terms of a pair of excited states, close to one another in energy, contribute to the spectrum  with a k〉} running over the first 10 excited states. To compare the results of the SOS procedure with those of CPP, we have numerically fitted Lorentzian lineshapes centered at each excitation energy to the CPP spectral points. The intensities obtained correlate well with the SOS results ,,2), withults see . The corBd-term dominated spectra with a large magnitude of the NSCD signal, we can discuss in more detail the information content of the NSCD spectrum for such systems. As a test case we have chosen cyt2, as it provides a simple spectrum dominated by just one couplet, for all the 13C nuclei. For this case we can neglect the Ba-term contribution altogether and approximate the Bd terms by including just the two interacting states - the first and the second one in eq.  of In this paper we have performed a computational study, using a recent implementation of the complex polarization propagator-quadratic response theory method, of the theoretically predicted, but not yet experimentally observed nuclear spin-induced circular dichroism effect. Via NSCD, a sensitive optical spectroscopy with atom-specific resolution might be realized, through the modulation of the dynamic dipolar polarizability of the molecule by magnetic hyperfine interaction localized at the nuclei. We have demonstrated on a set of 20 structures of nucleic acid bases that different molecular structures of related systems provide distinct NSCD features. In contrast to NMR spectroscopy, which provides nucleus-specific resolution with the spectral parameters determined by the electronic ground state of the systems, the shape of the NSCD spectrum is heavily correlated to the differences in the electron density between the ground and excited states. Molecules that undergo similar changes in density upon optical excitation give rise to qualitatively similar NSCD signals, even when their molecular structure is rather different, such as in the case of certain structures of thymine and cytosine. Consequently, there is no simple relation between the local chemical neighbourhood of the nucleus and its spectral response, in contrast to NMR.Bd- and Ba-terms. The Bd-term gives rise to couplet-like features in the spectra, which are particularly distinctive in molecules involving a pair of narrowly spaced excited states. The Bd-term is related to the transition moment of the magnetic hyperfine operator between the two excited states, and it provides a measure of the component of this moment perpendicular to the plane defined by cross product between the two electric dipole transition moment vectors between the ground state and the two excited states. Moreover, for systems dominated by the Bd-term, the sum-over-states procedure (with a limited range of states) provides a very good agreement with the rigorous response theory-based calculations. For the time being, an analogous physical interpretation relating the Ba-term to a small number of readily interpreted contributions, remains elusive to us.We have also performed a sum-over-states perturbational analysis of NSCD arising from the lowest excited states, in order to gain insight into the physical origins of the effect. It turns out that the NSCD signal can be understood as being composed from two qualitatively different contributions, which we name the Bd-terms.Due to its sensitivity to the structure of the excited states, NSCD shows potential for the identification of molecules based on their excitation properties. Moreover, direct insight into the relation between spatially extended electric dipole transition moments and the transition moment of the localized orbital magnetic hyperfine operator can be obtained in systems with narrowly spaced excited states, when NSCD is dominated by the Finally, we would like to briefly comment on the experimental feasibility of the NSCD measurement. Our results predict the magnitude of the NSCD effect to be on the order of tens to hundreds of microradians for fully polarized nuclei. Therefore, the NSCD effect is actually orders of magnitude stronger than the already experimentally measured NSOR. It is reasonable to expect it to be also measurable using a similar instrumentation. For example, the inherent shortness of a single measurement limited by nuclear magnetization relaxation time can be compensated for by repeating the data acquisition or by using spin-locking techniques. This approach is clearly viable as evidenced in previous experiments with NSOR. Additionally, NSCD is modulated at the Larmor frequency of the nuclear precession, while other potentially interfering effects like natural circular dichroism or classical magneto-optic effects are static in nature and can thus be easily separated and filtered out. We believe that a continued development of this branch of nuclear magneto-optic spectroscopy may provide novel opportunities for the investigation of molecular structure and properties.A development version of the DALTON program package333616et al.−1 was chosen to represent a reasonable situation for UV-VIS absorption bands in the aqueous phase. The NSCD signal was calculated in the wavelength range of 200–280 nm with the step of 2 nm, which covers the lowest optical excitation energies of the target systems and should be experimentally available to NSCD measurements in the future. For reasons of practicality of the forthcoming experiments, we were mainly interested in the NSCD response corresponding to the 13C (and 1H) nuclei as it is more difficult to maintain the macroscopic nuclear spin polarization for quadrupolar 17O and 14N nuclei due to their rapid relaxation, or the spin 15N due to its low abundance.The NSCD spectra were calculated with DALTON using the complex polarization propagator (CPP) method, also termed the damped response function formalism1639The residues of the linear and quadratic response functions for the involved electric dipole and orbital magnetic hyperfine operators required for the sum-over-states (SOS) approach were obtained from calculation performed by the adapted DALTON code.The excited-state differential densities were obtained using Turbomole 6.5Molecular graphics were created using the UCSF Chimera packageHow to cite this article: Štěpánek, P. et al. Relation between molecular electronic structure and nuclear spin-induced circular dichroism. Sci. Rep.7, 46617; doi: 10.1038/srep46617 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.