Molecular-Level Structural Analysis of siRNA-Loaded Lipid Nanoparticles by 1H NMR Relaxometry: Impact of Lipid Composition on Their Structural Properties.

1H NMR relaxometry was applied for molecular-level structural analysis of siRNA-loaded lipid nanoparticles (LNPs) to clarify the impact of the neutral lipids, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, on the physicochemical properties of LNP. Incorporating DSPC and cholesterol in ionizable lipid-based LNP decreased the molecular mobility of ionizable lipids. DSPC reduced the overall molecular mobility of ionizable lipids, while cholesterol specifically decreased the mobility of the hydrophobic tails of ionizable lipids, suggesting that cholesterol filled the gap between the hydrophobic tails of ionizable lipids. The decrease in molecular mobility and change in orientation of lipid mixtures contributed to the maintenance of the stacked bilayer structure of siRNA and ionizable lipids, thereby increasing the siRNA encapsulation efficiency. Furthermore, NMR relaxometry revealed that incorporating those neutral lipids enhanced PEG chain flexibility at the LNP interface. Notably, a small amount of DSPC effectively increased PEG chain flexibility, possibly contributing to the improved dispersion stability and narrower size distribution of LNPs. However, cryogenic transmission electron microscopy represented that adding excess amounts of DSPC and cholesterol into LNP resulted in the formation of deformed particles and demixing cholesterol within the LNP, respectively. The optimal lipid composition of ionizable lipid-based LNPs in terms of siRNA encapsulation efficiency and PEG chain flexibility was rationalized based on the molecular-level characterization of LNPs. Moreover, the NMR relaxation rate of tertiary amine protons of ionizable lipids, which are the interaction site with siRNA, can be a valuable indicator of the encapsulated amount of siRNA within LNPs. Thus, NMR-based analysis can be a powerful tool for efficiently designing LNP formulations and their quality control based on the molecular-level elucidation of the physicochemical properties of LNPs.

Feature Selection for the Interpretation of Antioxidant Mechanisms in Plant Phenolics.

Antioxidants, represented by plant phenolics, protect living tissues by scavenging reactive oxygen species through diverse reaction mechanisms. Research on antioxidants is often individualized, for example, focusing on the evaluation of their activity against a single reactive oxygen species or examining the antioxidant properties of compounds with similar structures. In this study, multivariate analysis was used to comprehensively examine antioxidant properties. Eighteen features were selected to explain the results of the antioxidant capacity tests. These selected features were then evaluated by supervised learning, using the results of the antioxidant capacity assays. Dimension-reduction techniques were also used to represent the compound space with antioxidants as a two-dimensional distribution. A small amount of data obtained from several assays provided us with comprehensive information on the relationships between the structures and activities of antioxidants.

Prediction and Chemical Interpretation of Singlet-Oxygen-Scavenging Activity of Small Molecule Compounds by Using Machine Learning.

A chemically explainable machine learning model was constructed with a small dataset to quantitatively predict the singlet-oxygen-scavenging ability. In this model, ensemble learning based on decision trees resulted in high accuracy. For explanatory variables, molecular descriptors by computational chemistry and Morgan fingerprints were used for achieving high accuracy and simple prediction. The singlet-oxygen-scavenging mechanism was explained by the feature importance obtained from machine learning outputs. The results are consistent with conventional chemical knowledge. The use of machine learning and reduction in the number of measurements for screening high-antioxidant-capacity compounds can considerably improve prediction accuracy and efficiency.

Effects of change in the formulation of lanthanum carbonate on laboratory parameters.

Lanthanum carbonate (LC) is available in the two formulations of chewable tablets and granules. In this study, we changed the formulation of LC from chewable tablet to granules, and compared the laboratory parameters for 3 months before and after changing formulation in 58 hemodialysis (HD) patients. We also surveyed patients about their preferences for the two formulations. The mean serum phosphorus (P) levels decreased significantly (P < 0.01) from 6.7 mg/dL to 6.4 mg/dL after the change. The levels for serum albumin and geriatric nutritional risk index increased significantly (P < 0.01). Serum calcium levels also increased significantly (P < 0.01), while serum intact parathyroid hormone levels decreased significantly (P < 0.01). In the survey, approximately half of the patients responded that the granules were easier to take than the chewable tablets. These findings suggest that changing the formulation of LC to granules may reduce serum P levels of the HD patients in clinical practices.

A comparison of systemic inflammation-based prognostic scores in patients on regular hemodialysis.

BACKGROUND/AIMS: Systemic inflammation-based prognostic scores have prognostic power in patients with cancer, independently of tumor stage and site. Although inflammatory status is associated with mortality in hemodialysis (HD) patients, it remains to be determined as to whether these composite scores are useful in predicting clinical outcomes. METHODS: We calculated the 6 prognostic scores [Glasgow prognostic score (GPS), modified GPS (mGPS), neutrophil-lymphocyte ratio (NLR), platelet lymphocyte ratio (PLR), prognostic index (PI) and prognostic nutritional index (PNI), which have been established as a useful scoring system in cancer patients. We enrolled 339 patients on regular HD (age: 64 ± 13 years; time on HD: 129 ± 114 months; males/females = 253/85) and followed them for 42 months. The area under the receiver-operating characteristics curve was used to determine which scoring system was more predictive of mortality. RESULTS: Elevated GPS, mGPS, NLR, PLR, PI and PNI were all associated with total mortality, independent of covariates. If GPS was raised, mGPS, NLR, PLR and PI were also predictive of all-cause mortality and/or hospitalization. GPS and PNI were associated with poor nutritional status. Using overall mortality as an endpoint, the area under the curve (AUC) was significant for a GPS of 0.701 (95% CI: 0.637-0.765; p < 0.01) and for a PNI of 0.616 (95% CI: 0.553-0.768; p = 0.01). However, AUC for hypoalbuminemia (<3.5 g/dl) was comparable to that of GPS (0.695, 95% CI: 0.632-0.759; p < 0.01). CONCLUSION: GPS, based on serum albumin and highly sensitive C-reactive protein, has the most prognostic power for mortality prediction among the prognostic scores in HD patients. However, as the determination of serum albumin reflects mortality similarly to GPS, other composite combinations are needed to provide additional clinical utility beyond that of albumin alone in HD patients.

Association of HCV core antigen seropositivity with long-term mortality in patients on regular hemodialysis.

Anti-hepatitis C virus (HCV) antibody seropositivity is independently associated with poor prognosis in hemodialysis (HD) patients. However, anti-HCV antibody cannot distinguish between patients with active infection and those who have recovered from infection. We therefore aimed in this study to examine the association of HCV core antigen (HCVcAg) seropositivity with mortality in HD patients. We first measured serum HCVcAg using an immunoradiometric assay and anti-HCV antibody in 405 patients on regular HD, and followed them for 104 months. There were 82 patients (20.2%) who had been positive for anti-HCV antibodies; 57 (69.5%) of these were positive for HCVcAg. During the follow-up, 29 patients were excluded, so we tested the association of HCVcAg seropositivity with all-cause, cardiovascular (CV) and non-CV mortalities in 376 patients. A total of 209 patients (55.6%) had expired during the observational period, 92 out of them due to CV causes. After adjusting for comorbid parameters, HCVcAg was independently associated with overall mortality (HR 1.61, 95% CI 1.05-2.47, p < 0.05). HCV infection was significantly related to liver disease-related mortality. Past HCV infection also contributed to CV mortality (HR 2.63, 95% CI 1.27-5.45, p < 0.01). In contrast, anti-HCV antibody and HCVcAg seropositivities did not associate with infectious disease-related and cancer-related (expect for hepatocellular carcinoma) mortality. It follows from these findings that HCVcAg serology is associated with all-cause and CV mortality in HD patients.

Influence of the assay for measuring serum albumin on corrected total calcium in chronic hemodialysis patients.

The Japanese Society for Dialysis Therapy guideline for secondary hyperparathyroidism recommends the use of albumin-corrected serum Ca as a therapeutic target in chronic hemodialysis patients; however, the assay used for albumin measurement may affect the corrected Ca level. In this study, we examined the impact of the albumin assay on corrected Ca levels in hemodialysis patients. We measured serum albumin using bromocresol green (BCG) and modified bromocresol purple (BCP) assays, and corrected Ca for albumin using Payne's formula in 422 hemodialysis patients (age 66±13 years; time on hemodialysis 116±111 months). Serum albumin values were 3.7±0.4 (1.4-4.6) g/dL by BCG and 3.3±0.4 (1.0-4.3) g/dL by modified BCP, with the differences between the two assays ranging from 0.0 to 0.6 with a mean of 0.35±0.09 g/dL. Serum C-reactive protein and globulin values were significantly higher in patients with differences in albumin greater than 0.5 g/dL (P<0.01). Based on the BCG method, 71 patients (16.8%) were classified with hypocalcemia, 51 (12.1%) with hypercalcemia, and 300 (70.0%) as normocalcemic. In contrast, when using modified BCP, 33 patients (7.9%) were labeled as hypocalcemic, while 92 (21.8%) were hypercalcemic. Depending on the use of either BCG or modified BCP, a discrepancy of classification was observed in 79 patients (18.7%): 41 patients were re-classified from normocalcemic to hypercalcemic, and 38 patients were re-classified from hypocalcemic to normocalcemic by selecting the modified BCP assay. These findings suggest that the type of assay used for albumin measurement has an impact on albumin-corrected Ca levels.

Modulatory role of dopamine D2 receptors and fundamental role of L-type Ca2+ channels in the induction of long-term potentiation in the basolateral amygdala-dentate gyrus pathway of anesthetized rats.

We have previously found that the induction of long-term potentiation in the synaptic pathway from the basolateral amygdala to the dentate gyrus (BLA-DG LTP) is regulated by L-type Ca(2+) channels, dopamine D(2) receptors and GABAergic inhibition. In the present study, we investigated possible relations among the three mechanisms by using anesthetized rats. Blockade of GABAergic inhibition with picrotoxin abolished both the inhibitory effect of the dopamine D(2) receptor antagonist chlorpromazine and the promoting effect of the dopamine D(2) receptor agonist quinpirole on the induction of BLA-DG LTP. However, the inhibitory effect of the L-type Ca(2+) channel blocker verapamil on BLA-DG LTP was not affected by picrotoxin. These results suggest that the role of dopamine D(2) receptors in the induction of BLA-DG LTP is modulatory and depends on GABAergic inhibition, whereas the role of L-type Ca(2+) channels is fundamental.

Basolateral amygdala D1- and D2-dopaminergic system promotes the formation of long-term potentiation in the dentate gyrus of anesthetized rats.

We have previously found that the induction of hippocampal long-term potentiation (LTP) is modulated by neuron activities in the basolateral amygdala (BLA). However, little is known about what neurotransmitter system in the BLA contributes to modulation of hippocampal LTP. In the present study, we investigated possible involvement of BLA dopaminergic system in the induction of LTP at the perforant path (PP)-dentate gyrus (DG) granule cell synapses of anesthetized rats. The induction of PP-DG LTP was significantly attenuated by intra-BLA injection of the D(1) receptor antagonist SCH23390 (2 or 6 nmol) or the D(2) receptor antagonists, chlorpromazine (30 or 100 nmol) or haloperidol (4.4 or 13.3 nmol). The effects of SCH23390 and haloperidol were abolished by concomitant intra-BLA injection of the D(1) receptor agonist SKF38393 (17 nmol) and the D(2) receptor agonist quinpirole (3 nmol), respectively. Furthermore, lesioning with 6-hydroxydopamine of the ventral tegmental area, the origin of the dopaminergic system projecting to the BLA, resulted in attenuated PP-DG LTP, which was restored by intra-BLA injection of SKF38393 or quipirole. These results suggest that the induction of PP-DG LTP is promoted by the BLA dopaminergic system via both D(1) and D(2) receptors.

A helix-to-coil transition at the epsilon-cut site in the transmembrane dimer of the amyloid precursor protein is required for proteolysis.

Processing of amyloid precursor protein (APP) by gamma-secretase is the last step in the formation of the Abeta peptides associated Alzheimer's disease. Solid-state NMR spectroscopy is used to establish the structural features of the transmembrane (TM) and juxtamembrane (JM) domains of APP that facilitate proteolysis. Using peptides corresponding to the APP TM and JM regions (residues 618-660), we show that the TM domain forms an alpha-helical homodimer mediated by consecutive GxxxG motifs. We find that the APP TM helix is disrupted at the intracellular membrane boundary near the epsilon-cleavage site. This helix-to-coil transition is required for gamma-secretase processing; mutations that extend the TM alpha-helix inhibit epsilon cleavage, leading to a low production of Abeta peptides and an accumulation of the alpha- and beta-C-terminal fragments. Our data support a progressive cleavage mechanism for APP proteolysis that depends on the helix-to-coil transition at the TM-JM boundary and unraveling of the TM alpha-helix.